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Evaluation of Genetic Diversity of Waxy Gene in Selected Rice Varieties by Using Microsatelilte Marker and Cleaved Amplified Polymorphic Sequence Marker

Nilufa Ferdous
Senior Scientific Officer
Grain Quality and Nutrition Division, BRRI

Abstract/ Executive Summary:

Identified four classes of alleles ranged from 100-120 bp in length and represented as class-I (100 bp), class-II (110 bp), class-III (116 bp) and class-IV (120 bp) respectively. Most of the high amylose content rice cultivars were grouped in Class-I allele (100bp). Furthermore, single nucleotide polymorphism were used to discriminate very low, low, intermediate and high amylose content rice cultivars. Cultivars of 11% or less amylose had AGTTATA sequence, which indicated very low amylose content cultivars. While, others had AGGTATA sequence, which indicated low, intermediate and high amylose containing cultivars.

Keywords: Rice, Amylose and Waxy gene

Location: Grain Quality and Nutrition Division Laboratory, BRRI HQ

Start Date: 01-07-2013

End Date: 01-06-2014

Program Area: Conservation and Biodiversity

Commodity: Rice

Objectives:

To identify amylose content at seedling stage.

Methodology:

Thirty five BRRI germplasm cultivars were collected from GRS division. For DNA isolation, rice cultivars were grown under Net house conditions and leaves of 3-4 weeks old seedlings were harvested. DNA was extracted from fresh leaves using the Cetyltrimethylammoniumbromide (CTAB) method (Doyle, 1991) and afterwards quantity of the extracted DNA was measured by Nanodrop Spectrophotometer and Agarose gel. Rice microsatellite marker RM190 (RM190-F: 5’-CTTTGTCTATCTCAAGACAC-3’ and RM190-R: 5’-TTGCAGATGTTCTTCCTGATG-3’) were used to amplify isolated DNA to find polymorphism among the cultivars. The product size of RM190 was 124bp. For analyzing the diversity among the cultivars after cutting with restriction enzyme AccI the following primer pair were used (RM190-F: 5’-CTTTGTCTATCTCAAGACAC-3’) and (GBSSW2-R: 5’- TTTCCAGCCCAACACCTTAC-3’). The product size was 252 bp, which generated two fragments of 124 bp and 128 bp after digestion with the restriction enzyme (AccI).

Source of Information: Proceedings of the BRRI Annual Internal Review, 2013-2014

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Four classes of allele were found among the cultivars on the basis of the molecular weight (band size) of the amplified Polymerase Chain Reaction (PCR) products. The range of the molecular weight of the amplified Polymerase Chain Reaction Among the cultivars, 26% of high amylose, 6% of low amylose and 3% of very low amylose were found in class-1. Similarly, 23% of high amylose, 9% of intermediate amylose, 3% of low amylose and 3% of the very low amylose were found in class-2. Similarly, 3% of the high amylose, 3% of intermediate amylose, and 3% of the very low amylose were found in class-3. On the other hand, no high or low amylose were found in Class-4 but 11% of the intermediate amylose and 9% of the very low amylose were placed in this class. Mostly, the cultivars with 100 bp allele showed high amylose content except 3 that fall in low and very low amylose group such as Khorma, Leda binni and Ranga binni. Similarly, cultivars with 110 bp allele also showed mostly high and intermediate amylose content except 2 varieties that fall in the low or very low amylose groups such as Kanai bansi and Cylindrical Tapi-629. Cultivars with the other two alleles (116 bp and 120 bp) mostly showed lower level of amylose content. G/T polymorphisms analysis of Waxy gene: All of the rice cultivars were non-glutinous type and 6 rice cultivars with very low amylose content were not digested by AccI digestion. Name of the varieties were Kala binni, Cylindrical Tapi-629, Ranga binni, Gohati binni, Binni-1 and Binni-2.

Knowledge Management: Report/Proceedings