Plant materials
Forty BC1F1 progenies were selected for this study from the backcross made between rice of F1 (Binadhan-5 x FL-478) and Binadhan-5. Binadhan-5 is a salt susceptible but one of the popular HYV of irrigated rice ecosystem in Bangladesh. On the other hand, FL-478 is one of the salt tolerant lines of IRRI. Binadhan-5 was the recurrent parent and FL-478 was the nonrecurrent donor line. F1 seeds were collected and grown for developing BC1F1 seedlings during Aman season 2010. F1 plants were crossed with Binadhan-5 following of backcrossing and 133 BC1F1 seeds were developed. Among these, 40 seedlings were selected to collect the leaf to make foreground selection genotypically at the molecular level (by using SSR markers).
Development of F1 population (Binadhan- 5 x FL 478) of rice
one rice genotype (FL-478) was selected as parent for transferring salt tolerant genes to high yielding rice genotype (Binadhan-5).
Production of BC1F1 plants:
Three sets of recurrent parental line (Binadhan- 5) and one set of F1 (Binadhan-5/FL-478) were seeded at an interval of 10 days starting from 11 June 2010.
Collection of leaf sample (Binadhan-5, FL-478, BC1F1 Lines) for DNA extraction
The seedlings of two Parents (Binadhan-5, FL-478), F1 and BC1F1 population rice germplasm were raised in pots at the glasshouse of BINA at the different growing season. Young vigorously growing fresh leaf samples from these seedlings were collected from 21-day-old seedlings to extract genomic DNA.
Genomic DNA extraction
Extraction of DNA was done using the mini preparation Cetyl Trimethyl Ammonium Bromide (CTAB) method from 3" young leaves tissues of 21-day old seedlings. Ten random primers viz. RM21, RM80, RM152, RM310, RM336, RM510, RM585, RM10720, RM28102, and RM28502 were used for surveying and among them; three primers (RM336, RM510 and RM585) showed polymorphisms during parental survey.
Amplification of SSR markers
PCR amplification of simple sequence repeats (SSR) was performed with 1.5 μl 10X PCR buffer, 0.75 μl dNTPs, 1.0 μl forward, 1.0 μl reverse primers, 0.5 μl Taq polymerase, 2.0 μl of each template DNA and 8.25 μl ddH2O using DNA thermal cycler.