saurav snp

Single Nucleotide Polymorphism Identification, Single Nucleotide Polymorphism Identification, Characterization, and Linkage Mapping in Characterization, and Linkage Mapping in QuinoaQuinoa

Saurav saha2014-11-106

Centre for Plant Biotechnology and Molecular BiologyCollege of Horticulture, Vellanikkara

Kerala Agriculture University

04/15/23 1

Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory

Single Nucleotide Polymorphism

Single nucleotide polymorphisms consist of a single change in the DNA code

SNPs occur with various allele frequencies. Those in

the 20-40% range are useful for genetic mapping

Those at frequencies between 1% and 20% may be

used with candidate gene approaches. Usually bi-allelic


Parents and population

• Eight quinoa accessions were used in this experiment

• Accessions represent the broad geographical distribution of quinoa

• Using this accessions as a parent four population are developed which is used for SNP detection and developed linkage map

• The population are following

Pop1 Pop39 Pop40 PopM3 PopGO

Population 1 and Pop39 are used for linkage mapping


Process of SNP detection • Genomic reductionThe principle this method restriction site conservation across

individuals, Removal of >90% of the genome to reduce the complexybity of

genome

Steps to be follow-Isolate the DNA from each individual four parentDouble-digest genomic DNA with two different restriction

enzyme ( EcoR1 and Bfa 1)


Cont...Double stranded adapters are ligated to the ends of

the digested DNA fragments.

Adaptor ligated to the end of the 6-base recognition site is end-labeled with a 5′-biotin molecule,

Adaptor on the 4-base recognition site is unlabeled.

.


Cont………• Those DNA segment are unleveled are removed using a

biotin-strepavidin paramagnetic bead separation.

• Specific MID barcode is ligated in the end of the DNA segment

• Equimolar amounts of each individual PCR sampler run in a gel

400-600 bp size DNA segment are selected for pyrosequencing sequencing


Assembly and SNP Detection• DNA reads were bio informatically separated into MID

barcode

• Contigs for each of the four mapping populations, generated by assembling the DNA read of the specific parent of a population


Total of 14,178 SNPs 3615 SNPs in 1888contigs in Pop39 low of 2092 SNPs in 995 contigs in PopM3

Base coverage>6x, (A/G or C/T type SNP are more


Steps of SNP discovery

Cluster refinement

Multiple alignment

SNP detection


SNP Assay Development

• Primer sets for 1248 putative SNPs were designed KASPar genotyping chemistry

• SNP was selected from the two population those are polymorphic

• Total of 511 (41%) SNPs produced clearly separated genotypic clusters


Diversity Panel Analysis• Diversity panel consisted of 113 accessions of C. quinoa

• Eight related Chenopodium taxa

• Out of the 511 SNP, 427 SNP was screened across the population

• 854 alleles were identified

• MAF ranged from 0.02 to 0.50

• SNP with a MAF ≥ 0.35 as highly polymorphic(46%)

• SNP with MAF ≥ 0.10 as polymorphic(90%)


Linkage Map Construction• Pop1 and Pop39, were used for linkage map construction

• Both populations were small (n = 61 and n = 67) and share a common parent (0654)

• Data of both population combine and construct a integrated linkage map (n = 128).

• 511 SNP locai are screened across the entire integrated mapping population using kasp genotiping chemistry


Conti…..

• Out of the 511 SNP 469(92%) genotype clusters that could that could easily scored

• LOD score 4.5, out of 469 SNP marker 451SNP(96)%

are grouped into 29 linkage grouped

• 20 linkage group linkage group >9 snp and 9 linkage group >5-6 SNP

• Total map consist of 1404 cM spanned by 451 SNP loci

• Each linkage group spanning 112 cM


Total 29 linkage group of a 451 SNP marker 20 are >9 SNP and 9 are 5-4 markerTotal 1404 cM map unit spent , AVG distance between two locai 3.3 cM

Total 29 linkage group of a 451 SNP marker 20 are >9 SNP and 9 are 5-4 markerTotal 1404 cM map unit spent , AVG distance between two locai 3.3 cM

saurav snp

Science

improvement laboratory

improvement laboratory

improvement laboratory

genomic dna

dna code snps

snp detection dna reads

digested dna fragments

dna segment equimolar