Molecular characterization of gynoecy in bitter gourd (Momordica charantia L.)

Abstract

Bitter gourd (Momordica charantia L.) is an important vegetable of tropics and sub-tropics of Asia belonging to Cucurbitaceae family. The immature fruits of bitter gourd are valued for its culinary and medicinal importance. Having highest amount of ascorbic acid and iron content, it is considered to be the most nutritive among all the cucurbitaceous vegetables. Heterosis is well exploited in bitter gourd for early maturity, increased yield and other agronomic traits through development of hybrids. However, the production of hybrids is labour-intensive involving manual bagging and hand pollination, thereby increasing the cost of seed production. The predominant sex form of bitter gourd is monoecious which bears separate male and female flowers on the same plant. However, gynoecious type bearing only female flowers were also reported in few locations of India, Japan and China. These gynoecious bitter gourd lines can be exploited as female parent to make hybrid seed production economical and easier, as it eliminates the need for emasculation and assisted pollination. It also aids in maintaining the genetic purity of hybrids and helps in harnessing the benefit of hybrid vigour including early maturity and high yield. Usually, the sex expression in cucurbits is highly influenced by environment and hormones, which makes the early phenotypic identification of gynoecy challenging. Identification of molecular markers tightly linked to gynoecy trait would ease the identification of gynoecious line in breeding programmes. In this study, 20 putative candidate genes governing sex expression were selected from different literatures and the gene sequences were retrieved from bitter gourd and cucumber genome assembly in NCBI GenBank database. Four ethylene biosynthesis genes (ACO1, ACS2, ACS3. ACS4 and ACS7), three MADS-box transcription factor encoding genes (AG6, MADS-boxTF23 and McAG2), one auxin related gene (CsARF5) and 12 WRKY transcription factor encoding genes comprise the list. A total of 20 gene-specific primer sets were designed from the selected region of each of the 20 genes. Genomic DNA was isolated from monoecious genotype, MC-136 and gynoecious, KAUMCGy-101. Efforts were made to amplify all the 20genes, however, only seven gene-specific primer sets designed from bitter gourd produced PCR amplification. The amplicons of expected product size from both the samples were sequenced. Sequence variation analysis was done by comparing the monoecious and gynoecious sequences generated to the reference sequence (monoecious) available in NCBI database for bitter gourd. Six valid variations including three SNPs and three In/Dels were found in AG6 and McAG2. All the variations except SNP1 of AG6 were present in non-coding regions. In/Del of 48 bp ([TC] 24 ) in AG6 gene caused a significant difference in the number of ‘TC’ repeats in the sequences which was used for the development of SSR marker. The marker showed clear polymorphism between monoecious and gynoecious genotypes used. It was further validated in three other monoecious lines namely, Priya, Priyanka and the wild type M. charantia var. muricata. Expected banding pattern showing polymorphism for gynoecious line was obtained for all the three monoecious lines screened. Thus, the study identified two potential candidate genes, AG6 and McAG2 for sex expression in bitter gourd. The SSR marker developed needs to be validated in large number of population and more number of varieties to confirm its use as a reliable polymorphic marker in marker-assisted selection of gynoecious lines of bitter gourd.