Xueyang Min

Simple sequence repeat (SSR) markers are commonly used for many genetic applications, such as map construction, fingerprinting, and genetic diversity analyses, due to their high reproducibility, polymorphism, and abundance. Endogenous miRNAs play essential roles in plant development and gene expression under diverse biotic and abiotic stress conditions. In the present study, we predicted 110 miRNA-SSR primer pairs from 287 precursor miRNAs. Among 110 primer pairs, 85 were successfully amplified and examined for transferability to other Gramineae and non-Gramineae species. The results showed that all 82 primer pairs yielded unambiguous and strong amplification, and across the 23 studied Cleistogenes accessions, a total of 385 alleles were polymorphic. The number of alleles produced per primer varied from 3 to 11, with an average of 4.69 per locus. The expected heterozygosity (He) ranged from 0.44 to 0.88, with an average of 0.74 per locus, and the PIC (Polymorphism Information Content) values ranged from 0.34 to 0.87, with an average of 0.69 per locus. Furthermore, 1422 miRNA target genes were predicted and analyzed using the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. In conclusion, the results showed that an miRNA-based microsatellite marker system can be applicable for genetic diversity and marker-assisted breeding studies.

Plant growth and development depends on its ability to maintain optimal cellular homeostasis during abiotic and biotic stresses. Cleistogenes songorica, a xerophyte desert plant, is known to have novel drought stress adaptation strategies and contains rich pools of stress tolerance genes. Proteins encoded by Late Embryogenesis Abundant (LEA) family genes promote cellular activities by functioning as disordered molecules, or by limiting collisions between enzymes during stresses. To date, functions of the LEA family genes have been heavily investigated in many plant species except perennial monocotyledonous species. In this study, 44 putative LEA genes were identified in the C. songorica genome and were grouped into eight subfamilies, based on their conserved protein domains and domain organizations. Phylogenetic analyses indicated that C. songorica Dehydrin and LEA_2 subfamily proteins shared high sequence homology with stress responsive Dehydrin proteins from Arabidopsis. Additionally, promoter regions of CsLEA_2 or CsDehydrin subfamily genes were rich in G-box, drought responsive (MBS), and/or Abscisic acid responsive (ABRE) cis-regulatory elements. In addition, gene expression analyses indicated that genes from these two subfamilies were highly responsive to heat stress and ABA treatment, in both leaves and roots. In summary, the results from this study provided a comprehensive view of C. songorica LEA genes and the potential applications of these genes for the improvement of crop tolerance to abiotic stresses.

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.