Insights Into the Genetic Diversity of Nordic Red Clover (Trifolium pratense) Revealed by SeqSNP-Based Genic Markers

Abstract

Red clover (Trifolium pratense) is one of the most important fodder crops worldwide. The knowledge of genetic diversity among red clover populations, however, is under development. This study provides insights into its genetic diversity, using single nucleotide polymorphism (SNP) markers to define population structure in wild and cultivated red clover. Twenty-nine accessions representing the genetic resources available at NordGen (the Nordic gene bank) and Lantmännen (a Swedish agricultural company with a red clover breeding program) were used for this study. Genotyping was performed via SeqSNP, a targeted genotype by sequencing method that offers the capability to target specific SNP loci and enables de novo discovery of new SNPs. The SNPs were identified through a SNP mining approach based on coding sequences of red clover genes known for their involvement in development and stress responses. After filtering the genotypic data using various criteria, 623 bi-allelic SNPs, including 327 originally targeted and 296 de novo discovered SNPs were used for population genetics analyses. Seventy-one of the SNP loci were under selection considering both Hardy-Weinberg equilibrium and pairwise FST distributions. The average observed heterozygosity (HO), within population diversity (HS) and overall diversity (HT) were 0.22, 0.21 and 0.22, respectively. The tetraploids had higher average HO (0.35) than diploids (0.21). The analysis of molecular variance (AMOVA) showed low but significant variation among accessions (5.4%; P < 0.001), and among diploids and tetraploids (1.08%; P = 0.02). This study revealed a low mean inbreeding coefficient (FIS = −0.04) exhibiting the strict outcrossing nature of red clover. As per cluster, principal coordinate and discriminant analyses, most wild populations were grouped together and were clearly differentiated from the cultivated types. The cultivated types of red clover had a similar level of genetic diversity, suggesting that modern red clover breeding programs did not negatively affect genetic diversity or population structure. Hence, the breeding material used by Lantmännen represents the major genetic resources in Scandinavia. This knowledge of how different types of red clover accessions relate to each other and the level of outcrossing and heterozygosity will be useful for future red clover breeding.

Novel GBS-Based SNP Markers for Finger Millet and Their Use in Genetic Diversity Analyses

“…The lower values in the latter case are most likely because most of the SNP markers used were located within genes ( Enyew et al, 2021 ) and hence are expected to be less diverse than GBS-based SNP markers. Similar bi-allelic SNP-based studies in noug ( Guizotia abyssinica ; Tsehay et al, 2020 ) and red clover ( Trifolium pratense ; Osterman et al, 2021 ) reported Ho values of 0.24 and 0.22, respectively. It is expected that noug and red clover have higher Ho values than finger millet and sorghum because both of these crops are strictly outcrossing, enabling a higher level of heterozygosity.…”

“…After seed germination, extra seedlings were removed, and only a single seedling was maintained for each accession. Three weeks after planting, the leaf tissue of each accession was separately sampled using the BioArk Leaf collection kit from LGC, Biosearch Technologies ( https://biosearchassets.blob.core.windows.net/assetsv6/guide_bioark-leaf-collection-kit.pdf ), as described in Osterman et al (2021) . Afterward, the samples were sent to LGC, Biosearch Technologies (Berlin, Germany) for genomic DNA extraction, followed by GBS-based genotyping.…”

Correlations of morpho-agronomic traits and forage quality properties in diverse red clover (Trifolium pratense L.) collections

“…The modern tetraploid red clover cultivars (2n = 4x = 28) have been developed using autopolyploidy from diploid genotypes through chromosome doubling (Taylor and Quesenberry, 1996). The cultivated types of red clover had a similar level of genetic diversity, implying that modern red clover breeding programs did not negatively affect either genetic diversity or population structure (Osterman et al, 2021).…”

Discovering candidate SNPs for resilience breeding of red clover

“…The accessions were planted and grown for two weeks in a greenhouse at the Swedish University of Agricultural Sciences (SLU, Alnarp, Sweden), as described in Osterman et al (2021). The BioArk leaf collection kit (LGC Biosearch Technologies) was used to collect ten 6 mm leaf discs (1 leaf disc/plant).…”

“…For instance, the publication of its reference genome (De Vega et al, 2015) has facilitated the discovery of quantitative trait loci (QTL) for various traits, and the "mining" of single nucleotide polymorphism (SNP) markers (Herrmann et al, 2008;Ergon et al, 2019;Li et al, 2019). In two recent papers on population genetics, SNPs were used to assess the population structure in individually genotyped red clover (Jones et al, 2020;Osterman et al, 2021). Jones et al studied 75 accessions from Europe, Asia, and Iberia where 70 were wild populations and five were commercially available breeding populations.…”