Development of genome-wide DNA polymorphism database for map-based cloning of rice genes

DNA polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. A genome-wide rice (Oryza sativa) DNA polymorphism database has been constructed in this work using the genomes of Nipponbare, a cultivar of japonica, and 93-11, a cultivar of indica. This database contains 1,703,176 single nucleotide polymorphisms (SNPs) and 479,406 Insertion/Deletions (InDels), approximately one SNP every 268 bp and one InDel every 953 bp in rice genome. Both SNPs and InDels in the database were experimentally validated. Of 109 randomly selected SNPs, 107 SNPs (98.2%) are accurate. PCR analysis indicated that 90% (97 of 108) of InDels in the database could be used as molecular markers, and 68% to 89% of the 97 InDel markers have polymorphisms between other indica cultivars (Guang-lu-ai 4 and Long-te-pu B) and japonica cultivars (Zhong-hua 11 and 9522). This suggests that this database can be used not only for Nipponbare and 93-11, but also for other japonica and indica cultivars. While validating InDel polymorphisms in the database, a set of InDel markers with each chromosome 3 to 5 marker was developed. These markers are inexpensive and easy to use, and can be used for any combination of japonica and indica cultivars used in this work. This rice DNA polymorphism database will be a valuable resource and important tool for map-based cloning of rice gene, as well as in other various research on rice (http://shenghuan.shnu.edu.cn/ricemarker).     

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.     

Genome-wide single nucleotide polymorphisms and insertion–deletions of Oryza sativa L. subsp. japonica cultivars grown near the northern limit of rice cultivation

Single nucleotide polymorphisms (SNPs) and insertions–deletions (InDels) are valuable molecular markers for molecular breeding among genetically closely related cultivars. Rice (Oryza sativa L. subsp. japonica) cultivars grown in Hokkaido (45–42°N), the northernmost region of rice paddy cultivation in Japan, have been bred for over 100 years for adaptation to low summer temperatures together with high yield and good eating quality. In this study, for 10 closely related rice cultivars released in Hokkaido and cultivar Koshihikari, we identified genome-wide SNPs and InDels by next-generation sequencing. More than 29 million reads from the Hokkaido cultivars, each 101 nucleotides long, were uniquely mapped to the Nipponbare reference genome. The average of the total nucleotide length of all uniquely mapped reads corresponded to 10.9 times (3,978 Mb with genome coverage of 90.7 %) the Nipponbare reference genome. An average of 99,955 putative SNPs (1.8 times the number in Koshihikari) and 14,617 putative InDels (also 1.8 times the number in Koshihikari) were detected in Hokkaido cultivars relative to the Nipponbare genome, which enabled analyses of the inheritance of pedigree haplotypes of four cultivars, SNPs and InDels among closely related Hokkaido cultivars, and haplotype blocks unique to Hokkaido cultivars. The comprehensive SNP and InDel data provide DNA marker resources and will facilitate quantitative trait locus analysis of biparental mapping of very closely related Hokkaido cultivars. Furthermore, the haplotype blocks unique to Hokkaido cultivars represent ideal genetic regions for improvement of cultivars to be grown near the northern and southern limits of rice cultivation.     

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 Indica and Japonica rice varieties, and for studying genetic relationships of wild rice species, particularly in terms of the Indica-Japonica differentiation. Translated from Journal of Fudan University (Natural Science), 2006, 45(3): 309–315 [译自: 复旦学报(自然科学版)]     

Development of Species-Specific InDel Markers in Citrus

Citrus taxonomy is complex owing to the existence of a wide range of species: Poncirus is used mainly for rootstock; Fortunella produces small fruit and edible pericarp; and Citrus comprises the most widespread fruit crop species worldwide. Rapidly increasing genome resources from different citrus species facilitate the development of convenient and genome-wide molecular markers that can be applied to both inter- and intra-species analyses. In this study, by comparing the genome sequences of four citrus species, a set of 1958 InDels were identified and 453 candidate InDels were converted into PCR-based markers. Among these candidate InDels, 268 (65%) exhibited length polymorphisms from 30 bp to 200 bp when applied to seven species from the genera Poncirus, Fortunella and Citrus. Seven InDel markers exhibited high intraspecific polymorphisms in a natural pummelo population. The results showed that the InDel markers are effective for both inter- and intra-specific variation and identification analyses. These InDel markers are expected to be applied to germplasm identification, phylogenetic analysis, genetic diversity evaluation and marker-assisted breeding in citrus.     

Genome-wide discovery of DNA polymorphism in Brassica rapa

Single nucleotide polymorphisms (SNPs) and/or insertion/deletions (InDels) are frequent sequence variations in the plant genome, which can be developed as molecular markers for genetic studies on crop improvement. The ongoing Brassica rapa genome sequencing project has generated vast amounts of sequence data useful in genetic research. Here, we report a genome-wide survey of DNA polymorphisms in the B. rapa genome based on the 557 bacterial artificial clone sequences of B. rapa ssp. pekinensis cv. Chiifu. We identified and characterized 21,311 SNPs and 6,753 InDels in the gene space of the B. rapa genome by re-sequencing 1,398 sequence-tagged sites (STSs) in eight genotypes. Comparison of our findings with a B. rapa genetic linkage map confirmed that STS loci were distributed randomly over the B. rapa whole genome. In the 1.4 Mb of aligned sequences, mean nucleotide polymorphism and diversity were θ = 0.00890 and π = 0.00917, respectively. Additionally, the nucleotide diversity in introns was almost three times greater than that in exons, and the frequency of observed InDel was almost 17 times higher in introns than in exons. Information regarding SNPs/InDels obtained here will provide an important resource for genetic studies and breeding programs of B. rapa.     

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).     

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.     

First-generation SNP/InDel markers tagging loci for pathogen resistance in the potato genome

A panel of 17 tetraploid and 11 diploid potato genotypes was screened by comparative sequence analysis of polymerase chain reaction (PCR) products for single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), in regions of the potato genome where genes for qualitative and/or quantitative resistance to different pathogens have been localized. Most SNP and InDel markers were derived from bacterial artificial chromosome (BAC) insertions that contain sequences similar to the family of plant genes for pathogen resistance having nucleotide-binding-site and leucine-rich-repeat domains (NBS-LRR-type genes). Forty-four such NBS-LRR-type genes containing BAC-insertions were mapped to 14 loci, which tag most known resistance quantitative trait loci (QTL) in potato. Resistance QTL not linked to known resistance-gene-like (RGL) sequences were tagged with other markers. In total, 78 genomic DNA fragments with an overall length of 31 kb were comparatively sequenced in the panel of 28 genotypes. 1498 SNPs and 127 InDels were identified, which corresponded, on average, to one SNP every 21 base pairs and one InDel every 243 base pairs. The nucleotide diversity of the tetraploid genotypes (pi = 0.72 x 10(-3)) was lower when compared with diploid genotypes (pi = 2.31 x 10(-3)). RGL sequences showed higher nucleotide diversity when compared with other sequences, suggesting evolution by divergent selection. Information on sequences, sequence similarities, SNPs and InDels is provided in a database that can be queried via the Internet.     

Genome-Wide Discovery of DNA Polymorphisms in Mei (<Emphasis Type="Italic">Prunus mume</Emphasis> Sieb. et Zucc.), an Ornamental Woody Plant,with Contrasting Tree Architecture and their Functional Relevance for Weeping Trait

Next-generation sequencing technologies provide opportunities to ascertain the genetic basis of phenotypic differences, even in the closely related cultivars via detection of large amount of DNA polymorphisms. In this study, we performed whole-genome re-sequencing of two mei cultivars with contrasting tree architecture. 75.87 million 100 bp pair-end reads were generated, with 92 % coverage of the genome. Re-sequencing data of two former upright mei cultivars were applied for detecting DNA polymorphisms, since we were more interested in variations conferring weeping trait. Applying stringent parameters, 157,317 mutual single nucleotide polymorphisms (SNPs) and 15,064 mutual insertions-deletions (InDels) were detected and found unevenly distributed within and among the mei chromosomes, which lead to the discovery of 220 high-density, 463 low-density SNP regions together with 80 high-density InDel regions. Additionally, 322 large-effect SNPs and 433 large-effect InDels were detected, and 10.09 % of the SNPs were observed in coding regions. 5.25 % SNPs in coding regions resulted in non-synonymous changes. Ninety SNPs were chosen randomly for validation using high-resolution melt analysis. 93.3 % of the candidate SNPs contained the predicted SNPs. Pfam analysis was further conducted to better understand SNP effects on gene functions. DNA polymorphisms of two known QTL loci conferring weeping trait and their functional effect were also analyzed thoroughly. This study highlights promising functional markers for molecular breeding and a whole-genome genetic basis of weeping trait in mei.     

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     

Development of InDel markers for Brassica rapa based on whole-genome re-sequencing

Genome-wide detection of short insertion/deletion length polymorphisms (InDels, <5 bp) in Brassica rapa (named the A genome) was performed by comparing whole-genome re-sequencing data from two B. rapa accessions, L144 and Z16, to the reference genome sequence of Chiifu-401-42. In total, we identified 108,558 InDel polymorphisms between Chiifu-401-42 and L144, 26,795 InDels between Z16 and Chiifu-401-42, and 26,693 InDels between L144 and Z16. From these, 639 InDel polymorphisms of 3–5 bp in length between L144 and Z16 were selected for experimental validation; 491 (77 %) yielded single PCR fragments and showed polymorphisms, 7 (1 %) did not amplify a product, and 141 (22 %) showed no polymorphism. For further validation of these intra-specific InDel polymorphisms, 503 candidates, randomly selected from the 639 InDels, were screened across seven accessions representing different B. rapa cultivar groups. Of these assayed markers, 387 (77 %) were polymorphic, 111 (22 %) were not polymorphic and 5 (1 %) did not amplify a PCR product. Furthermore, we randomly selected 518 InDel markers to validate their polymorphism in B. napus (the AC genome) and B. juncea (the AB genome), of which more than 90 % amplified a PCR product; 132 (25 %) showed polymorphism between the two B. napus accessions and 41 (8 %) between the two B. juncea accessions. This set of novel PCR-based InDel markers will be a valuable resource for genetic studies and breeding programs in B. rapa.     

An Improved Brassica rapa Genetic Linkage Map and Locus-specific Variations in a Doubled Haploid Population

In this study, we describe the construction of an improved Chinese cabbage genetic linkage map by integrating simple sequence repeats (SSRs) and insertion/deletion polymorphisms (InDels) into a previously published map of a doubled haploid (DH) population. The population was derived from a cross between the Chinese cabbage line BY (Brassica rapa ssp. pekinensis) and a European turnip line MM (Brassica rapa L. ssp. rapifera). A total of 629 markers were aligned to ten linkage groups, with a total map length of 1,173.8 cM, and an average distance between markers of 1.87 cm. Of the 126 SSRs and 133 InDels mapped, 46 and 34 were novel, respectively. A comparison of the linkage map with the B. rapa genome showed that more than 93 % of the markers, including 112 SSRs and 129 InDels, could be anchored unambiguously to a specific location on one of the ten chromosomes. In most cases, the order of markers on the linkage map and physical map was similar; however, the majority of linkage groups contained a number of markers whose positions were either transposed or had moved slightly forwards or backwards. During microspore culture, it was observed that 11 SSRs and one InDel showed either variation in size, or the appearance of new marker bands in the DH lines. As a first step to addressing this SSR/InDel marker instability, six SSR and one InDel loci were sequenced, which revealed that the size variation was due mainly to changes in repeat-motif number or to the insertion/deletion of new fragments of DNA.     

Selection of 29 highly informative InDel markers for human identification and paternity analysis in Chinese Han population by the SNPlex genotyping system

The interest of forensic researchers in single nucleotide polymorphism (SNP) has been attracted because of its potential advantages, such as low mutation rates, amenable to high-throughput automated platform and the improved application in the analysis of degraded samples. In this paper, 29 highly informative insertion/deletion (InDel, a special kind of SNP) markers were selected from the dbSNP () according to the given criteria. 109 unrelated Chinese Han subjects were genotyped for the 29 InDels with SNPlex genotyping system. The allele frequency data revealed that the combined power of discrimination for the 29 InDel markers was 0.999999999990867 and the combined probability of paternity exclusion (PE) was 0.9930. Sensitivity studies were performed to evaluate the flexibility of the SNPlex genotyping system on the set of 29 InDels. Highly reproducible results could be obtained with 40–100 ng genomic DNA and the proportion of total allele drop-in was significantly increased when the amount of DNA added to PCR was lower than 35 ng. These results suggested that the set of 29 InDels was useful in paternity analysis or human identification in the future.     

Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes

Blast resistance is one of the most important traits in rice breeding, and application of molecular markers for blast resistance breeding is likely to allow the rapid screening for the trait during early growth stages, without the need for inoculation of pathogen and phenotyping. Allele-specific PCR markers and insertion/deletion (InDel) markers, which genotype single-nucleotide polymorphisms and InDel polymorphisms, respectively, are useful tools for marker-assisted selections. We developed sets of allele-specific PCR and InDel markers for nine rice blast resistance genes—Piz, Piz-t, Pit, Pik, Pik-m, Pik-p, Pita, Pita-2, and Pib—which are commonly used in Japanese blast resistance rice breeding programs. For each resistance gene, we used the segregation information from thousands of progeny in several crosses or published gene locations to generate a marker that cosegregated with the gene and markers that closely flanked the gene on either side. The developed cosegregating markers uniquely discriminated among each of the lines with the individual resistance genes (except for Pita and Pita-2). Therefore, these markers will likely facilitate the development of multiline cultivars carrying one or a combination of these nine blast resistance genes. In addition, the systems we developed may be valuable tools in the quality control of seed production from blast-resistant multiline cultivars.     

Development of gene-based markers and construction of an integrated linkage map in eggplant by using Solanum orthologous (SOL) gene sets

We constructed an integrated DNA marker linkage map of eggplant (Solanum melongena L.) using DNA marker segregation data sets obtained from two independent intraspecific F(2) populations. The linkage map consisted of 12 linkage groups and encompassed 1,285.5 cM in total. We mapped 952 DNA markers, including 313 genomic SSR markers developed by random sequencing of simple sequence repeat (SSR)-enriched genomic libraries, and 623 single-nucleotide polymorphisms (SNP) and insertion/deletion polymorphisms (InDels) found in eggplant-expressed sequence tags (ESTs) and related genomic sequences [introns and untranslated regions (UTRs)]. Because of their co-dominant inheritance and their highly polymorphic and multi-allelic nature, the SSR markers may be more versatile than the SNP and InDel markers for map-based genetic analysis of any traits of interest using segregating populations derived from any intraspecific crosses of practical breeding materials. However, we found that the distribution of microsatellites in the genome was biased to some extent, and therefore a considerable part of the eggplant genome was first detected when gene-derived SNP and InDel markers were mapped. Of the 623 SNP and InDel markers mapped onto the eggplant integrated map, 469 were derived from eggplant unigenes contained within Solanum orthologous (SOL) gene sets (i.e., sets of orthologous unigenes from eggplant, tomato, and potato). Out of the 469 markers, 326 could also be mapped onto the tomato map. These common markers will be informative landmarks for the transfer of tomato's more saturated genomic information to eggplant and will also provide comparative information on the genome organization of the two solanaceous species. The data are available from the DNA marker database of vegetables, VegMarks (http://vegmarks.nivot.affrc.go.jp).     

Genome-wide DNA polymorphisms in elite indica rice inbreds discovered by whole-genome sequencing

Advances in next-generation sequencing technologies have aided discovery of millions of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels), which are an invaluable resource for marker-assisted breeding. Whole-genome resequencing of six elite indica rice inbreds (three cytoplasmic male sterile and three restorer lines) resulted in the generation of 338?million 75-bp paired-end reads, which provided 85.4% coverage of the Nipponbare genome. A total of 2?819?086 nonredundant DNA polymorphisms including 2?495?052 SNPs, 160?478 insertions and 163?556 deletions were discovered between the inbreds and Nipponbare, providing an average of 6.8 SNPs/kb across the genome. Distribution of SNPs and InDels in the chromosome was nonrandom with SNP-rich and SNP-poor regions being evident across the genome. A contiguous 4.3-Mb region on chromosome 5 with extremely low SNP density was identified. Overall, 83?262 nonsynonymous SNPs spanning 16?379 genes and 3620 nonsynonymous InDels in 2625 genes have been discovered which provide valuable insights into the basis underlying performance of the inbreds and the hybrids between these inbred combinations. SNPs and InDels discovered from this diverse set of indica rice inbreds not only enrich SNP resources for molecular breeding but also enable the study of genome-wide variations on hybrid performance.     

Molecular characterization of some Indian <Emphasis Type="Italic">Basmati</Emphasis> and other elite rice genotypes using fluorescent-AFLP

Cultivated rice is a high-volume, low-value cereal crop providing staple food to more than 50% of the world populace. A small group of rice cultivars, traditionally produced on the Indo-Gangetic plains and popularly known as Basmati, have exquisite quality grain characteristics and are a prized commercial commodity. Efforts to improve the yield potential of Basmati have led to the development of several crossbred Basmati-like cultivars. In this study we have analysed the genetic diversity and interrelationships among 33 rice genotypes consisting of the traditional Basmati, improved Basmati-like genotypes developed in India and elsewhere, American long-grain rice and a few non-aromatic rice using a DNA marker-based approach - fluorescent-amplified fragment length polymorphism (f-AFLP). Using a set of nine primer-pairs we scored a total of 10,672 data points over all of the genotypes in the size range of 75-500 bp. The scored data points corresponded to a total of 501 AFLP markers (putative loci/genome landmarks) of which 327 markers (65%) were polymorphic. The f-AFLP marker data, which were analysed using different clustering algorithms and principal component analysis, indicate that: (1) considerable genetic variability exists in the analysed genotypes; (2) traditional Basmati cultivars could be distinctly separated from the crossbred Basmati-like genotypes as well as from the non-aromatic rice; (3) the crossbred Basmati-like cultivars from the subcontinent and elsewhere are genetically very distinct; (4) f-AFLP-based clustering, in general, conforms to the putative pedigree of the improved genotypes. Moreover, analysis to ascertain the scope of AFLP as a technique suggests that the polymorphism revealed by three selective primer-pair combinations is sufficient to obtain reliable estimates of genetic diversity for the type of material used in this study. However, its utility to identify group-specific DNA markers was discounted due to a low frequency of observed group-specific discrete markers.     

Genome-wide Investigation of Intron Length Polymorphisms and Their Potential as Molecular Markers in Rice (Oryza sativa L.)

Intron length polymorphisms (ILPs) have been used as geneticmarkers in some studies. However, a systematic investigationand large-scale exploitation of ILP markers has not been reported.In this study, we performed a genome-wide search of ILPs betweentwo subspecies (indica and japonica) in rice using the draftgenomic sequences of cultivars 93-11 (indica) and Nipponbare(japonica) and 32 127 full-length cDNA sequences of Nipponbareobtained from public databases. We identified 13 308 putativeILPs. Based on these putative ILPs, we developed 5811 candidateILP markers via electronic-PCR with primers designed in flankingexons. We further conducted experiment to verify the candidateILP markers. Out of 215 candidate ILP markers tested on 93-11,Nipponbare and their hybrid, we successfully exploited 173 codominantILP markers. Further analyses on 10 rice accessions showed thatthese ILP markers were widely applicable and most (71.1%) exhibitedsubspecies specificity. This feature suggests that ILPs wouldbe useful for the studies of genome evolution and inter-subspeciesheterosis and for cross-subspecies marker-assisted selectionin rice. In addition, by testing 51 pairs of the ILP primerson five Gramineae plants and three dicot plants, we found anotherdesirable characteristic of rice ILP markers that they havehigh transferability to other plants.