Cross-species transferability of EST-SSR markers developed from the transcriptome of Melilotus and their application to population genetics research

Abstract

Melilotus is one of the most important legume forages, but the lack of molecular markers has limited the development and utilization of Melilotus germplasm resources. In the present study, 151 M clean reads were generated from various genotypes of Melilotus albus using Illumina sequencing. A total of 19,263 potential EST-SSRs were identified from 104,358 unigene sequences. Moreover, 18,182 primer pairs were successfully designed, and 550 primer pairs were selected using criteria of base repeat type, fragment length and annealing temperature. In addition, 550 primer pairs were screened by using PCR amplification products and used to assess polymorphisms in 15 M. albus accessions. A total of 114 primer pairs were detected as being highly polymorphic, and the average polymorphism information content (PIC) value was 0.79. Furthermore, those 114 polymorphic primer pairs were used to evaluate the transferability to 18 species of the genus Melilotus, and 70 EST-SSR markers were found to be transferable among the 18 Melilotus species. According to the UPGMA dendrogram and STRUCTURE analysis, the 18 Melilotus species were classified into three clusters. This study offers a valuable resource for the genetic diversity and molecular assisted breeding of germplasm resources in the genus Melilotus.

Figure 1

EST-SSR marker variations of 18 Melilotus species using primers 86, 170, 281 and 547. Each accession includes three individual plants; the letter ‘M’ denotes the molecular markers, which are 200 bp and 150 bp (top to bottom) with primer 86, primer 170, primer 281 and primer 547 (top to bottom).

Figure 2

Comparative electropherogram analysis of four EST-SSR loci (primers 21, 31, 61 and 392) among different accessions of M. albus. The primer 21 had trinucleotide repeats of (TTC)₄, (TTC)₅, and (TTC)₇. (TCT)₄, and (TCT)₇ were obtained using primer 31. Primer 61 can generate tandem repeats of (GATTA)₄ and (GATTA)₅. (GA)₇, (GA)₈ and (GA)₁₂ were obtained by using primer 392.

Figure 3

Cluster analysis of 18 species of the Melilotus genus based on 70 EST-SSR markers.

Figure 4

Genetic structure of 54 individuals for 18 Melilotus species as inferred by STRUCTURE with the EST-SSR marker data set. Histogram of the STRUCTURE analysis for the model with K = 3 (showing the highest ΔK). The smallest vertical barre presents one individual. The assignment proportion of each individual into Cluster I, Cluster II and Cluster III is shown along the y-axis.