Novel miRNA-SSRs for Improving Seed Hardness Trait of Pomegranate (Punica granatum L.)

Abstract

Present research discovered novel miRNA-SSRs for seed type trait from 761 potential precursor miRNA sequences of pomegranate. SSR mining and BLASTx of the unique sequences identified 69 non-coding pre-miRNA sequences, which were then searched for BLASTn homology against Dabenzi genome. Sixty three true pri-miRNA contigs encoding 213 pre-miRNAs were predicted. Analysis of the resulting sequences enabled discovery of SSRs within pri-miRNA (227) and pre-miRNA sequences (79). A total of 132 miRNA-SSRs were developed for seed type trait from 63 true pri-miRNAs, of which 46 were specific to pre-miRNAs. Through ePCR, 123 primers were validated and mapped on eight Tunisia chromosomes. Further, 80 SSRs producing specific amplicons were ePCR-confirmed on multiple genomes i.e. Dabenzi, Taishanhong, AG2017 and Tunisia, yielding a set of 63 polymorphic SSRs (polymorphism information content ≥0.5). Of these, 32 miRNA-SSRs revealed higher polymorphism level (89.29%) when assayed on six pomegranate genotypes. Furthermore, target prediction and network analysis suggested a possible association of miRNA-SSRs i.e. miRNA_SH_SSR69, miRNA_SH_SSR36, miRNA_SH_SSR103, miRNA_SH_SSR35 and miRNA_SH_SSR53 with seed type trait. These miRNA-SSRs would serve as important genomic resource for rapid and targeted improvement of seed type trait of pomegranate.

Chromosome-specific potential intron polymorphism markers for large-scale genotyping applications in pomegranate

“…The NJ tree based on 24 PIP markers divided 31 genotypes into two separated groups constituting wild and cultivated types. These findings strongly corroborate the clustering patterns recorded previously in pomegranates using SSRs and miRNA-SSRs (Patil et al, 2020a , b , 2022 ). It was interesting to note that cluster 2 was found to be more diverse by including a few introduced exotic pomegranate accessions like Nimali (Srilanka), Kandhari (Afghanistan), GR Pink (Russia), and Shirin Anar (Russia).…”

“…The PCA plot also grouped 31 genotypes into two major clusters as that of the NJ tree. The PCos 1 accounted for a higher proportion of the variance of 19.9%, separating wild type from cultivars in pomegranate as reported in previous studies (Patil et al, 2020a(Patil et al, , 2022. Overall, the NJ tree and the factorial analysis showed strong agreements based on the PIP markers.…”

“…Patil et al (2021) also found moderate genetic variability among 30 pomegranate genotypes using 16 chromosome-specific HvSSR markers. Similarly, the utility of gene-derived miRNA-based SSR markers for genetic diversity study was demonstrated in pomegranate in an earlier study (Patil et al, 2020b(Patil et al, , 2022.…”

“…Similarly, Patil et al ( 2021 ) identified the 265 most informative SSR markers with an average PIC value of 0.46 through ePCR validation of in silico -designed markers across multiple pomegranate genomes. Recently, Patil et al ( 2022 ) reported the identification of 77 polymorphic miRNA-SSRs for seed hardness breeding through multiple genome-ePCR confirmations in pomegranate.…”

“…Similarly, Patil et al ( 2020b ) also performed the GO analysis for miRNA target genes to elucidate the importance of gene-derived miRNA-SSRs markers for trait mapping in pomegranate. In a recent study, we performed GO for 727 gene targets of miRNAs that are part of seed development (Patil et al, 2022 ) and observed biological processes to be the most abundant category. Binding and catalytic activity had the highest representation in the molecular functions category.…”