Diversity analyses in alfalfa have mainly evaluated genetic relationships of cultivated germplasm, with little known about variation in diploid germplasm in the M. sativa-falcata complex. A collection of 374 individual genotypes derived from 120 unimproved diploid accessions from the National Plant Germplasm System, including M. sativa subsp. caerulea, falcata, and hemicycla, were evaluated with 89 polymorphic SSR loci in order to estimate genetic diversity, infer the genetic bases of current morphology-based taxonomy, and determine population structure. Diploid alfalfa is highly variable. A model-based clustering analysis of the genomic data identified two clearly discrete subpopulations, corresponding to the morphologically defined subspecies falcata and caerulea, with evidence of the hybrid nature of the subspecies hemicycla based on genome composition. Two distinct subpopulations exist within each subsp. caerulea and subsp. falcata. The distinction of caerulea was based on geographical distribution. The two falcata groups were separated based on ecogeography. The results show that taxonomic relationships based on morphology are reflected in the genetic marker data with some exceptions, and that clear distinctions among subspecies are evident at the diploid level. This research provides a baseline from which to systematically evaluate variability in tetraploid alfalfa and serves as a starting point for exploring diploid alfalfa for genetic and breeding experiments.
Improving drought tolerance of crop plants is a major goal of plant breeders. In this study, we characterized biomass and drought-related traits of 220 Medicago truncatula HapMap accessions. Characterized traits included shoot biomass, maximum leaf size, specific leaf weight, stomatal density, trichome density and shoot carbon-13 isotope discrimination (δ(13) C) of well-watered M. truncatula plants, and leaf performance in vitro under dehydration stress. Genome-wide association analyses were carried out using the general linear model (GLM), the standard mixed linear model (MLM) and compressed MLM (CMLM) in TASSEL, which revealed significant overestimation of P-values by CMLM. For each trait, candidate genes and chromosome regions containing SNP markers were found that are in significant association with the trait. For plant biomass, a 0.5 Mbp region on chromosome 2 harbouring a plasma membrane intrinsic protein, PIP2, was discovered that could potentially be targeted to increase dry matter yield. A protein disulfide isomerase-like protein was found to be tightly associated with both shoot biomass and leaf size. A glutamate-cysteine ligase and an aldehyde dehydrogenase family protein with Arabidopsis homologs strongly expressed in the guard cells were two of the top genes identified by stomata density genome-wide association studies analysis.
Association mapping enables the detection of marker-trait associations in unstructured populations by taking advantage of historical linkage disequilibrium (LD) that exists between a marker and the true causative polymorphism of the trait phenotype. Our first objective was to understand the pattern of LD decay in the diploid alfalfa genome. We used 89 highly polymorphic SSR loci in 374 unimproved diploid alfalfa (Medicago sativa L.) genotypes from 120 accessions to infer chromosome-wide patterns of LD. We also sequenced four lignin biosynthesis candidate genes (caffeoyl-CoA 3-O-methyltransferase (CCoAoMT), ferulate-5-hydroxylase (F5H), caffeic acid-O-methyltransferase (COMT), and phenylalanine amonialyase (PAL 1)) to identify single nucleotide polymorphisms (SNPs) and infer within gene estimates of LD. As the second objective of this study, we conducted association mapping for cell wall components and agronomic traits using the SSR markers and SNPs from the four candidate genes. We found very little LD among SSR markers implying limited value for genomewide association studies. In contrast, within gene LD decayed within 300 bp below an r2 of 0.2 in three of four candidate genes. We identified one SSR and two highly significant SNPs associated with biomass yield. Based on our results, focusing association mapping on candidate gene sequences will be necessary until a dense set of genome-wide markers is available for alfalfa.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-012-1854-2) contains supplementary material, which is available to authorized users.
The Medicago sativa–falcata complex includes the economically important forage legume alfalfa and its primary gene pool. The complex includes both diploid and tetraploid taxa, which are usually designated as distinct subspecies. The relationships among wild diploid members of the complex have been clarified using molecular markers, but the relationship among unimproved tetraploid germplasm is poorly understood. Our aim was to investigate the population genetic structure of the tetraploid Medicago sativa–falcata complex to deduce the amount and pattern of genetic diversity using genome‐wide simple‐sequence repeat (SSR) markers. We used 70 tetraploid accessions (280 genotypes) from the USDA National Plant Germplasm Collection that were collected from throughout the entire natural distribution range of the species and that represented putative wild populations. Population genetic analyses were conducted to determine the patterns of demarcation among accessions and germplasm groups. Model‐based cluster analysis indicated that tetraploid alfalfa has two main groups corresponding to the subspecies sativa and falcata. Medicago sativa subsp. ×varia produced a hybrid pattern in between M. sativa subsp. sativa and M. sativa subsp. falcata. The studies also revealed that there is a spatial genetic structure among subsp. falcata accessions, implying that extensive sampling from different localities for curation of alfalfa genetic resources is important.
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