Maria J. Monteros

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.

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 r2 = 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

BackgroundAlfalfa, 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.ResultThe 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.ConclusionWe 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.

Lignin Modification Leads to Increased Nodule Numbers in Alfalfa

Reducing lignin content in stems and roots of alfalfa results in an increased nodule number phenotype. 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.

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

BackgroundAlfalfa (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.ResultsA 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.ConclusionsThe 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.

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.

QTL Mapping of Aluminum Tolerance in Tetraploid Alfalfa

Aluminum (Al) toxicity in acid soils is one of the factors limiting crop production. Alfalfa (Medicago sativa L.) is one of the most important forage legumes worldwide and is susceptible to Al toxicity. Al tolerance in alfalfa was identified in a diploid Medicago sativa subs. caerulea accession (Sledge et al., 2002) and has been successfully integrated at the tetraploid level in the Al-tolerant genotype Altet-4. The goals of this study are to identify and confirm quantitative trait loci (QTL) for Al-tolerance in tetraploid alfalfa. Two populations of at least 190 individuals each were developed from crosses between Altet-4 (Al-tolerant) and the Al-susceptible genotypes 95-608 derived from CUF-101, and NECS141, a semi-dormant breeding line developed in Iowa. The parental lines and the progeny from the mapping populations were screened using a callus bioassay and a whole plant assay. Genetic linkage maps constructed using EST-SSR markers (Sledge et al., 2005) were developed and used to identify QTL associated with Al tolerance. Comparison of QTL identified using different screening methods will be discussed. The long-term goal of this research is to use molecular markers associated with the Al-tolerance trait to accelerate the development of alfalfa cultivars with improved productivity in acidic and Al-toxic soils.

High Density Array for SNP Genotyping and Mapping in Tetraploid Alfalfa

The development of an Illumina iSelect Infinium array that includes approximately 9,277 SNPs identified from sequencing 27 diverse alfalfa (Medicago sativa L.) genotypes can be used to accelerate genome-wide association, mapping and molecular breeding efforts in alfalfa. Individuals from a mapping population obtained from a cross between Altet-4 and NECS-141 segregating for aluminum tolerance, fall regrowth, lignin content and other agronomic characteristics were genotyped using the developed array. The dosage status (nulliplex, simplex, duplex, triplex, or quadruplex) of an individual was successfully distinguished using GenomeStudio software for most of the SNPs evaluated. A total of 3,701 SNPs were polymorphic and segregating in the progeny of the mapping population. The number of SNP with segregation ratios suitable for mapping in tetraploid alfalfa were 1,738 SNPs for simplex × nulliplex, 582 SNPs for duplex × nulliplex, and 426 SNPs for simplex × simplex. TetraploidMap software was used to integrate SNPs from the array with an existing framework map. The colinearity between the resulting linkage map and the Medicago truncatula physical map was evaluated. The resulting high-density linkage map in alfalfa generated from this study can be useful to guide the ongoing assembly of the tetraploid alfalfa genome sequence and to identify genic SNPs in relevant loci associated with key traits of agronomic importance. The iSelect Infinium assay represents a platform for high-throughput SNP genotyping in alfalfa that greatly improves genotyping efficiency and enables detection of allelic dosage in a tetraploid species.

Identification of QTLs Associated with Morphological and Agronomic Traits in White Clover ( Trifolium repens L.)

White clover (Trifolium repens L.) is an important cool-season perennial forage legume species used in pastures to improve forage quality. The identification of molecular markers linked to morphological and agronomic traits could facilitate the development of superior white clover cultivars. The objectives of this study were to map quantitative trait loci (QTL) associated with morphological and agronomic traits using a F1 population from a double pseudo-testcross between two highly heterozygous genotypes. Phenotypic data was collected from multiple field locations and years for morphological traits (leaf length and width, petiole length, stolon diameter and inter-node length), and for growth traits (plant spreading, plant height, and stolon number). Analysis of variance indicated there were significant effects from location, genotype, and genotype × location for the traits evaluated. Correlation coefficients showed that growth traits in the field were highly correlated with each other. Broad sense heritability estimates for all traits evaluated were less than 25%. The population was genotyped using simple sequence repeat (SSR) markers and multiple QTL model (MQM) analysis was used to identify 37 QTLs on eight linkage groups associated with five morphological traits and four plant growth traits. The consistent location of QTLs for the same traits or highly correlated traits across different locations and years indicates the potential value of utilizing marker-assisted breeding for white clover improvement.

Evaluation and Utilization of Morphological Variation in a Medicago truncatula Core Collection

Medicago truncatula is a model species for legume biology and has been used to develop tools for molecular genetics and genomics. Nested core collections representing the existing genetic diversity from the USDA germplasm collection have previously been identified using molecular markers. The practical value of nested core collections is that they allow implementation of efficient strategies to characterize phenotypic variation compared to random selection of accessions from the whole collection or using geographic stratification to select accessions for evaluation. The goals of this research were to efficiently use the M. truncatula core collections to characterize the natural existing variation associated with morphological traits. We assayed variation in pods (length, number of coils, direction of coiling, number of pods per raceme), leaves (morphology and pigmentation patterns), and roots (morphology and biomass) from accessions included in the nested core collections. A remarkable natural variation was identified for the morphological characteristics evaluated in pods, leaves and roots. Accessions with contrasting phenotypes for multiple traits are currently being used to develop mapping populations. The M. truncatula core collections are publicly available and should enable researchers to efficiently evaluate genetic variation for additional traits of interest.