Patil Prakash G, Sharma Jyotsana, Nanjundappa Manjunatha, Singh N V, Bohra Abhishek, Gunnaiah Raghavendra, Jamma Shivani M, Vinayaka Jeer, Sangnure Vipul R, Marathe R A
Biotechnology and Plant Pathology, ICAR-National Research Centre on Pomegranate (NRCP), Solapur, 413255 India.
State Agriculture Biotechnology, Centre, Centre for Crop & Food Innovation, Murdoch University, Perth, Western Australia.
3 Biotech. 2022 Jul;12(7):153. doi: 10.1007/s13205-022-03209-z. Epub 2022 Jun 22.
This study reports genome wide characterization and development of first set of microsatellite markers through in silico analysis of eight sequenced pv. strains available in the public database. SSR survey resulted in identification of ~ 4638 perfect SSRs, with mean marker frequency 901 SSRs/Mb and densitiy of 11,006 bp/Mb aross the eight genomes. Frequency distribution graphs revealed hexa-nucleotide repeats were more prominent fowllowed by tri-, tetra-, di- and penta-nucleotides in the analysed genomes. We desinged 2927 SSR primers that are specific to the strain LMG 859 and ePCR confirmed on seven other genomes. This resulted in identification of 542 informative SSRs that are producing single amplicons, from which 66 primers were successfully validated through wet lab experiments on eight isolates of pomegranate. Furthermore, utility of these SSRs were demostrated by analysing molecular diversity among 22 isolates using 20 Xap_SSR primers. SSRs revealed moderate genetic diversity among isolates (61%) and grouped 11 isolates that are repersenting six different states into one cluster. This proved the earlier evidence of wider spread of ST3 type Xap acoss India using Multi locus Sequence Typing (MLST) technique. In summary, Xap_SSR will serve as powerful genomics tools that would helps in monitoring of population dynamics, taxonomy, epidomology and quarantine aspects in bacterial blight pathogen through development of microsatellite based Multilocus Variable number of Tandem repeat analysis (MLVA) in future.
The online version contains supplementary material available at 10.1007/s13205-022-03209-z.
本研究通过对公共数据库中八个已测序的pv. 菌株进行电子分析，报告了全基因组特征以及第一套微卫星标记的开发情况。SSR调查共鉴定出约4638个完美SSR，八个基因组的平均标记频率为901个SSR/Mb，密度为11,006 bp/Mb。频率分布图显示，在所分析的基因组中，六核苷酸重复最为突出，其次是三核苷酸、四核苷酸、二核苷酸和五核苷酸。我们设计了2927个针对菌株LMG 859的SSR引物，并在其他七个基因组上通过电子PCR进行了验证。这导致鉴定出542个产生单扩增子的信息性SSR，其中66个引物通过对八个石榴分离株的湿实验室实验成功验证。此外，通过使用20个Xap_SSR引物分析22个分离株之间的分子多样性，证明了这些SSR的实用性。SSR显示分离株之间存在中等程度的遗传多样性（61%），并将代表六个不同邦的11个分离株归为一个聚类。这证实了早期使用多位点序列分型（MLST）技术得出的ST3型Xap在印度广泛传播的证据。总之，Xap_SSR将成为强大的基因组学工具，未来通过开发基于微卫星的多位点可变串联重复分析（MLVA），有助于监测细菌性疫病病原体的种群动态、分类学、流行病学和检疫方面。
在线版本包含可在10.1007/s13205-022-03209-z获取的补充材料。
