Abstract
Microsatellite (simple sequence repeats, SSRs) marker is one of the most widely used markers in marker-assisted breeding. As one type of functional markers, MicroRNA-based SSR (miRNA-SSR) markers have been exploited mainly in animals, but the development and characterization of miRNA-SSR markers in plants are still limited. In the present study, miRNA-SSR markers for Medicago truncatula (M. truncatula) were developed and their cross-species transferability in six leguminous species was evaluated. A total of 169 primer pairs were successfully designed from 130 M. truncatula miRNA genes, the majority of which were mononucleotide repeats (70.41%), followed by dinucleotide repeats (14.20%), compound repeats (11.24%) and trinucleotide repeats (4.14%). Functional classification of SSR-containing miRNA genes showed that all targets could be grouped into three Gene Ontology (GO) categories: 17 in biological process, 11 in molecular function, and 14 in cellular component. The miRNA-SSR markers showed high transferability in other six leguminous species, ranged from 74.56% to 90.53%. Furthermore, 25 Mt-miRNA-SSR markers were used to evaluate polymorphisms in 20 alfalfa accessions, and the polymorphism information content (PIC) values ranged from 0.39 to 0.89 with an average of 0.71, the allele number per marker varied from 3 to 18 with an average of 7.88, indicating a high level of informativeness. The present study is the first time developed and characterized of M. truncatula miRNA-SSRs and demonstrated their utility in transferability, these novel markers will be valuable for genetic diversity analysis, marker-assisted selection and genotyping in leguminous species.
Highlights
Studying the genetic diversity and population structure of germplasm resources is important for understanding the genetic basis of traits, and necessary for the discovery of new germplasm characteristics in order to develop and utilize germplasm resources for plant improvement [1,2,3,4]
DNA-based markers can be divided into two types: one type is non-polymerase chain reaction (PCR)-based markers (RFLP, restriction fragment length polymorphism) and the other type includes PCR-based markers (RAPD, random amplified polymorphic DNA; AFLP, amplified fragment length polymorphism; SSR, simple sequence repeat; SNP, single nucleotide polymorphism; and ILP, intron length polymorphism)
When SSRs are located in the intergenic region of the genome, such as expressed sequence tags (ESTs), transcriptome sequences, and miRNA genes, the SSR markers derived from these regions could be acted as “functional genetic markers” and widely used for marker assisted breeding and genomic selection [9,13]
Summary
Studying the genetic diversity and population structure of germplasm resources is important for understanding the genetic basis of traits, and necessary for the discovery of new germplasm characteristics in order to develop and utilize germplasm resources for plant improvement [1,2,3,4]. MiRNAs are a new class of small, endogenous and non-coding RNAs; they have been a significant avenue of research due to their critical role in development, differentiation, growth, metabolism and disease-resistance processes and in response to biotic and abiotic stresses through post-transcriptional regulation of gene expression in plants [14,15,16,17,18]. A large number of SSR markers derived from M. truncatula have been developed and applied in legume crops, including alfalfa [33,34]. This study reports the first genome-wide identification and development of SSR markers based on M. truncatula miRNAs; the markers’ potential for transferability and genetic diversity assessments in six leguminous analysed
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