Molecular Characterization of Cytoplasmic Male Sterile Moricandia Arvensis Brassica Juncea and Tagging of Male Fertility Restorer Gene

Abstract

Cytoplasmic male sterile (mori) Brassica juncea carries unaltered mt genome of Moricandia arvensis, and the fertility restoration is gametophytic, governed by a single nuclear gene. This fertility restorer locus is unique in that it is capable of restoring male fertility to two other alloplasmic CMS systems of B. juncea. It was also found that male sterility and fertility was associated with changes in the transcript pattern of mitochondrial atp a gene. The present investigations were aimed at molecular tagging of the restorer gene and further characterization of cytoplasmic male sterility with respect to atp a gene transcripts.As a first step toward cloning of this restorer gene, molecular tagging of the Rf locus was attempted using RAPD analysis. A BClFl population segregating for male sterility/fertility was used for tagging following bulk segregant analysis (BSA) method. In the present study, RAPD analysis revealed low polymorphism and was found unsuitable for gene tagging. Since RAPD technique did not yield high polymorphism between male fertile and male sterile bulks, AFLP technique was explored to tag fertility restorer locus. Out of 64 EcoRI/MseI primer combinations tested in the bulks, five combinations gave polymorphic amplification patterns. Further testing of these primers in individual plants showed four amplicons associated with the male fertility trait. Polymorphic amplicons were cloned and used for designing SCAR primers. One of the SCAR primers generated amplicons in almost all the fertile plants. Linkage analysis using MAPMAKER showed two AFLP and one SCAR markers linked to the male fertility gene with a map distance ranging from 0.6 to 2.9 cM.