Previously we conducted a pilot study and demonstrated the high frequency of single nucleotide polymorphisms and their possible uses as genetic markers for corn. In an effort to isolate a large number of SNPs from maize, eight genotypes representing more than 90% of allelic diversity within the test population that were considered for pilot study, were further selected. A 'resequencing' strategy of isolation of SNPs was adopted and the sequences in the DuPont/Pioneer EST database served as initial templates for primer design. The primer pairs designed from 3�untranslated regions of the genes of interest were amplified from the genomic DNA extracted from the 8 preselected genotypes. The PCR products (an average length of 350 bases) were sequenced using dideoxy terminator chemistry and their sequences aligned to identify the polymorphic sites. The Phred/Phrap/Consed suite of software was custom modified so that the SNPs and small insertion/deletions (indels) are tagged and a file of polymorphic sites is generated. At this context, it needs to be borne in mind that the term "SNP" is being used very loosely here; polymorphisms involving more than just single nucleotides are identified during the process, this included indels as stated above, and also some novel 'Miniature Inverted Repeat Transposable Elements (MITES)'.

To date we have designed primers from more than 700 genes/ESTs of interest. PCR, sequencing, scoring of SNPs and cataloguing was finished for 530 loci, from which we identified variants in 311 loci. The remaining 219 loci were either monomorphic, primers failed to amplify, or the majority of the genotypes did not sequence well. A total of 1655 polymorphic sites were identified within the 311 loci, spanning 107,606 base regions of the maize genome, and catalogued. Indels accounted for 27 percent of all the total variations observed and the rest were comprised of transitions and transversions (SNPs), transitions being 25 percent more than expected. An average of 5.3 variants was detected per locus, although there were hot spots of mutations in many loci. Our results from this large-scale study confirmed the high rate of SNP polymorphism (1/80 bp) and the high rate of indels (1/240 bp). Out of 311 loci for which we have SNP information, 164 of them could be easily mapped due to the fact that they are also polymorphic between the parents (B73 and MO17) of a recombinant inbred mapping population. 41 percent of 168 loci also result in length difference that could be visualized on a polyacrylamide gel to generate mapping data. An example of haplotypes found in one of the loci is depicted in Table1.

Apart from adding SNPs from additional loci, we are also evaluating high-density high throughput SNP genotyping using different approaches available commercially. SNPs will allow higher throughput; low cost multiplexed genotyping for molecular breeding, genetic diagnostics and research applications.

Table 1: The major haplotypes found in the region spanning intronIV of Globulin1-S locus among the 8 genotypes analyzed for SNP.
 

		Polymorphic base position
Haplotype	Genotype
	Name/No.	57	130	165	232	236	274	374
		Exon IV			Intron IV			Exon V
H1	3	T	T	A	T	T	ND	C
	4	T	T	A	T	T	ND	C
	6	T	T	A	T	T	ND	C
	8	T	T	A	T	T	ND	C
	B73	T	T	A	T	T	ND	C
H2	MO17	C	T	A	C	C	D	C
	7	C	T	A	C	C	D	C
H3	5	C	C	T	T	T	D	T

Notes: 1. Sequence length covered: 403 bp. 2. Only the polymorphic positions are shown. 3. ND = No deletion
 
 

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