Comprehensive Characterization and Validation of Chromosome-Specific Highly Polymorphic SSR Markers From Pomegranate (Punica granatum L.) cv. Tunisia Genome

Prakash Goudappa Patil; Nripendra Vikram Singh; Abhishek Bohra; Keelara Puttaswamy Raghavendra; Rushikesh Mane; Dhananjay M Mundewadikar; Karuppannan Dhinesh Babu; Jyotsana Sharma

doi:10.3389/fpls.2021.645055

Comprehensive Characterization and Validation of Chromosome-Specific Highly Polymorphic SSR Markers From Pomegranate (Punica granatum L.) cv. Tunisia Genome

Front Plant Sci. 2021 Mar 16:12:645055. doi: 10.3389/fpls.2021.645055. eCollection 2021.

Authors

Prakash Goudappa Patil¹, Nripendra Vikram Singh¹, Abhishek Bohra², Keelara Puttaswamy Raghavendra³, Rushikesh Mane¹, Dhananjay M Mundewadikar¹, Karuppannan Dhinesh Babu¹, Jyotsana Sharma¹

Affiliations

¹ ICAR-National Research Centre on Pomegranate, Solapur, India.
² ICAR-Indian Institute of Pulses Research, Kanpur, India.
³ ICAR-Central Institute for Cotton Research, Nagpur, India.

Abstract

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.

Keywords: chromosome; genome; hypervariable SSR; polymorphism; pomegranate.