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Molecular Characterization of Brassica Oil Crop Varieties of Bangladesh Using Ssr Technique

L. Rahman
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

M. R. Molla
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

M. N. Islam
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

The use of Simple Sequence Repeats (SSR) for identification of polymorphism in the varieties of cultivated Brassica species have been used by many researchers of present day. According to many authors the identification and differentiation of the varieties through DNA fingerprinting using microsatellites (SSRs) are quite effective, when the variety specific primers are used. This is gaining importance particularly when the distinguishing a variety from others using morphological traits are becoming difficult due to use of limited elite varieties for new varieties. The variations at DNA level that are conserved in B.napus B.juncea and B.rapa have been reported by Szewe-McFaden, (1996) . In one case 17 primers amplified products in three species and among them 13 detected variation between and within species. According to them once the moderate number of primer pairs is accessible to the users' community, SSR genotyping may provide useful method for characterization, conservation and utilization of agricultural crop diversity. In the present study a total of 21 varieties: 7 of Brassica napus varieties, 5 of Brassira juncea varieties and 9 of Brassica rapa varieties released or registered in Bangladesh have been used to characterize those groups. DNA was extracted from a total of 21 varieties following standard protocol using SDS and Phenol: Chloroform: IAA followed by alcohol precipitation. DNA was confirmed using 1% agarose gel and quantified at 260nm spectrophotometrically (Specrronicf Genesis TM). A set of five microsatellite loci (B.n.12A, B.n.35D, B.n.38A, B.n.59A I and B.n.68/1) have been selected from the literature cited by Szewc-McFadden et al. (1996) to estimate the potential of these marker for variety identification. Finally three primers, B.n.12A, B.n.38A and B.n.59A I were selected based on their performance for SSR data analysis. Upon PCR amplification, the alleles were separated on polyacrylamide gel using a sequencing gel electrophoresis system and visualized by silver-staining method. In this study it was found that all three primers have shown variable polymorphism in different species. The genetic distance indicated differentiation of 5 varieties of Indian mustard into two main clusters: Rai 5, Daulat. BARI sarisha-10 and BARI sarisha-11 grouped in cluster I and Shambol alone formed cluster 2. Shambol showed highest genetic distance values (0.4055) with Daulat, BAR I sarisha-I 0 and BARI sarisha-11 .In case of B.rapa the genetic distance indicated differentiation of 9 varieties of rape seed into two main clusters: Tori-7. BARI sharisha-9 and BARI sharisha-12 grouped in cluster 2 while others in cluster I. In cluster I Agrani and Sarnpad grouped together in sub-cluster I and with minimal genetic distance (0.000). The highest genetic distance value (3.860) was observed between Sampad and Tori- 7. In case of varieties of B.napus" species one of the three primers (B.n.12A) has identified more than two alleles in one location of similar genome sets of four varieties viz; BlNA Sharisa 5,7.8, and 13. This is possibly due to the unstable accumulation of micro-mutants in the genomes during the process of selection from mutation generation.

Keywords: Molecular characterization, Brassica oil crops varieties, SSR technique

Location: Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Mustard

Objectives:

To identify polymorphism in the varieties of cultivated Brassica species.

Methodology:

Twenty one varieties of three Brassica species including 5 of Brassica juncea, 9 of B.rapa and 7 of B.napus were selected for microsatellite analysis. Seeds were supplied by Oilseed Research center of BARI, Plant Breeding division of BINA and the Department of genetics and plant breeding of BAU. Seeds were germinated and grown at aseptic condition. Fresh leaf samples of 12-days-old seedling were used as the source of genomic DNA. Leaf tissues were cut into small pieces, homogenized and digested with extraction buffer (50 mM Tris-HCI, 25 mM EDT A, 300 'mM NaCI and 1 % SDS, pH 8.0). After incubation for 20 minutes at 65°C with intermittent swirling, the mixture was emulsified with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1 v/v/v). DNA was precipitated using two volume of absolute alcohol in presence of 0.3M sodium acetate and pelleted by centrifugation. The pellets were then washed with 70% ethanol, air dried and re-suspended in an appropriate volume of TE buffer (10 mM Tris- HCI, 1 mM EDT A, pH=8.0). DNA quality was checked by electrophoresis in a minigel and quantification was accomplished using a spectrophotometer (Spectronic" Genesis P", Spectronic Instruments Inc., USA). A set of five microsatellite loci ( B.n.12A, B.n.35D, B.n.38A, B.n.59Al and B.n.68/l) have been selected from the literature cited by Szewc-Mcf'adden et al. (1996) to estimate the potential of these marker for variety identification. Finally three primers, B.n.12A, B.n.38A and B.n.59Al were selected based on their performance for SSR data analysis. Three developed rice microsatellite primer pairs (RM 11, RM 151 and RM 153) were used in the analysis . Polymerase Chain Reactions were done in a volume of 10 III containing lOX PCR Buffer, 0.25 mM each of the dNTPs, 2.5 JlM of each primer, 1 unit ampli Taq DNA polymerase, 50 ng template DNA and a suitable amount of sterilize deionized water. Amplification were carried out in a oil free thermal cycler (Thermal cycler gradient, Eppendorf) with the following program: Initial denaturation at 94DC for 3 min followed by 35 cycles at 95 DC for 30 see, 58 DC for 45 see, and 72 DC for 1 min and a final cycle at 72 DC for 7 min. PC~ products were checked in 2% agarose gel. PCR products were separated on 6% denatured polyacrylamide gel containing 19: 1 Acrylamide : Bis acrylamide and 7M urea. Electrophoresis was carried out on Sequi Gen GT eletrophoresis cell (Bio-Rad laboratories, USA). Gels were stained with silver using the Pro mega Silver Stquence" protocol with some modifications (28). Digital images of gels wre made using an A4 scanner. The size (in nucleotides) of the most intensely amplified band for each microsatellite marker was determined based on its migration relative to molecular weight (mw) size markers (100bp DNA ladder, Genei, India). The polymorphism information content (PIC) was calculated according to Neis statistics (Nei, 1973): PIC=1-L:(P/), where Pi is the frequency of the ith allele detected in the germ plasm. Allele frequency was determined using software POPGENE (version 1.31) (Yeh et al., 1999).

Source of Information: PROCEEDINGS OF THE INTERNATIONAL CONFERENCE ON PLANT BREEDING AND SEED FOR FOOD SECURITY, ICPBSFS: 102-108, 2009

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The genetic distance indicated differentiation of 5 varieties of Indian mustard into two main clusters: Rai 5, Daulat. BARI sarisha-10 and BARI sarisha-11 grouped in cluster I and Shambol alone formed cluster 2. Shambol showed highest genetic distance values (0.4055) with Daulat, BARI sarisha-1 0 and BARI sarisha-11 .In case of B.rapa the genetic distance indicated differentiation of 9 varieties of rape seed into two main clusters: Tori-7. BARI sharisha-9 and BARI sharisha-12 grouped in cluster 2 while others in cluster I. In cluster I Agrani and Sarnpad grouped together in sub-cluster I and with minimal genetic distance (0.000). The highest genetic distance value (3.860) was observed between Sampad and Tori- 7. In case of varieties of B.napus" species one of the three primers (B.n.12A) has identified more than two alleles in one location of similar genome sets of four varieties viz; BlNA Sharisa 5,7.8, and 13. This is possibly due to the unstable accumulation of micro-mutants in the genomes during the process of selection from mutation generation.

Knowledge Management: Report/Proceedings