Development of SCAR marker(s) for aphid tolerance in Brassica juncea (L.) Czern. & Coss
| dc.contributor.advisor | Singh, Ravi Shankar | |
| dc.contributor.author | Rekha, Kumari | |
| dc.date.accessioned | 2019-06-26T06:24:59Z | |
| dc.date.available | 2019-06-26T06:24:59Z | |
| dc.date.issued | 2018-06 | |
| dc.description.abstract | Brassica juncea (L.) Czern. & Coss. commonly known as “Indian mustard” is predominant member of Brassicaceae family in the Indian subcontinent. The mustard aphid, Lipaphis erysimi (Kalt.) is one of the major insect-pest of rapeseed-mustard. There are few reports on molecular markers related to aphid resistance/tolerance in Brassica species, which are not good enough for high level of confirmation. Sequence Characterized Amplified Region (SCAR) markers are important markers used for tagging of a gene or to link a specific trait. In the present study, for the development of SCAR markers for aphid tolerance/susceptibility derived from RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeat) markers, eight different genotypes of B. juncea comprising tolerant (IC-399802, IC-491089, IC-312545, IC-312553) and susceptible (IC-385686, IC-264131, IC-426392, Laxmi) identified previously and available were used. Firstly, all the eight genotypes were grown in RBD design with three replications and the data was recorded on five observations, and on the basis of aphid infestation index, tolerant level of genotypes, IC 491089, IC 312545, IC 385686 and IC 312553 was re-confirmed. Then, RAPD and ISSR fingerprinting was done with these genotypes, Out of 51 RAPD marker, only thirteen were found to be polymorphic while out of twelve ISSR markers eight were polymorphic. We obtained three primers OPE 16 (RAPD), UBC 839 (ISSR) and UBC 864 (ISSR) producing bands which could discriminate tolerant genotypes from susceptible ones for aphid tolerance. One RAPD primer, OPE 16 produced two bands of size ~600bp and ~300 bp, which could discriminate tolerant genotypes from susceptible ones for aphid tolerance. UBC 839 yielded ~800 bp unique band in bulk tolerant while UBC 864 yielded three bands of ~1200 bp, ~1000 bp, and ~500 bp in tolerant genotypes. Total six unique bands were selected, which were either present in susceptible or tolerant genotypes for developing SCAR markers. These bands were ligated into pTZ57R/T cloning vector and transformed into DH5α E.coli cells, the transformed colonies were identified by Blue-white screening on X-gal-Ampicillin-LB Agar plates. The insert in the transformed colonies were confirmed by double digestion and by colony PCR, then plasmids were isolated and sequenced. BLASTN analysis of these SCAR sequences was done to see if any of these related to resistance. The sequences of BJSCAR2, BJSCAR3, BJSCAR4 and BJSCAR5 showed high similarity with nucleotide sequences of A. thaliana, B. rapa, B. napus and other Brassica spp. but none specifically related to resistance. BJSCAR1 did not show any match in GenBank nucleotide database, this could be a novel sequence. The sequences obtained were used to design different sets of SCAR primers. Out of seven sets of SCAR primers obtained from SCAR marker sequences, BJSCAR1-F1 and BJSCAR1-R1 yielded a prominent unique bands in all the four susceptible genotypes as well as in the bulk susceptible and was absent in the tolerant genotypes. This primer set also did not show any amplification in B. fruticulosa, a highly tolerant to aphid (used as control), thereby confirms this SCAR primer set’s discriminatory power for aphid susceptible and tolerant genotypes of mustard. Other SCAR primers (BJSCAR2-F1 and BJSCAR2-R1 for SCAR2; BJSCAR3-F1, BJSCAR3-R1 for SCAR3; BJSCAR4-F1, BJSCAR4-R1 for SCAR4; BJSCAR5-F1, BJSCAR5-R1 for SCAR5) could not show clear cut polymorphism between susceptible and tolerant genotypes. This indicates that probably polymorphism of RAPD/ISSR markers from which these markers were derived lost upon conversion into SCAR marker. In conclusion, in the present study, we developed one SCAR marker and validated in different susceptible/tolerant genotypes of B. juncea. This marker distinguished susceptible and tolerant genotypes. This was also tested with B. fruiticulosa, an aphid tolerant genotypes, which further confirms its discriminatory power. We further suggests that it could be further refined using more aphid resistant genotypes for its wider applicability in Brassica spp. | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810110030 | |
| dc.language.iso | en | en_US |
| dc.pages | 91 | en_US |
| dc.publisher | Department of Plant Breeding and Genetics, BAU, Sabour | en_US |
| dc.sub | Plant Breeding | en_US |
| dc.subject | null | en_US |
| dc.theme | Molecular biology and Genetic Engineering | en_US |
| dc.these.type | M.Sc | en_US |
| dc.title | Development of SCAR marker(s) for aphid tolerance in Brassica juncea (L.) Czern. & Coss | en_US |
| dc.type | Thesis | en_US |