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Agronomic Performance and Genetic Diversity of Common Bean (Phaseolus Vulgaris) Varieties in Haiti

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Title:
Agronomic Performance and Genetic Diversity of Common Bean (Phaseolus Vulgaris) Varieties in Haiti
Creator:
Mainviel, Riphine
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[Gainesville, Fla.]
Florida
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University of Florida
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english
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Horticultural Sciences
Committee Chair:
Meru,Geoffrey Mugambi
Committee Co-Chair:
Vallejos,Carlos E
Committee Members:
Colbert,Raphael Wesly

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agronomic -- bean -- breeding -- genetic
Horticultural Sciences -- Dissertations, Academic -- UF
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theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
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Horticultural Sciences thesis, M.S.

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Abstract:
Common bean (Phaseolus vulgaris L.) is one of the essential food legumes in the world and plays a vital role in providing nutritional security and revenue for low-income families in the Caribbean, including Haiti. Despite its economic importance, bean production in Haiti is constrained by many biotic and abiotic factors that limit yield. Besides, most bean cultivars currently in the market are low-yielding, averaging 0.6 tons/ hectare, which is well below the world average (0.86 tons/ ha). The current study aimed to evaluate the agronomic performance of 13 elite dry bean-breeding lines currently under development by the USAID-AREA and the Legume Innovation Lab programs at a highland location in Haiti. Moreover, the study aimed to determine the genetic diversity among a collection of 92 Haitian bean cultivars using genotyping by sequencing. Analysis of variance and trait mean separation was done in R statistical package, while a UPGMA dendrogram based on genetic dissimilarity matrix was constructed using Darwin software. Significant differences were observed for most of the recorded parameters. The yield of bean varieties ranged from 0.48 to 1.24 tons/ha, with a mean of 0.89 t/ha. Among the traits measured, the number of pods per plant and number of seeds per pod showed the highest correlation with dry seed yield, thus may be used for indirect selection of seed yield in common bean. Genetic diversity analysis revealed a prevalence of Mesoamerican gene pool within the accession collection, with a few of Andean origin, and some level of admixture. ( en )
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In the series University of Florida Digital Collections.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2019.
Local:
Adviser: Meru,Geoffrey Mugambi.
Local:
Co-adviser: Vallejos,Carlos E.
Statement of Responsibility:
by Riphine Mainviel.

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AGRONOMIC PERFORMANCE AND GENETIC DIVERSITY OF COMMON BEAN ( PHASEOLUS VULGARIS ) VARIETIES IN HAITI By RIPHINE MAINVIEL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2019

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2019 Riphine Mainviel

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To my husband, family and mentor s

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4 ACKNOWLEDGMENTS First, I would like to express my sincere gratitude to the Almighty God for giving me life, health, intelligence, and the means necessary to carry out this work. I want to thank the USAID and UF/ IFAS global (AREA) for granting me this scho larship to pursue graduate studies and for providing funding for this research project. I want to express my gratitude to my supervisor Dr. Geoffrey Meru, for his support, advice, and mostly his patience during my academic career at the University of Flori da. I am very grateful to every member of my master committee, namely Dr. Geoffrey Meru, Dr. Raphael Colbert, and Dr. C. Eduardo Vallejos, for their kind guidance for my research project. I much appreciate the support from Area project coordinators includi ng Dr. Rosalie Koenig and Dr. Lemane Delva. I also thank all the members of my lab, including Marie Darline Dorval, Vincent N. Michael, Yuqing Fu, and Alexis Ramos, for their help and encouragement. I am grateful to agronomist Jean Joany Moline for his sup port with the field trial in Haiti. I want to thank the employees of the National Seed Service of the Ministry of Agriculture of Haiti, including Dr. Emmanuel Prophete and a gronomist Gasner Demosthene for their support. I want to express my gratitude to my father Alix Mainviel, my brothers and sisters and my cousin Wilson Minviel, for their love and precious help. I also thank all my friends, especially Elsie Gaspard and Tamar Zamor, for their encouragement. Lastly, my most sincere appreciation for my lovin g husband Josue St Fort whose support was invaluable throughout my graduate program.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ...... 4 LIST OF TABLES ................................ ................................ ................................ ................ 7 LIST OF FIGURES ................................ ................................ ................................ .............. 8 LIST OF ABBREVIATIONS ................................ ................................ ................................ 9 ABSTRACT ................................ ................................ ................................ ........................ 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ........ 13 Significance of the Study ................................ ................................ ............................ 14 Objectives ................................ ................................ ................................ .................... 15 2 LITERATURE REVIEW ................................ ................................ .............................. 16 Environmental Adaptation ................................ ................................ .......................... 16 Nutritional Value of Common Bean ................................ ................................ ............ 17 Bean Production in Haiti ................................ ................................ ............................. 17 Production Constraints in Haiti ................................ ................................ ................... 18 Bean Breeding in Haiti ................................ ................................ ................................ 19 Genetic Di versity of Common Bean ................................ ................................ ........... 20 3 AGRONOMIC PERFORMANCE OF COMMON BEAN VARIETIES IN HAITI ......... 27 Background ................................ ................................ ................................ ................. 27 Materials and Methods ................................ ................................ ............................... 28 Plant Materials ................................ ................................ ................................ ...... 28 Experimental Site and Research Design ................................ ............................. 28 Data Analysis ................................ ................................ ................................ ........ 29 Results ................................ ................................ ................................ ........................ 29 Agronomic Traits ................................ ................................ ................................ .. 29 Phenological and Morphological Traits ................................ ............................... 30 Qualitative Traits ................................ ................................ ................................ .. 31 Evaluation of Common Bean Genotypes for Disease Resistance ..................... 31 Phenotypic Correlations ................................ ................................ ....................... 32 Principal Component Analysis ................................ ................................ ............. 32 Discussion ................................ ................................ ................................ ................... 33 Limitations ................................ ................................ ................................ ................... 36 4 GENETIC DIVERSITY AMONG 92 COMMON BEAN VARIETIES COLLECTED ACROSS HAITI ................................ ................................ ................................ ........... 54

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6 Background ................................ ................................ ................................ ................. 54 Materials and Methods ................................ ................................ ............................... 56 Plant Materials ................................ ................................ ................................ ...... 56 Seed Germination and DNA Extraction ................................ ............................... 56 Genotyping by Sequencing ................................ ................................ .................. 57 Data Analysis ................................ ................................ ................................ ........ 57 Results ................................ ................................ ................................ ........................ 58 Genotyping ................................ ................................ ................................ ........... 58 Population Structure ................................ ................................ ............................. 58 Phylogenetic Tree ................................ ................................ ................................ 59 Principal Component Analysis ................................ ................................ ............. 59 Disc ussion ................................ ................................ ................................ ................... 59 5 SUMMARY AND CONCLUSIONS ................................ ................................ ............. 66 LIST OF REFERENCES ................................ ................................ ................................ ... 68 BIOGRAPHICAL SKETCH ................................ ................................ ................................ 75

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7 LIST OF TABLES Table page 2 1 New bean varieties released in Haiti since 2006 ................................ ................... 25 3 1 Common bean accessions used in the study ................................ ........................ 40 3 2 Phenological (P), morphological (M), qualitative (Q), and agronomic (A) traits measured in the study ................................ ................................ ............................ 41 3 3 Multiple comparison of means for agronomic traits of the genotypes .................. 42 3 4 Multiple comparison of means for the phenological traits of 23 genotypes .......... 44 3 5 Multiple comparison of means for morphological traits of 23 genotypes .............. 45 3 6 Qualitative traits mea sured for the genotypes ................................ ....................... 48 3 7 Multiple comparison of means for disease scoring of the bean cultivars ............. 49 3 8 Mean value and standard deviation of diseases scored during the field trial ....... 50 3 9 PCA for the phenotypic traits. ................................ ................................ ................ 52

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8 LIST OF FIGURES Figure page 2 1 Bean planted area in Haiti ................................ ................................ ...................... 26 3 1 Location of the experimental site. ................................ ................................ .......... 38 3 2 Rainfall of the experimental site. ................................ ................................ ............ 39 3 3 Temperature of the experimental site. ................................ ................................ ... 39 3 4 Variation in yield and yield components between cultivars of Mesoamerican and Andean gene pools in the current study. ................................ ........................ 43 3 5 Maturity of the genotypes based on difference between days to 50% flowering and to 50% maturity ................................ ................................ ................ 46 3 6 Leaf area index recorded for the genotypes ................................ .......................... 47 3 7 Disease incidence according to bean type ................................ ............................ 51 3 8 Phenotypic correlation ................................ ................................ ............................ 52 3 9 PCA plot of the phenotypic traits ................................ ................................ ............ 53 4 1 Mean L(K) (SD) over 20 runs for each K value ................................ ................... 62 4 2 ................................ ................................ ................................ ................... 62 4 3 Population structure inferred by Bayesian approach based on 1115 SNPs for k=2, Mesoamerican (red), Andean (green). ................................ .......................... 63 4 4 Phylogenetic tree obtained for 96 lines of common bean and 1115 SNPs. ......... 63 4 5 Two dimension plot obtained from principal component analysis (PCA) for 96 lines of common bean and 1115 SNPs based on gene pool. ............................... 64 4 6 Two dimension plot obtained from principal component analysis (PCA) for 96 lines of common bean and 1115 SNPs based on bean market type. ................... 65

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9 LIST OF ABBREVIATIONS ANOVA Analysis of Variance AREA Appui a la Recherche et au Dveloppement Agricole (Support to Agricultural Research and Development) ARS Agricultural Research Service BCMV Bean Common Mosaic Virus BCMNV Bean Common Mosaic Necrosis Virus BGMV Bean Golden Mosaic Virus BGYMV Bean Golden Yellow Mosaic Virus BL Breeding Lines GBS Genotyping by Sequencing CC Cultivar Check CIAT International Center for Tropical Agriculture FAMV Faculty of Agronomy and Veterinary Medicine IFAS Institute of Food and Agricultural Sciences LAI Leaf Area Index LBP Legume Breeding Program NGS Next Generation Sequencing ORE Organization for Rehabilitation of the Environment PCA Principal Component Analysis RAPD Random Amplified Polymorphic DNA SNP Single Nucleotide Polymorphism SNS National Seed Service SRB Sample Resuspension Buffer TREC Tropical Research and Education Center

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10 UF University of Florida UPGMA Unweighted Pair Group Method with Arithmetic mean UPR University of Puerto Rico USAID United States Agency for International Development USDA United States Department of Agriculture

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A GRONOMIC PERFORMANCE AND GENETIC DIVERSITY OF COMMON BEAN ( PHASEOLUS VULGARIS ) VARI E TIES IN HAITI By Riphine Mainviel August 2019 Chair: Geoffrey Meru Major: Horticultural Science s Common bean ( Phaseolus vulgaris L.) is one of the essential food legumes in the world and plays a vital role in providing nutritional security and reve nue for low income families in the Caribbean, including Haiti. Despite its economic importance, bean production in Haiti is constrained by many biotic and abiotic factors that limit yield. Besides, most bean cultivars currently in the market are low yieldi ng, averaging 0.6 tons/ hectare, which is well below the world average (0.86 tons/ ha). The current study aimed to evaluate the agronomic performance of 13 elite dry bean breeding lines currently under development by the USAID AREA and the Legume Innovatio n Lab programs at a highland location in Haiti. Moreover, the study aimed to determine the sequencing. Analysis of variance and trait mean separation was done in R stat istical package, while a UPGMA dendrogram based on genetic dissimilarity matrix was constructed using Darwin software. Significant differences were observed for most of the recorded parameters. The yield of bean varieties ranged from 0.48 to 1.24 tons/ha, with a mean of 0.89 t/ha. Among the traits measured, the number of pods per plant and number of seeds per pod showed the highest correlation with dry seed yield, thus may

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12 be used for indirect selection of seed yield in common bean. Genetic diversity analys is revealed a prevalence of Mesoamerican gene pool within the accession collection, with a few of Andean origin, and some level of admixture.

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13 CHAPTER 1 INTRODUCTION Common bean ( Phaseolus vulgaris ) is a highly nutritive legume crop consumed by millions of people worldwide as a significant source of protein (Beebe et al. 2000). Other economically important Phaseolus species include P. lunatus (lima bean), P. coccineus (runner bean) and P. acutifolius (tep ary bean). Phaseolus legume crops belong to Fabaceae family (Yuste Lisbona et al. 2014) and are cultivated for their edible seeds and pods or unripe fruit (Beebe et al., 2013). They share this family with other important crops such as peanut ( Arachis hypo gaea ), soybean ( Glycine max ), broad bean ( Vicia faba ), lentil ( Lens culinaris ), sweet pea ( Lathyrus odoratus ), cowpea ( Vigna unguiculata ), chickpea ( Cicer arietinum ) and alfalfa ( Medicago sativa ). Phaseolus vulgaris originated from Central America and was first cultivated in Central Mexico The Mesoamerican populations were migrated to South America through different migration events (Bitocchi et al., 2012). Currently, it constitutes a significant food crop in the tropical, subtropical, and temperate region s of Africa, Europe, Asia, and the Americas (Wortmann, 2006). In 2017, the global area planted with bean was estimated at more than 36.4 million hectares, with a total production of 31.4 million tons (FAOSTAT, 2019). This crop is primarily produced in Lati n America and Eastern and Southern Africa, where it is critical to nutritional security and income generation ( Raatz et al, 2019 ). Many biotic and abiotic factors limit common bean production in Haiti. Biotic factors include viral, fungal, and bacterial d iseases, as well as insect pests, while abiotic factors include mineral toxicity, drought stress, high temperature, flooding, and nutritional deficiencies. Over the last two decades, collaborative breeding efforts have

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14 led to the recent release of superior disease resistant and early maturing bean varieties such as PR1146 138 and XRAV 40 4 in Haiti (Beaver et al., 2014, 2016). Nevertheless, the common bean cultivars in Haiti are still low yielding, averaging 0.6 tons per ha in 2017. Also, there is a lack of knowledge on the genetic diversity of common bean varieties grown in Haiti, information that is necessary for breeding and conservation efforts. Significance of the Study Common bean is the most important source of protein in Haiti, especially for the resource poor. Due to its economic and nutrition value, current breeding efforts are generated towards addressing production challenges in Haiti, including poor yield, biotic, and abiotic pressure. Towards this end, many initiatives have led to the rel ease of improved cultivars exhibiting resilience to pests and diseases. However, more cultivars showing wide adaptability in various agroecological zones of the country are needed. Furthermore, knowledge of the genetic diversity of common bean accessions grown across the country is lacking. Therefore, the goal of the current study was to evaluate the agronomic performance of 13 elite common bean breeding lines currently under development by the UF AREA and the Legume Innovation Lab programs and determine t he genetic diversity among 92 common bean accessions in Haiti. This project will aid the selection of advanced breeding lines for release to growers and elucidate the genetic structure of common bean accessions grown in Haiti for breeding and conservation purposes.

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15 Objectives This research project has two main objectives : i) d etermine the agronomic performance of 13 advanced breeding lines of common beans currently under development by the UF AREA and the Legume Innovation Lab programs ; and ii) d etermine the genetic diversity among 92 bean genotypes collected from different agro ecological zones across Haiti using genotyping by sequencing

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16 CHAPTER 2 LITERATURE REVIEW Environmental A daptation Common bean is a widely adaptable, warm season herbaceous plant with distribution in tropical, subtropical, and temperate environments (Heuz et al. 2015). The crop can grow within a wide altitude range from the sea level up to 2200 to 3000 m. The optimum annual rainfall required for the crop is between 500 and 1500 mm but can grow within a wide precipitation range (300 to 4300 mm). Common bean can tolerate environmental temperature up to 35 o C but prefers a range between 15 o C and 23 o C ( Wortmann 2006; Heu z et al. 2015). It can be cultivated in diverse types of soils, but ideally in well drained silt loam, sandy loam or clay loam soils with high organic content and a pH between 4 and 9 (Ecoport, 2013). Mineral deficiencies may occur in acidic sandy soils ( Mo and Mg) and calcareous Soils (Zn) (Heuz et al., 2015; Ecoport, 2013; Wortmann, 2006). Despite its adaptation to diverse growing conditions, common bean shows sensitivity to certain elements such as B, Mn, Al, and high level s of Na. The life cycle of t he determinate common bean varies between 60 to 90 days, whereas that of the indeterminate climbing types can extend up to 300 days (Heuz et al. 2015). The yield of common bean varies depending on the varieties snap bean or dry beans. For snap bean varie ties, where the green pod is harvested at about 25 to 30 days after flowering, the yield obtained can vary between 5 to 7.5 t ons /ha. On the other hand, the average yield of dr y bean s is estimated at 0.5 to 1.5 tons/ ha, but higher yields up to 2.8 5 tons/ ha have been reported (Heuz et al., 2015; Ecoport, 2013; Wortmann, 2006).

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17 Nutritional V alue of C ommon B ean Common bean is rich in protein (22 27% dry matter), as well as starch (39 47% dry matter) (Heuz et al. 2015). It is considered a valuable source of essential vitamins and minerals (iron and zinc), soluble fiber, and is of low fat content (Zargar et al., 2014, Keller et al., 2015). Furthermore, beans contain a lot of bioactive compounds, including enzyme inhibitors, phytates, lectins, oligosaccharides, and diverse phenolic substances (Zargar et al., 2014). It has been observed that the presence of polyphenolic compounds in common bean reduces the risk for several disorders, inc luding cardiovascular disease, diabetes, colon cancer, and obesity (Zargar et al., 2017). Based Bean P roduction in Hai ti Common bean has been domesticated for over 8000 years from a wild vining plant distributed in the highlands of Middle America (Mesoamerican) and the Andes, to a significant leguminous food crop, widely adapted to a wide range of environments across the world (Gmez et al., 2004). In the Caribbean, common bean was introduced during the pre Columbian era through trade by Taino and Arawak tribes from the Mesoamerican and Andean centers of origin (COG), respectively (Gepts et al., 1988). Since then, common b ean has become the most important source of protein in the Caribbean, including Haiti, especially for the resource poor who cannot afford animal derived protein foods. The demand for common beans in Haiti has increased exponentially over the last half cent ury has demonstrated by a dramatic increase in production between 1961 (89,000 ha; 37,500 tons) and 2017 (171,850 ha; 111,398 tons) (FAOSTAT, 2019). However, within the same period, there was little increase in

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18 yield (0.42 tons ha 1 in 1961 to 0.65 tons ha 1 in 2017) (FAOSTAT, 2019), meaning that most of the observed shift in production was due to increased acreage. The current common bean production in Haiti is below the world average (0.86 tons ha 1 in 2017) (FAOSTAT, 2019) In Haiti, common bean is grown in two main agro ecological zones; lowlands and highlands (Colbert, 2017). The most consumed seed types in Haiti are black, red mottled, yellow, white, and pinto (Beaver et al., 2012). The locally produced beans are preferred over the imported bean. Commo n b ean is grown in diverse areas across all departments of Haiti, but most predominantly in the West region followed by Artibonite and Center regions (Fig 2 1). The UF AREA bean breeding program conducts trials across locations in the West Department, incl uding Duvier a highland site with an altitude of 887m and Cabaret a lowland location with an elevation of 51m. Production Constraints in Haiti Common bean production is affected by both biotic and abiotic factors wherever it is grown. According to Hnatuszk o Konka et al. (2014), beans yield is limited by six major diseases and several hostile abiotic conditions. In the Caribbean, diseases limiting bean yield include those caused by viral (Bean Golden Yellow Mosaic Virus, Bean Common Mosaic Virus, Bean Common Mosaic Necrosis Virus), fungal (e.g. rust caused by Uromyces appendiculatus web blight caused by Thanatephorus cucumeris and root and stem rots caused by Rhizoctonia solani Fusarium solani and Macrophomina phaseolina ), and bacterial (bacterial blight c aused by Xanthomonas axonopodis pv. Phaseoli ) (Beaver et al., 2012) pathogens. The major insect pests include leafhoppers ( Empoasca kraemeri ), leaf beetles ( Cerotoma spp.), lepidoptera

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19 ( Hedylepta indicata and Etiella zinckenella ) and bruchids ( Acanthoscelides obtectus and Zabrotes subfasciatus ) (Beaver et al., 2012). Abiotic stress factors limiting common bean production in Haiti include mineral toxicity (e.g. B, Mn, and Al), drought and heat stress, flooding, low pH and poor soil fertility/ nu tritional deficiencies (Colbert, 2017). In most cases, growers are unable to correct for these factors due to lack of awareness/ education, as well as lack of capital to buy agricultural inputs. Natural disasters such as earthquakes and hurricanes also ha rm bean production in Haiti, but losses incurred vary from year to year. Between 2000 and 2017, about eight significant hurricanes have hit Haiti during bean growing seasons, causing devastating crop loss and field abandonment due to population displacemen ts. Besides, socio economic factors such as high cost of inputs and inaccessibility to the local market (e.g. poor infrastructure/ roads) negatively affect the value of bean production in Haiti. Bean Breeding in Haiti Due to the agronomic and economic impo rtance of common bean in Haiti and around the world, several international collaborative initiatives have been undertaken to mitigate production challenges. Through the Medicago Genome Consortium and the International Conferences on Legume Genomics and Gen etics, legume scientists discuss, prioritize and harmonize efforts in legume genomics and genetics to provide pragmatic solutions (Hnatuszko Konka et al., 2014). In this context, an International consortium aimed to establish the best framework for advanci ng knowledge on bean was established in 2000 in Sevilla, Spain. Bean researchers at the Escuela Agricola Pan Americana (Zamorano), the Universities of Puerto Rico and Nebraska, and the

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20 USDA ARS have collaborated with CIAT, the Instituto Dominicano de Inves tigaciones Agropecuarias y Forestales and the National Seed Service of the Ministry of Agriculture of the Republic of Haiti to develop and release bean cultivars and improved germplasm for the Caribbean with the support from the Bean/Cowpea CRSP, the Dry G rain Pulse CRSP and recently the USAID AREA and the Legume Innovation Lab programs (Beaver et al., 2012; Colbert et al., 2017). The primary focus of this great collaborative effort has been the development of high yielding, disease, and insect resistant cu ltivars adapted to the wide environmental variation in Haiti. Through these efforts, several bean cultivars have been developed and released to growers across the Caribbean, including Haiti. A non exhaustive list of these cultivars is provided in Table 2 1. Genetic Diversity of Common Bean According to Schmutz et al. (2014), common beans are diploids organism (2n = 22) with a genome size of about 587 Mb. The small genome, coupled with a low index of genome duplication (most loci are a single copy), makes common bean a suitable experimental organism (Mller et al., 2014; Vidak et al., 2017). The recent availability of a draft genome for common bean has created opportunities for further inquiry into the genetic mechanisms underlying economically important traits such as yield and resistance to biotic and abiotic stress (Ariani et al., 2016; Meziadi et al., 2016; Schmutz et al. 2014). Phenotypic and molecular markers have been extensively used for analysis of genetic diversity in common bean (Miklas et al., 2006; Kwak and Gepts, 2009; Blair et al., 2009), and have shown evidence of 2 major gene pools, Andean and Mesoamerican, which were domesticated independently (Kwak and Gepts, 2009; Bitocchi et al., 2012; Schmutz et al., 2014; Ariani et al., 2016). The Mesoamerican gene

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21 pool is adapted to lower altitudes and higher temperatures than the Andean gene pool (Ariani et al., 2016). The gene pools are further divided into six races according to morphological criteria and agro ecological adaptation, three each for Mesoamerican (Durango, Jalisco, and Mesoamerica) and Andean (Chile, Nueva Granada and Peru) (Blair et al., 2009). Several authors have examined genetic diversity among common bean landraces in Central America and Caribbean regions. Gomez et al. (2004) evaluated the pattern of genetic diversity among nine red seeded landraces from Nicaragua with molecular and phenotypic markers and fo und that variation uncovered at the molecular level were due to the difference among and within landraces, while differences at the phenotypic level were attributed to adaptation to agro ecological zones. For each landrace, twelve individuals were genotype d with seven bean microsatellite markers, while fourteen phenotypic traits were measured in two zones. These results implied that molecular differentiation was due to a founder effect, whereas the phenotypic variation was due to the effect of adaptation. W hile investigating the genetic diversity among 65 common bean landraces in the Caribbean using morphological and molecular markers, Durn et al. (2005) found that the accessions could be grouped into Mesoamerican and Andean gene pools. Mesoamerican phenoty pes comprised all the red mottle lines from Haiti and three landraces from the Dominican Republic collected near the Haitian border, while Andean phenotypes consisted of lines from Puerto Rico and the Dominican Republic. Blair and Lorigados (2016) evaluate d the diversity among 210 common bean landraces in Cuba using 36 SSR markers and found the majority of the genotypes to be Mesoamerican, with a few of Andean origin. The level of introgression

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22 between the two gene pools was lower than that previously obser ved in germplasm from other secondary centers of diversity, thus implying that Cuban beans are most likely derived from race Mesoamerica and race Nueva Granada, with a little mixing from other races. Beyond the Caribbean, genetic diversity among common be an landraces has also been reported. For example, using morphological and microsatellite markers, Asfaw et al. (2009) evaluated the genetic diversity and population structure among 192 common bean landraces from East African (Ethiopia and Kenya) highlands. The study revealed considerable genetic and phenotypic diversity that corresponded to the two recognized gene pools (Andean and Mesoamerican) with little introgression between these groups. Moreover, it was observed that the genetic divergence was slightl y higher for the Ethiopian landraces compared to Kenyan landraces and that Mesoamerican genotypes were more diverse than the Andean genotypes. Becerra et al. (2010) characterized 237 Chilean common bean landraces using microsatellite markers and found that Andean genotypes were predominant. Race Chile was found closely related to races Nueva Granada and Peru of the Andean gene pool, but further differentiated from the race Mesoamerica of Mesoamerican gene pool. The substantial genetic differences between th e two races (Chile and Mesoamerica) suggested the potential value for novel allele transfer between the two gene pools, although it may be challenging to combine genetic crosses due to hybridization barriers. Maras et al. (2015) reported high genetic diver sity among 119 common bean landraces in the Western Balkans using 13 SSR markers where Andean genotypes were more prevalent. In 2018, Campa et al. conducted a molecular characterization of a Spanish

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23 diversity panel of 308 common bean lines through Genotypi ng by Sequencing. The panel was characterized by 3,099 single nucleotide polymorphisms, which revealed a wide genetic variation and a low level of redundant material within the Spanish bean panel. The two main gene pools were identified through Structure, cluster, and principal component analysis. However, most of the lines (70%) were associated with the Andean gene pool. Furthermore, lines showing intergene pool introgression were also observed, which suggest the use of the two gene pools in the breeding o f snap bean cultivars. Accessions with similar genetic profile were identified which may need to be removed to maximize the panel diversity. The usefulness of the panel for future GWAS was also validated through association mapping of determinacy. Similar ly, an in depth genome characterization of a Brazilian common bean core collection was conducted by Valdisser et al. 2017, using DArTseq high density SNP genotyping. In this study, 6,286 SNPs were genotyped in genic (43.3%) and non genic regions (56.7%) wh ich allowed the genetic subdivision based on the two main gene pools (K=2), and grain types (k=3 and k=5). A total of 83% of all SNPs were polymorphic in the Andean gene pool while 91% were polymorphic within the Mesoamerican gene pool, while 26% of all SN Ps were able to distinguish the gene pools. The findings of this study showed that the DArTseq approach could generate a large set of useful SNPs for common bean with complete genome coverage, where both coding and non coding regions are represented. This approach also allowed accurate evaluation of genetic diversity in the Brazilian beans collection. An optimization of genotyping by sequencing (GBS) data in common bean was conducted by Schrder et al., 2016. The main goal of this project was to imp rove the

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24 quality and the coverage of GBS data in common bean ( Phaseolus vulgaris L.) to increase the number of SNPs available for genome wide association studies (GWAS). Twenty five common bean genotypes from the Mesoamerican gene pool were used for compar ison of 2 libraries by using the standard ApeK1 fragments and MseI/Taq I double digest fragments. The results revealed an increase of 3.8 to 12.5 fold in SNPs based on a minimum coverage (3X, 5X, and 8X).

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25 Table 2 1. New bean varieties released in Hait i since 2006 Variety Market type Characteristics Country/Institution collaboration Author, year PR1146 138 yellow Resistance BGYMV (bgm), BCMV, BCMNV (bc 3) UPR / SNS Haiti / USDA ARS Beaver et al., 2016 XRAV 40 4 black Resistance BGYMV (bgm), BCMV, BCMNV (bc 3), earliness UPR / UNL EEA/ USDA ARS / IDIAF Rep Dom. /EAP Zamorano / SNS Haiti Beaver et al., 2014 PR0633 10 Red mottled Resistance BGYMV (bgm), BCMV, BCMNV (bc 3) (I) UPR / USDA ARS / IDIAF Rep. Dom. /SNS Haiti Prophete et al., 2013 PR0737 1 Red mottled Resistance BGYMV (bgm), BCMV, BCMNV (bc 3) UPR / USDA ARS / IDIAF Rep. Dom./SNS Haiti Prophete et al., 2013 PR9745 232 Red mottled Resistance to BGYMV (bgm) and BCMV (I) CIAT / UPR /IDIAF Rep. Dom./ SNS Haiti Blair et al., 2006 RMC 3 Red mottled Resistance to BGYMV (bgm) and BCMV (I) CIAT/ UPR/ IDIAF Rep. Dom./ SNS Haiti Blair et al., 2006

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26 Figure 2 1 Bean planted area in Haiti

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27 CHAPTER 3 AGRONOMIC PERFORMANCE OF COMMON BE AN VARIETIES IN HAITI Background Common bean is one of the most essential food legumes in the world and plays an important role in providing nutritional security and revenue for low income families in the Caribbean countries, including Haiti. It is a main con stituent of the daily diet in Haiti, where consumers primarily prefer black, red mottled, yellow, white and pinto bean (Beaver et al., 2012). Despite its economic importance, bean production is constrained by many biotic stresses including viral, fungal an d bacterial diseases, as well as insect pests. Abiotic factors limiting bean production include extreme pH, low soil fertility, salinity, drought and heat stress (Colbert, 2017). These factors, combined with the variable agro ecological zones in the countr y, emphasize the need to develop widely adapted bean germplasm with acceptable biotic and abiotic resilience. Currently, most of the common bean cultivars grown in Haiti are low yielding, averaging 0.6 tons/ ha, a value significantly less than the world av erage 0.86 tons/ha Over the last two decades, collaborative breeding efforts have led to recent release of superior bean varieties, seven of which have been released since 2006 (PR1146 138, XRAV 40 4, PR0633 10, PR0737 1, AIFI Wuriti, PR9745 232 and RMC 3) (Beaver et al., 2 014; Beaver et al., 2016; Blair et al., 2006; Prophete et al., 2013; Rosas et al., 2008). These lines exhibit superior agronomic performance over traditional landraces for traits such as earliness (XRAV 40 4), tolerance to drought and low soil fertility (A IFI Wuriti), and improved resistance to viral diseases (BGYMV, BCMV and BCMNV). Currently, breeding efforts are underway to develop more high yielding

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28 cultivars through a partnership between USAID AREA project and the Legume Innovation Lab. The goal of thi s study was to evaluate the agronomic performance and phenotypic diversity among 13 advanced common bean breeding lines and identify the best lines for further selection and release to growers in Haiti. Materials and Methods Plant M aterial s Thirteen adva nced breeding lines from UF AREA and the Legume Innovation Lab programs, as well as 12 cultivar checks, were used in the study. This germplasm pool consisted of twelve b lack, five r ed mottled, one r ed, one p into, one b eige, and five w hite bean types (Table 3.1). Experimental S ite and R esearch D esign The experiment was conducted at Duvier, a highland location in the w est region of Haiti (Figure 3.1). This trial was performed during the first bean cropping season of the year (April to July 2018). During this year, the experimental site received a monthly rainfall that ranged from 0.55 to 150.1 mm (Fig 3.2) and the temperature bet ween 23 to 29 o C (Fig 3.3). The soil preparation was conducted using conventional tillage, and the bean lines were sowed manually in three replicated plots in a randomized complete block design. Each replicate or plot consisted of 62 individuals. For each plot, the genotypes were planted in two rows of 31 plants in a 3.6 m 2 area. In row spacing was 10 cm, while between row spacing was 60 cm and the distance between the block and the plots was one m eter The plots were maintained using hand weeding and bas ic pest management practices. Phenotypic assessment of bean lines was done by measuring eleven agronomic traits, according to Gomez et al., 2004 (Table 3.2). Besides, leaf area index, which corresponds to the ratio of leaf surface area over a total ground surface

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29 area, was determined by using LAI meter: AccuPAR LP 80. The diseases that were more prevalent during the experiment including Powdery Mildew, Bean Common Mosaic Virus (BCMV), Bean Golden Mosaic Virus (BGMV) were evaluated using a scale of 1 to 9 ac cording to CIAT where 1 to 3 is resistant, 4 to 6 intermediate and 7 to 9 susceptible. Data A nalysis All data collected were subjected to a nalysis of v ariance (ANOVA) using R 3.6.0. The assumptions required for linear models, including normality, independ ence of errors, and linearity were tested before running the ANOVA test. The three traits were considered as co factors in the model to account for the influence of growth habit, gene pool, and disease incidence on seed yield. The least square means multip le comparison test was used to identify significant differences between the genotypes for the different phenotypic traits assessed. Pearson correlation was performed for the phenotypic traits. Furthermore, the mean values of these traits were used to perfo rm principal component analysis (PCA) to identify patterns of phenotypic variation. Results This project aimed to assess the agronomic performance of 13 advanced breeding lines of common bean along with 12 cultivar checks. However, two cultivar checks (Lo cal 4 red and Local 6 Pinto) did not germinate well in the field. Thus, the results are presented for the remaining 23 genotypes. Agronomic T raits Analysis of variance and mean separation revealed significant differences (p < 0.05) among the genotypes for most of the traits evaluated (Table 3.3). The yield ranged from 0.48 to 1.24 tons ha 1. Overall, the Mesoamerican bean types (black,

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30 white, and bei ge) were significantly higher in yield, pods/ plant, seeds/pod than their Andean counterparts except for seed weight (Figure 3 4). The three varieties that had highest yields included two cultivar checks, Verano and LORE 234 Local, and the breeding line P R1627 8. The genotypes showed a significant difference in the number of pods per plant with a range of 4.33 to 8.87. The number of seeds per pod ranged from 3.15 to 5.73 and differed significantly (P<0.05) among the bean cultivars. The value recorded for 1 00 seed weight showed statistically significant differences among the genotypes and ranged between 12.62 and 28.26 g (Table 3.3). Phenological and M orphological T raits The phenological traits assessed in this experiment included days to 50% flowering, da ys to 50% maturity, and earliness that was calculated based on the difference between day to 50% flowering and to 50% maturity. The accessions showed significant differences (P<0.05) for the three parameters (Table 3 4). The number of days to flowering ran ged from 37 to 43 days, the number of days to maturity from 56 to 68 days and earliness ranged from 19 to 27 days after flowering. The earliest varieties included the breeding line PR1423 153 and two cultivar checks LORE 234 local, and local three red, all of which reached physiological maturity within 19 to 20 days from flowering (Fig 3 5). The accessions also showed significant differences for the morphological traits evaluated, including stem length, pod length, and pod width (Table 3 5). The value recor ded for the stem length ranged from 25.30 cm to 38.67 cm. The pod length for the bean genotypes ranged from 8.02 to 11.77 cm, and the range for pod width varied from 1.00 to 1.31 cm. Leaf area index exhibited a significant difference among the genotypes an d ranged from 1.13 to 2.65 (Fig 3 6).

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31 Qualitative T raits Three types of wing petal color were recorded for the genotypes in the experiment, including white, purple, and pink. From the 23 genotypes, 2 had pink flowers, 10 had white flowers, and 11 had purple flowers (Table 3 6). Based on the results, all the whit e beans had white flowers. However, white colored flowers were also found for colored seeds, such as black and red seeded types in the current experiment. The common bean genotypes used in the study had either determinate or indeterminate type II growth h abit. However, the indeterminate type II was more prevalent, with 74% of the genotypes showing this growth habit (Table 3 6). E valuation of C ommon B ean G enotypes f or D isease R esistance The most prevalent diseases in the field were evaluated during the field trial. The diseases that were assessed include powdery mildew, Bean Golden Mosaic Virus (BGMV), and Bean Common Mosaic Virus (BCMV) using a scale of 1 to 9. The genotypes showed a significant difference in their response to different diseases (Table 3 7). Based on the scale of CIAT for evaluation of powdery mildew, 14 varieties were found resistant, eight varieties intermediate and one susceptible (Table 3 8). Significant differences were recorded in the response of the genotypes to viral diseases: BG MV and BCMV. Among the lines, 16 showed resistance to BGMV, while seven were intermediate for strength. In contrast to the other diseases, the plots were more affected by BCMV with only seven varieties showing resistance, 14 intermediate and two susceptibl e. On average, the white beans were less affected by both fungal and viral diseases evaluated in the field, followed by black beans (Fig 3 7)

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32 Phenotypic C orrelation s The Pearson correlation analysis between phenotypic traits is presented in Figure 3 8. Flowering time (days to flowering) was positively correlated with the number of days to 50% maturity, number of pods per plant, number of seeds per pod, yield, LAI, and growth habit, but it was not significantly correlated with earliness. A significant pos itive correlation was observed between days to 50% maturity and earliness. Seed yield was positively associated with the number of pods per plant, number of seeds per pod and growth habit, with the strongest correlation with the number of pods per plant. S eed weight was positively correlated with stem length, pod length and pod width, whereas it was negatively correlated with the number of seeds per pod, number of pods per plant, flowering time and growth habit. A positive correlation was recorded between l eaf area index and 50% maturity, the number of pods per plant, stem length, and flowering time. P rincipal Component Analysis The total proportion explained by the 11 PCs and corresponding vector loadings are presented in Table 3 9. The first principal component (PC1) captured the maximum amount of variation (53%) and separated the genotypes mainly based on growth habit, seed wei ght, number of seeds per pod, number of pods per plant, stem length, pod length, and pod width. On the other hand, the second PC explains 17.44% of the variation and separated the varieties mainly on leaf area index, day to flowering, and maturity. The thi rd PC captured about 13% of the variation and separated the genotypes based on yield, pods per plant, pod length. Together, PC1, PC2, and PC3 explained 83.29% of the total phenotypic variation. A PCA plot based on PC1 and PC2

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33 revealed three major bean type clusters (Fig 3 9). The red mottled accessions formed a distinct cluster and were characterized by high seed weight, pod length, and pod width. Black beans formed the largest cluster and varied widely in yield, pods per plant, seeds per pods, days to matu rity, and leaf area index. On the other hand, the white beans formed a tight cluster that showed less variation for the traits assayed. The accessions of b eige and r ed bean type were respectively clustered close to the Mesoamerican and Andean bean types. Discussion In the current study, the agronomic performance of 13 advanced breeding lines of common bean was evaluated in a highland location in Haiti (Duvier). Considerable variation was observed among the genotypes for most of the assessed parameters. Ov erall, Mesoamerican cultivars outperformed Andean cultivars in all agronomic traits, except for seed weight. The difference in agronomic performance between the two gene pools is expected and has been widely reported in the literature (Singh et al., 1991a; White & Gonzles 1990; White et al., 1992; Sexton et al., 1994). As expected, seed yield was highly influenced by growth habit and disease incidence. When these traits were considered as cofactors in the model, no significant differences were observed for yield across the genotypes. The yield was highly correlated with the number of pods per plant as well as the number of seeds per pod, indicating that these traits may be used for indirect selection of dry seed yield. Significant positive correlations betw een these traits have been previously reported for common bean (Mebrahtu et al., 1991). These observations are expected because the number of pods per plant and number of seeds per pod is the most critical components of seed yield in common bean (Ambachew et al., 2015; Mebrahtu et al., 2001). The relationship among

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34 the three traits may indicate pleiotropic or genetic linkage for the underlying loci (Dilday et al., 1990), thus allowing for tandem selection of the traits (Ambachew et al., 2015). Significant n egative correlations were found between seed size (seed weight) and number of pods per plant ( 0.31) and the number of seeds per pod ( 0.59). This result confirms previous observation that yield difference between Andean and Mesoamerican gene pools is due to seed size (White and Gonzles,1990). The accessions differed significantly in phenological and morphological traits. Within the Mesoamerican gene pool, the cultivar LORE 234 Local ( b lack b ean) had the least flowering and maturity time, while within the Andean gene pool, Local3 Red was the best cultivar for the two traits. Therefore, the two cultivars may be used to improve earliness in the Haiti bean breeding program. As expected, days to 50% flowering and days to 50% maturity were positively correlated (Zeven et al., 1999). However, the number of days to 50% flowering was not significantly correlated to earliness. This situation may be explained by variation in the growth habit of the accessions. Some bean accessions with indeterminate growth habit may take longer to mature, despite reaching 50% flowering relatively early compared to a determinate variety. Therefore, the number of days the plant takes to reach maturity after flowering is a better indicator of earliness than the number of days from sowing to maturity. Similarly, day to flowering was positively correlated with the number of seeds per pod and number of pods per plant, further confirming the phenological distinction between the Andean and Mesoamerican gene pools. A positive and significant co rrelation was recorded between stem length, pod length, and pod width, with the highest correlation between pod length and pod width. Zeven et al. (1999) reported a

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35 positive correlation between pod length and pod width in common bean. A similar relationshi p was observed between pod length and width, and with seed weight, and may be explained by the fact that the larger seeded bean types which have higher seed weight require longer and larger pods to the contrary to the small seeded types. Similar to findin gs by Zeven et al. (1999), white bean accessions exclusively had white flower color. On the contrary, white flowers were found across the other bean types. Indeterminate growth habit was observed in 74% of the genotypes (Mesoamerican), while the rest (26%; Andean) were determinate type. As with previous studies, growth habit was positively correlated with pods per plant and seeds per pod (Kornegay et al, 1992). Significant differences in disease severity were observed among the cultivars. The breeding line s (PR1423 99, PR1423 110, PR1423 153, PR1564 20, PR1564 3, PR1564 53, PR1627 10 and PR1627 8) were resistant in their response to powdery mildew, thus may be used as the source of resistance in the breeding program. For BGMV, 9 of the 13 breeding lines wer e found resistant, and five were intermediate The response of the breeding lines to BGMV could be explained by the presence of bgm gene (Table 3 1). As previously revealed by Urrea et al., 1994; Velez et al. 1, 998 and Blair et al., 2007, the bgm that is located on Pv03 confer resistance to chlorosis inducing of BGMV infection. Eleven of the breeding lines (74%) were found to have intermediate to high resistance to BCMV. This was expected because they carry I and bc 3 genes. The I gene that has a nearly t erminal position on Pv02 (Vallejos et al., 2000), and bc 3 gene located on Pv06 (Johnson et al., 1997) are recognized to confer resistance to all known strains of BCMV and BCMNV in common bean (Mukeshimana

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36 et al., 2005). Two of the breeding lines were rate d as susceptible to BCMV, indicating that the resistance alleles in these lines was not fixed, and may have been lost through repeated selfing in the breeding program. Since the current experiment was conducted in one location, it is necessary to confirm r esistance responses across all the genotypes in multiple environments in the future. As expected, the principal component analysis revealed distinct clustering according to bean type. The red mottled accessions formed a different cluster characterized by high seed weight, pod length and pod width, while black and white beans formed separated unique clusters. In conclusion, the results reported in this study showed a significant difference among the genotypes for most of the traits measured. The PR1627 8 (white bean) outperformed the other breeding lines in yield and disease resistance; therefore, it is a candidate for release to growers. Furthermore, the cultivars checks, including LORE 234 local, and Local 3 red could be used as a source of earliness for breeders. Since the data used was only for one season and one location, further trials are necessary to validate the results. Based on phenotypic relationships, the number of pods per plant and number of seeds per pod may be useful traits for indirect s election for seed yield in common bean Limitations The current project has its limitations, which may affect its reproducibility. This research was conducted under conditions typical for Haitian farmers (agriculture with low input). The goal was to select varieties that can thrive under different biotic and abiotic stress with minimal input. The field trial was rainfed with no fertilizer application. In addition, no pesticides were applied during the experiments, and the varieties differed

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37 in their respons e to the diseases assessed (powdery mildew, BCMV, and BGMV). All these cofounding factors ought to be considered when interpreting the results reported here. The growth habit of the different gene pools may also affect the yield of the genotypes. For insta nce, the Mesoamerican lines with their indeterminate growth habit usually have a higher yield than the Andean lines that have determinate growth habit. However, to reduce the effect of some of the cofounding factors, disease incidence, as well as growth ha bit, were included in the ANOVA model for yield.

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38 Figure 3 1. Location of the experimental site

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39 Figure 3 2 Rainfall of the experimental site Data adapted from W orldweatheronline.com Figure 3 3. Temperature of the experimental site Data adapted from Worldweatheronline.co m

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40 Table 3 1. Common bean accessions used in the study Breeding line Market type Characteristics Country/Institution collaboration PR1423 99 Black bgm, I, bc3, CBB Univ. of Puerto Rico & UF Haiti AREA LBP PR1423 110 Black bgm, I, bc3, CBB Univ. of Puerto Rico & UF Haiti AREA LBP PR1423 117 Black bgm, I, bc3, CBB Univ. of Puerto Rico & UF Haiti AREA LBP PR1423 153 Black bgm, I, bc3, CBB Univ. of Puerto Rico & UF Haiti AREA LBP XRAV 40 4 (Sankara) Black bgm, I, bc3, CBB Univ. of Puerto Rico, Univ. of Nebraska, USDA ARS, Univ. Zamorano & National Seed Service Haiti MEN2201 64 Black bgm, I, bc3, PR1564 20 Black bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP PR1564 53 Black bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP PR1564 3 Black bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP PR1627 8 White bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP PR1627 10 White bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP PR1627 13 White bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP Verano White bgm, I, bc3, PR1654 1 Red Mottled bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP PR1654 2 Red Mottled bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP PR1654 3 Red Mottled bgm, I, bc3, Univ. of Puerto Rico & UF Haiti AREA LBP Badillo Red Mottled Salagnac local Black LORE 234 local Black ORE Haiti & UF Haiti AREA LBP Local1 Red mottled Local2 Black Local3 Red Local4 White Local5 Beige Local6 Pinto

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41 Table 3 2. Phenological (P), morphological (M), qualitative (Q), and agronomic (A) traits measured in the study Character classification Description Day to flowering (DAP) P Number of days from sowing to the stage when 50% of the sampled plants have begun to flower Physiological maturity (DAP) P Number of days from sowing until 50% of the sampled plants have changed the color of their pods Stem length (cm) M The distance from the ground surface to the tip of the main guide at flowering. Sample size: 10 plants plot 1 Pod length, cm M Exterior distance from the pod apex to the peduncle. Sample size: 30 pods pl ot 1 Pod width, cm M Distance from the right angle to the sutures as the middle of the pod. Sample size: 30 pods plot 1 Growth habit Q Determined according to Muoz et al (1993). Sample size: 10 plants plot 1 Wing petal color Q Determined in freshly opened flowers according to Muoz et al (1993). Sample size: 10 plants plot 1 Pods plant 1 A Average number of fertile pods plant 1. Sample size: 10 plants plot 1 Seeds pod 1 A Average number of seeds pod 1 Sample size: 30 pods plot 1 100 seed weight, g A Average 100 seed weight (14% moisture) Yield per plot, kg ha 1 A Determined on the basis of the total number of harvested plants plot 1

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42 Table 3 3 M ultiple comparison of mean s for agronomic traits of the genotypes Agronomic traits Lines Market type Varieties Yield (tons/ha) Pods/plant Seeds/pod 100 seeds weight CC Be Local5 Beige 0.83 ns 6.47abcd 4.67cdef 15.87ab CC Bl Local2 Black 0.92 ns 6.03abcd 5.30def 16.93ab CC Bl LORE 234 Local 1.24 ns 8.87d 4.92def 16.43ab CC Bl MEN2201 64 0.96 ns 6.47abcd 4.98def 16.76ab BL Bl PR1423 110 0.78 ns 6.10abcd 5.27def 14.11ab BL Bl PR1423 117 0.76 ns 5.77abcd 5.25def 14.61ab BL Bl PR1423 153 0.99 ns 6.93abcd 5.72f 14.54ab BL Bl PR1423 99 0.88 ns 6.10abcd 5.73f 14.54ab BL Bl PR1564 20 0.82 ns 7.20abcd 4.48cde 14.34ab BL Bl PR1564 3 0.97 ns 7.90bcd 5.55ef 12.62a BL Bl PR1564 53 1.02 ns 7.10abcd 5.38def 15.17ab CC Bl Salagnac local 0.94 ns 6.97abcd 4.67bcd 16.57ab CC Bl XRAV 40 4 0.78 ns 5.63abcd 5.00abc 15.96ab CC R Local3 Red 0.88 ns 5.00ab 3.58abc 28.26ab CC R M Badillo 1.01 ns 5.40abc 4.31bcd 23.55ab CC R M Local1 RM 0.48 ns 4.33a 3.15a 18.96ab BL R M PR1654 1 0.61 ns 5.67abcd 3.30ab 18.40ab BL R M PR1654 2 0.66 ns 5.70abcd 3.33ab 19.55ab BL R M PR1654 3 0.73 ns 4.80ab 3.35def 26.12cd BL W PR1627 10 0.98 ns 7.57abcd 4.83def 15.63d BL W PR1627 13 0.94 ns 7.47abcd 4.95def 14.77bc BL W PR1627 8 1.16 ns 8.47cd 5.30def 15.10abc CC W VERANO 1.16 ns 8.70cd 4.65cdef 15.90bc CC: cultivar check, BL: breeding line, Be: beige, Bl: black, R: red, RM: red mottled ; W: white; ns: no signifance

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43 Figure 3 4. Variation in yield and yield components between cultivars of Mesoa m erican and Andean gene pool s in the current study

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44 Table 3 4 Multiple comparison of mean s for the phenological traits of 23 genotype s Phenological traits Lines Bean type Varieties 50% flowering 50%Maturity Earliness CC Be Local5 Beige 39.33abcd 61.00abcd 21.67abc CC Bl Local2 Black 43.00e 67.67g 24.67abc CC Bl LORE 234 Local 37.00a 56.00a 19.00a CC Bl MEN2201 64 37.33ab 59.00abc 21.67abc BL Bl PR1423 110 41.00cde 61.67bcde 20.67ab BL Bl PR1423 117 40.33abcde 61.00abcd 20.67ab BL Bl PR1423 153 40.00abcde 59.00abc 19.00a BL Bl PR1423 99 40.67bcde 65.67defg 25.00abc BL Bl PR1564 20 41.00cde 65.00defg 24.00abc BL Bl PR1564 3 42.33de 66.33fg 24.00abc BL Bl PR1564 53 41.67de 68.00g 26.33bc CC Bl Salagnac local 41.67de 62.00cdef 20.33ab CC Bl XRAV 40 4 37.33ab 59.00abc 21.67abc CC R Local3 Red 37.00a 56.67ab 19.67a CC R M Badillo 41.00cde 64.00cdefg 23.00abc CC R M Local1 RM 37.00a 64.33defg 27.33c BL R M PR1654 1 37.67abc 61.67bcde 24.00abc BL R M PR1654 2 39.00abcd 64.00cdefg 25.00abc BL R M PR1654 3 39.67de 62.00cdef 22.33abc BL W PR1627 10 42.00de 68.33g 26.33bc BL W PR1627 13 41.33de 67.67g 26.33bc BL W PR1627 8 42.33de 67.00fg 24.67abc CC W VERANO 41.33de 66.00defg 24.67abc CC: cultivar check, BL: breeding line, Be: beige, Bl: black, R: red, RM: red mottled W: White

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45 Table 3 5 Multiple comparison of means for morphological traits of 23 genotypes Morphological Traits Lines Bean type Varieties Stem Length (cm) Pod length (cm) Pod width (cm) CC Be Local5 Beige 34.59abc 8.94abcd 1.11abcdefg CC Bl Local2 Black 31.74abc 8.89abcd 1.09abcdef CC Bl LORE 234 Local 33.72abc 8.94abcd 1.10abcdef CC Bl MEN2201 64 26.42a 8.75abcd 1.08abcd BL Bl PR1423 110 27.43ab 8.55abc 1.00a BL Bl PR1423 117 27.08a 8.44ab 1.05abc BL Bl PR1423 153 25.30a 8.91abcd 1.04ab BL Bl PR1423 99 26.35a 9.07abcd 1.06abcd BL Bl PR1564 20 27.34ab 8.02a 1.08abcd BL Bl PR1564 3 28.80abc 8.92cd 1.08abcd BL Bl PR1564 53 26.24a 8.49ab 1.13abcdefg CC Bl Salagnac local 38.22bc 9.37bcd 1.17bcdef CC Bl XRAV 40 4 27.83abc 8.55abc 1.06abcd CC R Local3 Red 34.21abc 11.24e 1.31f CC R M Badillo 38.67c 11.77e 1.24ef CC R M Local1 RM 33.29abc 9.08abcd 1.17cdef BL R M PR1654 1 34.69abc 9.86d 1.19defg BL R M PR1654 2 35.88abc 9.83cd 1.21efg BL R M PR1654 3 34.19abc 9.83cd 1.24fg BL W PR1627 10 26.70a 8.07ab 1.13bcdef BL W PR1627 13 26.38a 8.10ab 1.10abcde BL W PR1627 8 26.56a 8.85abcd 1.10abcde CC W VERANO 28.25abc 8.05a 1.13bcdef CC: cultivar check, BL: breeding line, Be: beige, Bl: black, R: red, RM: red mottled, W: White

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46 Figure 3 5. Maturity of the genotype s based on difference between day s to 50% flowering and to 50% maturity

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47 Figure 3 6. Leaf area index recorded for the genotypes

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48 Table 3 6 Qualitative traits measured for the genotypes Bean type Lines growth habit wing petal color Beige Local 5 Beige Indeterminate type II white Black Local 2 Black Indeterminate type II purple Black LORE 234 local Indeterminate type II purple Black MEN2201 64 Indeterminate type II purple Black PR1423 110 Indeterminate type II purple Black PR1423 117 Indeterminate type II purple Black PR1423 153 Indeterminate type II purple Black PR1423 99 Indeterminate type II purple Black PR1564 20 Indeterminate type II purple Black PR1564 3 Indeterminate type II purple Black PR1564 53 Indeterminate type II purple Black Salagnac local Indeterminate type II pink Black XRAV 40 4 Indeterminate type II purple Red Local 3 Red Determinate white Red Mottled Badillo Determinate white Red Mottled Local 1 Red Mottled Determinate white Red Mottled PR1654 1 Determinate pink Red Mottled PR1654 2 Determinate white Red Mottled PR1654 3 Determinate white White PR1627 10 Indeterminate type II white White PR1627 13 Indeterminate type II white White PR1627 8 Indeterminate type II white White VERANO Indeterminate type II white

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49 Table 3 7 Multiple comparison of mean s for disease scoring of the bean cultivars Varieties Mildew BGMV BCMV Badillo 6.00ef 1.67ab 4.67bcdef Local 1 Red Mottled 5.67def 2.33abc 6.33fg Local 2 Black 3.00abc 1.00a 5.00cdefg Local 3 Red 8.00f 5.33e 6.33fg Local 5 Beige 3.00abc 3.67bcde 6.00efg LORE 234 local 3.00abc 5.00de 5.67defg MEN 2201 64 4.00abcde 2.67abcd 3.00abcdef PR1423 110 3.33abcd 6.00e 2.33abcd PR1423 117 3.67abcde 5.33e 2.67abcde PR1423 153 3.33abcd 6.00e 1.00a PR1423 99 3.00abc 4.67cde 6.00efg PR1564 20 2.00a 1.67ab 5.67defg PR1564 3 2.67ef 1.33ab 6.00efg PR1564 53 2.00a 1.67ab 5.33cdefg PR1627 10 2.33a 2.00ab 2.33abcd PR1627 13 3.67abcde 1.67ab 4.00abcdefg PR1627 8 2.33a 1.33ab 1.33ab PR1654 1 5.00bcde 1.33ab 6.67g PR1654 2 5.33cde 1.33ab 6.00efg PR1654 3 6.00ef 1.67ab 6.67g Salagnac local 3.00abc 2.00ab 6.00efg VERANO 2.67ab 1.00a 2.00abc XRAV 40 4 2.67ab 2.33abc 5.00cdefg

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50 Table 3 8 M ean value and standard deviation of disease s scored during the field trial Var P Mildew category BGMV Category BCMV Category Badillo 6.00 0.0 I 1.67 1.54 R 4.67 1.54 I Local 1 Red Mottled 5.67 0.15 I 2.33 0.57 R 6.33 0.57 I Local 2 Black 3.00 1.0 R 1.00 0.0 R 5.00 1.00 I Local 3 Red 8.00 1.0 S 5.33 0.57 I 6.33 0.57 I Local 5 Beige 3.00 1.0 R 3.67 1.52 I 6.00 0.00 I LORE 234 local 3.00 0.0 R 5.00 0.0 I 5.67 1.52 I MEN 2201 64 4.00 1.0 I 2.67 0.57 R 3.00 1.00 R PR1423 110 3.33 0.57 R 6.00 1.00 I 2.33 2.30 R PR1423 117 3.67 0.57 I 5.33 1.54 I 2.67 2.08 R PR1423 153 3.33 0.57 R 6.00 1.00 I 1.00 0.00 R PR1423 99 3.00 1.0 R 4.67 0.57 I 6.00 1.00 I PR1564 20 2.00 0.0 R 1.67 0.57 R 5.67 0.57 I PR1564 3 2.67 0.57 R 1.33 0.57 R 6.00 0.00 I PR1564 53 2.00 0.0 R 1.67 1.54 R 5.33 0.57 I PR1627 10 2.33 0.57 R 2.00 1.00 R 2.33 2.30 R PR1627 13 3.67 2.0 I 1.67 0.57 R 4.00 1.73 I PR1627 8 2.33 0.57 R 1.33 1.00 R 1.33 0.57 R PR1654 1 5.00 0.0 I 1.33 0.57 R 6.67 0.57 S PR1654 2 5.33 0.57 I 1.33 0.57 R 6.00 1.00 I PR1654 3 6.00 1.0 I 1.67 0.57 R 6.67 0.57 S Salagnac local 3.00 1.0 R 2.00 1.00 R 6.00 1.00 I VERANO 2.67 0.57 R 1.00 0.00 R 2.00 1.73 R XRAV 40 4 2.67 0.57 R 2.33 0.57 R 5.00 1.73 I

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51 Figure 3 7. Disease incidence according to bean ty pe

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52 Figure 3 8. Phenotypic correlation Table 3 9 PCA for the phenotypic traits PC1 PC2 PC3 PC4 PC5 PC6 Variation explained (%) 52.99 17.44 12.86 6.52 4.95 2.52 Day to flowering 0.25 0.45 0.11 0.44 0.20 0.40 Day to maturity 0.17 0.58 0.16 0.31 0.28 0.01 Stem length 0.32 0.19 0.20 0.37 0.37 0.61 Pod length 0.34 0.08 0.31 0.18 0.45 0.26 Pod width 0.35 0.23 0.26 0.05 0.30 0.02 Pods/plants 0.31 0.05 0.42 0.35 0.37 0.13 Seeds/pod 0.36 0.12 0.14 0.23 0.47 0.23 Seed weight 0.37 0.03 0.23 0.26 0.12 0.19 Yield (tonnes/ha) 0.22 0.00 0.69 0.01 0.08 0.21 Leaf Area Index 0.09 0.56 0.15 0.54 0.23 0.48 Growth Habit 0.39 0.14 0.06 0.08 0.15 0.16

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53 Table 3 9. Continued PC7 PC8 PC9 PC10 PC11 Variation explained (%) 1.40 0.72 0.46 0.10 0.06 Day to flowering 0.11 0.53 0.08 0.07 0.16 Day to maturity 0.19 0.54 0.21 0.19 0.17 Stem length 0.03 0.30 0.23 0.12 0.13 Pod length 0.45 0.02 0.20 0.50 0.01 Pod width 0.20 0.22 0.69 0.28 0.12 Pods/plants 0.32 0.26 0.16 0.12 0.49 Seeds/pod 0.00 0.24 0.15 0.48 0.44 Seed weight 0.54 0.19 0.32 0.13 0.50 Yield (tonnes/ha) 0.07 0.16 0.38 0.10 0.48 Leaf Area Index 0.21 0.17 0.01 0.03 0.02 Growth Habit 0.52 0.27 0.29 0.59 0.02 Figure 3 9. PCA plot of the phenotypic traits

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54 CHAPTER 4 GENETIC DIVERSITY AMONG 92 COMMON BEAN VARIETIES COLLECTED ACROSS HAITI Background Common bean ( Phaseolus vulgaris L.) is an important food legume in the world superseded in economic importance only by soybean ( Glycine max L.) and peanut ( Arachis hypogea L.). The genus Phaseolus consists of ov er 30 species (Debouck, 1991), five of which are domesticated; P. acutifolious A. Gray (tepary bean), P. coccineus L. (runner bean), P. lunatus L. (lima bean), P. polyanthus Greenman (year long bean), and P. vulgaris L. (common bean or snap bean) (Debouck, 1999). Among these, common bean is most cultivated across the globe and forms an essential source of nutrition and income in Latin America and Eastern and Southern Africa (Broughton et al., 2003). Common bean is postulated to have independently originate d from two centers of diversity, which led to the formation of Mesoamerican and Andean gene pools (Blair et al., 2009). The delineation between the two gene pools is supported by distinct molecular signatures observed in phaseolin seed proteins (Gepts et a l., 1986), allozymes (Singh et al. 1991), morphological traits (Singh et al., 1991), and DNA markers (Becerra et al., 1994). The gene pools are further divided into six races according to morphological criteria and agro ecological adaptation, three each fo r Mesoamerican (Durango, Jalisco, and Mesoamerica) and Andean (Chile, Nueva Granada, and Peru) (Blair et al., 2009). Knowledge and understanding of the genetic diversity among bean germplasm collection are essential for conservation efforts, as well as for broadening of the genetic base of varieties. Although many bean varieties are grown in Haiti, little information is available on their genetic structure, thus hindering germplasm improvement efforts.

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55 Besides, most growers do not identify varieties by name, instead of by market class, thus making it difficult to determine the diversity of cultivated common bean in the co untry. Characterization of germplasm using DNA molecular markers provides quantitative estimates of genetic diversity (Manifesto et al., 2001). A diverse number of markers are applied in assessing genetic diversity in beans including amplified fragment len gth polymorphisms, simple sequence repeats (SSR) and resistance gene analogs (Blair et al., 2013, Grisi et al., 2007; Yu et al., 2000) and single nucleotide polymorphism (SNPs) (Campa et al, 2018; Valdisser et al, 2017). DNA sequencing has become feasible with the advancement of next generation sequencing (NGS) technologies. NGS technologies such as genotyping by sequencing (GBS) have made DNA sequencing faster and cost effective. The GBS method has been extensively used for species with high diversity and large genome (Campa et al., 2018). GBS method generates thousands of SNP markers across the genome and has been used extensively in common bean for genome wide association study (GWAS), high density linkage map construction and diversity study (Hart et al ., 2015; Katuuramu et al., 2018, Bhakta et al., 2015, Campa et al, 2018). The goal of the current study was to determine the genetic diversity among 92 bean genotypes collected from different agro ecological zones across Haiti using GBS technology.

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56 Mat erials and Method s Plant M aterials Seeds of ninety two lines of beans were collected from different agro ecological zones of Haiti. Twenty three of these lines were obtained from the USAID AREA and the Legume Innovation Lab programs. In addition, four control genotypes that are commonly used for discriminating Mesoamerican and Andean gene pools in previous diversity studies were included (Asfaw et al. 2009; Blair et al. 2009, 2016). The controls are G4494 (Calima) and G19833 (Chaucha Chuga) for the Ande an gene pool and Dorado (DOR 364) and G5773 (ICA Pijao) for the Mesoamerican gene pool (Blair and Lorigados, 2016). Seed G ermination and DNA E xtraction For each genotype, twenty five seeds were germinated in cells filled with Jolly Gardener PRO LINE C/B Growing Mix medium in the greenhouse (22 32C) at the University of Florida Tropical Research and Education Center, Homestead, F lorida At the two leaf stage, one leaf of about 2 cm was collected from 20 individuals of each genotype and immediately fro zen in liquid nitrogen. DNA was extracted using a modified protocol that combined a differential centrifugation step from the nuclear fraction protocol (Bhakta et al., 2015), and the Flavorgen Biotech DNA isolation Kit. 100 mg of ground tissue was placed i n 2 ml Eppendorf tube using 1500 ul of sample resuspension buffer (SRB) with the inclusion of 0.5 % Triton X 100 and 1% of beta mercaptoethanol. The tubes were spun at 1500 rpm for 10 minutes. Subsequently, the supernatant was discarded, and the pellet was used for the DNA extraction using the Flavorgen kit. The use of differential step with the inclusion of Triton X 100 detergent allowed to remove the cytoplasmic DNA and obtain high quality of nuclear DNA, which is required for

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57 sequencing. The detergent di ssolves the plastid, and mitochondrial membranes, therefore, allow the release of cytoplasmic DNA in the buffer solution (Bhakta et al., 2015). The DNA quality was evaluated by using 0.8% agarose gel, and the concentration was assessed using Fisher Nanodro p One and Qubit 4 fluorometer. Genotyping by S equencing Genotyping by sequencing was conducted according to Schrder et al 2016, at Georgia Genomics and Bioinformatics Core at the University of Georgia using double digestion with MseI and Taq I restrictio n enzymes. This method was used because it has been shown to improve the quality and the coverage of GBS data in common bean (Schrder et al., 2016). The GBS library was prepared by ligating the digested DNA to unique nucleotide adapters (barcodes) followe d by PCR with flow cell attachment site tagged primer. Illumina NextSeq 150x High output Flow Cell was used to perform the sequencing. Demultiplexing with quality filtering was conducted by using Stacks. The sequencing reads were aligned to the P. Vulgaris L. reference genome sequence using Burrow Wheelers Alignment (BWA) tools. The reference based pipeline in Stacks was used for the extraction of single nucleotide polymorphism (SNP). Data filtration was performed in Tassel 5.2.52 by considering missing dat a inferior to 70%, and minor allele frequency (MAF>0.01). Data A nalysis The population structure analysis was conducted with Structure v2.3.4. The Structure parameters used were admixture model with independent allele frequencies, a burn in period of 1000 and 5000 Markov Chain Monte Carlo (MCMC) iterations with 20 replications for each hypothetical number of subpopulations (k) between 1 and 5. The

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58 optimum K value was calculated according to Evanno et al. (2005) using Structure Harvester. A new burn in peri od of 10000 and 30000 MCMC iterations were conducted for the optimum K value to assign accessions to subpopulations (Campa et al., 2018). Cluster analysis and principal component analysis (PCA) was conducted by using Darwin 6.0.021. Results Genotyping T he goal of this project was to assess the genetic diversity of 92 common bean lines from Haiti using Next Generation Sequencing technology. Genotyping was conducted using Illumina NextSeq. Sequencing of the GBS library yielded approximately 454,854,048 rea ds, while the Q30 value exceeded 80%. A total of 27,823 SNPS were identified. After filtering for missing data and minor allele frequency, a total of 1,115 SNPs distributed across the eleven chromosomes was selected. The number of SNPs per chromosome rang ed from 62 on chromosome Pv6 to 169 on chromosome Pv11, with an average of 98 markers/ chromosome. Population S tructure The Structure v2.3.4 software was used for testing a hypothetical number of optimal amount of two subpopulations (Fig 4 1 and Fig 4 2 ). The two main groups identified (Fig 4 3 ) included a group of 8 lines closely related to the controls G4494 and G19833 from the Andean gene pool and a group of 84 lines closely related to Mesoamerican controls (DOR 364 and G5773). However, 12 lines showed some level of admixture between the two gene pools.

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59 Phylogenetic T ree A dendrogram was constructed using UPGMA in Darwin using 1,115 SNPs. Figure 4 4 showed the phylogenetic tree obtained. From this tree, two main groups were obtained, a small group that included 2 Andean check cultivars G4494 and G19833 and eight other lines, and a large group that included 2 Mesoamerican controls (DOR 364 and G5773, in addition to 84 other lines which are predominantly small seeded black beans. A certain level of admixture was found in 2 lines in the Andean and ten lines in the Mesoamerican subpopulation Principal C omponent A nalysis A two dimensional plot obtained i n the principal component analysis (PCA) is presented in Figure 4 5 The first component PC1 accounted for 10.7% of the variances and distinguished the two main groups, Andean and Mesoamerican, as two separate clusters. The lines that showed introgression were identified through Structure and are mainly clustered in the intersection between the two main groups. On the other hand, the second PC accounted for 2.49% of the variance and revealed more diversity within the Mesoamerican than within the Andean gene pool. According to the PCA in Figure 4 6 which displayed the genotypes based on market type, the Mesoamerican group contained diverse bean types, including small seeded black beans which are prevalently grown and consumed in Haiti, yellow bean, white, and red mottled whereas the Andean group was composed mostly of large seeded red and red mottled beans. Discussion This project aimed to determine the genetic diversity among a collection of common bean accessions in Haiti and is the first of a kind to use genome wide based molecular markers derived through GBS.

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60 The accessions included in the current study are a good representation of the different agro ecological regions in the country since they were collected from small scale farmers across the country who typically save seed for subsequent growing seasons. The bean pool also contained lines from different breeding programs such as ORE (Organization for Rehabilitation of the Environment), University of Puerto Rico and UF Haiti AREA legume breeding program, University of Nebraska, USDA ARS, University Zamorano and National Seed Service (SNS) Haiti. Structure, c luster and p rincipal c omponent a nalysis (PCA) revealed the presence of the two main gene pools, Andean and Mesoamerican, in the Haitian bean collection. The separation between the two groups was confirmed by the presence of the Andean controls G4494 and G19833 in the Andean subgroup (Fig 4 4 ), and the Mesoamerican controls (DOR 364 and G5773) in the Mesoamerican subpopulation. Separation of the two gene pools has been observed in bean collections from the Carribean using RAPD and phaseolin analysis markers (Dur n et al. 2005, Castieiras et al., 1991, Lioi et al., 1990). Most of the lines (87%) collected clustered within the Mesoamerican gene pool. This result indicates that growers may prefer accessions of the Mesoamerican gene pool (black beans, yellow beans, white beans, and red mottled) due to higher yields. Additionally, this gene pool is typically well adapted to high temperature conditions in Haiti (Blair and Lorigados, 2016). In the current study, red mottled bean types clustered in both gene pools. Howe ver, the majority of these accessions were genetically similar to the Mesoamerican controls. These results confirmed previous findings that found a

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61 predominance of Mesoamerican type red mottled lines in Haiti, the Dominican Republic, and Cuba (Blair and Lo rigados, 2016; Durn et al., 2005). Although, the population structure analysis revealed two main subpopulations in the bean collection evaluated, 12 cultivars (about 13%) showed some level of introgression between the two gene pools. The level of in trogression found between the two gene pools in Haiti is lower than that reported in other countries including China, Italy, Ethiopia, Kenya, Spain, Portugal, Rwanda, which represent secondary centers of diversity for common beans (Asfaw et al, 2009; Blair et al, 2010, Masi et al.,2009; Martins et al, 2006; Rodio et al, 2003; Santalla et al, 2010; Sicard et al, 200 5 ; Zhang et al, 2008). Previous molecular characterization studies of common bean landraces and cultivars from the Caribbean revealed possible introgression between the two gene pools (Durn et al., 2005). Similar admixture levels were found among common bean accessions from Cuba (Blair and Lorigados, 2016) and Brazil (Blair et al., 2013; Burle et al., 2010). The admixture observed between the t wo gene pools might be expected due to inter gene pool breeding efforts that primarily utilize the Mesoamerican genetic background to improve specific characteristics of Andean bean lines (Beaver, 1999; Durn et al. 2005). Besides Haiti, an active breedin g program in the tropics, particularly in the highlands of eastern and southern Africa have resulted in increased admixture between the gene pools (Blair and Obrigado 2016 ; Daz et al. 2011; Blair et al., 2010 ; Daz and Blair, 2006). The level of inter ge ne pool introgression is lower in regions of common bean domestication, including the Mesoamerican region of Central America and Mexico, and the Andes mountains of South America (Blair and Obrigados, 2016; Avila et al., 2012; Blair et al., 2011).

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62 F igure 4 1. Me a n L(K) ( SD ) over 20 runs for each K value Figure 4 2

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63 Figure 4 3 Population structure inferred by Bayesian approach based on 1115 SNPs for k=2, Mesoamerican (red), Andean (green) Figure 4 4 Phylogenetic tree obtained for 96 lines of common bean and 1115 SNPs

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64 Figure 4 5 Two dimension p lot obtained from p rincipal c omponent a nalysis (PCA) for 96 lines of common bean and 1115 SNP s based on gene pool

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65 Figure 4 6 Two dimension plot obtained from p rincipal c omponent a nalysis (PCA) for 96 lines of common bean and 1115 SNP s based on bean market ty pe

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66 CHAPTER 5 SUMMARY AND CONCLUSIONS In the current study we evaluate d the agronomic performance of 13 elite common bean breeding lines currently under development by the UF AREA and the Legume Innovation Lab programs in Haiti T he results reported in this study showed a significant difference among the genotypes for most of the traits measured. The PR1627 8 (white bean) outperformed the other breeding lines in yield and disease resistance; therefore, it is a candidate for release to growers. Furthermore, the cultivars checks, including LORE 234 local, and Local 3 red could be used as a source of earliness for breeders. Based on phenotypic relationships, the number of pods per plant and number of seeds per pod may be useful traits for indirect selection for seed yield in common bean However, the data used w ere only for one s eason and one location ; therefore, further trials are necessary to validate the results. Besides this current project has some limitations that need to be considered when interpreting the results reported here This research was conducted under conditions typical for Haitian farmers (agriculture with low input) with the goal to select varieties that can thrive under different biotic and abiotic stres s with minimal input. The field trial was rainfed with no ferti lizer and pesticide application s Furthermore, the varieties differed in their response to the diseases assessed (powdery mildew, BCMV, and BGMV). The growth habit of the different gene pools may also affect the yield of the genotypes. Nevertheless to red uce the effect of some of the cofounding factors, disease incidence, as well as growth habit, were included in the ANOVA model for yield.

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67 The genetic diversity was also assessed among a collection of 92 common bean lines from Haiti, which included landr aces, breeding lines and released cultivars. The GBS revealed 27,823 SNPs, among which 1,115 were used for the diversity analysis after filtering for missing data and minor allele frequency. S tructure, cluster, and principal component analyses revealed th e presence of the two main gene pools in the bean population in Haiti. From these two subpopulations, the Mesoamerican gene pool was predominant with about 87% of the accessions collected, thus reflect the preference of this bean type among Haiti consumers However, 12 lines showed some level of introgression between the two main gene pools. The principal component analysis revealed more extensive genetic diversity within the Mesoamerican than within the Andean gene pool in the accession collection

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68 LIST OF REFERENCES Ambachew, D., Mekbib, F., Asfaw, A., Beebe, S. E., & Blair, M. W. (2015). Trait associations in common bean genotypes grown under drought stress and field infestation by BSM bean fly. The Crop Journal, 3(4), 305 316. Ariani, A., y Teran, J. C. B. M., & Gepts, P. (2016). Genome wide identification of SNPs and copy number variation in common bean ( Phaseolus vulgaris L.) using genotyping by sequencing (GBS). Molecular breeding 36 (7), 87. Asfaw, A. Blair, M. W., & Almekinders, C. (2009). Genetic diversity and population structure of common bean ( Phaseolus vulgaris L.) landraces from the East African highlands. Theoretical and Applied Genetics 120 (1), 1 12. Avila, T., Blair, M. W., Reyes, X., & Ber tin, P. (2012). Genetic diversity of bean (Phaseolus) landraces and wild relatives from the primary centre of origin of the Southern Andes. Plant Genetic Resources 10 (1), 83 92. Beaver, J. S. (1999). Improvement of large seeded race Nueva Granada cultivar s. In Common bean improvement in the twenty first century (pp. 275 288). Springer, Dordrecht. Beaver J.S., Zapata M. Alameda M. Porch T. Rosas J.C. Godoy Lutz G. & Prophete E. ( 2012 ) Common bean improvement in the Caribbean. Beaver, J. S., Prophete, E. H., Rosas, J. C., Lutz, G. G., Steadman, J. R., & Porch, T. G. (2014). Release of'XRAV 40 4'black bean ( Phaseolus vulgaris L.) cultivar. Journal of Agriculture of the University of Puerto Rico 98 (1), 83 87. Beaver, J. S., Prophete, E., Dmost hne, G., & Porch, T. G. (2016). Registration of PR1146 138 Yellow Bean Germplasm Line. Journal of Plant Registrations 10 (2), 145 148. Becerra Velasquez, V. L., & Gepts, P. (1994). RFLP diversity of common bean ( Phaseolus vulgaris ) in its centres of origin. Genome 37 (2), 256 263. Becerra Velsquez, V. L., Paredes, M., Rojo, C., Diaz, L. M., & Blair, M. W. (2010). Microsatellite marker characterization of Chilean Common Bean ( Phaseolus vulgaris L.) germplasm. Beebe, S., Skroch, P. W., Tohme, J., Duque M. C., Pedraza, F., & Nienhuis, J. (2000). Structure of genetic diversity among common bean landraces of Middle American origin based on correspondence analysis of RAPD. Crop science 40 (1), 264 273.

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69 Beebe, S., Rao, I., Blair, M., & Acosta, J. (2013). P henotyping common beans for adaptation to drought. Frontiers in physiology 4 35. Bhakta, M. S., Jones, V. A., & Vallejos, C. E. (2015). Punctuated distribution of recombination hotspots and demarcation of pericentromeric regions in Phaseolus vulgaris L. PLoS One, 10(1), e0116822. Bitocchi, E., Nanni, L., Bellucci, E., Rossi, M., Giardini, A., Zeuli, P. S., ... & Papa, R. (2012). Mesoamerican origin of the common bean ( Phaseolus vulgaris L.) is revealed by sequence data. Proceedings of the National Academy of Sciences 109 (14), E788 E796. Blair, M. W., Giraldo, M. C., Buendia, H. F., Tovar, E., Duque, M. C., & Beebe, S. E. (2006). Microsatellite marker diversity in common bean ( Phaseolus vulgaris L.). Theoretical and Applied Genetics, 113(1), 100 109. Blair, M. W., Rodriguez, L. M., Pedraza, F., Morales, F., & Beebe, S. (2007). Genetic mapping of the bean golden yellow mosaic geminivirus resistance gene bgm 1 and linkage with potyvirus resistance in common bean ( Phaseolus vulgaris L.). Theoretical and Applied Genetics 114 (2), 261 271. Blair, M. W., Daz, L. M., Buenda, H. F., & Duque, M. C. (2009). Genetic diversity, seed size associations and population structure of a core collection of common b eans ( Phaseolus vulgaris L.). Theoretical and Applied Genetics 119 (6), 955 972. Blair, M. W., Gonzlez, L. F., Kimani, P. M., & Butare, L. (2010). Genetic diversity, inter gene pool introgression and nutritional quality of common beans ( Phaseolus vulgaris L.) from Central Africa. Theoretical and Applied Genetics 121 (2), 237 248. Blair, M. W., Daz, L. M., Gill Langarica, H. R., Rosales Serna, R., Mayek Perez, N., & Acosta Gallegos, J. A. (2011). Genetic relatedness of Mexican common bean cultivars reveale d by microsatellite markers. Crop science 51 (6), 2655 2667. Blair, M. W., Izquierdo, P., Astudillo, C., & Grusak, M. A. (2013). A legume biofortification quandary: variability and genetic control of seed coat micronutrient accumulation in common beans. Fr ontiers in plant science, 4, 275. Blair, M. W., & Lorigados, S. M. (2016). Diversity of common bean landraces, breeding lines, and varieties from Cuba. Crop Science 56 (1), 322 330. Broughton, W. J., Hernandez, G., Blair, M., Beebe, S., Gepts, P., & Vander leyden, J. (2003). Beans (Phaseolus spp.) model food legumes. Plant and soil 252 (1), 55 128.

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75 BIOGRAPHICAL SKETCH Riphine Mainviel was born in Petit Gove, Haiti. She earned a Bachelor of Science degree in Agr iculture with a major in n atural r esources and e nvironment at the Faculty of Agronomy and Veterinary Medicine (FAMV) of the State University of Haiti in 2014. U pon graduation, she worked for the Ministry of Agriculture of Haiti in the Mitigation and Natural Disaster Program from 2015 to 2017. She was granted a scholarship in 2017 from UF Horticultural Sciences De partment, University of Florida. She was working with Dr. Geoff r ey Meru at TREC/ Homestead along with Dr. C. Eduardo Vallejos and Dr. Raphael Colbert as her committee members. Her primary research involved analyzing agronomic performance and genetic divers ity of common bean varieties in Haiti.