MSc.

Chilli is one of the most important condiments in India and our country is
the largest producer, contributing 25% of the world production. During 2010-11,
India produced 0.8 mt of dry chilli from an area of 0.93 mha. Bacterial wilt
(caused by Ralstonia solanacearum) is a major reason for the lower productivity
of this crop, causing up to complete losses under severe infections.
The study entitled “Identification of AFLP marker linked with bacterial
wilt resistance in chilli (Capsicum annuum L.)” was carried out at the Centre for
Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture
during the period 2011-2013. The objective of the study was to identify AFLP
marker linked with bacterial wilt resistance in chilli (Capsicum annuum L.).
Three chilli genotypes Ujwala, Anugraha and Pusa Jwala and their
progenies were used in the study. Anugraha is a chilli variety with resistance to
bacterial wilt disease. Pusa Jwala is the near isogenic line (NIL) of Anugraha,
both differing only in the gene for resistance to bacterial wilt. Ujwala was the
donor parent for resistance while developing Anugraha from Pusa Jwala through
back crossing programme.
Pusa Jwala and Anugraha varieties were screened in open field with
artificial inoculation to confirm the disease reaction. Highly resistant Anugraha
plant was crossed with the pollen from most susceptible Pusa Jwala plant. The F1
seeds were harvested and this generation was field screened to observe the
disease reaction. Previous report on bacterial wilt resistance points to monogenic
homozygous recessive condition for resistance. Accordingly, all the F1 plants are
supposed to be susceptible; but our screening had shown the resistance in F1
generation to be nearly 50 per cent. The reason for the deviation from the
expected ratio is attributed to the selection of a heterozygous (Rr) plant as male
parent. Further the F1 plants were raised in pots, selfed seeds were harvested and
further the pots were infected with bacteria and F2 seeds harvested from
susceptible F1s were used for raising the F2 population. F2 population was used as
segregating population for generating the susceptible and resistant bulks in bulk
segregant analysis (BSA) using AFLP method.
Screening of 200 F2 plants was done along with parents Anugraha and
Pusa Jwala using leaf cutting and pin pricking methods. F2 segregating plants
have shown 69.5 per cent wilt incidence, pointing to ~3:1 ratio, confirming that
the resistance is governed by homozygous recessive condition.
DNA extraction was done from parents and all the 200 F2 plants and
Ujwala by CTAB method (Rogers and Bendich, 1994). Good quality of DNA
with UV absorbance ratio (A260/280) ranged 1.8- 2.0 were used for further
AFLP analysis. DNA from 9 highly susceptible and 9 highly resistant F2 plants
was separately pooled for developing bulk. BSA was carried out with AFLP
analysis the DNA using resistant parent (Anugraha), susceptible parent (Pusa
Jwala), resistance donor (Ujwala), F2 resistant bulk and F2 susceptible bulk.
The AFLP was performed using the standard kit provided by Chromus
Biotech, Bangalore, following the restriction digestions using frequent and rare
cutters, adapter ligation, pre-amplification and selective amplification using the
primer combination EcoACT+ MseCAC. The amplified PCR products were
separated by capillary electrophoresis on an ABI Prism 310 Genetic Analyzer
along with GeneScan™ 500 LIZ® size standard and data generated were
collected by Data Collection 2.1 software. Totally, 124 bands ranging 50-500bp
were amplified. Among them, three polymorphic bands with 103, 118 and 161 bp
linked with the resistance allele and three polymorphic bands with 183, 296 and
319 bp linked with susceptible allele were identified. Due to the repeatable nature
of AFLP, these 6 markers could be directly employed in MAS breeding
programmes.