Patil Prakash Goudappa, Singh Nripendra Vikram, Bohra Abhishek, Raghavendra Keelara Puttaswamy, Mane Rushikesh, Mundewadikar Dhananjay M, Babu Karuppannan Dhinesh, Sharma Jyotsana
ICAR-National Research Centre on Pomegranate, Solapur, India.
ICAR-Indian Institute of Pulses Research, Kanpur, India.
Front Plant Sci. 2021 Mar 16;12:645055. doi: 10.3389/fpls.2021.645055. eCollection 2021.
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 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.
对‘突尼斯’基因组（296.85 Mb）进行的简单序列重复（SSR）调查共鉴定出365,279个完美SSR，分布在八条染色体上，平均标记密度为1,230.6个SSR/Mb。我们发现随着染色体长度缩短，标记密度增加，染色体长度与SSR丰度呈正相关趋势。从1号染色体（55.56 Mb）挖掘出的SSR数量最多（60,708个），而最短的8号染色体（27.99 Mb）的标记密度最高（1,294.62个SSR/Mb）。此外，我们根据SSR基序的长度将所有SSR基序分为三大类。在八条染色体中，III类的SSR基序数量最多（301,684个，占82.59%），其次是II类（31,056个，占8.50%）和I类（5,003个，占1.37%）。对染色体内部SSR基序类型分布的研究表明，每条染色体上六核苷酸重复序列最为丰富，其次是二核苷酸重复序列，这些结果与‘突尼斯’基因组的整体特征一致。关于主要重复类型，AT/AG最为常见（14.16%），其次是AAAAAT/AAAAAG（7.89%）、A/C（7.54%）、AAT/AAG（5.23%）、AAAT/AAAG（4.37%）和AAAAT/AAAAG（1.2%）类型。我们通过电子聚合酶链反应（ePCR）在‘突尼斯’基因组中设计并验证了总共3,839个I类SSR，发现1,165个（30.34%）SSR产生单一扩增子。然后，我们从经ePCR验证的I类SSR中选择了906个高度可变的SSR（> 40 nt），并在多个石榴基因组草图中进行了验证，得到了265个高度多态性SSR的子集。其中，235对引物通过湿实验室实验在六个石榴基因型上进行了验证。我们在六个基因型上发现了221个（94%）多态性SSR，其中187个SSR的多态信息含量（PIC）值≥ 0.5。通过使用跨越八条石榴染色体的16个HvSSR分析30个石榴基因型的遗传多样性，证明了所开发SSR的实用性。总之，我们开发了一套全面的高度多态性全基因组SSR。这些染色体特异性SSR将成为未来石榴遗传研究、种质管理和基因组辅助育种的有力基因组工具。
