Development of SSR markers

Construction of Library

Genomic DNA from different arabica varieties were used to construct partial genomic library. About 10mg DNA was cut by 24U Sau3AI (NEB) overnight at 370C.Following electrophoresis in 1% agarose gel (1x TAE), 500 to 1500bp fragments were selected and DNA was extracted by Gene Clean (Amersham Pharmacia).

DNA fragments were ligated to BamHI (NEB) cut dephosphorylated (Shrimp Alkaline Phosphates, USB) pBluescriptKS(+) vector and inserted into Escherichia coli DHa10B by electroporation. The transformants were grown overnight at 370C on LB solid medium containing 100ug/ml ampicillin, 50ug/ml X-gal, 0.5 mM IPTG. The following day, white colonies were transferred to 96 well microtiter plates (Nalge Nunc International, USA) in the presence of ampicillin in liquid medium, and bacteria were grown overnight at 370C.

Generation of filters containing arrays of plasmid clones

A replica of original plate was transferred to Hybond N+(Amersham) membranes with replicator (Nalge Nunc International, USA). The bacterial colonies were grown on the membrane on LB agar with ampicilline at 370C overnight. The membranes were transferred to Whatman 3MM filter paper saturated with 10% SDS side up for 4 minutes. The membrane are than transferred to filter paper saturated with a denaturing solution (0.5 N NaOH, 1.5M NaCl,H2O) for 10 minutes, submerge in excess of 1x neutralizing solution (0,5M Tris-Cl pH 7.4,1.5M Nacl,H2O) for 5 min and washed in 2x SSC for another 5 min. The filters were air-dried and UV- cross-linked the immobilized DNA to the filters.

Isolation of clones containing repeat motif

The membranes containing approximately 10,000 arrays plasmid clones were screened for repeat motifs with the synthetic Oligonucleotide probe of (CA)15, (GA)15, (CAA)10, (GGT)10, (ATT)10, (AGG)10, (AGA)10, (ACT)10 and (CATA)8. These probes were used in different combination according to their Tm value. For labeling the probe, 10 pmol Oligonucleotide was added to 50Uci g32P dATP and 20U polynucleotide kinase (New England Biolabes) at 370C for 30 min. Prehybridization for 1-2h was done at a 550C in hybridization buffer (6xSSC; 5xDenhardts solution 50x: 5g of ficoll, 5g of BSA, 5g of PVP to 500ml by H2O and 0.5% SDS). Hybridization was conducted for 4 to 6 hr. at 550C. After hybridization membranes were washed with 6xSSC for 20 to 30 min in 55 to 600C till the radioactive count come down subsequently. The membrane were placed on Whatmant 3MM paper for a few seconds to remove excess solution and then immediately wrapped in plastic wrap and exposed to Phosphoimager screen for 4 hr. All the positive clones were identified from the screening and placed in another microtiter plate.

PCR amplification and sequencing of the clones

Positive clones were grown in 3mL LB containing ampicillin overnight. The plasmid was extracted by Alkaline-Lysis method (Sambrook et al, 1989). Quality of the plasmid clones were checked through PCR amplification with M13 universal primers and running it in 1.5%agarose gel. The plasmid clones, which gave clear single bands, were sequenced with an automatic ABI PRISM 377 DNA sequencer using the Big Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, Calif.), for both forward and reverse strands.

Primer design and evaluation

The repeat regions have been identified by repeat finding program (http://ars-genome.cornell.edu/cgi-bin/rice/ssrtool.pl), tandem repeat finder Version 2.02(Dr. Gary Benson, 1999) and manual screening. PCR primers were design flanking microsatellite repeat sequences containing more than 16bp using the PRIMER 3 program (S.Lincoln, M.Daly, and E.Lander, Cambridge, Mass.) combined with manual design. The aim of the primer selection procedure was to produce well-matched primer, 17-22 nucleotides long, devoid of consecutive tracts of a single nucleotide, with a GC content around 50%(Tm approximately 600C), and preferably G-or C-rich at the 3’ end. Primers that met these requirements were preferentially selected to produce a PCR product in the range of 150 to 250bp. Primer were synthesized by automated oligosynthasizer (Applied Biosystems 394 DNA/RNA synthesizer) with the forward primer of each pair being chemically 5’ end labeled with the fluorescent dyes 6-FAM or HEX from Applied Biosystems (ABI).