Development of genome-wide insertion/deletion markers in rice based on graphic pipeline platform

DNA markers play important roles in plant breeding and genetics.The Insertion/Deletion(InDel) marker is one kind of co-dominant DNA markers widely used due to its low cost and high precision.However,the canonical way of searching for InDel markers is time-consuming and laborintensive.We developed an end-to-end computational solution(InDel Markers Development Platform,IMDP) to identify genome-wide InDel markers under a graphic pipeline environment.IMDP constitutes assembled genome sequences alignment pipeline(AGA-pipe) and next-generation resequencing data mapping pipeline(NGS-pipe).With AGA-pipe we are able to identify 12,944 markers between the genome of rice cultivars Nipponbare and 93-11.Using NGS-pipe,we reported 34,794 InDels from re-sequencing data of rice cultivars Wu-Yun-Geng7 and Guang-Lu-Ai4.Combining AGApipe and NGS-pipe,we developed 205,659 InDels in eight japonica and nine indica cultivars and 2,681 InDels showed a subgroup-specific pattern.Polymerase chain reaction(PCR)analysis of subgroup-specific markers indicated that the precision reached 90%(86 of 95).Finally,to make them available to the public,we have integrated the InDels/markers information into a website(Rice InDel Marker Database,RIMD,http://202.120.45.71/).The application of IMDP in rice will facilitate efficiency for development of genome-wide InDel markers,in addition it can be used in other species with reference genome sequences and NGS data.     

Discovery of genome-wide DNA polymorphisms in a landrace cultivar of Japonica rice by whole-genome sequencing

Molecular breeding approaches are of growing importance to crop improvement. However, closely related cultivars generally used for crossing material lack sufficient known DNA polymorphisms due to their genetic relatedness. Next-generation sequencing allows the identification of a massive number of DNA polymorphisms such as single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) between highly homologous genomes. Using this technology, we performed whole-genome sequencing of a landrace of japonica rice, Omachi, which is used for sake brewing and is an important source for modern cultivars. A total of 229 million reads, each comprising 75 nucleotides of the Omachi genome, was generated with 45-fold coverage and uniquely mapped to 89.7% of the Nipponbare genome, a closely related cultivar. We identified 132,462 SNPs, 16,448 insertions and 19,318 deletions between the Omachi and Nipponbare genomes. An SNP array was designed to validate 731 selected SNPs, resulting in validation rates of 95 and 88% for the Omachi and Nipponbare genomes, respectively. Among the 577 SNPs validated in both genomes, 532 are entirely new SNP markers not previously reported between related rice cultivars. We also validated InDels on a part of chromosome 2 as DNA markers and successfully genotyped five japonica rice cultivars. Our results present the methodology and extensive data on SNPs and InDels available for whole-genome genotyping and marker-assisted breeding. The polymorphism information between Omachi and Nipponbare is available at NGRC_Rice_Omachi (http://www.nodai-genome.org/oryza_sativa_en.html).     

Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.).

We searched the genomes of eight rice cultivars (Oryza sativa L. ssp. japonica and ssp. indica) and a wild rice accession (Oryza rufipogon Griffith) for nucleotide polymorphisms, and identified 7805 polymorphic loci, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), in predicted intergenic regions. Polymorphisms are useful as DNA markers for genetic analysis or positional cloning with segregating populations of crosses. Pairwise comparison between cultivars and a neighbor-joining tree calculated from SNPs agreed very well with relationships between rice strains predicted from pedigree data or calculated with other DNA markers such as p-SINE1 and simple sequence repeats (SSRs), suggesting that whole-genome SNP information can be used for analysis of evolutionary relationships. Using multiple SNPs to identify alleles, we drew a map to illustrate the alleles shared among the eight cultivars and the accession. The map revealed that most of the genome is mono- or di-allelic among japonica cultivars, whereas alleles well conserved among modern japonica paddy rice cultivars were often shared with indica cultivars or wild rice, suggesting that the genome structure of modern cultivars is composed of chromosomal segments from various genetic backgrounds. Use of allele-sharing analysis and association analysis were also tested and are discussed.     

Differentiation of Indica-Japonica rice revealed by insertion/deletion (InDel) fragments obtained from the comparative genomic study of DNA sequences between 93-11 (Indica) and Nipponbare (Japonica)

DNA polymorphisms from nucleotide insertion/deletions (InDels) in genomic sequences are the basis for developing InDel molecular markers.To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species,we studied 49 Indica,43 Japonica,and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs.Results indicated that of the 45 InDel primer pairs,41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%.The scatter plotting data of the principal component analysis (PCA) indicated that:(i) the InDel primer pairs can easily distinguish Indica from Japonica rice varieties,in addition to revealing their genetic differentiation;(ii) the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties;and (iii)the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types.It is concluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying lndica and Japonica rice varieties,and for studying genetic relationships of wild rice species,particularly in terms of the Indica-Japonica differentiation.     

The Organelle Genomes of Hassawi Rice (Oryza sativa L.) and Its Hybrid in Saudi Arabia: Genome Variation, Rearrangement, and Origins

Hassawi rice (Oryza sativa L.) is a landrace adapted to the climate of Saudi Arabia, characterized by its strong resistance to soil salinity and drought. Using high quality sequencing reads extracted from raw data of a whole genome sequencing project, we assembled both chloroplast (cp) and mitochondrial (mt) genomes of the wild-type Hassawi rice (Hassawi-1) and its dwarf hybrid (Hassawi-2). We discovered 16 InDels (insertions and deletions) but no SNP (single nucleotide polymorphism) is present between the two Hassawi cp genomes. We identified 48 InDels and 26 SNPs in the two Hassawi mt genomes and a new type of sequence variation, termed reverse complementary variation (RCV) in the rice cp genomes. There are two and four RCVs identified in Hassawi-1 when compared to 93-11 (indica) and Nipponbare (japonica), respectively. Microsatellite sequence analysis showed there are more SSRs in the genic regions of both cp and mt genomes in the Hassawi rice than in the other rice varieties. There are also large repeats in the Hassawi mt genomes, with the longest length of 96,168 bp and 96,165 bp in Hassawi-1 and Hassawi-2, respectively. We believe that frequent DNA rearrangement in the Hassawi mt and cp genomes indicate ongoing dynamic processes to reach genetic stability under strong environmental pressures. Based on sequence variation analysis and the breeding history, we suggest that both Hassawi-1 and Hassawi-2 originated from the Indonesian variety Peta since genetic diversity between the two Hassawi cultivars is very low albeit an unknown historic origin of the wild-type Hassawi rice.     

Independent domestication of Asian rice followed by gene flow from japonica to indica

Results from studies on the domestication process of Asian rice Oryza sativa have been controversial because of its complicated evolutionary history. Previous studies have yielded two alternative hypotheses about the origin(s) of the two major groups of O. sativa: japonica and indica. One study proposes a single common wild ancestor, whereas the other suggests that there were multiple domestication events of different types of wild rice. Here, we provide clear evidence of the independent domestication of japonica and indica obtained via high-throughput sequencing and a large-scale comparative analysis of two wild rice accessions (W1943 and W0106) and two cultivars (a japonica cultivar called "Nipponbare" and an indica cultivar called "Guangluai-4"). The different domestication processes of the two cultivar groups appear to have led to distinct patterns of molecular evolution in protein-coding regions. The intensity of purifying selection was relaxed only in the japonica group, possibly because of a bottleneck effect. Moreover, a genome-wide comparison between Nipponbare, Guangluai-4, and another indica cultivar (93-11) suggests multiple hybridization events between japonica and indica, both before and after the divergence of the indica cultivars. We found that a large amount of genomic DNA, including domestication-related genes, was transferred from japonica to indica, which might have been important in the development of modern rice. Our study provides an overview of the dynamic process of Asian rice domestication, including independent domestication events and subsequent gene flow.     

Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance

Next‐generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole‐genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought‐tolerant Nagina 22 and salinity‐tolerant Pokkali). More than 356 million 90‐bp paired‐end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1 784 583 nonredundant single‐nucleotide polymorphisms (SNPs) and 154 275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401 683 and 662 509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One‐fourth of the SNPs and InDels were detected in genic regions, and about 3.5% of the total SNPs resulted in nonsynonymous changes. Large‐effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high‐throughput genotyping and molecular breeding for abiotic stress traits in rice.     

Development of PCR-based SNP markers for rice blast resistance genes at the<Emphasis Type="Italic"> Piz</Emphasis> locus

We assessed the utility of single-nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms (InDels) as DNA markers in genetic analysis and breeding of rice. Toward this end, we surveyed SNPs and InDels in the chromosomal region containing the Piz and Piz-t rice blast resistance genes and developed PCR-based markers for typing the SNPs. Analysis of sequences from a blast-susceptible Japanese cultivar and two cultivars each containing one of these genes revealed that SNPs are abundant in the Piz and Piz-t regions (on average, one SNP every 248 bp), but the number of InDels was much lower. The dense distribution of SNPs facilitated the generation of SNP markers in the vicinity of the genes. For typing these SNPs, we used a modified allele-specific PCR method. Of the 49 candidate allele-specific markers, 33 unambiguously and reproducibly discriminated between the two alleles. We used the markers for mapping the Piz and Piz-t genes and evaluating the size of DNA segments introgressed from the Piz donor cultivar in Japanese near-isogenic lines containing Piz. Our findings suggest that, because of its ability to generate numerous markers within a target region and its simplicity in assaying genotypes, SNP genotyping with allele-specific PCR is a valuable tool for gene mapping, map-based cloning, and marker-assisted selection in crops, especially rice.Communicated by D.J. Mackill     

The rice mitochondrial genomes and their variations

Based on highly redundant and high-quality sequences, we assembled rice (Oryza sativa) mitochondrial genomes for two cultivars, 93-11 (an indica variety) and PA64S (an indica-like variety with maternal origin of japonica), which are paternal and maternal strains of an elite superhybrid rice Liang-You-Pei-Jiu (LYP-9), respectively. Following up with a previous analysis on rice chloroplast genomes, we divided mitochondrial sequence variations into two basic categories, intravarietal and intersubspecific. Intravarietal polymorphisms are variations within mitochondrial genomes of an individual variety. Intersubspecific polymorphisms are variations between subspecies among their major genotypes. In this study, we identified 96 single nucleotide polymorphisms (SNPs), 25 indels, and three segmental sequence variations as intersubspecific polymorphisms. A signature sequence fragment unique to indica varieties was confirmed experimentally and found in two wild rice samples, but absent in japonica varieties. The intersubspecific polymorphism rate for mitochondrial genomes is 0.02% for SNPs and 0.006% for indels, nearly 2.5 and 3 times lower than that of their chloroplast counterparts and 21 and 38 times lower than corresponding rates of the rice nuclear genome, respectively. The intravarietal polymorphism rates among analyzed mitochondrial genomes, such as 93-11 and PA64S, are 1.26% and 1.38% for SNPs and 1.13% and 1.09% for indels, respectively. Based on the total number of SNPs between the two mitochondrial genomes, we estimate that the divergence of indica and japonica mitochondrial genomes occurred approximately 45,000 to 250,000 years ago.