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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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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)
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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.
.
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
Steps of SNP discovery
Cluster refinement
Multiple alignment
SNP detection
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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%)
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
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
Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory