Identification of microRNAs from Amur grape (Vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics

Abstract

Background: MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species.

Results: A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism.

Conclusions: Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.

Figure 1

Comparison of abundance levels of conserved and non-conserved va-miRNAs identified by deep sequencing.

Figure 2

3'RACE and 5' RACE PCR products of seven amplified va-miR-SNPs shown in an ethidium bromide-stained agarose gel. Sizes of the molecular weight markers of the bottom and the top bands are 50 bp and 100 bp, respectively. Lanes 1-7, 1'-7' are 3'RACE (up) and 5'RACE (down) products of va-miR156a-SNP, va-miR166a-SNP, va-miR166h-SNP, va-miR169b-SNP, va-miR169l-SNP, va-miR169o-SNP and va-miR171c/d-SNP, respectively. The sizes of 3'RACE products are about 83 bp while the size of 5'RACE products are about 57 bp.

Figure 3

Number distribution of miR-SNP of diverse conserved miRNAs familes in Amur grape.

Figure 4

Comparison of polymorphisms of va-miR160a, b, c, d, e and AK373591.1 binding sites, respectively. Va-miR160a, va-miR160b, va-miR160c, va-miR160d, va-miR160e respectively showed wild type sequences; miR160a-SNP, miR160b-SNP, miR160c-SNP, miR160d-SNP, miR160e-SNP exhibited SNP types of these miRNAs.

Figure 5

Real-time RT-PCR expression profiles of va-miRNAs with three frequencies from deep sequencing sRNA library in seven tissues of Amur grape. I, II, III denote va-miRNAs with high, moderate and low frequency in the deep sequenced sRNA library, respectively; the red arrows denote the flower- and/or berry-specific miRNAs. Lanes: young leaf (4 cm in diameter), large leaf (8 cm in diameter), stem (0.2 cm in diameter), tendril (0.1 cm in diameter), inflorescence (0.2 cm in diameter per grain), flower (0.25 cm in diameter per grain), young berry (0.8 cm in diameter) and large berry (1.5 cm in diameter). Each reaction was repeated three times and the template amount was corrected by 5.8 s rRNAs.

Figure 6

Mapping of mRNA cleavage sites by RNA ligase-mediated 5' RACE. Each top strand (black) depicts a miRNA-complementary site in the target mRNA, and each bottom strand (pink) depicts the miRNA. Watson-Crick pairing (vertical dashes), G:U wobble pairing (circles) and mismatched bases pairing (X) are indicated. Arrows indicate the 5' termini of mRNA fragments isolated from Amur grape, as identified by cloned 5'RACE products, with the frequency of clones shown. Only cloned sequences that matched the correct gene and had 5' ends within a 100 nt window centered on the miRNA complementary site are counted. Partial mRNA sequences from target genes were aligned with the miRNAs. Numbers indicate the fraction of cloned PCR products terminating at different positions.