1k-RiCA (1K-Rice Custom Amplicon) a novel genotyping amplicon-based SNP assay for genetics and breeding applications in rice

Abstract

Background: While a multitude of genotyping platforms have been developed for rice, the majority of them have not been optimized for breeding where cost, turnaround time, throughput and ease of use, relative to density and informativeness are critical parameters of their utility. With that in mind we report the development of the 1K-Rice Custom Amplicon, or 1k-RiCA, a robust custom sequencing-based amplicon panel of ~ 1000-SNPs that are uniformly distributed across the rice genome, designed to be highly informative within indica rice breeding pools, and tailored for genomic prediction in elite indica rice breeding programs.

Results: Empirical validation tests performed on the 1k-RiCA showed average marker call rates of 95% with marker repeatability and concordance rates of 99%. These technical properties were not affected when two common DNA extraction protocols were used. The average distance between SNPs in the 1k-RiCA was 1.5 cM, similar to the theoretical distance which would be expected between 1,000 uniformly distributed markers across the rice genome. The average minor allele frequencies on a panel of indica lines was 0.36 and polymorphic SNPs estimated on pairwise comparisons between indica by indica accessions and indica by japonica accessions were on average 430 and 450 respectively. The specific design parameters of the 1k-RiCA allow for a detailed view of genetic relationships and unambiguous molecular IDs within indica accessions and good cost vs. marker-density balance for genomic prediction applications in elite indica germplasm. Predictive abilities of Genomic Selection models for flowering time, grain yield, and plant height were on average 0.71, 0.36, and 0.65 respectively based on cross-validation analysis. Furthermore the inclusion of important trait markers associated with 11 different genes and QTL adds value to parental selection in crossing schemes and marker-assisted selection in forward breeding applications.

Conclusions: This study validated the marker quality and robustness of the 1k-RiCA genotypic platform for genotyping populations derived from indica rice subpopulation for genetic and breeding purposes including MAS and genomic selection. The 1k-RiCA has proven to be an alternative cost-effective genotyping system for breeding applications.

Keywords: Amplicon-based next generation sequencing; Breeding and genotyping; Genomic selection; Indica; Marker-assisted selection (MAS); Oryza sativa; SNP fingerprinting; Single nucleotide polymorphism (SNP).

Fig. 1

1k-RiCA SNPs physical positions. Genome-wide physical position distribution of 995 SNPs from the 1k-RiCA-assay across all rice chromosomes. SNPs designed from the C6AIR (Thomson et al. 2017) are represented in blue color, SNPs from the ‘3000 rice genomes’ are in yellow (Mansueto et al. , and Wang et al. 2018), and trait-markers are in green

Fig. 2

Principal component analysis of 283 O. sativa accessions using the 1k-RiCA genotypic data. Principal component analysis of 283 rice accessions genotyped with 895 SNP markers using the 1k-RiCA. Subpopulation classification for 185 accessions as indica (ind), aus (aus), tropical japonica (trop), temperate japonica (temp), aromatic (aro), and japonica (jap) was defined based of Wang et al. (2018) and McCouch et al. (2016) classification

Fig. 3

Principal component analysis of 431 O. sativa S. indica accessions using the 1k-RiCA genotypic data. PCA on 431 ‘Indica’ accessions including 177 diverse lines (ind), 41 ‘black rice’ (black rice), and 213 lines derived from 7 bi-parental families from IRRI’s Favorable Environments Breeding Program (Family). Diverse lines were color and coded as open maroon circles ('ind' - indica), as solid black squares ('black rice'). Elite breeding lines were classified by family (‘Family’) based on their pedigree data. Blue dots ('Family-1'), open blue triangles ('Family-2'), open magenta diamonds ('Family-3'), open inverted brown triangles ('Family-4'), green crosses ('Family-5'), ochre dots ('Family-6') and yellow diamonds ('Family-7')

Fig. 4

Polymorphic SNPs distribution across pairwise combinations of a) indica x indica, japonica x japonica b), and c) indica x japonica. Distribution of polymorphic markers between pairs of accessions from a) indica by indica (ind by ind), b) japonica by japonica (jap by jap), and c) indica by japonica (ind by jap) using 218 ‘indica’and 57 ‘japonicas’ accessions. The average number of polymorphic markers for each class combination is determined by a dotted line

Fig. 5

Prediction abilities of genomic selection models for FLW, GY and PH based on 5 fold cross-validation using the 1k-RiCA. Average predictive abilities across 5 fold stratified cross validations experiments (k = 5) using 353 rice lines measured for flowering time (FLW), grain yield (GY), and plant height (PH) for seven different statistical models; Pedigree BLUP (Pedigree), BayesA, BayesB, BayesC, BayesLasso (Bayes L), rrBLUP (ridge regression) and RKHS using genomic and pedigree relationship matrices (RKHS G + A)