Pooled DNA sequencing in hairy vetch (Vicia villosa Roth) reveals QTL for seed dormancy but not pod dehiscence.

Introduction: Hairy vetch (Vicia villosa Roth) is a promising legume cover crop, but its use is limited by high rates of pod dehiscence and seed dormancy. Methods: We used phenotypically contrasting pooled DNA samples (n=24 with 29-74 individuals per sample) from an ongoing cover crop breeding program across four environments (site-year combinations: Maryland 2020, Maryland 2022, Wisconsin 2021, Wisconsin 2022) to find genetic associations and genomic prediction accuracies for pod dehiscence and seed dormancy. We also combined pooled DNA sample genetic association results with the results of a prior genome-wide association study. Results and discussion: Genomic prediction resulted in positive predictive abilities for both traits between environments and with an independent dataset (0.34-0.50), but reduced predictive ability for DNA pools with divergent seed dormancy in the Maryland environments (0.07-0.15). The pooled DNA samples found six significant (false discovery rate q-value<0.01) quantitative trait loci (QTL) for seed dormancy and four significant QTL for pod dehiscence. Unfortunately, the minor alleles of the pod dehiscence QTL increased the rate of pod dehiscence and are not useful for marker-assisted selection. When combined with a prior association study, sixteen seed dormancy QTL and zero pod dehiscence QTL were significant. Combining the association studies did not increase the detection of useful QTL.

Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection.

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Genome-wide association mapping in hairy vetch (Vicia villosa) discovers a large effect locus controlling seed dormancy.

Hairy vetch (Vicia villosa Roth), a winter-hardy annual legume, is a promising cover crop. To fully leverage its potential, seed production and field performance of V. villosa must be improved to facilitate producer adoption. Two classic domestication traits, seed dormancy (hard seed) and dehiscence (pod shatter), are selection targets in an ongoing breeding program. This study reports a genome-wide association study of 1,019 V. villosa individuals evaluated at two sites (Knox City, Texas and Corvallis, Oregon) for the proportion of dormant seed, visual pod dehiscence scores, and two dehiscence surrogate measures (force to dehiscence and pod spiraling score). Trait performance varied between sites, but reliability (related to heritability) across sites was strong (dormant seed proportion: 0.68; dehiscence score: 0.61; spiraling score: 0.42; force to dehiscence: 0.41). A major locus controlling seed dormancy was found (q-value: 1.29 × 10-5; chromosome 1: position: 63611165), which can be used by breeding programs to rapidly reduce dormancy in breeding populations. No significant dehiscence score QTL was found, primarily due to the high dehiscence rates in Corvallis, Oregon. Since Oregon is a potentially major V. villosa seed production region, further dehiscence resistance screening is necessary.

A reference assembly for the legume cover crop hairy vetch (Vicia villosa).

Vicia villosa is an incompletely domesticated annual legume of the Fabaceae family native to Europe and Western Asia. V. villosa is widely used as a cover crop and forage due to its ability to withstand harsh winters. Here, we generated a reference-quality genome assembly (Vvill1.0) from low error-rate long-sequence reads to improve the genetic-based trait selection of this species. Our Vvill1.0 assembly includes seven scaffolds corresponding to the seven estimated linkage groups and comprising approximately 68% of the total genome size of 2.03 Gbp. This assembly is expected to be a useful resource for genetically improving this emerging cover crop species and provide useful insights into legume genomics and plant genome evolution.

Transcript profiling of hairy vetch (Vicia villosa Roth) identified interesting genes for seed dormancy.

Hairy vetch, a diploid annual legume species, has a robust growth habit, high biomass yield, and winter hardy characteristics. Seed hardness is a major constraint for growing hairy vetch commercially. Hard seeded cultivars are valuable as forages, whereas soft seeded and shatter resistant cultivars have advantages for their use as a cover crop. Transcript analysis of hairy vetch was performed to understand the genetic mechanisms associated with important hairy vetch traits. RNA was extracted from leaves, flowers, immature pods, seed coats, and cotyledons of contrasting soft and hard seeded "AU Merit" plants. A range of 31.22-79.18 Gb RNA sequence data per tissue sample were generated with estimated coverage of 1040-2639×. RNA sequence assembly and mapping of the contigs against the Medicago truncatula (V4.0) genome identified 76,422 gene transcripts. A total of 24,254 transcripts were constitutively expressed in hairy vetch tissues. Key genes, such as KNOX4 (a class II KNOTTED-like homeobox KNOXII gene), qHs1 (endo-1,4-ß-glucanase), GmHs1-1 (calcineurin-like metallophosphoesterase), chitinase, shatterproof 1 and 2 (SHP1, SHP2), shatter resistant 1-5 (SHAT1-5)(NAC transcription factor), PDH1 (prephenate dehydrogenase 1), and pectin methylesterases with a potential role in seed hardness and pod shattering, were further explored based on genes involved in seed hardness from other species to query the hairy vetch transcriptome data. Identification of interesting candidate genes in hairy vetch can facilitate the development of improved cultivars with desirable seed characteristics for use as a forage and as a cover crop.

Intraspecific Variation for Leaf Physiological and Root Morphological Adaptation to Drought Stress in Alfalfa (Medicago sativa L.).

Drought stress reduces crop biomass yield and the profitability of rainfed agricultural systems. Evaluation of populations or accessions adapted to diverse geographical and agro-climatic environments sheds light on beneficial plant responses to enhance and optimize yield in resource-limited environments. This study used the morphological and physiological characteristics of leaves and roots from two different alfalfa subspecies during progressive drought stress imposed on controlled and field conditions. Two different soils (Experiments 1 and 2) imposed water stress at different stress intensities and crop stages in the controlled environment. Algorithm-based image analysis of leaves and root systems revealed key morphological and physiological traits associated with biomass yield under stress. The Medicago sativa subspecies (ssp.) sativa population, PI478573, had smaller leaves and maintained higher chlorophyll content (CC), leaf water potential, and osmotic potential under water stress. In contrast, M. sativa ssp. varia, PI502521, had larger leaves, a robust root system, and more biomass yield. In the field study, an unmanned aerial vehicle survey revealed PI502521 to have a higher normalized difference vegetation index (vegetation cover and plant health characteristics) throughout the cropping season, whereas PI478573 values were low during the hot summer and yielded low biomass in both irrigated and rainfed treatments. RhizoVision Explorer image analysis of excavated roots revealed a smaller diameter and a narrow root angle as target traits to increase alfalfa biomass yield irrespective of water availability. Root architectural traits such as network area, solidity, volume, surface area, and maximum radius exhibited significant variation at the genotype level only under limited water availability. Different drought-adaptive strategies identified across subspecies populations will benefit the plant under varying levels of water limitation and facilitate the development of alfalfa cultivars suitable across a broad range of growing conditions. The alleles from both subspecies will enable the development of drought-tolerant alfalfa with enhanced productivity under limited water availability.

Phenotyping seedlings for selection of root system architecture in alfalfa (Medicago sativa L.).

BACKGROUND: The root system architecture (RSA) of alfalfa (Medicago sativa L.) affects biomass production by influencing water and nutrient uptake, including nitrogen fixation. Further, roots are important for storing carbohydrates that are needed for regrowth in spring and after each harvest. Previous selection for a greater number of branched and fibrous roots significantly increased alfalfa biomass yield. However, phenotyping root systems of mature alfalfa plant is labor-intensive, time-consuming, and subject to environmental variability and human error. High-throughput and detailed phenotyping methods are needed to accelerate the development of alfalfa germplasm with distinct RSAs adapted to specific environmental conditions and for enhancing productivity in elite germplasm. In this study methods were developed for phenotyping 14-day-old alfalfa seedlings to identify measurable root traits that are highly heritable and can differentiate plants with either a branched or a tap rooted phenotype. Plants were grown in a soil-free mixture under controlled conditions, then the root systems were imaged with a flatbed scanner and measured using WinRhizo software. RESULTS: The branched root plants had a significantly greater number of tertiary roots and significantly longer tertiary roots relative to the tap rooted plants. Additionally, the branch rooted population had significantly more secondary roots > 2.5 cm relative to the tap rooted population. These two parameters distinguishing phenotypes were confirmed using two machine learning algorithms, Random Forest and Gradient Boosting Machines. Plants selected as seedlings for the branch rooted or tap rooted phenotypes were used in crossing blocks that resulted in a genetic gain of 10%, consistent with the previous selection strategy that utilized manual root scoring to phenotype 22-week-old-plants. Heritability analysis of various root architecture parameters from selected seedlings showed tertiary root length and number are highly heritable with values of 0.74 and 0.79, respectively. CONCLUSIONS: The results show that seedling root phenotyping is a reliable tool that can be used for alfalfa germplasm selection and breeding. Phenotypic selection of RSA in seedlings reduced time for selection by 20 weeks, significantly accelerating the breeding cycle.

Four chromosome scale genomes and a pan-genome annotation to accelerate pecan tree breeding.

Genome-enabled biotechnologies have the potential to accelerate breeding efforts in long-lived perennial crop species. Despite the transformative potential of molecular tools in pecan and other outcrossing tree species, highly heterozygous genomes, significant presence-absence gene content variation, and histories of interspecific hybridization have constrained breeding efforts. To overcome these challenges, here, we present diploid genome assemblies and annotations of four outbred pecan genotypes, including a PacBio HiFi chromosome-scale assembly of both haplotypes of the 'Pawnee' cultivar. Comparative analysis and pan-genome integration reveal substantial and likely adaptive interspecific genomic introgressions, including an over-retained haplotype introgressed from bitternut hickory into pecan breeding pedigrees. Further, by leveraging our pan-genome presence-absence and functional annotation database among genomes and within the two outbred haplotypes of the 'Lakota' genome, we identify candidate genes for pest and pathogen resistance. Combined, these analyses and resources highlight significant progress towards functional and quantitative genomics in highly diverse and outbred crops.

Genotype- and tissue-specific miRNA profiles and their targets in three alfalfa (Medicago sativa L) genotypes.

BACKGROUND: Alfalfa (Medicago sativa L.) is a forage legume with significant agricultural value worldwide. MicroRNAs (miRNAs) are key components of post-transcriptional gene regulation and essentially regulate many aspects of plant growth and development. Although miRNAs were reported in alfalfa, their expression profiles in different tissues and the discovery of novel miRNAs as well as their targets have not been described in this plant species. RESULTS: To identify tissue-specific miRNA profiles in whole plants, shoots and roots of three different alfalfa genotypes (Altet-4, NECS-141and NF08ALF06) were used. Small RNA libraries were generated and sequenced using a high-throughput sequencing platform. Analysis of these libraries enabled identification of100 miRNA families; 21 of them belong to the highly conserved families while the remaining 79 families are conserved at the minimum between M. sativa and the model legume and close relative, M. truncatula. The profiles of the six abundantly expressed miRNA families (miR156, miR159, miR166, miR319, miR396 and miR398) were relatively similar between the whole plants, roots and shoots of these three alfalfa genotypes. In contrast, robust differences between shoots and roots for miR160 and miR408 levels were evident, and their expression was more abundant in the shoots. Additionally, 17 novel miRNAs were identified and the relative abundance of some of these differed between tissue types. Further, the generation and analysis of degradome libraries from the three alfalfa genotypes enabled confirmation of 69 genes as targets for 31 miRNA families in alfalfa. CONCLUSIONS: The miRNA profiles revealed both similarities and differences in the expression profiles between tissues within a genotype as well as between the genotypes. Among the highly conserved miRNA families, miR166 was the most abundantly expressed in almost all tissues from the three genotypes. The identification of conserved and novel miRNAs as well as their targets in different tissues of multiple genotypes increased our understanding of miRNA-mediated gene regulation in alfalfa and could provide valuable insights for practical research and plant improvement applications in alfalfa and related legume species.

The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies.

BACKGROUND: Alfalfa (Medicago sativa L.) is the primary forage legume crop species in the United States and plays essential economic and ecological roles in agricultural systems across the country. Modern alfalfa is the result of hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp. falcata. Due to its large and complex genome, there are few genomic resources available for alfalfa improvement. RESULTS: A de novo transcriptome assembly from two alfalfa subspecies, M. sativa ssp. sativa (B47) and M. sativa ssp. falcata (F56) was developed using Illumina RNA-seq technology. Transcripts from roots, nitrogen-fixing root nodules, leaves, flowers, elongating stem internodes, and post-elongation stem internodes were assembled into the Medicago sativa Gene Index 1.2 (MSGI 1.2) representing 112,626 unique transcript sequences. Nodule-specific and transcripts involved in cell wall biosynthesis were identified. Statistical analyses identified 20,447 transcripts differentially expressed between the two subspecies. Pair-wise comparisons of each tissue combination identified 58,932 sequences differentially expressed in B47 and 69,143 sequences differentially expressed in F56. Comparing transcript abundance in floral tissues of B47 and F56 identified expression differences in sequences involved in anthocyanin and carotenoid synthesis, which determine flower pigmentation. Single nucleotide polymorphisms (SNPs) unique to each M. sativa subspecies (110,241) were identified. CONCLUSIONS: The Medicago sativa Gene Index 1.2 increases the expressed sequence data available for alfalfa by ninefold and can be expanded as additional experiments are performed. The MSGI 1.2 transcriptome sequences, annotations, expression profiles, and SNPs were assembled into the Alfalfa Gene Index and Expression Database (AGED) at http://plantgrn.noble.org/AGED/ , a publicly available genomic resource for alfalfa improvement and legume research.

Root Traits and Phenotyping Strategies for Plant Improvement.

Roots are crucial for nutrient and water acquisition and can be targeted to enhance plant productivity under a broad range of growing conditions. A current challenge for plant breeding is the limited ability to phenotype and select for desirable root characteristics due to their underground location. Plant breeding efforts aimed at modifying root traits can result in novel, more stress-tolerant crops and increased yield by enhancing the capacity of the plant for soil exploration and, thus, water and nutrient acquisition. Available approaches for root phenotyping in laboratory, greenhouse and field encompass simple agar plates to labor-intensive root digging (i.e., shovelomics) and soil boring methods, the construction of underground root observation stations and sophisticated computer-assisted root imaging. Here, we summarize root architectural traits relevant to crop productivity, survey root phenotyping strategies and describe their advantages, limitations and practical value for crop and forage breeding programs.

Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting.

Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

Lignin modification leads to increased nodule numbers in alfalfa.

Reduction of lignin levels in the forage legume alfalfa (Medicago sativa) by down-regulation of the monolignol biosynthetic enzyme hydroxycinnamoyl coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) results in strongly increased digestibility and processing ability of lignocellulose. However, these modifications are often also associated with dwarfing and other changes in plant growth. Given the importance of nitrogen fixation for legume growth, we evaluated the impact of constitutively targeted lignin modification on the belowground organs (roots and nodules) of alfalfa plants. HCT down-regulated alfalfa plants exhibit a striking reduction in root growth accompanied by an unexpected increase in nodule numbers when grown in the greenhouse or in the field. This phenotype is associated with increased levels of gibberellins and certain flavonoid compounds in roots. Although HCT down-regulation reduced biomass yields in both the greenhouse and field experiments, the impact on the allocation of nitrogen to shoots or roots was minimal. It is unlikely, therefore, that the altered growth phenotype of reduced-lignin alfalfa is a direct result of changes in nodulation or nitrogen fixation efficiency. Furthermore, HCT down-regulation has no measurable effect on carbon allocation to roots in either greenhouse or 3-year field trials.

Development of an alfalfa SNP array and its use to evaluate patterns of population structure and linkage disequilibrium.

A large set of genome-wide markers and a high-throughput genotyping platform can facilitate the genetic dissection of complex traits and accelerate molecular breeding applications. Previously, we identified about 0.9 million SNP markers by sequencing transcriptomes of 27 diverse alfalfa genotypes. From this SNP set, we developed an Illumina Infinium array containing 9,277 SNPs. Using this array, we genotyped 280 diverse alfalfa genotypes and several genotypes from related species. About 81% (7,476) of the SNPs met the criteria for quality control and showed polymorphisms. The alfalfa SNP array also showed a high level of transferability for several closely related Medicago species. Principal component analysis and model-based clustering showed clear population structure corresponding to subspecies and ploidy levels. Within cultivated tetraploid alfalfa, genotypes from dormant and nondormant cultivars were largely assigned to different clusters; genotypes from semidormant cultivars were split between the groups. The extent of linkage disequilibrium (LD) across all genotypes rapidly decayed to 26 Kbp at r(2) = 0.2, but the rate varied across ploidy levels and subspecies. A high level of consistency in LD was found between and within the two subpopulations of cultivated dormant and nondormant alfalfa suggesting that genome-wide association studies (GWAS) and genomic selection (GS) could be conducted using alfalfa genotypes from throughout the fall dormancy spectrum. However, the relatively low LD levels would require a large number of markers to fully saturate the genome.

Prevalence of single nucleotide polymorphism among 27 diverse alfalfa genotypes as assessed by transcriptome sequencing.

BACKGROUND: Alfalfa, a perennial, outcrossing species, is a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, particularly if many genome-wide markers are available. Transcriptome sequencing enables efficient high-throughput discovery of single nucleotide polymorphism (SNP) markers for a complex polyploid species. RESULT: The transcriptomes of 27 alfalfa genotypes, including elite breeding genotypes, parents of mapping populations, and unimproved wild genotypes, were sequenced using an Illumina Genome Analyzer IIx. De novo assembly of quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of individual reads to the contigs enabled the detection of 872,384 SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants at about 95% of SNP sites identified among five wild, unimproved genotypes are still present in cultivated alfalfa, and all four US breeding programs also contain a high proportion of these SNPs. Thus, little evidence exists among this dataset for loss of significant DNA sequence diversity from either domestication or breeding of alfalfa. Structure analysis indicated that individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa) were clearly separated. CONCLUSION: We used transcriptome sequencing to discover large numbers of SNPs segregating in elite breeding populations of alfalfa. Little loss of SNP diversity was evident between unimproved and elite alfalfa germplasm. The EST and SNP markers generated from this study are publicly available at the Legume Information System ( http://medsa.comparative-legumes.org/) and can contribute to future alfalfa research and breeding applications.

High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.).

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is available to authorized users.

Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most common type of sequence variation among plants and are often functionally important. We describe the use of 454 technology and high resolution melting analysis (HRM) for high throughput SNP discovery in tetraploid alfalfa (Medicago sativa L.), a species with high economic value but limited genomic resources. RESULTS: The alfalfa genotypes selected from M. sativa subsp. sativa var. 'Chilean' and M. sativa subsp. falcata var. 'Wisfal', which differ in water stress sensitivity, were used to prepare cDNA from tissue of clonally-propagated plants grown under either well-watered or water-stressed conditions, and then pooled for 454 sequencing. Based on 125.2 Mb of raw sequence, a total of 54,216 unique sequences were obtained including 24,144 tentative consensus (TCs) sequences and 30,072 singletons, ranging from 100 bp to 6,662 bp in length, with an average length of 541 bp. We identified 40,661 candidate SNPs distributed throughout the genome. A sample of candidate SNPs were evaluated and validated using high resolution melting (HRM) analysis. A total of 3,491 TCs harboring 20,270 candidate SNPs were located on the M. truncatula (MT 3.5.1) chromosomes. Gene Ontology assignments indicate that sequences obtained cover a broad range of GO categories. CONCLUSIONS: We describe an efficient method to identify thousands of SNPs distributed throughout the alfalfa genome covering a broad range of GO categories. Validated SNPs represent valuable molecular marker resources that can be used to enhance marker density in linkage maps, identify potential factors involved in heterosis and genetic variation, and as tools for association mapping and genomic selection in alfalfa.

SNP assay to detect the 'Hyuuga' red-brown lesion resistance gene for Asian soybean rust.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi Syd., has the potential to become a serious threat to soybean, Glycine max L. Merr., production in the USA. A novel rust resistance gene, Rpp?(Hyuuga), from the Japanese soybean cultivar Hyuuga has been identified and mapped to soybean chromosome 6 (Gm06). Our objectives were to fine-map the Rpp?(Hyuuga) gene and develop a high-throughput single nucleotide polymorphism (SNP) assay to detect this ASR resistance gene. The integration of recombination events from two different soybean populations and the ASR reaction data indicates that the Rpp?(Hyuuga) locus is located in a region of approximately 371 kb between STS70887 and STS70923 on chromosome Gm06. A set of 32 ancestral genotypes which is predicted to contain 95% of the alleles present in current elite North American breeding populations and the sources of the previously reported ASR resistance genes (Rpp1, Rpp2, Rpp3, Rpp4, Rpp5, and rpp5) were genotyped with five SNP markers. We developed a SimpleProbe assay based on melting curve analysis for SNP06-44058 which is tighly linked to the Rpp?(Hyuuga) gene. This SNP assay can differentiate plants/lines that are homozygous/homogeneous or heterozygous/heterogeneous for the resistant and susceptible alleles at the Rpp?(Hyuuga) locus.

Genome-wide identification of microsatellites in white clover (Trifolium repens L.) using FIASCO and phpSSRMiner.

BACKGROUND: Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover. RESULTS: Genomic libraries containing simple sequence repeat (SSR) sequences were constructed using a modified Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) procedure and phpSSRMiner was used to develop the microsatellite markers. SSR motifs were isolated using two biotin-labeled probes, (CA)17 and (ATG)12. The sequences of 6,816 clones were assembled into 1,698 contigs, 32% of which represented novel sequences based on BLASTN searches. Approximately 32%, 28%, and 16% of these SSRs contained hexa-, tri-, and di-nucleotide repeats, respectively. The most frequent motifs were the CA and ATG complementary repeats and the associated compound sequences. Primer pairs were designed for 859 SSR loci based on sequences from these genomic libraries and from GenBank white clover nucleotide sequences. A total of 191 SSR primers developed from the two libraries were tested for polymorphism in individual clones from the parental genotypes GA43 ('Durana'), 'SRVR' and six F1 progeny from a mapping population. Ninety two percent produced amplicons and 66% of these were polymorphic. CONCLUSION: The combined approach of identifying SSR-enriched fragments by FIASCO coupled with the primer design and in silico amplification using phpSSRMiner represents an efficient and low cost pipeline for the large-scale development of microsatellite markers in plants.The approach described here could be readily adapted and utilized in other non-related species with none or limited genomic resources.