IN SILICO DEVELOPMENT OF CONSERVED INTRON SCANNING PRIMERS (CISPs) FOR THE DETECTION OF SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs) IN PIGEONPEA (Cajanus cajan L.)
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Date
2010-07-25
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Pigeonpea (Cajanus cajan. L), a member of fabaceae is a nutritious tropical and
subtropical crop. It is often considered as an ‘orphan crop’ due to limited resources for genome
characterization. With the availability of complete genome sequence information of Medicago
truncatula, Conserved Intron Scanning Primer (CISP) approach was used for targeted discovery
of functional and anonymous Single Nucleotide Polymorphisms (SNPs) in pigeonpea. A total of
307 candidate Cajanus-Medicago CISPs were developed by BLAST aligning 10,000 pigeonpea
ESTs downloaded from NCBI database, with the Medicago genome. Random 192 CISPs
representing loci distributed over eight chromosomes of Medicago were tested on ten members
of legume family. Total amplification of CISP loci was ranged from 70.83% (Urdbean, TAU 1)
to 96.88% (Medicago, EC 547749). Very low and high rate of single copy amplification was
observed in groundnut (TMV 2, 25.52 %) and Medicago (EC 547749, 75.52%). The study
demonstrated the success of cross taxon primers such as CISPs suggesting their potential use in
comparative legume genomics. Among 192 CISPs, selected 118 primers which produced
prominent single copy amplification in BRG 2 were used to genotype other varieties of
pigeonpea. Out of 118 CISPs, 96.61 % of amplification was observed in ICP 7035, BRG 1and
GS 1, where as 98.30 % amplification was observed in BRG3. With respect to single copy
amplification among the selected pigeonpea genotypes, the success rate of 77.97% (ICP 7035) –
83.89% (BRG 3, ICPL 87119) was observed. Sequencing of single copy amplicons from 36
CISPs produced across 8 pigeonpea varieties revealed 42 SNPs and 7 INDELs. We designed 16
SNAP primer pairs corresponding to 8 SNP sites using Web SNAPER
(http://ausubellab.mgh.harvard.edu/). Development of more CISPs are required for molecular
genetic diversity study in pigeonpea.
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