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, 8 (3), e60176

Identification of SNPs in Closely Related Temperate Japonica Rice Cultivars Using Restriction Enzyme-Phased Sequencing


Identification of SNPs in Closely Related Temperate Japonica Rice Cultivars Using Restriction Enzyme-Phased Sequencing

Sang-Ic Kim et al. PLoS One.


Very low polymorphism in the germplasm typically used by breeding programs poses a significant bottleneck with regards to molecular breeding and the exploitation of breeding materials for quantitative trait analyses. California rice cultivars, derived from a very small base of temperate japonica germplasm and having a relatively brief breeding history, are a good example. In this study, we employed a reduced representation sequencing approach called Restriction Enzyme Site Comparative Analysis (RESCAN) to simultaneously identify and genotype single nucleotide polymorphisms (SNPs) in forty-five rice cultivars representing the majority of the 100 year-old breeding history in California. Over 20,000 putative SNPs were detected relative to the Nipponbare reference genome which enabled the identification and analysis of inheritance of pedigree haplotypes. Haplotype blocks distinguishing modern California cultivars from each other and from the ancestral short grain temperate japonica cultivars were easily identified. Reduced representation sequencing methods such as RESCAN are a valuable alternative to SNP chip genotyping and low coverage whole genome sequencing.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Distribution of RESCAN reads generated from two size selection methods.
In silico NlaIII digestion of total Nipponbare genome (black line) showed that about 13% of total restriction fragments exist in the range of target size (200–300 bp). About 51% of the reads from the gel electrophoresis (LabChip) method were in the target range compared to 27% of the reads from the SPRI (AMPure) method.
Figure 2
Figure 2. Composition of SNP types in California cultivars.
SNPs were grouped into three categories: rare SNPs found in less than 15 cultivars, medium frequency SNPs found in between 16–30 cultivars and common SNPs found in more than 30 cultivars.
Figure 3
Figure 3. High-density haplotype maps of 45 California cultivars generated by RESCAN.
(A) short grain (B) medium grain (C) long grain Regions in blue are monomorphic with regard to the Nipponbare reference genome. Regions in red represent haplotype blocks and white areas represent missing data.
Figure 4
Figure 4. Phylogenic analysis of 45 California cultivars.
(A) Unrooted neighbor-joining tree showing genetic relationships among the cultivars based on C.S. Chord distance calculated using 4,469 SNPs (B) Principal-coordinates analysis (PCA) plot showing that temperate japonica medium grain group is clearly distinguished from tropical japonica group. The X and Y-axes show PC1 and PC2, respectively, of the PCA analysis of the SSR data. (C) Population structure analysis plots (K = 4) generated using the STRUCTURE program. The colors of the numbers represent the respective groups: red, temperate japonica (short grain); black, temperate japonica (medium grain); blue, tropical japonica (long grain) respectively.
Figure 5
Figure 5. Evaluation of pedigrees of California cultivars derived from bi-parental crosses.
Grey areas represent SNPs shared the parents and the descendent cultivars. Red areas represent haplotype blocks shared with the female parent blue areas represent those shared with the male parent. Green areas are haplotypes that are not derived from either parent and may represent scoring errors or differences between the accessions used in this study and those used originally for the breeding of the cultivars. The semidwarf-1(sd-1) and glabrous-1 (gl-1) loci are indicated with an asterisk (*).

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Grant support

This work was supported by USDA-ARS CRIS Project 5306-21000-017-00D ( and California Rice Research Board Project RB-3 ( The California Rice Research Board is a non-profit, California rice grower-funded body which awards grants primarily to public research institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.