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Validation of Molecular Markers for Use in Mas/mabc Program for Salinity Tolerance

M.S. Rahman
Senior Scientific officer
Plant Physiology Division, BRRI, Gazipur

A.Ferdousi

M.R.Islam
Principle Scientific Officer
Plant Breeding Division, BRRI, Gazipur

M.A. Salam
Chief scientific Officer
Plant Breeding Division, BRRI, Gazipur

M.S.Islam
Chief scientific Officer
Plant Physiology Division, BRRI, Gazipur

M.A.H. Molla

Z.I. Seraj
Proffesor
Department of Biochemistry and Molecular Biology, Dhaka University

Abstract/ Executive Summary:

Traditional breeding approaches to develop salt tolerant varieties have been applied for many years, but progress has been slow. Marker assisted selection (MAS) has been recently adopted to improve the power and efficiency of breeding programs. Therefore, an experiment was designed to find out tightly linked and unlinked markers for use in MAS/MABC scheme for ‘Saltol’ introgression in Plant Physiology Division, BRRI, Gazipur with the collaboration of Dhaka university. IR6O494 x BR29 populations (F6) were screened for tolerance arid sensitivity to salt stress. A total of 978 progenies were screened. Also seedlings were scored according to the scoring system. Out of 978 seedlings 325 were scored 3/4 and considered tolerant, 423 scored 5 considered to be moderately tolerant, 167 were scored 6/7 considered as moderately susceptible and 63 were scored 9, which were considered susceptible. Out of 7 markers studied, R.M1287 and lM493 were found significant at 1% level and can be used in foreground selection as most tightly linked to the Saltol. CP06224 and RM10793 were found significant at 5% level, in spite of that the following two markers are very difficult to score. RM3627 was found insignificant meaning that it has no linkage (unlinked) with Saltol and can be used in recombinant selection because of its close proximity towards Saltol, But interesting thing is that the physical position of two marker AP3206-41 8 and Met. Syn. U NC is within the Saitol but has no linkage with the trait that means the QTL is not continuous, there are several loci contributing for this tolerance.

Keywords: Rice, Salinity tolerance, Molecular markers, MAS/MABC.

Location: Plant Physiology Division, BRRI, Gazipur

Start Date: 00-00-2007

End Date: 00-00-2008

Program Area: Conservation and Biodiversity

Commodity: Rice

Objectives:

To find out tightly linked and unlinked markers for use in MAS/MABC scheme for ‘Saltol’ introgression_.

Methodology:

F₆ breeding population of the cross between a salt tolerant IR line IR60494 and BRRI dhan29 a farmers popular BRRI variety were mapped. The phenotypic screening for the salinity tolerance at seedling stage was done by the method described by Gregorio et al. (1997). The complete sequence of rice (Nipponbare) has recently become available from International Rice Genome Sequencing Project (IRGSP), (http://rgp.dna.affrc.go.jp/cgi-bin/statusdb/seqcollab.pl). The Microsatellite/SSR markers, RM23 and RM9 were identified as the flanking marker for ‘Saltol’ locus, (Bonilla et al., 2002). The region was further reduced to 5.1 cM by using RILs (Elahi et al 2004). SSR, BAC clone and PAC clone markers within and flanking the ‘Saltol’ locus was designed (www.genome.wi.mit.edu/cgi-bin/primer/primer3www.cg). Some Rice Microsatellite (RM) primers were taken from ‘Nature’ publication. In this study fifteen markers were tested to find polymorphism between IR60494 and BR29. Newly developed leaves were collected after establishment of the screened seedlings of transplanted rice in the field for above purpose. About 1.0 g fresh leaf was collected and wrapped with foil paper as well as masking tape and marked with a marker pen and stored immediately in Liquid Nitrogen. Then again the leaves in the laboratory were packed and sealed in a plastic bag and stored into -80⁰C. Genomic DNA of the selected populations was isolated separately along with the parents and standard checks. DNA was extracted using two protocols i.e. CTAB method described by Doyle and Doyle (1990) and the DNeasy® plant mini kit from QIAGEN (www.qiagen.com). Other procedures like quality checking, quantification and storage of DNA; PCR amplification, PAGE run, image analysis ect, all of these were followed as per standard followed in the molecular laboratory. Markers scoring in Agarose and Acrylamide gels were done manually with the help of Alpha Imager software (www.alphaimager.com). Single marker analysis was done through ANOVA to find out the marker-trait linkage by CROPSTAT 7.0 software developed by IRRI Biometrics unit (http://www.irri.org).

Source of Information: Proceedings of the BRRI Annual Internal Review for 2007-2008

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Out of 978 seedlings 325 were scored 3/4 and considered tolerant, 423 scored 5 considered to be moderately tolerant, 167 were scored 6/7 considered as moderately susceptible and 63 were scored 9, which were considered susceptible. None of the progenies scored I (highly tolerant). A total of 15 SSR, STS and designed primers saturating the ‘Saltol’ locus and flanking the region were tested in 1R60494 and BRRI dhan29 for polymorphism. Out of 15 markers seven were found to be distinctly polymorphic between 1R60494 and BR29 and were used to test the breeding populations. One hundred ninety eight tolerant and sensitive extremes were chosen for molecular analysis. Out of seven markers RM493 and RM 1287 were significant at 1% and CP06224 and RM10793 were significant at 5% level of probability, respectively and marker RM3627, AP3206-4 18 and Methionine synthetase were insignificant. Significance at 1% and 5% level means that the markers were tightly linked with the trait and therefore co-segregate with the trait/QTL. These markers can then he used to locate the trait/QTL in the population. However, insignificance of markers implies that markers are loosely associated or unlinked with the trait/QTL and thus recombination can frequently happen between the marker and trait/QTL.

Knowledge Management: Report/Proceedings