Molecular characterization of shattering in weedy rice (Oryza sative f. spontanea) biotypes of kerala

Abstract

The study entitled “Molecular characterization of shattering in weedy rice (Oryza sativa f. spontanea) biotypes of Kerala” was conducted at the Integrated Biotechnology Block, College of Agriculture, Vellayani, Thiruvananthapuram during 2015-2017. The objective of the study was isolation and sequencing of genes related to shattering viz. sh4 and qsh1 in weedy rice biotypes and characterization by expression profiling and phylogenetic analysis.

Degenerate primers were designed for sh4 and qsh1 genes based on the gene sequences retrieved from NCBI database (sh4 and qsh1 forward and reverse primers) which were used to isolate and identify the genes. Both genomic DNA and Total RNA of weedy rice and cultivated rice Uma were isolated. PCR was done using both genomic DNA and cDNA. Amplification was obtained in two sets of designed primers i.e. sh4 (FW2 x RW2) and qsh1 (FW2 x RW2). When DNA was used as template for PCR, two of the sh4 primers and one of the qsh1 primers designed for shattering yielded amplicons at the expected size of 649bp, 696bp and 747bp respectively. PCR in cDNA samples of RNA amplicon of size ~690bp with the primer combination of sh4 (FW2 x RW2) in flag leaf was observed. Also, an amplicon of size ~750bp in flag leaf cDNA was produced using degenerate qsh1 FW2 x RW2 primers. None of these primers produced an amplification in any of the cDNA samples of cultivated rice Uma. The tBLASTx programme showed that the amplicon of size ~690bp was similar to shattering 4 (sh4) genes of Oryza populations, whereas the amplicon of size ~750bp belonged to qsh1 gene for putative transcription factor qsh1 of rice populations and mRNA of Oryza sativa japonica group.

The result clearly revealed that the gene responsible for shattering viz., sh4 and qsh1 are present in both weedy rice and cultivated variety Uma as evidenced from the PCR amplification of genomic DNA. However, the genes responsible for shattering investigated in the present study viz., sh4 and qsh1 were not expressed in any of the growth stages in cultivated rice variety Uma.

The NCBI conserved Domain Search programme, showed that the qsh1 FW2 x RW2 sequence of amplicon at ~750bp (obtained by reaction using weedy rice flag leaf cDNA) belonged to homeobox kn domain. The sequence of the amplicons of qsh1 obtained was the partial fragments of the first ever qsh1 gene for putative transcription factor qsh1 to be isolated from Oryza sativa f. spontanea.

Phylogenetic tree constructed for sh4 FW2 x RW2 sequence, had highest similarity to shattering (sh4) gene of Oryza meridionalis voucher and also had relationship with Oryza rufipogon and Oryza sativa. However, the weedy rice qsh1 FW2 x RW2 query sequence was closely related to qsh1 gene of Oryza rufipogon, together with Oryza sativa indica group and Oryza sativa japonica group. Semi quantitative analysis with sh4 and qsh1 gene primers showed that the sh4 gene was expressed in flag leaf and grains and qsh1 gene was expressed only in the flag leaf.

From this study it can be inferred that the gens responsible for shattering in weedy rice biotypes of Kerala viz. sh4 and qsh1 are present both in cultivated rice and weedy rice. However, the genes are expressed only in weedy rice during the flowering stage.