USDA Core Collection Research Articles

Dissecting the Genetic Diversity of USDA Cowpea Germplasm Collection Using Kompetitive Allele Specific PCR-Single Nucleotide Polymorphism Markers.

Cowpea (Vigna unguiculata L. Walp) is an important grain legume crop of the subtropics, particularly in West Africa, where it contributes to the livelihoods of small-scale farmers. Despite being a drought-resilient crop, cowpea production is hampered by insect pests, diseases, parasitic weeds, and various abiotic stresses. Genetic improvement can help overcome these limitations, and exploring diverse cowpea genetic resources is crucial for cowpea breeding. This study evaluated the genetic diversity of 361 cowpea accessions from the USDA core collection for the species using 102 Kompetitive Allele Specific PCR (KASP) single nucleotide polymorphism (SNP) markers. A total of 102 KASP-SNP was validated in the germplasm panel, and 72 showed polymorphism across the germplasm panel. The polymorphism information content (PIC) of all SNPs ranged from 0.1 to 0.37, with an average of 0.29, while the mean observed heterozygosity was 0.52. The population structure revealed three distinct populations that clustered into two major groups after phylogenetic analysis. Analysis of molecular variance (AMOVA) indicated greater genetic variation within populations than among populations. Although cowpea generally has a narrow genetic diversity, the accessions used in this study exhibited considerable variation across geographical regions, sub-species, and improvement status. These results indicated that the selected KASP genotyping assay can provide robust and accurate genotyping data for application in the selection and management of cowpea germplasm in breeding programs and genebanks.

Genome-Wide SNP Identification and Association Mapping for Seed Mineral Concentration in Mung Bean (Vigna radiata L.).

Mung bean (Vigna radiata L.) quality is dependent on seed chemical composition, which in turn determines the benefits of its consumption for human health and nutrition. While mung bean is rich in a range of nutritional components, such as protein, carbohydrates and vitamins, it remains less well studied than other legume crops in terms of micronutrients. In addition, mung bean genomics and genetic resources are relatively sparse. The objectives of this research were three-fold, namely: to develop a genome-wide marker system for mung bean based on genotyping by sequencing (GBS), to evaluate diversity of mung beans available to breeders in the United States and finally, to perform a genome-wide association study (GWAS) for nutrient concentrations based on a seven mineral analysis using inductively coupled plasma (ICP) spectroscopy. All parts of our research were performed with 95 cultivated mung bean genotypes chosen from the USDA core collection representing accessions from 13 countries. Overall, we identified a total of 6,486 high quality single nucleotide polymorphisms (SNPs) from the GBS dataset and found 43 marker × trait associations (MTAs) with calcium, iron, potassium, manganese, phosphorous, sulfur or zinc concentrations in mung bean grain produced in either of two consecutive years’ field experiments. The MTAs were scattered across 35 genomic regions explaining on average 22% of the variation for each seed nutrient in each year. Most of the gene regions provided valuable candidate loci to use in future breeding of new varieties of mung bean and further the understanding of genetic control of nutritional properties in the crop. Other SNPs identified in this study will serve as important resources to enable marker-assisted selection (MAS) for nutritional improvement in mung bean and to analyze cultivars of mung bean.

Is the USDA core collection of common bean representative of genetic diversity of the species, as assessed by SNP diversity?

AbstractCore collections are envisioned to be a representative subset of larger germplasm collections. They were introduced to facilitate the characterization and use of these germplasm collections. The common bean (Phaseolus vulgaris L.) core collection of the USDA Western Regional Plant Introduction Station was one of the first collections to be established in the early 1990s. Here, we evaluate the representativity of this common bean collection in light of the availability of a single nucleotide polymorphism (SNP) platform and new information about genetic diversity of the species, including phaseolin and seed type data. The SNP diversity was studied with a combination of STRUCTURE, principal coordinate analysis (PCoA), and neighbor‐joining analysis (NJA). STRUCTURE analyses were conducted for K (number of subpopulations) = 3 and K = 7, based on the ad hoc statistic ΔK. The K = 3 analysis recognized the split between Andean and Mesoamerican domesticates and the subdivision of the Mesoamerican domesticates into high‐ (Durango/Jalisco) and low‐altitude (Mesoamerica) ecogeographic races. The K = 7 analysis further subdivided the Andean group identified for K = 3, as well as the high‐altitude group from the Mesoamerican gene pool. It also identified smaller groups consisting of Mesoamerican wild beans. The PCoA and NJA confirmed the STRUCTURE results and highlighted the existence of presumed hybridization among groups. Our results suggest that this core collection should be updated by adding domesticated categories, developing a separate wild common bean core collection, and developing cores for specific purposes.

USDA Core Collection of Common Bean Needs to Be Updated

USDA Core Collection of Common Bean Needs to Be Updated

Assessing the potential for soybean yield improvement through plant architectural modification

AbstractSoybean yield potential can be increased by increasing the number of pods per plant, which can be achieved by increasing the number of nodes per plant while decreasing internode length to prevent lodging due to excessive height. In an effort to discover the genetic variability for this trait, a genome wide association study (GWAS) was performed on 451 diverse lines from the USDA core collection for height, internode length, and the number of nodes. The QTL signifying Dt1 was found correlating to height and quantity of nodes, but no significant QTL for internode spacing were uncovered. From our evaluation, this suggests that genomic selection for variation plant height is feasible; however, no specific genetic trait was detected to select for internode spacing.

Genome wide association study discovers genomic regions involved in resistance to soybean cyst nematode (Heterodera glycines) in common bean.

Common bean (Phaseolus vulgaris L.) is an important high protein crop grown worldwide. North Dakota and Minnesota are the largest producers of common beans in the USA, but crop production is threatened by soybean cyst nematode (SCN; Heterodera glycines Ichinohe) because most current cultivars are susceptible. Greenhouse screening data using SCN HG type 0 from 317 plant introductions (PI’s) from the USDA core collection was used to conduct a genome wide association study (GWAS). These lines were divided into two subpopulations based on principal component analysis (Middle American vs. Andean). Phenotypic results based on the female index showed that accessions could be classified as highly resistant (21% and 27%), moderately resistant (51% and 48%), moderately susceptible (27% and 22%) and highly susceptible (1% and 3%) for Middle American and Andean gene pools, respectively. Mixed models with two principal components (PCs) and kinship matrix for Middle American genotypes and Andean genotypes were used in the GWAS analysis using 3,985 and 4,811 single nucleotide polymorphic (SNP) markers, respectively which were evenly distributed across all 11 chromosomes. Significant peaks on Pv07, and Pv11 in Middle American and on Pv07, Pv08, Pv09 and Pv11 in Andean group were found to be associated with SCN resistance. Homologs of soybean rhg1, a locus which confers resistance to SCN in soybean, were identified on chromosomes Pv01 and Pv08 in the Middle American and Andean gene pools, respectively. These genomic regions may be the key to develop SCN-resistant common bean cultivars.

Molecular Characterization of Mung Bean Germplasm from the USDA Core Collection Using Newly Developed KASP‐based SNP Markers

Mung bean [Vigna radiata (L.) Wilczek] is an important, nutritious legume food crop for tropical and subtropical countries with high value in Asia and worldwide potential. However, the genetic improvement of mung bean is slowed by low polymorphism of previous molecular markers, even microsatellites. The goals of this study were to develop Kompetitive Allele Specific Polymerase Chain Reaction (KASP)‐based single‐nucleotide polymorphism (SNP) markers and use them for characterization of 94 cultivated mung bean genotypes from the USDA originating in 27 countries across 10 regions of the world, all being cultivars rather than wild accessions. We targeted 42 SNPs from previous sequencing and converted them into 20 robust KASP assays. Of these, 18 were polymorphic among the mung bean cultivars, with 38 alleles total and 1.9 alleles per locus average. The polymorphism information content of the newly developed markers ranged from zero for monomorphic markers to 0.375 for the most diverse biallelic polymorphic marker (MBkSNP_39) and averaged 0.250 across all loci. Population structure analysis grouped 90% of the accessions into two subpopulations, with 10% belonging to an admixture group, but did not follow geographic origins of the germplasm, suggesting no clear center of origin and blending of the subpopulations. An analysis of molecular variance revealed 22% of genetic variation among subpopulations and 78% within subpopulations. The first two axes of region‐wide principle coordinate analysis explained 81.26% variation of total variation, indicating the existence of genetic diversity among groups. The SNP markers of this study can be used in molecular breeding of mung bean and are the first to work with KASP detection.

Association Studies and Legume Synteny Reveal Haplotypes Determining Seed Size in Vigna unguiculata

Highly specific seed market classes for cowpea and other grain legumes exist because grain is most commonly cooked and consumed whole. Size, shape, color, and texture are critical features of these market classes and breeders target development of cultivars for market acceptance. Resistance to biotic and abiotic stresses that are absent from elite breeding material are often introgressed through crosses to landraces or wild relatives. When crosses are made between parents with different grain quality characteristics, recovery of progeny with acceptable or enhanced grain quality is problematic. Thus genetic markers for grain quality traits can help in pyramiding genes needed for specific market classes. Allelic variation dictating the inheritance of seed size can be tagged and used to assist the selection of large seeded lines. In this work we applied 1,536-plex SNP genotyping and knowledge of legume synteny to characterize regions of the cowpea genome associated with seed size. These marker-trait associations will enable breeders to use marker-based selection approaches to increase the frequency of progeny with large seed. For 804 individuals derived from eight bi-parental populations, QTL analysis was used to identify markers linked to 10 trait determinants. In addition, the population structure of 171 samples from the USDA core collection was identified and incorporated into a genome-wide association study which supported more than half of the trait-associated regions important in the bi-parental populations. Seven of the total 10 QTLs were supported based on synteny to seed size associated regions identified in the related legume soybean. In addition to delivering markers linked to major trait determinants in the context of modern breeding, we provide an analysis of the diversity of the USDA core collection of cowpea to identify genepools, migrants, admixture, and duplicates.

Genetic variability of isoflavones in the USDA red clover core collection

Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones known for their human health benefits. The objectives of this study were: i) to quantify, using HPLC analysis, isoflavones in 77 accessions from the USDA core collection and a Brazilian line; ii) to verify possible relationships depending on their origin, improvement status or maturity type and; iii) to verify the seasonal variation. The isoflavone mean contents were 29.27 µg g-1 of dry material for daidzein, 163.69 µg g-1 for genistein, 11353.29 µg g-1 for formononetin and 6568.8 µg g-1 for biochanin A. Clustering was mainly influenced by the total amount of isoflavones and partially due to maturity type, improvement status and geographic origin. The seasonal evaluation demonstrated an increase of concentration during winter, and decrease during spring. These results highlighted accessions that can be used to develop new varieties with low or high isoflavones concentration.

Using minimum DNA marker loci for accurate population classification in rice (Oryza sativa L.)

Because of the rich diversity among rice accessions grown around the world in distinct environments, traditional methods using morphology, cross compatibility and geography for classifying rice accessions according to different sub-populations have given way to use of molecular markers. Having a few robust markers that can quickly assign population structure to germplasm will facilitate making more informed choices about genetic diversity within seedbanks and breeding genepools. WHICHLOCI is a computer program that selects the best combination of loci for population assignment through empirical analysis of molecular marker data. This program has been used in surveys of plant species, for fish population assignment, and in human ancestry analysis. Using WHICHLOCI, we ranked the discriminatory power of 72 DNA markers used to genotype 1,604 accessions of the USDA rice core collection, and developed panels with a minimum number of markers for population assignment with 99% or higher accuracy. A total of 14 markers with high discriminatory power, genetic diversity, allelic frequency, and polymorphic information content were identified. A panel of just four markers, RM551, RM11, RM224 and RM44, was effective in assigning germplasm accessions to any of five sub-populations with 99.4% accuracy. Panels using only three markers were effective for assignment of rice germplasm to specific sub-populations, tropical japonica,temperate japonica,indica, aus, and aromatic. Assignment to tropical japonica,temperate japonica, or indica sub-populations was highly reliable using 3–4 markers, demonstrated by the high correlation with assignment using 72 markers. However, population assignment to aus and aromatic groups was less reliable, possibly due to the smaller representation of this material in the USDA core collection. More reference cultivars may be needed to improve population assignment to these two groups. This study demonstrated that a small number of DNA markers is effective for classification of germplasm into five sub-populations in rice. This will facilitate rapid screening of large rice germplasm banks for population assignment at a modest cost. The resulting information will be valuable to researchers to verify population classification of germplasm prior to initiating genetic studies, maximizing genetic diversity between sub-populations, or minimizing cross incompatibility while maximizing allelic diversity within specific sub-populations.

Genetic diversity and relatedness of rice cultivars resistant to straighthead disorder

With 4 figures and 3 tables SummaryStraighthead, a physiological disorder of rice (Oryza sativa L.), has been reported in numerous rice‐growing countries for its severe impact on yield reduction. Genetic diversity, relatedness and structure of parental germplasm are important for breeders to design strategies in breeding programmes. We analysed 1002 rice accessions that were sampled from the USDA core collection, evaluated for straighthead and genotyped with 72 molecular markers. Forty‐two accessions were resistant to straighthead with no yield reduction under severe infestation. The resistant cultivars originated from 15 countries in 10 geographic regions worldwide. The accessions had great diversification indicated by 15 phenotypic characteristics including all types of grain and cooking quality. The number of alleles averaged 5.9, and the 72 molecular markers were highly informative among the 42 resistant accessions. Thirty resistant accessions were identified as indica type and were clearly classified in two groups. Ten resistant cultivars were identified as temperate japonica and the other two were positioned between temperate and tropical japonica rice. Breeders worldwide could use these findings on phenotypic diversification and genetic relatedness among the straighthead resistant accessions in order to make germplasm choices for transferring straighthead resistance to their local cultivars, serving various needs on grain, cooking quality and genetic classification in their programmes.

Infection and development of Phoma medicaginis on moderately resistant and susceptible alfalfa genotypes

In North America, spring black stem and leaf spot, caused by Phoma medicaginis, is one of the most important diseases of alfalfa (Medicago sativa). Symptoms appear initially as small, dark brown to black spots that eventually enlarge and on leaves the spots are associated with chlorosis and defoliation. The infection process of P. medicaginis was previously investigated on susceptible alfalfa genotypes, but little is known about this process with alfalfa plants with differing degrees of resistance. We used light and scanning electron microscopy to follow infection and development of P. medicaginis from pycnidiospore deposition on leaf surfaces through pycnidia formation on selected susceptible and resistant alfalfa genotypes from the USDA core collection. In both old and young leaves, spore germination, penetration, development of mycelium, and pycnidia formation were delayed by approximately 24 h on moderately resistant alfalfa genotypes compared with susceptible genotypes. A similar delay in visible symptom development occurred until about 192 h after inoculation, after which the amount of chlorosis in all genotypes was similar.

Characterization of a USDA Kentucky Bluegrass (Poa pratensis L.) Core Collection for Reproductive Mode and DNA Content by Flow Cytometry

Kentucky bluegrass (Poa pratensis L.) is an important turf and forage grass species with a facultative apomictic breeding behavior. In this study, mature seed and leaf tissue from 38 accessions of a USDA core collection of Kentucky bluegrass were analyzed with flow cytometry to characterize the reproductive mode and DNA content for each accession. Major reproductive pathways for each accession were determined based upon the presence and the position of the peaks observed and the known methods of reproduction for Kentucky bluegrass. While the majority of the accessions exhibited facultative apomictic reproductive behavior with a combination of reduced, zygotic and unreduced, parthenogenic embryo production, obligate sexual or obligate apomictic accessions were also found to be present in this core collection. In addition, reduced, parthenogenic and unreduced zygotic embryos were also detected in several accessions. Flow cytometric analysis of somatic tissue revealed a large range of DNA variation within this core collection. We also examined the sensitivity of flow cytometry in analyzing bulked samples containing a large number of plants with varied DNA content and determined that flow cytometry can effectively detect a plant having a different DNA content within a 15-plant bulk sample. Overall the combination of mature seed and somatic tissue analysis generated important information for the Kentucky bluegrass core collection and can be an effective and affordable tool to characterize even greater numbers of Kentucky bluegrass accessions.

Biomass production and related characters in the core collection of Pisum germplasm

Retention of residue on the soil surface following harvest is an effective method of reducing soil erosion from both wind and water. The pea crop produces small amounts of residue to effectively reduce soil erosion. Severe erosion occurs in pea production areas such as the Palouse Region of the US Pacific Northwest (PNW) when low residue crops such as spring pea or lentil are followed by fall-sown wheat. The current study was conducted to determine the range of total aboveground biomass (TAB), seed yield, and straw (residue) production from the plant identification (PI) accessions that comprise the core collection of Pisum germplasm. In addition, the potential for increasing seed yield and straw production simultaneously was evaluated. Three hundred and ninety PI accessions were screened in the field in 1996 and 1997. The variation for TAB, seed yield and straw production among the PI accessions exceeded that of the controls both years. Seed yield was positively correlated with straw production (r = 0.81, p< 0.01) indicating that seed and straw production can be increased simultaneously through positive selection for both traits. Significant favorable variation is present among accessions in the USDA core collection of Pisum germplasm which could be used to increase both seed yield and total biomass production of adapted breeding lines.