Dissanayaka, D.

Jha, A.

Warkentin, T.

English

University of Saskatchewan Research Archive

Genome wide association study for Fe, Zn, and Se concentration in field pea (Pisumsativum L.)Dilanganie Dissanayaka*, Ambuj Jha , Tom WarkentinSoils and Crops Conference6th March 2018- SaskatoonIntroduction▪ Iron (Fe), Zinc (Zn) and Selenium (Se) are among the twenty essential nutrients which comprise the basis of all human nutrition (WHO).▪ According to FAOSTAT (2012):✓ 60%  Fe deficient✓ 30%  Zn deficient  ✓ 15%  Se deficient ▪ Micronutrient malnutrition?✓Diet supplementation✓ Food fortification✓ Bio-fortification: Bio-fortification is the practice of deliberately increasing the content of an essential nutrient in a crop through agronomic practices, conventional plant breeding, or modern biotechnology.Strategies to overcome the micronutrient malnutrition▪ Pulses including field pea contain significant proportions of the recommended daily allowance (RDA) of micronutrients (Ray et al. 2014).▪ Field pea (Pisum sativum L.) is one of the major pulse crops in the world with annual production of 10 million tonnes (FAOSTAT 2012).▪ Canada is at the top of the field pea producing countries.Biofortification potential of Field peaObjectives1) Evaluation of diverse pea accessions for Fe, Zn and Se concentration.2) Genome wide association study (GWAS) to identify SNPs associated with Fe, Zn and Se concentration.3) Validation of identified trait-associated SNP markersMethodology1) Phenotyping▪ Table 1 : Details of seed samples for evaluation of Fe, Zn, and Se concentration - 177 pea accessions Developed at the Crop Development Centre (CDC), University of Saskatchewan.Year Fargo, North DakotaSaskatoon,SaskatchewanRosthern, SaskatchewanSample number2013 - 2 replicates - 177 x 2 = 3542014 2 replicates - - 177 x 2 = 3542015 - 2 replicates - 177 x 2 = 3542016 - 2 replicates 2 replicates 177 x 4 = 708▪ Grinding the seed samples:- using the cyclone sample mill (UDY Corporation, Fort Collins, Colorado-USA).▪ Sample digestion:- using a Vulcan-84 automatic digester (Thavarajah et al. 2007).▪ Micronutrient concentration analysis:- using an atomic absorption spectrophotometer (Diapari et al. 2015).2) Soil Analysis▪ Correlation analysis of Fe, Zn, and Se concentration in seeds with Fe, Zn, and Se concentration in soil. - 2017 GWAS panel in Saskatoon, SK and Rosthern, SK.- 10 randomly selected accessions- 2 biological replicates- 3 depths (A, B, C horizons)- soil pH, texture, particle size, organic and inorganic C,   plant available nutrients including N, P, and K.3) Association Study▪ Genotyping: Genotyping-by-sequencing* (Elshire et al. 2011).▪ Population structure: (Software : STRUCTURE) ▪ Kinship, GWAS: (Software : TASSEL) ▪ Identifying SNPs associated with Fe, Zn, and Se concentration.*Gali et al. (unpublished)Table 2 : Summary of the analysis of variance (ANOVA) of zinc (Zn) concentration in seeds of field pea across Rosthern, SK Saskatoon, SK and Fargo, ND in 2013, 2014, 2015, and 2016.Y –Year; L – Location; G – Genotype; CV – Coefficient of variation; Pr - ProbabilityPr < 0.001 is represented with ***Trait Year Location Mean ± SD(ppm)CV (%) Range(ppm)Effects           F value                      pry L G G*Y G*LZn 2013 Saskatoon 28.34 ± 3.98 14.0 16.78-39.89 . . 4.1*** . .2014 Fargo, ND 23.22 ± 4.66 20.1 12.83-43.46 . . 3.24*** . .2015 Saskatoon 29.62 ± 3.66 12.3 16.64-44.08 . . 2.53*** . .2016 Saskatoon 32.79 ± 4.4913.719.75-44.33 . . 4.28*** . .2016 Rosthern 33.55 ± 4.92 14.7 21.97-51.47 . . 5.16*** . .2016 Ros + Sas 33.17 ± 4.72 14.2 19.75-51.47 . 10.58*** 6.55*** . 1.032013/2015/2016 Saskatoon 30.26 ± 4.46 14.8 16.64-44.33 209.27*** . 5.72*** 1.28 .Results - PhenotypingZincIronTable 3: Summary of the analysis of variance (ANOVA) of iron (Fe) concentration in seeds of field pea across Rosthern, SK Saskatoon, SK and Fargo, ND in 2013, 2014, 2015, and 2016.Trait Year LocationMean ± SD(ppm) CV%Range(ppm) Effects           F value                      pry L G G*Y G*LFe 2013 Saskatoon 45.95 ± 6.46 14.1 31.41-68.96 . . 4.51*** . .2014 Fargo, ND 52.95 ± 8.97 17.0 36.72-90.53 . . 5.05*** . .2015 Saskatoon 48.80 ± 6.16 12.6 37.4-74.21 . . 2.05*** . .2016 Saskatoon 50.81 ± 6.68 13.2 29.22-74.76 . . 1.97*** . .2016 Rosthern 53.81 ± 6.66 12.4 39.73-74.52 . . 3.31*** . .2016 Ros + Sas 52.31 ± 6.83 13.1 29.22-74.76 . 61.39*** 4.03*** . 0.982013/2015/2016 Saskatoon 48.51 ± 6.74 13.9 29.22-74.76 86.06*** . 4.84*** 1.03 .Y –Year; L – Location; G – Genotype; CV – Coefficient of variation; Pr - ProbabilityPr < 0.001 is represented with ***SeleniumTable 4: Summary of the analysis of variance (ANOVA) of selenium (Se) concentration in seeds of field pea across Rosthern, SK Saskatoon, SK and Fargo, ND in 2013, 2014, 2015, and 2016.Trait Year LocationMean ± SD(ppm) CV%Range(ppm)Effects           F value                      pry L G G*Y G*LSe 2013 Saskatoon 1.09 ± 0.85 78.3 0.25-6.12 . . 0.98 . .2014 Fargo,ND 0.41 ± 0.09 22.6 0.22-0.88 . . 2.04*** . .2015 Saskatoon 1.73 ± 0.76 44.2 0.27-8.75 . . 1.07 . .2016 Saskatoon 0.96 ± 0.37 39.0 0.33-3.28 . . 0.98 . .2016 Rosthern 0.58 ± 0.3 51.8 0.06-1.9 . . 0.8 . .2016 Ros/Sas 0.77 ± 0.39 50.5 0.06-3.28 . 230.5*** 0.99 . 0.822013/2015/2016 Saskatoon 1.26 ± 0.77 61.2 0.25-8.75 126.98*** . 1 0.97 .Y –Year; L – Location; G – Genotype; CV – Coefficient of variation; Pr - ProbabilityPr < 0.001 is represented with ***Results – Soil AnalysisLocation Micronutrient A horizon B horizon C horizon AverageRosthern Se -0.30 0.36 0.57* 0.47*Zn 0.03 0.43 -0.26 0.31Fe 0.25 0.26 0.21 0.25Saskatoon Se -0.07 -0.05 0.76*** 0.20Zn 0.50 0.44 -0.13 0.53*Fe 0.62 0.49 0.40 0.59**Table 8: Pearson’s correlation coefficient (r) between the micronutrient concentration of soil and seeds for Se, Zn, and Fe in A, B, C, and the total depth of A,B, and C horizons in Rosthern and Saskatoon, 2017.Significance: * at 0.05, ** at 0.01, *** at 0.001 Results - Association studyFig 1: Phylogenetic clustering of 175 GWAS accessions based on 14391 SNPs*Fig 2: Heat map image based on Kinship analysis of 175 GWAS accessions**Gali et al. (unpublished)Trait Marker P value -log 10Fe Sc_219_42293 5.57E-07 6.25**Sc_219_42326 5.57E-07 6.25**Sc_6182_63782 2.15E-06 5.67*Zn Sc_8032_15394 1.44E-07 6.84**Sc_1512_36017 1.57E-06 5.80*Sc_1340_418746 3.28E-06 5.48*Table 7: SNP markers associated with Fe and Zn concentration in seeds of field pea across Rosthern, SK Saskatoon, SK and Fargo, ND in 2013, 2014, 2015, and 2016 (after Bonferroni correction was applied)Significance: at 0.05* - >5.46at 0.01** > 6.16at 0001*** >7.16Fig 3: Manhattan plot of Fe in Saskatoon,2013 Fig 4: Manhattan plot of Zn  in Rosthern,2016 ▪ Genotypes differed significantly (P=0.001) for Fe and Zn in all location-years, but not for Se. ▪ Soil and seed Fe and Zn concentration is significantly correlated (P=0.05) in Saskatoon but not in Rosthern. Soil and seed Se concentration is significantly correlated (P=0.05) in Rosthern but not in Saskatoon.▪ After Bonferroni correction, 3 markers were detected for Fe concentration and 3 markers for Zn concentration (P=0.05). Few other less significant markers – for all three micronutrientsConclusions▪ Convert identified SNPs to KASP markers▪ Validation of identified SNP markers using two recombinant inbred line populations segregating for seed micronutrient concentration➢PR-02➢PR-07Future workAcknowledgement▪ Supervisor : Dr. Tom Warkentin▪ Dr. Ambuj Jha▪ Dr. Kishore Gali▪ Committee membersDr. Tim Sharbel (chair)Dr. Rosalind BueckertDr. Bunyamin Tar’anThank you!Questions? Comments?

Genome wide association study for Fe, Zn, and Se concentration in field pea (Pisum sativum L.)

eCommons@USASK

https://harvest.usask.ca/bitstream/10388/8673/1/D.%20Dissanayaka%20et%20al.%2c%202018.pdf

Genome wide association study for Fe, Zn, and Se concentration in field pea (Pisum sativum L.)

Abstract

Similar works

Full text

Available Versions

University of Saskatchewan Research Archive

eCommons@USASK