Tagging of bacterial wilt resistance gene in Solanum melongena var.insanum by molecular markers

Abstract

The study entitled ‘Tagging of bacterial wilt resistance gene in Solanum melongena var. insanum by molecular markers’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2009-2011.Bacterial wilt caused by Ralstonia solanacearum (Smith 1896, Yabuuchi et al., 1995) is an important problem in cultivation of tropical and subtropical crops like potato, brinjal, chilli, tomato etc. World wide approach to control the disease is to use resistant varieties. Gopimony (1983) reported Solanum melongena var. insanum (wild variety) is resistant to bacterial wilt and controlling gene is monogenic and dominant.This investigation was taken up for tagging of bacterial wilt resistance gene in Solanum melongena var. insanum by RAPD through bulked segregant analysis as reported by Michelmore et al., (1991). The genotypes used for the study were resistant variety Solanum melongena var. insanum I.C. number 421463, susceptible variety Pusa Purple Long and segregating F2 population for bacterial wilt incidence. To raise segregating F2 population F1 was raised by controlled crossing of Solanum melongena var. insanum I.C. number 421463 with pollen grains of Pusa Purple Long. Then F1 plant was selfed to get F2 population.The parents and F2 genotypes were screened for bacterial wilt incidence by stem puncture method of artificial inoculation using R. solanacearum inoculum. In all cases death of plants were confirmed by ooze test. The genotypes were classified against bacterial wilt incidence according to classification of Mew and Ho (1976). The variety Solanum melongena var. insanum I. C. number 421463 was resistant, Pusa Purple Long and F2 were susceptible. Ratio of susceptible to resistant in F2 generation was 2.7:1. So it can assume that bacterial wilt resistant character in resistant parent can be homozygous and recessive.Genomic DNA for RAPD analysis was isolated by modified CTAB method (1994). Good quality DNA with an absorbance ratio of 1.8-2.0 was used for RAPD analysis. PCR reaction mixtures and conditions for DNA amplification were standardized. Ninty eight primers belonging to series A, OPA, OPAG, OPAH, OPB, OPC, OPF, OPP, OPU, RY and RN were initially screened with DNA of resistant and susceptible parents to select primers with polymorphism. Out of ninty eight primers tested thirty, were reported as bacterial wilt specific. Seventeen primers were selected for BSA based on polymorphism. None of wilt specific primers showed polymorphism.Bulked segregate analysis was done with seventeen selected primers for polymorphism. The genotypes used for the study were susceptible parent, resistant parent, five resistant and five susceptible F2 progenies. Among the tested primers only OPP-14 has recorded polymorphism between resistant and susceptible genotypes in BSA. It has produced 470 bp polymorphic amplicon in resistant parent and resistant bulk. Co-segregation analysis was done with OPP-14 primer with individuals of susceptible and resistant bulk. In co-segregation analysis OPP-14 specific amplicon got amplified not only in resistant parent, resistant bulked but also in three susceptible F2s also. The polymorphic band produced by OPP-14 primer in resistant parent and resistant bulk was eluted from resistant parent and cloned in pGEM-T vector, and was transformed into E. coli JM 109 cells. Recombination of the insert was confirmed through colony PCR reaction with universal T7 and SP6 primers. The cloned fragment was sequenced to obtain the nucleotide sequence information and was named as W-3. The sequence obtained after vector screening was named as “Sol-3”, was subjected to Blastn search. Blastn search revealed significant levels of homology with AC237888 Solanum tuberosum clone RH183L22 present in NCBI database. The sequence was also analysed using EMBOSS Transeq and Blastp. Analysis revealed homology with ABC948993 polyprotein (Oryza australiesis). ORF analysis revealed the longest ORF of 108 bp was on +3 frame which revealed significant level of homology with putative retrotransposon protein in Solanum demissum.Two sets STS primers were designed using Primer3Plus. The efficiency of STS primer set BWRGB-2 to distinguish resistant and susceptible genotypes was tested and it amplified a fragment of 200 bp in all the genotypes tested