Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

Abstract

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity.

Inter-species Transferability of Microsatellite Markers Derived from Wild Relatives to Cultivated Species of Finger Millet

“…Whereas in other types, AGG & AAG in tri, AAAG in tetra and CGTCA in penta type were most frequent in rest of the species. These results were in accordance with the previous reports in finger millet and other major crops as the mining for repeats in genomic region yielded 39 to 66% of AG/CT repeats [9,13]. The comparative low number of di-nucleotide SSRs was attributed to the reason that these repeats were often present in close vicinity of other repeats, especially penta nucleotides forming compound SSRs.…”

“…Although large and diverse germplasm collections are available in both public and private organizations, no concerted efforts have been made to assess the diversity of this species at the molecular level and integrate these results with morphological characterization in breeding programmes for higher yield and disease resistance [6]. The past decade has witnessed continuous efforts towards developing both conventional and EST-SSR markers in finger millet [7,8,5,9]. Studies on comparative genomics have indicated high genomic colinearity between rice and finger millet [10] and cross-transferability of SSR markers from major cereal crops to E. coracana indicated 57% reproducible cross-species/genus amplicons [11] and 73-95% cross-amplification in finger millet from pearl millet SSRs [12].…”

Gene Discovery and Advances in Finger Millet [Eleusine coracana (L.) Gaertn.] Genomics—An Important Nutri-Cereal of Future

“…Out of 92, 80 SNP markers were polymorphic. However, SNP markers also resulted in a low PIC value of 0.29 revealing narrow genetic base of finger millet as reported with SSRs (Gimode et al, 2016). …”

“…Kumar et al (2016b) identified 23,000 SNPs through genotyping by sequencing (GBS) of 113 diverse finger millet genotypes. Similarly, 23285 SNPs were generated using next-generation sequencing of two cultivated finger millet genotypes, and 92 SNP markers were validated further for genetic diversity in cultivated and wild species of finger millet (Gimode et al, 2016). Out of 92, 80 SNP markers were polymorphic.…”

Genetic Characterization of Landraces and Varieties using Functional Microsatellite Markers in Eleusine Coracana (L.)

“…The knowledge of genetic diversity estimated by molecular markers is essential to select genetically distinct germplasm for hybridization programs. In the absence of finger millet genome sequence, attempts have been made to develop microsatellite markers and single nucleotide polymorphism (SNPs) (Gimode et al, 2016 Microsatellites (also called as simple sequence repeats: SSRs) are still the cost-effective and robust markers that offer high reproducibility and polymorphism. Currently, there are < 300 informative microsatellites in finger millet and a couple of SNP studies (Sood et al, 2016).…”