This document describes research on identifying single nucleotide polymorphisms (SNPs) in quinoa to develop genetic linkage maps. Researchers used eight quinoa accessions to develop mapping populations. Genomic DNA from parents was digested and sequenced to discover 14,178 SNPs across populations. Primer sets were designed for 1,248 SNPs, of which 511 produced clear clusters. These SNPs were screened in a diversity panel of 113 accessions to identify polymorphic markers. Two populations totaling 128 plants were used to construct an integrated linkage map with 29 linkage groups spanning 1,404 cM and containing 451 SNP markers.

1. Single Nucleotide Polymorphism Identification,Single Nucleotide Polymorphism Identification,
Characterization, and Linkage Mapping inCharacterization, and Linkage Mapping in
QuinoaQuinoa
Saurav saha
2014-11-106
Centre for Plant Biotechnology and Molecular Biology
College of Horticulture, Vellanikkara
Kerala Agriculture University
06/28/15 1

2. 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

3. 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

4. Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
Process of SNP detection
• Genomic reduction
The 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 parent
Double-digest genomic DNA with two different
restriction enzyme ( EcoR1 and Bfa 1)

5. 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.
.

6. 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

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

8. 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

9. Beltsville Area / Soybean Genomics and Improvement LaboratoryBeltsville Area / Soybean Genomics and Improvement Laboratory
Total of 14,178 SNPs 3615 SNPs in 1888
contigs in Pop39 low of 2092 SNPs in 995 contigs in PopM3
Base coverage>6x, (A/G or C/T type SNP are more

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

11. 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

12. 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%)

13. 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

14. 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

15. 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 marker
Total 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 marker
Total 1404 cM map unit spent , AVG distance between two locai 3.3
cM

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