ANALYSIS OF CYTOPLASMIC MALE STERILE AND RESTORER INBRED LINES AND PREDICTION OF HETEROSIS IN BRASSICA NAPUS HYBRIDS USING MOLECULAR MARKERS

Brassica napus is an important oilseed crop throughout the world. Development of hybrid varieties for commercial production is of interest to plant breeders and producers as compared to open pollinated cultivars due to significant positive heterosis in hybrids. Overall objectives of this project were to efficiently produce high yielding, better quality hybrids in Brassica napus using both classical and molecular marker technology. As a first step, genetic diversity among 60 accessions including maintainers and restorers of the Cytoplasmic Male Sterile (CMS) lines along with some Open Pollinated (OP) cultivars were analyzed. Using 20 Sequence Related Amplified Polymorphism (SRAP) primer combinations 162 polymorphic markers were obtained. Unweighted Pair Group Method using Arithmetic Averages (UPGMA) cluster analysis placed all accessions into 5 main clusters that were mostly according to their pedigree and country of origin. The presence of maintainers and restorers in different clusters indicated sufficient diversity that can be exploited for hybrid seed production in B. napus. Loss of markers due to inbreeding depression between open pollinated cultivars and their respective inbred lines that were selfed for 5-7 generations were also noted. Based on the cluster analysis 12 maintainer and 8 restorer lines from different clusters were selected for production of 96 hybrids that were evaluated in the field for agronomic characters. Based on Mid Parent and High Parent Heterosis, 6 hybrids with seed yield more than 2000 kg ha-1and oil content more than 42% were selected for large scale evaluation. Results also indicated a moderate increase in seed yield (1553 kg ha-I) from inter cluster crosses than from intra-cluster crosses (1352 kg ha-I). Correlation co-efficient and regression analyses indicated only a moderate relationship between genetic distance resulted from molecular markers and seed yield (kg ha-I). Using three F2 and one backcross population, it was found that the cytoplasmic male sterility is conditioned by a single dominant restorer gene. To enhance the selection of restorer lines at early seedling stage, 3 molecular markers linked to the fertility restorer gene in CMS background with 1 % recombination were found. By chromosome walk towards the direction of the restorer gene, a 100% linked marker to the restorer gene was detected. Sequence analysis and NCBI database search indicated homology with pentatricopeptide (PPR) proteins that have functions in almost all stages of gene expression, including messenger RNA (mRNA) transcription, splicing, processing, editing, translation and stability. To gain some insight into the causes of male sterility, transcripts from two CMS lines CMS-ARITI21 and CMS-ARIT2 along with their respective maintainer lines were analyzed using primers from known mitochondrial genes. Sequence analysis indicate that CMS-ARIT121 exactly match with polima CMS while CMS-ARIT2 was quite unique.Overall, these results provide opportunities for plant breeders to efficiently select for new restorer and CMS lines and exploit the available genetic diversity and heterosis for successful hybrid seed production in B. napus