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Foreground Selection Through Ssrs Markers for the Development of Salt Tolerant Rice Variety

Dr. Lutful Hassan
Professor
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

U. Mondal*
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. S. R. Khanom
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S. N. Begum
Plant Breeding Division, Bangladesh, Institute of Nuclear Agriculture, Mymensingh-2202, Bangladesh, *E-mail:upamamondal@gmail.com

Abstract/ Executive Summary:

Salinity is a great problem for rice production worldwide incurring substantial yield loss; a great threat towards food security. Marker-assisted backcrossing is one of the feasible methods to develop a new salt tolerant rice cultivar to cope with the challenge. The study was focused on to introgress salt tolerant genes from a tolerant rice line, FL-478 to Binadhan-7, an early, agronomically suitable and susceptible variety. Backcrossing was done during boro season; where Binadhan-7 was the recurrent parent and FL-478 was the donor parent. 141 BC₁F₁ lines were developed, which were subjected to foreground selection; the first level of selection of marker assisted backcrossing program. The aim of foreground selection was to identify the introgressed lines. 141 BC₁F₁ populations were evaluated with tightly linked salt tolerant markers; AP3206f, RM3412b and RM336. A total of 47 heterozygous BC₁F₁ lines were selected finally, which have alleles of both of the parents. Those introgressed lines could be efficiently used in further development of a stable early salt tolerant rice variety.

Keywords: Foreground selection, SSRs markers, Marker-assisted backcrossing

Location: Mymensingh

Start Date: 00-00-2012

End Date: 00-00-0000

Program Area: Variety and Species

Commodity: Rice

Objectives:

To introgress salt tolerant genes from a tolerant rice line, FL-478 to Binadhan-7, an early, agronomically suitable and susceptible variety

Methodology:

Plant materials: FL-478 (IR 66946-3R-178-1-1) was used as one of the parent for transferring salt tolerant QTL as it contains Saltol QTL on chromosome 1. It is a recombinant inbred line derived from (Pokkali C IR 29). It has a high level of seedling stage salinity tolerance in rice. Binadhan-7 is an early, high yielding transplanted aman variety was used as another parent. It is susceptible to salinity. A backcrossed program was conducted where, Binaadhan-7 was used as recurrent parent and FL-478 was the non-recurrent donor parent. From that crossing program 23 F₁ seeds were produced. The F₁seeds were backcrossed with Binadhan-7 to make BC₁F₁ seeds. Cultural management: For the ease of handling, the experiment was conducted on the plastic pot (bucket 10 L). Recommended cultural operations were followed as and when necessary to ensure the normal plant growth and development. Development of backcross seeds: Crossing scheme: Synchronization of flowering time is the most important operation for MABC program. To produce BC₁F₁ seeds, F₁seeds were seeded in two sets at 14 days interval and recurrent parents were staggered in four sets starting from 15 January, 2012. First set of recurrent parent was seeded 7 days before the seeding F₁ generation. Second and third set were seeded in same time of F₁generation and finally fourth set was seeded in 7 days after the seeding of F₁ generation. Raising of backcross plants: 201 mature backcrossed seeds were collected 21 days after dusting. Seeds were dried into a seed dryer at 65°C for seven days and then set for germination. Among the 201 seeds, 171 sprouted seeds were grown in the pot. The seeds were seeded in two sets in order to ease the work pressure. Among those, 141 seedlings were survived and leaves were collected for foreground selection .Collection of leaf sample and DNA extraction: Samples were collected from young, vigorous leaves from 25 days old seedlings (Binadhan-7, FL-478, BC₁F₁ population) to extract genomic DNA.NA. The collected leaf samples were placed in an ice box and finally the samples were stored in (-) 80º C freezer.Using the Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method, DNA was extracted from the leaves collected from at least 2-3 seedling of each genotype. The simplified mini scale procedure for DNA isolation in PCR analysis developed at IRRI was followed. The quality and quantity of the isolated DNA was sufficient for PCR analysis. Quantified DNA samples from each genotype were subjected to PCR amplification with SSR primers. Polymorphism survey: Polymorphism survey of two parents and BC₁F₁ populations were carried out using 7 foreground markers (RM585, RM336, RM3412b, RM10748, RM8094, AP3206 and AP3206f). Out of 7 markers, 3 markers (AP3206f, RM3412b and RM336) showed clear polymorphisms which were used in genotyping the foreground selection of the 141 BC₁F₁ rice lines for salt tolerant genotypes. PCR array: The PCR cocktail had total volume of 12.75 μL/reaction containing 1.5 μL 10X TB buffer (containing 200mM Tris- HCl pH 8.3, 500mM KCl, 15mM MgCl₂), 0.75 μL of 1mM dNTP, 1.0 μL each of 5μM forward and reverse primers, and 0.25 μL of Taq DNA polymerase with required ddH₂O. 2 μL genomic DNA (5–25 ng of DNA Template) was added PCR cocktail. The steps of PCR reactions were: initial denaturation for 5 minutes at 94°C, then annealing at 55°C for 1 minute. Polymerization for 2 minutes at 72°C to complete a cycle and cycle was repeated for 34 times. The final extension duration was 7 minutes at 72°C. then, PCR products were mixed with 3 μl of 2X gel loading dye. Polymorphisms in the PCR products were analyzed by electrophoresis using mini vertical polyacrylamide gels for high throughput manual genotyping. The gels were stained in ethidium bromide and documented using BIOMETRA Gel Documentation System. Data analysis: Scoring of bands: The pattern of bands obtained after amplification with the primers was scored with reference to two parents. The band having same level of FL-478 was scored as ‘T’ which indicated the homozygous allele of the tolerant parent for a particular microsatellite marker. Similarly, the bands with similar level of Binadhan-7 was scored as ‘S’. The heterozygous alleles having both the bands of two parents were scored as ‘H’. Allele scoring: With the help of Alpha Ease FC 5.0 software, the size (in nucleotide base pairs) of the amplified band for each SSR marker was determined based on its migration relative to 20bp DNA Ladder (a molecular weight size marker). Analysis of SSR Data: The summary statistics including the number of alleles per locus, major allele frequency, gene diversity and Polymorphism Information Content (PIC) values were determined using POWER MARKER version 3.23 (Liu and Muse 2005), a genetic analysis software. Polymorphism Information Content (PIC) value described and modified by Anderson E. (1993) for self-pollinated species. Estimation of gene and genotypic frequencies: The proportions of different alleles of a gene present in a Mendelian population are known as gene frequency. Gene frequencies in a population can be readily estimated by the total number of each of the alleles of the gene present in these individuals and their ratio to the total number for all the alleles of the gene is estimated. The proportions of different genotypes for a gene in a population are known as genotypic frequencies for that gene; often they are also called zygotic frequencies.

Source of Information: J. Bangladesh Agril. Univ. 11(1):. 67–72, 2013, ISSN 1810-3030

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Funding Source:

Budget:

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Achievement/Findings:

After conducting foreground selection, 47 BC₁F₁lines were selected for further recombinant and background selection to minimize ‘linkage drag’ while recovering more genetic background of recurrent parent. Three foreground markers AP3206f, RM3412b and RM336 showed polymorphism among the rice lines, could facilitate selection, mapping, cloning genes, QTL analysis and so on, which in turn increase rice production in saline environments.

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