Genetic diversity, population structure, and taxonomic confirmation in annual medic (Medicago spp.) collections from Crimea, Ukraine

Abstract

Annual medic (Medicago spp.) germplasm was collected from the Crimean Peninsula of Ukraine in 2008 to fill gaps in geographic coverage in the United States department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS) temperate-adapted forage legume collection. A total of 102 accessions across 10 Medicago species were collected. To assess genetic diversity, population structure, and to confirm taxonomic identities, the collections were phenotypically and genetically characterized. Phenotyping included the use of 24 descriptor traits while genetic characterization was accomplished using a 3K Diversity Array Technologies (DArTag) panel developed for alfalfa (Medicago sativa L.). For both field and molecular characterizations, a reference set of 92 geographically diverse and species-representative accessions were obtained from the NPGS collection. Phenotypic descriptors showed consistency among replicated plants within accessions, some variation across accessions within species, and evident distinctions between species. Because the DArTag panel was developed for cultivated alfalfa, the transferability of markers to the species being evaluated was limited, resulting in an average of ~1,500 marker loci detected per species. From these loci, 448 markers were present in 95% of the samples. Principal component and phylogenetic analysis based on a larger set of 2,396 selected markers clustered accessions by species and predicted evolutionary relationships among species. Additionally, the markers aided in the taxonomic identity of a few accessions that were likely mislabeled. The genotyping results also showed that sampling individual plants for these mostly self-pollinating species is sufficient due to high reproducibility between single (n=3) and pooled (n=7) biological replicate leaf samples. The phenotyping and the 2,396 Single Nucleotide Polymorphism (SNP) marker set were useful in estimating population structure in the Crimean and reference accessions, highlighting novel and unique genetic diversity captured in the Crimean accessions. This research not only demonstrated the utility of the DArTag marker panel in evaluating the Crimean germplasm but also highlighted its broader application in assessing genetic resources within the Medicago genus. Furthermore, we anticipate that our findings will underscore the importance of leveraging genetic resources and advanced genotyping tools for sustainable crop improvement and biodiversity conservation in annual medic species.

Keywords: DArTag genotyping; diversity; germplasm; legume; marker.

Figure 1

Summary information for Medicago spp. germplasm accessions used in the study. (A) Map showing the collection sites for most of the accessions used in the study. The inset shows information only for the accessions collected from Crimea, Ukraine. (B) Counts for accessions and number of samples genotyped from Crimea, Ukraine (Collection Mission) and the NPGS reference subset (Genebank). Each accession had three single-plant samples (biological replicates, n=3) and one bulked (n=7) sample. *10 plants for each accession were phenotyped in the field.

Figure 2

Illustration of the target and off-target SNPs extracted from microhaplotypes based on pairwise alignment with reference microhaplotypes and extraction of collapsed read counts from the individual read counts.

Figure 3

Representative phenotypic characteristics for the ten annual medic (Medicago spp.) species evaluated. (A) shoot architecture; (B) leaflets; (C) fruit/pods, and (D) seeds.

Figure 4

Principal component analysis based on 18 phenotypic descriptor traits of 175 accessions.

Figure 5

Upset plot of the intersection of polymorphic SNPs across different species. The horizontal bars represent total number of polymorphic SNPs found among the accessions within the corresponding species. The vertical bar plots represent the number of SNPs either unique to the specific species or common markers between two species indicated below the bars. The first ten vertical bars from the left to right represent SNPs unique to the species as indicated by the red dot whereas the remaining bars represent number of SNPs shared between two species indicated by red dot and the line segment.

Figure 6

Summary of missing marker loci across different species. (A) Boxplot representing the distribution of the number of missing marker loci across 11 Medicago species. The letters in the boxplot indicate the grouping of species with significantly different missing rates evaluated by Games-Howell test (α < 0.05). The line and numbers with gradient color below the x-axis represents F_ST values between M. sativa and the 10 diploid Medicago species. The x-axis represents different Medicago species. The primary y-axis denotes the number of missing marker loci whereas the secondary y-axis represents F_ST value calculated between M. sativa and the 10 diploid Medicago species. (B) Statistics of missing rate of marker loci in each species.

Figure 7

Correlations of read counts per marker loci among three single-plant samples and the bulked sample for each of the 192 accessions. (A) Heatmap representing correlations among single-plant samples (P1, P2, and P3) and the bulked sample for each of the 192 accessions. (B) Histogram of correlation coefficients among the single-plant samples and bulked samples. (C) Correlation matrix among three single-plant samples and the bulked sample of a selected M. arabica (W6 33642) accession.

Figure 8

Scatter plot of sample-wise microhaplotype numbers in the single-plant (median of the three single-plant samples; blue dots) and bulked (red dots) samples for 181 accessions. The trend (blue curve) of microhaplotype numbers among single-plant samples was generated using a LOWESS regression.

Figure 9

Population structure analysis of the accessions used in the study. (A) Principal component analysis using estimated allele dosages in 192 accessions. Possible mislabeled or misidentified accessions are listed in the figure. (B) Phylogenetic tree representing relationships among the 192 accessions. Different colors of accessions and branches represent unique species. The colors in the outer concentric circle represents the collection accession source. The numbers on the branches represent the bootstrap values (>50%). (C) Heatmap of pairwise F_ST values among all 10 species used in the study and M. sativa accessions from Zhao et al. (2023). (D) Simplified species tree representing genetic relatedness of the Medicago species under study based on the neighbor joining tree constructed using pairwise F_ST comparison with M. sativa as the root.