MSc

Chilli or hot pepper (Capsicum annuum L.) is one of the most important commercially available universal spice crop which is grown throughout the world. Among the various diseases affecting chilli, Bacterial wilt (BW) is one of the most
dangerous disease which drastically limits its cultivation. The disease is caused by
Ralstonia solanacearum which is a soil-borne bacterium. The loss in yield is estimated about 30-100 per cent due to BW in chilli. For managing the disease several chemical,
biological and cultural methods were practiced but development of resistant variety through conventional breeding was found to be effective. Anugraha and Ujwala are two bacterial wilt resistant varieties released by KAU. Earlier attempts were made to develop molecular markers such as RAPD, AFLP, SSR in chilli and SCAR in eggplant linked with this trait. The present study was conducted to validate the reported molecular markers such as RAPD, SCAR, AFLP and SSR linked for bacterial wilt disease resistance through bulk segregant analysis method so as to enable marker assisted selection in breeding programme. In the present investigation, the resistant variety Anugraha was crossed with Pusa Jwala which was a high yielding variety but susceptible for BW disease. The F1 population was raised and self-pollinated to get F2 seeds. The F2 seeds were raised in pots and inoculated with bacterial suspension for selecting resistant and susceptible plants. Resistant variety Ujwala was used as resistance donor in the study.The genomic DNA was isolated from parent genotypes and F2 segregating population. The isolated DNA was purified by RNase treatment to obtain good quality DNA which was further utilized in Bulk Segregant Analysis (BSA). For BSA, the isolated genomic DNA from Ujwala, Anugraha, Pusa Jwala and bulked DNA of 5 resistant and 5 susceptible F2 segregating plants were used for validation of markers. The protocols for RAPD, SCAR and SSR had been modified and used in the study whereas, AFLP protocol
was developed for validation studies. The results of RAPD analysis revealed a unique band of size 1.24 Kb which was
present only in resistant donor parent Ujwala which was absent in other genotypes. The amplicon of size 1.24 Kbp was eluted, reamplified and sequenced but the sequence information obtained was only 481 bp. The nucleotide sequence and amino acid sequences were annotated using bioinformatics tools BLASTn and BLASTp respectively. The result of BLASTn analysis revealed that the query sequence of size 481 bp was
showing homology similarity with mitochondrial genome of Capsicum annuum cultivar Jeju where as BLASTp analysis showed homology similarity with hypothetical protein
(Vicia faba). The results of SCAR and SSR markers analysis showed monomorphic band of size 350 bp and 250 bp respectively in all the samples which reveals that SCAR and SSR markers were unable to characterize the resistant and susceptible genotypes. AFLP analysis was done by using genomic DNA (500 ng) with steps involved viz., Restriction of genomic DNA and ligation with adaptors, pre amplification, selective amplification. The selective amplified product was electrophoresed in 6 per cent Urea PAGE. The result of AFLP analysis revealed that reported polymorphic band of size 100
to 200 bp were absent in all the genotypes and shown only monomorphic bands.Reported molecular markers selected were not able to distinguish susceptible and resistant genotypes to bacterial wilt. Attempts can be made to get full sequence
information of RAPD amplicon of 1.24 kb and can be analysed with bioinformatics tools for confirmation.