Molecular cloning and development of RAPD-SCAR markers for the selection of thermo-tolerant line of tropical tasar silkworm

IF 0.6 Q4 ENVIRONMENTAL SCIENCES

Journal of environmental biology Pub Date : 2023-06-03 DOI:10.22438/jeb/44/3(si)/jeb-24

I. G. Prabhu, M. Manjappa, M. M. Baig, N. Kumar, A. Sinha, S. Kutala

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Abstract

Aim: To develop random ampliﬁed polymorphic DNA (RAPD) -sequence-characterized ampliﬁed region (SCAR) markers for selecting thermo-tolerant line of tropical tasar silkworm. Methodology: In the environmental chamber, Daba BV cocoons of A. mylitta were exposed to high temperature at 46°C/4 hr for 3 days. After the emergence, genomic DNA was extracted from thermo-tolerant moths and thermo-susceptible pupae. The genomic DNA was amplified using 30 RAPD random decamer primers. The improved RAPD fragments that can differentiate thermo-tolerant and susceptible line of A. mylitta were eluted, cloned in pJET1.2 vector and sequenced. SCAR markers were developed based on the sequence and validated in the subsequent generations. Results: Among 30 RAPD primers, OPK04, OPAJ15 and OPA17 generated polymorphic bands to differentiate thermo-tolerant and susceptible line of A. mylitta. These polymorphic bands were eluted, cloned and sequenced. Sequencing of three cloned fragments revealed that clone PB1 comprised of 1412 bp, clone PB2 comprised of 704 bp and clone PB3 comprised of 931 bp. Sequence specific stable multiplex SCAR markers TT-PB1, TT-PB2 and TT-PB3 were designed and synthesized. PCR amplification was performed using DNA templates of 10 thermo-tolerant and 10 thermo-susceptible samples. SCAR marker TT-PB1 was observed to be more specific to thermo-tolerant line of tropical tasar silkworm and validation with 25 samples each in next generation also supported the specificity of TT-PB1. Interpretation: Among three SCAR markers, TT-PB1 showed more specificity for selecting the thermo-tolerant line of A. mylitta. Therefore, the study provides an effective and precise PCR-based molecular marker system for selecting thermo-tolerant lines in tropical tasar silkworm to overcome seed crop loss due to high temperature stress in tasar rearing hotter zones. Key words: Antheraea mylitta, cloning, RAPD, SCAR marker, Thermo-tolerance

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热带柞蚕耐热品系RAPD-SCAR标记的克隆与开发

目的:建立随机扩增多态性DNA (RAPD) -序列特征扩增区(SCAR)标记，用于热带柞蚕耐热品系的选育。方法:在环境室中，将mylitta的Daba BV茧暴露于46℃/4 h的高温下，持续3天。羽化后，从耐热蛾和热易感蛾蛹中提取基因组DNA。利用30条RAPD随机引物扩增基因组DNA。利用改进的RAPD片段进行了洗脱，并在pJET1.2载体上进行了克隆和测序。SCAR标记是基于该序列开发的，并在后代中得到验证。结果:在30条RAPD引物中，OPK04、OPAJ15和OPA17产生了多态条带，可以区分密枝耐高温系和敏感系。对这些多态性条带进行洗脱、克隆和测序。3个克隆片段测序结果显示，克隆PB1全长1412 bp，克隆PB2全长704 bp，克隆PB3全长931 bp。设计并合成了序列特异性的多重SCAR标记TT-PB1、TT-PB2和TT-PB3。采用10份耐热和10份热敏感样品的DNA模板进行PCR扩增。SCAR标记TT-PB1对热带柞蚕耐高温品系的特异性更强，对下一代各25个样品的验证也支持了TT-PB1的特异性。解释:在3个SCAR标记中，TT-PB1对杨梅耐高温品系的选择具有更强的特异性。因此，本研究为热带沙蚕耐热品系的选择提供了一种有效、精确的分子标记系统，以克服高温胁迫对热带沙蚕造成的种子损失。关键词:柞蚕，克隆，RAPD, SCAR标记，耐热性

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