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Paul Skroch and Jim Nienhuis

The genetic variation in a population of one hundred Snap Bean varieties, including processing and garden types, was studied using RAPD markers. All one hundred genotypes were distinguished by unique combinations of banding patterns. These unique “fingerprints” were tested for repeatability. Certain bands were very reliable and can be used for varietal identification. The RAPD marker data was also used to estimate genetic relationships among a subset of the one hundred lines. The results of the analysis agreed with known pedigree information. These markers will allow more precise monitering and control of germplasm by those who are involved with the breeding and production of superior seed.

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James Nienhuis, Paul Skroch, and Steve Beebe

Nuñas are a type of common bean (Phaseolus vulgaris) that possess the unusual characteristic of popping or expanding their cotyledonary tissue when heated. Numerous landraces of nuña beans were domesticated in the Andean region of South America (Peru, Bolivia, and Ecuador) and have been grown and consumed in this region since antiquity. The practical consideration in the domestication of nuñas in the high Andes was likely due to the greater energy efficiency in cooking toasted vs. boiled seeds.The Phaseolus germplasm bank at CIAT (Centro Internacional de Agricultura Tropical) has developed a core collection of Andean beans that includes numerous nuña landraces. Based on the wide range of phaseolin types observed among nuña landraces, it has been hypothesized that nuñas may represent a greater source of genetic diversity compared to other landraces and cultivars of common bean. Eighty nuña accessions and 120 nonpopping common bean accessions were randomly sampled from the CIAT Andean germplasm core collection. The 200 accessions were characterized for 140 mapped RAPD markers. The objectives of our research were to 1) understand the genetic structure of nuña bean accessions relative to other Andean common beans, and 2) to measure the genetic distance and genetic diversity between nuña and other Andean bean populations.

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James Nienhuis, Jan Tivang, Paul Skroch, and Joao B. dos Santos

Knowledge of relative genetic distance among genotypes is useful in a breeding program because it permits organization of germplasm resources. Genetic distance (GD) was estimated among 65 Phaselous lunatus L.. accessions, which included 4 large-seeded and 7 small-seeded cultivars and 54 germplasm accessions (landrace's) from the Caribbean and North, Central, and South America. Based on 125 polymorphic random amplification polymorphic DNA (RAPD) bands, two major clusters, which generally correspond in seed size and geographic region to [be Mesoamerican and Andean gene pools, were observed among the landraces (GD = 0.726 ± 0.041). Four Fordhook cultivars and a landrace from the United States formed a separate cluster that is more distantly related to the small- (GD) = 0.561 ± 0.039) than to the large-seeded cluster (GD = 0.303 ± 0.022). The mean GD between the Andean and Mesoamerican (0.726), Mesoamerican and Fordhook (0.561), and Andean and Fordhook (0.303) clusters were all significant. The significant GD between the Andean and Mesoamerican groups supports the hypothesized existence of two major gene pools in lima bean. The RAPD marker diversity of the Mesoamerican group was the largest (0.1 10), followed by the Andean (0.097) and Ford hook (0.062) groups. The plot of the relationship between the coefficient of variation (cv) and sample size (number of bands) indicates that cvs as low as 10% for estimating CD between Andean and Mesoamerican lima bean accessions can be achieved by sampling as few as 100 bands.

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Geunhwa Jung, Paul Skroch, James Nienhuis, and Dermot Coyne

One of the highest levels of common bacterial blight (CBB) resistance identified in Phaseolus vulgaris is found in XAN-159, which was developed for leaf resistance to CBB through six generations of pedigree selection of progenies derived from the interspecific cross [(`Pinto UI 114' × PI 319441) × P. acutifolius PI 319443] × `Masterpiece'. A RAPD genetic linkage map was previously constructed in a recombinant inbred population derived from the common bean cross PC-50 × XAN-159 for identification of genomic regions associated with bacterial disease resistance in XAN-159. To confirm that chromosomal regions associated with CBB resistance in XAN-159 were introgressed from tepary bean, we investigated the parentage of each genomic interval in XAN-159 by studying the genomic constitutions of the four different parents involved in the pedigree. The results indicate that all genomic regions associated with CBB resistance contain intervals derived exclusively from tepary bean. The uniqueness of marker polymorphisms associated with resistance to CBB in XAN-159 will allow the application of marker assisted selection for these resistance genes in most populations of common bean.

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Mario Crespo, James Nienhuis, Jan Tivang, and Paul Skroch

Knowledge of relative genetic distance among genotypes is useful in a breeding program because it permits organization of germplasm resources. Genetic distance (GD) was estimated among 113 faba bean, Vicia faba L. genotypes, which included three botanical varieties from different geographical areas around the world. The genotypes included 87 accessions from Bolivia, 14 accessions from the Middle East and North Africa, five accessions from Australia, and seven commercial varieties from Europe. Twenty-three RAPD primers were scored yielding four to 13 polymorphic bands resulting in a total of 165 bands. Our objective was to determine genetic relationships among accessions and cultivars as measured by RAPD markers. The genetic relationships were estimated using the ratio of discordant to total bands scored. A multidimensional scaling (MDS) plot indicated four clusters corresponding to: i) European commercial cultivars; ii) the Middle East, North Africa, and Australian accessions; iii) the Bolivian highland landraces; and iv) the Bolivian collection maintained in a valley environment. A permutation test confirmed the four clusters (P < 0.01). Sampling variance results indicated that a CV of 10% could be obtained with as few as 148 bands between groups. Selection and drift appears the main cause of divergence of two populations in the Bolivian faba bean collection. The results of this study indicated that RAPDs are a powerful tool for evaluation of germplasm conservation methods in faba bean.

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James Nienhuis, Joao B. dos Santos, Paul Skroch, and Jan Tivang

Genetic distance was calculated among 92 tomato, Lycopersicon esculentum, genotypes, which included open pollinated cultivars and commercial hybrids of both fresh market and processing types. Over 800 were screened, and 44 10-mer primers which had clear banding patterns and were polymorphic among cultivars were selected. From each of the primers an average of five polymorphic bands were classified for their presence or absence for each genotype. Genetic distance was calculated as the ratio of discordant to total bands scored. A multidimensional scaling (MDS) plot indicated that the processing cultivars, `UC82' derivatives and fresh market types generally formed separate clusters. Within groups, genetic distance corresponded to known pedigree relationships. The genetic distance between duplicate samples of 10 genotypes ranged from 0.01 to 0.05. The results of this study indicated that RAPDs provide a high degree of resolution for estimating genetic relationships among tomato cultivars.

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Jan G. Tivang, Neal DeVos, James Nienhuis, and Paul Skroch

Individual heads (capitula) from five discrete artichoke, Cyara scolymus L., populations were evaluated using RAPD markers. One vegetatively-propagated cultivar; Green Globe; two seed-propagated cultivars, Imperial Star and Big Heart XR-1; and two breeding populations were examined. Twenty-seven RAPD primers were scored yielding 2 to 16 polymorphic bands resulting in a total of 178 bands. Our objective was to determine if RAPD markers could be used to distinguish between and within populations. The genetic relationships among populations as well as among individuals within each population were estimated using the ratio of discordant to total bands scored. Data reduction (MDS) provided a plot indicating five clusters corresponding to the five populations. Confirmation of the presence of five discrete clusters was obtained by analysis of variance of the marker frequencies. The genetic diversity index (GDI) was calculated for each populations as the pooled variance of band frequency for each population. The GDI values were highly correlated to the mean genetic distance within each population. The homogeneity of variance for the GDI values associated with each population were compared using the Siegel-Tukey test for homogeneity of spread.

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Jan Tivang, Paul W. Skroch, James Nienhuis, and Neal De Vos

The magnitude of genetic differences among and heterogeneity within globe artichoke cultivars is unknown. Variation among individual heads (capitula) from three artichoke cultivars and two breeding populations were evaluated using RAPD markers. One vegetatively propagated cultivar (`Green Globe'), two seed-propagated cultivars (`Imperial Star' and `Big Heart') and two breeding populations were examined. Two to thirteen polymorphic bands were observed for 27 RAPD primers, which resulted in 178 scored bands. Variation was found within and among all cultivars, and breeding populations indicating that all five groups represent heterogeneous populations with respect to RAPD markers. The genetic relationships among individual genotypes were estimated using the ratio of discordant bands to total bands scored. Multidimensional scaling of the relationship matrix showed five independent clusters corresponding to the three cultivars and two breeding populations. The integrity of the five clusters was confirmed using pooled chi-squares for fragment homogeneity. Average gene diversity (Hs) was calculated for each population sample, and a one-way analysis of variance showed significant differences among populations. `Big Heart' had an Hs value equivalent to the two breeding populations, while clonally propagated `Green Globe' and seed propagated `Imperial Star' had the lowest Hs values. The RAPD heterogeneity observed within clonally propagated `Green Globe' is consistent with phenotypic variability observed for this cultivar. Overall, the results demonstrate the utility of the RAPD technique for evaluating genetic relationships and contrasting levels of genetic diversity among populations of artichoke genotypes.

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D. Michael Benson, Paul R. Fantz, and Walter A. Skroch

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Julie Villand, James Nienhuis, Paul Skroch, and Jan Tivang

Precise cultivar descriptions are necessary to support Plant Variety Protection and utility applications for patent protection. However, accurate discrimination among cultivars is contingent upon the dependability of the method used to delineate lines. The efficiency and reliability of Amplified Fragment Length Polymorphisms (AFLPs), Random Amplified Polymorphic DNAs (RAPDs), microsatellite polymorphisms, and phenotypic traits were studied in order to determine a method's ability to accurately predict pedigree relationships among a set of 20 California processing tomato cultivars. All molecular marker and phenotypic trait data sets were independently produced using identical cultivar seed sources. Data was reduced to a genetic distance measure and presented as a multidimensional scaling (MDS) plot. Principal component analysis using the scored quantitative phenotypic traits was computed and is compared to molecular marker data results. Experimental error, sampling variance, and independence of scored bands for each molecular marker technique are presented. These estimates should assist breeders to determine a sufficient level of characterization, determine a minimum distance considered to be unique, and defend pedigree relationships.