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Genetic Diversity of BRRI Varieties Using Microsatellite Markers

M S Ali
BRRI, Gazipur

H A Rabbi
BRRI, Gazipur

N Sultana
BRRI, Gazipur

H A Hossain
BRRI, Gazipur

M Islam
BRRI, Gazipur

M E Hoque
BRRI, Gazipur

Abstract/ Executive Summary:

Genetic diversity of 50 BRRI developed modern rice varieties was studied through DNA profiling to protect them from biopiracy. Simple sequence repeat (SSR/microsatellites) markers were chosen due to their comparative superiority over other markers for this purpose. A total of 110 microsatellite markers were used to characterize and discriminate the test materials. Fifty-one primers showed polymorphism giving 191 alleles. The number of alleles per locus ranged from two (RM17, RM119, RM174, RM346, RM348, RM431, RM482, RM495 and RM517) to eight (RM310, RM219), where average number of allele was 3.745. The polymorphism information contents (PIC) lied between 0.106 (RM348) to 0.80 (RM310). Most robust marker was RM310 since it provided the highest PIC value (0.8023). Pair-wise genetic dissimilarity co-efficient values detected BR5 and BR21 as the most genetically distant varieties, whereas BR22 and BR23 were the closest. UPGMA clustering system grouped BRRI varieties into seven major clusters at 0.49 similarity co-efficient. Cluster 1 was identified as the largest cluster comprising 18 varieties. Cluster 3 and cluster 6 were the smallest with only two varieties. All the aromatic varieties (BRRI dhan34, BRRI dhan37, BRRI dhan38 and BR5) grouped in the same cluster indicating their genetic closeness. The information obtained from DNA fingerprinting studies helps to distinctly identify and characterize 50 BRRI varieties using 51 different microsatellite markers. This information can be used in background selections during backcross breeding programmes. This study inferred that most of the BRRI varieties have narrow genetic base and hence inclusion of new landraces in future breeding programmes is recommended to broaden it.

Keywords: Genetic Diversity, BRRI Varieties, Microsatellite Markers

Location: Biotechnology Division, BRRI, Gazipur

Start Date: 15-01-2011

End Date: 20-10-2011

Program Area: Variety and Species

Commodity: Rice

Objectives:

To protect the intellectual property rights (IPR) of our breeders by DNA fingerprinting and to assess the genetic variability among 50 BRRI developed rice varieties

Methodology:

Fifty BRRI released rice varieties were taken. The seeds were obtained from Biotechnology Division, BRRI, Gazipur. They were germinated in petri dishes and sown in earthen pots to raise seedlings. Fresh leaf samples of 21-day-old rice seedlings were used as the source of genomic DNA. DNA was isolated following CTAB method with minor modifications described by Zheng et al. (1995). At first leaf tissue were cut into small pieces, homogenized and digested with extraction buffer (1M Tris, 0.5M EDTA, 5M NaCl and 20% SDS, pH 8.0). After incubation for 20 min at 65°C with intermittent swirling, the mixture was emulsified with chloroform: IAA mix (24: 1 mixture of chloroform and isoamyl alcohol). After centrifugation, the upper aqueous layer was removed into a different tube and cold ethanol was added. After centrifugation a small pellet was visible. The pellets were then washed with 70% ethanol, dried by a concentrator and resuspended in an appropriate volume of TE buffer (1M Tris, 0.5M EDTA, pH 8.0). DNA quality was checked by agarose gel electrophoresis with lambda DNA (50 ng/µl) and quantification was done using a spectrophotometer (Nano drop 1000 V3.6, USA). PCR was carried out in 10 µ l reactions volume containing 1 µ l of MgCl2 free 10 PCR buffer with (NH4)2SO4, 1.2 µ l of 25 mM MgCl2, 0.2 µ l of 10 mM dNTPs, 0.2 µ l of 5 U/µ l Taq DNA polymerase, 0.5 µ l of 10 µ M forward and reverse primers (Promega corporation, USA) and 3 µ l (10 ng) of DNA using a 96 well thermal cycler. Amplification were carried out in a thermal cycler (G-strom, GSI, England ) with the following program: 94°C for 5 min (initial denaturation) followed by 35 cycles of 94°C for 1 min (denaturation), 55°C for 1 min (annealing), 72°C for 2 min (extension) with a final extension for 7 min at 72°C. The annealing temperatures were adjusted based on the specific requirements of each primer combination. After amplification, PCR products were mixed with gel loading dye (bromophenol blue, xylene cyanol and sucrose), and electrophoresed using vertical polyacrylamide gels (8% denatured polyacrylamide gel containing 19: 1 acrylamide: bhisacrylamide) for manual genotyping. Four µ l of the amplification products were resolved by running the gel in 1 TBE buffer for 1.5 to 2.5 hrs (depending on the allele size) at around 90 volts and 500 mA electricity (CBS scientific, USA). The gels were stained in 1 µ g/ml ethidium bromide and documented using UVPRO (Uvipro Platinum, EU) gel documentation unit. A total of 51 SSR markers (distributed across the 12 chromosomes) with clear amplifications were selected for genetic diversity analysis.

Source of Information: BANGLADESH RICE JOURNAL 16(1):105-109, June 2012 ISSN 1025-7330 (Bangladesh Rice J.)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

We can conclude that the genetic information gathered here provides unique DNA profiles for BRRI released rice varieties, which will serve as a strong weapon to provide our breeders IPR and further it will be of greater help in background selections during back cross breeding programmes. Moreover, these data will help the breeders to select parents for future breeding programmes as the identification and use of diverse genetic resources is a prerequisite for plant improvement. This study concludes that most BRRI varieties have narrow genetic base. So it is recommended to use genetically diverse varieties for future breeding programme or include new and untouched landraces to incorporate new genes and broaden genetic base.

Knowledge Management: Journal