MSc

The development of hybrids with desirable heterosis is a major goal in plant breeding. In
traditional hybridization, following the emasculation which is a labour intensive process, pollen is
manually transferred to the female organs. The higher cost of the hybrid seeds, considerably owing
to the labour costs for emasculation, is the major constraint in achieving more rapid adoption of
vegetable hybrid technology. Use of male sterile parent is understood to reduce the cost of hybrid
seed production by avoiding the manual emasculation. In this context, the recently reported male
sterile line of ridge gourd from Kerala Agricultural University, is commercially very important.
A system to confirm the male sterility at an early phase of parent itself, is very important
since any kind of fertility restoration at a later phase will fail the hybridization programme.
A molecular marker associated with the male sterility will be enormously useful at this phase, to
confirm the male sterility of the female parent used in the hybridization programme. The study
entitled “Molecular characterization of male sterility in ridge gourd Luffa acutangula (L.) Roxb.”
was carried out at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,
Kerala Agricultural University, with the objective to identify the molecular marker/s linked with the
male sterility in the newly reported sterile line of ridge gourd from KAU, using SSR and ISSR
marker systems.
Plant materials used in this study were three male fertile lines, Haritham, Deepthi, and
Arka Sumeet and the Haritham (male sterile line of KAU). In the male sterile line, unopened
rudimentary male buds with poorly developed anthers containing shrunken, non viable pollen grains
were observed. In male fertile plants, the anthers were well developed with viable pollen grains.
Total genomic DNA was extracted from the young leaves, using CTAB method and
SSR and ISSR marker systems were employed for characterizing the male sterility. SSR primers are
highly genus specific and since genomic data on Luffa is lacking, suitability of SSR primers from
the related genus such as Citrullus and Cucumis was attempted. Initial attempts on electrophoresis
of SSR amplicons on 2 per cent agarose gel were successful for few primers only. Thus, to resolve
the small fragments, 10 per cent native PAGE was subsequently used. In SSR assay 25 primers
were screened, among which 14 were selected. Total genomic DNA of the male fertile and sterile
lines were amplified with these SSR primers. The primers CI1-21, DE0144 and CsWCT25 have
yielded distinctly polymorphic bands associated with the male sterility with 125, 50, 350 bp sizes,
respectively.
In ISSR assay, among the 49 primers screened initially, 16 were selected for the study.
UBC841 has yielded a distinctly polymorphic band for male fertility at 1185bp. This marker was
missing in the male sterile line. Dendrogram was generated based on the ISSR study in order to
study genetic relationship among the ridge gourd lines. Haritham (fertile) and Haritham (male
sterile) showed 82 per cent similarity. The universal mitochondrial primers were additionally
screened in this study and among them; Rps14 gave polymorphic band linked with male sterility, at
80 bp size.
Direct sequencing with NGS platform was done of the identified polymorphic bands, it
was failed with CI1-21, Rps14, DE0144 and UBC841. Only with CsWCT25 sequence of 225 bp
was obtained. CsWCT25 is already reported to be tightly linked with gynoecy in cucumber, SCAR
primers are also designed using this sequence. These markers will have wide application in marker
assisted selection and hybrid seed production.