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Journal of Environmental Biology

pISSN: 0254-8704 ; eISSN: 2394-0379 ; CODEN: JEBIDP
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    Abstract - Issue May 2023, 44 (3)                                     Back


nstantaneous and historical temperature effects on a-pinene

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

 

I.G. Prabhu1*, Manjappa2, M.M. Baig1, N. Kumar1, A.K. Sinha3 and S.Kutala1

 

1Central Tasar Research & Training Institute, Central Silk Board, Ranchi – 835 303, India

2Central Sericultural Research & Training Institute, Central Silk Board, Mysore – 570 008, India

3Ranchi University, Ranchi - 834 008, India

*Corresponding Author Email : immanual.gilwish@gmail.com                    *ORCiD: https://orcid.org/0009-0003-7069-1150

 

Received: 07.12.2022                                                                                                 Revised: 10.05.2023                                                                           Accepted: 12.05.2023

 

 

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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