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Multiple sequence alignment and phylogenetic analysis of wheat pathogens using conserved genes for identification and development of diagnostic markers
Cereal Research Communications ( IF 1.6 ) Pub Date : 2021-07-09 , DOI: 10.1007/s42976-021-00193-7
Sangeeta Gupta 1 , Rashmi Aggarwal 1 , Sapna Sharma 1 , Malkhan S. Gurjar 1 , Bishnu M. Bashyal 1 , Mahender S. Saharan 1 , Shweta Agarwal 1
Affiliation  

Molecular markers are fast, reliable and robust for early detection of pathogens. They need unique stable regions specific to pathogens for developing good diagnostics. The multiple sequence alignment and phylogenetic studies of conserved loci such as Internal Transcribed Spacer (ITS), Translation elongation factor (TEF-1α), β-tubulin gene and glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of wheat pathogens identified intergeneric (5–30%) and intrageneric (2–24%) variability with multiple sequence polymorphism (5–250 bp) for selection of pathogen-specific regions. ITS region precisely showed specificity for Tilletia indica and storage pathogens (Aspergillus spp., Penicillium spp. and Fusarium spp.), while TEF-1α gene distinctly differentiated 5 Fusarium species with high potential (10%) and found highly suitable for developing qPCR or LAMP assays for detection of Fusaruim head blight disease. β-tubulin gene showed, enormous variabilities among species of Puccinia (15%) and Aspergillus (23%) for developing their diagnostics. GAPDH gene with the highest intergenic sequence variability (> 30%) found suitable for developing markers at generic level. Further, it has differentiated different species of Aspergilli and Bipolaris. The variability among different pathogens enabled the selection and development of appropriate diagnostic marker such as, PCR, qPCR, RPA, LAMP assay, macro- or micro-array. As a proof of concept, a PCR-based diagnostics (MN754026) for Tilletia indica was developed, which amplified a band of 148 bp only in Tilletia indica and detected as little as 10 pg of DNA. Developed maker has tremendous potential for timely management of this quarantined pathogen.



中文翻译:

利用保守基因对小麦病原菌进行多序列比对和系统发育分析,用于诊断标记的鉴定和开发

分子标记快速、可靠且稳健,可用于病原体的早期检测。他们需要特定于病原体的独特稳定区域来开发良好的诊断方法。对小麦病原体的内部转录间隔区 (ITS)、翻译延伸因子 ( TEF-1α )、β-微管蛋白基因和 3-磷酸甘油醛脱氢酶基因 ( GAPDH)等保守位点的多序列比对和系统发育研究确定了属间性 (5 –30%) 和属内 (2–24%) 变异性,具有多序列多态性 (5–250 bp),用于选择病原体特异性区域。ITS 区域精确地显示出对Tilletia indica和贮藏病原体(Aspergillus spp.,青霉属 和镰刀菌属),而TEF-1α基因可明显区分 5种具有高潜力 (10%) 的镰刀菌属物种,并且发现非常适合开发 qPCR 或 LAMP 检测以检测镰刀菌枯萎病。β-微管蛋白基因显示,锈菌属(15%) 和曲霉属(23%) 在开发诊断方面存在巨大差异。发现具有最高基因间序列变异性 (> 30%) 的GAPDH基因适合开发通用水平的标记。此外,它还区分了不同种类的曲霉双极. 不同病原体之间的差异使得能够选择和开发适当的诊断标记,例如 PCR、qPCR、RPA、LAMP 检测、宏或微阵列。作为概念证明,开发了一种用于籼稻的基于 PCR 的诊断 (MN754026) ,它仅在籼稻中扩增了 148 bp 的条带,并检测到低至 10 pg 的 DNA。开发的制造商在及时管理这种隔离的病原体方面具有巨大的潜力。

更新日期:2021-07-09
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