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Front Genet ; 13: 866504, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35495126


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.

Front Genet ; 12: 704075, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394192


Here we report on comprehensive chloroplast (cp) genome analysis of 16 pomegranate (Punica granatum L.) genotypes representing commercial cultivars, ornamental and wild types, through large-scale sequencing and assembling using next-generation sequencing (NGS) technology. Comparative genome analysis revealed that the size of cp genomes varied from 158,593 bp (in wild, "1201" and "1181") to 158,662 bp (cultivar, "Gul-e-Shah Red") among the genotypes, with characteristic quadripartite structures separated by a pair of inverted repeats (IRs). The higher conservation for the total number of coding and non-coding genes (rRNA and tRNA) and their sizes, and IRs (IR-A and IR-B) were observed across all the cp genomes. Interestingly, high variations were observed in sizes of large single copy (LSC, 88,976 to 89,044 bp) and small single copy (SSC, 18,682 to 18,684 bp) regions. Although, the structural organization of newly assembled cp genomes were comparable to that of previously reported cp genomes of pomegranate ("Helow," "Tunisia," and "Bhagawa"), the striking differences were observed with the Lagerstroemia lines, viz., Lagerstroemia intermedia (NC_0346620) and Lagerstroemia speciosa (NC_031414), which clearly confirmed previous findings. Furthermore, phylogenetic analysis also revealed that members outside the genus Punica were clubbed into a separate clade. The contraction and expansion analysis revealed that the structural variations in IRs, LSC, and SSC have significantly accounted for the evolution of cp genomes of Punica and L. intermedia over the periods. Microsatellite survey across cp genomes resulted in the identification of a total of 233 to 234 SSRs, with majority of them being mono- (A/T or C/G, 164-165 numbers), followed by di- (AT/AT or AG/CT, 54), tri- (6), tetra- (8), and pentanucleotides (1). Furthermore, the comparative structural variant analyses across cp genomes resulted in the identification of many varietal specific SNP/indel markers. In summary, our study has offered a successful development of large-scale cp genomics resources to leverage future genetic, taxonomical, and phylogenetic studies in pomegranate.

Front Plant Sci ; 12: 645055, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33796127


The simple sequence repeat (SSR) survey of 'Tunisia' genome (296.85 Mb) identified a total of 365,279 perfect SSRs spanning eight chromosomes, with a mean marker density of 1,230.6 SSRs/Mb. We found a positive trend in chromosome length and the SSR abundance as marker density enhanced with a shorter chromosome length. The highest number of SSRs (60,708) was mined from chromosome 1 (55.56 Mb), whereas the highest marker density (1,294.62 SSRs/Mb) was recorded for the shortest chromosome 8 (27.99 Mb). Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. Across the eight chromosomes, the class III had maximum number of SSR motifs (301,684, 82.59%), followed by the class II (31,056, 8.50%) and the class I (5,003, 1.37%). Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides, and these results are consistent with 'Tunisia' genome features as a whole. Concerning major repeat types, AT/AG was the most frequent (14.16%), followed by AAAAAT/AAAAAG (7.89%), A/C (7.54%), AAT/AAG (5.23%), AAAT/AAAG (4.37%), and AAAAT/AAAAG (1.2%) types. We designed and validated a total of 3,839 class I SSRs in the 'Tunisia' genome through electronic polymerase chain reaction (ePCR) and found 1,165 (30.34%) SSRs producing a single amplicon. Then, we selected 906 highly variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across multiple draft genomes of pomegranate, which provided us a subset of 265 highly polymorphic SSRs. Of these, 235 primers were validated on six pomegranate genotypes through wet-lab experiment. We found 221 (94%) polymorphic SSRs on six genotypes, and 187 of these SSRs had ≥ 0.5 PIC values. The utility of the developed SSRs was demonstrated by analyzing genetic diversity of 30 pomegranate genotypes using 16 HvSSRs spanning eight pomegranate chromosomes. In summary, we developed a comprehensive set of highly polymorphic genome-wide SSRs. These chromosome-specific SSRs will serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate.