Development of scar marker for authentication of gender in kodampuli (Garcinia gummi-gutta var.gummigutta)

Abstract

The diabetes and cardiovascular disease are the two serious obesity related life-style diseases, spreading at alarming rate throughout the world, especially in thickly populated third world countries in which India occupies the prime position. The fleshy fruit rind of Kodampuli (Garcinia gummi-gutta) is the richest natural source of anti-obesity metabolite hydroxyl citric acid (HCA). Which inhibit the conversion of carbohydrate to fats without affecting Kreb’s cycle through an enzyme ATP citrate lyase. Since Kodampuli is a polygamodioecious tree, it takes 8 to 12 years to identify the female trees. No significant reports are available for sex determination in Kodampuli on the basis of physiological, biochemical or molecular characters. Sex identification, lack of orthotropic shoots for grafting, prolonged seed dormancy, poor seed germination and lack of awareness of its pharmaceutical significance are hindering the extensive cultivation of this backyard companion crop in Kerala and other coastal regions of country. In the present study, an attempt was made to develop simple PCR based technique which can use for gender diagnostic in this plant. DNA samples were extracted from field grown 15 to 20 years old 25 male and 25 female trees and were bulked to 5 samples each by sex type. Earlier reported RAPD primers viz. Kit C1, Kit C8 and Kit C9 were screened but no significant polymorphism was observed. So a total of random 46 decamer primers were tested and six primers were selected for further analysis. On rescreening of the six selected primes viz. RN 5, RN 9, RN 10, RY 5, RY 18 and OPAH 12 only OPAH 12 reproduce male specific band in bulked and individual samples. Random amplified polymorphic DNA (RAPD) fragments were generated in the both bulks in order to identify markers that were polymorphic between male and female plants. A 550 base-pair (bp) male-specific DNA fragment generated with the OPAH-12 primer was identified. The polymorphic male specific band produced by OPAH 12 primer was eluted and cloned in pGEM-T vector, and transformed into E. coli JM 109 cells. Cloned cells were subjected to blue-white screening and transformed one was sent for sequencing The sequence obtained after vector screening was subjected to nucleotide blast search and ORF finder. It does not reveal any significant levels of homology and reading frame. Two pairs of SCAR primer were designed on the basis of sequence. These SCAR primers were checked for male and female samples but no polymorphic band was observed. The future line of work can be to screen the male and female genotypes with more number of primers to obtain larger base pair polymorphic band. That can used to convert this dominant marker to co-dominant one like SCAR marker. SCAR marker would be successfully employed in breeding experiments for Marker Assisted Selection.