Large-scale deployment of a rice 6 K SNP array for genetics and breeding applications

Abstract

Background: Fixed arrays of single nucleotide polymorphism (SNP) markers have advantages over reduced representation sequencing in their ease of data analysis, consistently higher call rates, and rapid turnaround times. A 6 K SNP array represents a cost-benefit "sweet spot" for routine genetics and breeding applications in rice. Selection of informative SNPs across species and subpopulations during chip design is essential to obtain useful polymorphism rates for target germplasm groups. This paper summarizes results from large-scale deployment of an Illumina 6 K SNP array for rice.

Results: Design of the Illumina Infinium 6 K SNP chip for rice, referred to as the Cornell_6K_Array_Infinium_Rice (C6AIR), includes 4429 SNPs from re-sequencing data and 1571 SNP markers from previous BeadXpress 384-SNP sets, selected based on polymorphism rate and allele frequency within and between target germplasm groups. Of the 6000 attempted bead types, 5274 passed Illumina's production quality control. The C6AIR was widely deployed at the International Rice Research Institute (IRRI) for genetic diversity analysis, QTL mapping, and tracking introgressions and was intensively used at Cornell University for QTL analysis and developing libraries of interspecific chromosome segment substitution lines (CSSLs) between O. sativa and diverse accessions of O. rufipogon or O. meridionalis. Collectively, the array was used to genotype over 40,000 rice samples. A set of 4606 SNP markers was used to provide high quality data for O. sativa germplasm, while a slightly expanded set of 4940 SNPs was used for O. sativa X O. rufipogon populations. Biparental polymorphism rates were generally between 1900 and 2500 well-distributed SNP markers for indica x japonica or interspecific populations and between 1300 and 1500 markers for crosses within indica, while polymorphism rates were lower for pairwise crosses within U.S. tropical japonica germplasm. Recently, a second-generation array containing ~7000 SNP markers, referred to as the C7AIR, was designed by removing poor-performing SNPs from the C6AIR and adding markers selected to increase the utility of the array for elite tropical japonica material.

Conclusions: The C6AIR has been successfully used to generate rapid and high-quality genotype data for diverse genetics and breeding applications in rice, and provides the basis for an optimized design in the C7AIR.

Keywords: CSSL development; High-throughput genotyping; O. rufipogon; Oryza sativa; Rice diversity; SNP fingerprinting; Single nucleotide polymorphism (SNP).

Fig. 1

Distribution of SNP distance to its neighboring SNP. More than 50% of SNPs are within 60 kb from a neighboring SNP. The average spacing between SNPs is 79 kb. About 10% of SNPs are >220 kb to the neighboring SNP

Fig. 2

Distribution of the number of polymorphic markers found in pairwise comparisons across diverse germplasm. The number of polymorphic SNPs/pairwise comparison is shown along the x-axis and its count along the y-axis. Panels display the following groups: a All pairwise comparisons (n = 33,413), b Indica vs. Japonica accessions (n = 13,392), c Cultivated vs. wild accessions (n = 5336), d Indica vs. Indica accessions (n = 7628), e Japonica vs. Japonica accessions (n = 5780), and f Wild vs. wild accessions (n = 255). The mean polymorphic SNPs/pairwise comparison are: 1972 (all pairs), 2541 (Indica vs. Japonica), 1987 (cultivated vs. wild), 1347 (within Indica), 1394 (within Japonica), 1413 (within wilds)

Fig. 3

Distribution of the number of polymorphic markers found in pairwise comparisons across the five rice subgroups: indica, aus, aromatic, temperate japonica and tropical japonica. The number of polymorphic SNPs is indicated in the heatmap index

Fig. 4

Distance tree constructed using the Neighbor-Joining method based on 232 Oryza sativa, 23 Oryza rufipogon, 2 O. meriodionalis (AA genome) and 1 Oryza officinalis (outgroup) accessions.A total of 4940 SNP data points from the C6AIR was used for the analysis and the number at the nodes indicate boot- strap value (100 replicates). The colors in the tree correspond to species and subpopulations; Oryza officinalis indicated in light green; O. meriodionalis in dark green; Oryza rufipogon in black, and the five subpopulations of Oryza sativa are in orange (aus), red (indica), purple (aromatic or Group V), cyan (tropical japonica) and blue (temperate japonica)

Fig. 5

Tracking QTL introgressions on chromosomes 6 and 9 of rice using the C6AIR. a QTL for Sub1 was previously mapped to chromosome 9 at 6,388,840–6,658,011 bp (MSUv7), (Xu et al. ; Septiningsih et al. 2009). Two C6AIR markers that localize close to Sub1 (~1.5 cM region) can be used to track the presence or absence of the QTL for development of Sub1 introgression lines (blue box = Sub1, gray boxes = nearby markers). b Table shows genotypes of Xa7 and Sub1 predictive markers in popular rice varieties (control) and their derived introgression lines that carry Xa7 and Sub1. c C6AIR genotype calls around the Sub1 QTL region (~1.8 Mb) Sub1 donors, Sub1-introgression lines and wild-type recurrent parents. Introgression size of varieties carrying a functional SUB1A gene vary, i.e. BR11-Sub1 and Sambha-Mahsuri-Sub1 have the smallest introgressed regions among the SUB1 varieties (blue box = Sub1 genotype, white box = wild type genotype, gray boxes = missing data). d QTL for Xa7 is located on chromosome 6 at MSU7 position 27,963,796–28,082,632 bp (Chen et al. 2008). Five C6AIR markers localize close to Xa7 and one SNP is within the Xa7 region. These markers can be used to track the presence or absence of the QTL for development of Xa7 introgression lines (purple box = Xa7, gray boxes = nearby markers)

Fig. 6

Comparison of the use of 384-SNP Golden Gate assay (OPA 6.1) and C6AIR genotyping platforms for foreground and background selection. CSSLs were developed between the elite tropical japonica variety Cybonnet as the recurrent parent, and O. rufipogon, IRGC105567, as the donor parent. a Distribution of 260 informative SNPs across 12 rice chromosomes detected using the 384-SNP assay (OPA 6.1) (left) and 1868 polymorphic SNPs using the C6AIR (right). The red bar indicates 5 Mb target introgression from the donor parent in the background of Cybonnet. Using the 384-OPA, an average 5 Mb region harbors ~1–3 informative SNPs; using the C6AIR, a 5 Mb region harbors ~5–8 informative SNPs. b Graphical representation of CSSL selection targeting overlapping introgressions on Chromosome 1 in the Cybonnet X IRGC105567 library using the 384-OPA 6.1 platform (38 polymorphic SNPs detected) (pink), and the C6AIR (221 polymorphic SNPs) (purple). Blue line indicates a region of putative incompatibility or sterility identified in this population