Evolution of the Grain Dispersal System in Barley.

About 12,000 years ago in the Near East, humans began the transition from hunter-gathering to agriculture-based societies. Barley was a founder crop in this process, and the most important steps in its domestication were mutations in two adjacent, dominant, and complementary genes, through which grains were retained on the inflorescence at maturity, enabling effective harvesting. Independent recessive mutations in each of these genes caused cell wall thickening in a highly specific grain "disarticulation zone," converting the brittle floral axis (the rachis) of the wild-type into a tough, non-brittle form that promoted grain retention. By tracing the evolutionary history of allelic variation in both genes, we conclude that spatially and temporally independent selections of germplasm with a non-brittle rachis were made during the domestication of barley by farmers in the southern and northern regions of the Levant, actions that made a major contribution to the emergence of early agrarian societies.

Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.

Fine mapping of Rf5 region for a sorghum fertility restorer gene and microsynteny analysis across grass species.

Cytoplasmic male sterility (CMS) is widely used to control pollination in the production of commercial F1 hybrid seed in sorghum. So far, 6 major fertility restorer genes, Rf1 to Rf6, have been reported in sorghum. Here, we fine-mapped the Rf5 locus on sorghum chromosome 5 using descendant populations of a 'Nakei MS-3A' × 'JN43' cross. The Rf5 locus was narrowed to a 140-kb region in BTx623 genome (161-kb in JN43) with 16 predicted genes, including 6 homologous to the rice fertility restorer Rf1 (PPR.1 to PPR.6). These 6 homologs have tandem pentatricopeptide repeat (PPR) motifs. Many Rf genes encode PPR proteins, which bind RNA transcripts and modulate gene expression at the RNA level. No PPR genes were detected at the Rf5 locus on the corresponding homologous chromosome of rice, foxtail millet, or maize, so this gene cluster may have originated by chromosome translocation and duplication after the divergence of sorghum from these species. Comparison of the sequences of these genes between fertile and CMS lines identified PPR.4 as the most plausible candidate gene for Rf5.

Investigation of the Genetic Diversity of a Rice Core Collection of Japanese Landraces using Whole-Genome Sequencing.

The Rice Core Collection of Japanese Landraces (JRC) consisting of 50 accessions was developed by the genebank at the National Agriculture and Food Research Organization (NARO) in 2008. As a Japanese landrace core collection, the JRC has been used for many research projects, including screening for different phenotypes and allele mining for target genes. To understand the genetic diversity of Japanese Landraces, we performed whole-genome resequencing of these 50 accessions and obtained a total of 2,145,095 single nucleotide polymorphism (SNPs) and 317,832 insertion-deletions (indels) by mapping against the Oryza sativa ssp. japonica Nipponbare genome. A JRC phylogenetic tree based on 1,394 representative SNPs showed that JRC accessions were divided into two major groups and one small group. We used the multiple genome browser, TASUKE+, to examine the haplotypes of flowering genes and detected new mutations in these genes. Finally, we performed genome-wide association studies (GWAS) for agronomical traits using the JRC and another core collection, the World Rice Core Collection (WRC), comprising 69 accessions also provided by the NARO genebank. In leaf blade width, a strong peak close to NAL1, a key gene for the regulation of leaf width, and, in heading date, a peak near HESO1 involved in flowering regulation were observed in GWAS using the JRC. They were also detected in GWAS using the combined JRC + WRC. Thus, JRC and JRC + WRC are suitable populations for GWAS of particular traits.

Development of an SSR marker-based genetic linkage map and identification of a QTL associated with flowering time in Eustoma.

Lisianthus (Eustoma grandiflorum) is an important floricultural crop cultivated worldwide. Despite its commercial importance, few DNA markers are available for molecular genetic research. In this study, we constructed a genetic linkage map and to detect quantitative trait loci (QTLs) for important agronomic traits of lisianthus. To develop simple sequence repeat (SSR) markers, we used 454-pyrosequencing technology to obtain genomic shotgun sequences and subsequently identified 8263 putative SSRs. A total of 3990 primer pairs were designed in silico and 1189 unique primer pairs were extracted through a BLAST search. Amplification was successful for more than 1000 primer pairs, and ultimately 278 SSR markers exhibited polymorphism between the two lisianthus accessions evaluated. Based on these markers, a genetic linkage map was constructed using a breeding population derived from crosses between the two accessions, for which flowering time differed (>140 days when grown under 20°C). We detected one QTL associated with flowering time (phenotypic variance, 27%; LOD value, 3.7). The SSR marker located at this QTL may account for variation in flowering time among accessions (i.e., three accessions whose nodes of the first flower were over 30 had late-flowering alleles of this QTL).

Elucidation of the origin of 'agriocrithon' based on domestication genes questions the hypothesis that Tibet is one of the centers of barley domestication.

Wild barley forms a two-rowed spike with a brittle rachis whereas domesticated barley has two- or six-rowed spikes with a tough rachis. Like domesticated barley, 'agriocrithon' forms a six-rowed spike; however, the spike is brittle as in wild barley, which makes the origin of agriocrithon obscure. Haplotype analysis of the Six-rowed spike 1 (vrs1) and Non-brittle rachis 1 (btr1) and 2 (btr2) genes was conducted to infer the origin of agriocrithon barley. Some agriocrithon barley accessions (eu-agriocrithon) carried Btr1 and Btr2 haplotypes that are not found in any cultivars, implying that they are directly derived from wild barley through a mutation at the vrs1 locus. Other agriocrithon barley accessions (pseudo-agriocrithon) carried Btr1 or Btr2 from cultivated barley, thus implying that they originated from hybridization between six-rowed landraces carrying btr1Btr2 and Btr1btr2 genotypes followed by recombination to produce Btr1Btr2. All materials we collected from Tibet belong to pseudo-agriocrithon and thus do not support the Tibetan Plateau as being a center of barley domestication. Tracing the evolutionary history of these allelic variants revealed that eu-agriocrithon represents six-rowed barley lineages that were selected by early farmers, once in south-eastern Turkmenistan (vrs1.a1) and again in the eastern part of Uzbekistan (vrs1.a4).

Structural features of two major nucleolar organizer regions (NORs), Nor-B1 and Nor-B2, and chromosome-specific rRNA gene expression in wheat.

The reference genome sequence of wheat 'Chinese Spring' (CS) is now available (IWGSC RefSeq v1.0), but the core sequences defining the nucleolar organizer regions (NORs) have not been characterized. We estimated that the total copy number of the rDNA units in the wheat genome is 11 160, of which 30.5%, 60.9% and 8.6% are located on Nor-B1 (1B), Nor-B2 (6B) and other NORs, respectively. The total length of the NORs is estimated to be 100 Mb, corresponding to approximately 10% of the unassembled portion of the genome not represented in RefSeq v1.0. Four subtypes (S1-S4) of the rDNA units were identified based on differences within the 3' external transcribed spacer regions in Nor-B1 and Nor-B2, and quantitative PCR indicated locus-specific variation in rDNA subtype contents. Expression analyses of rDNA subtypes revealed that S1 was predominantly expressed and S2 weakly expressed, in contrast to the relative abundance of rDNA subtypes in the wheat genome. These results suggest a regulation mechanism of differential rDNA expression based on sequence differences. S3 expression increased in the ditelosomic lines Dt1BL and Dt6BL, suggesting that S3 is subjected to chromosome-mediated silencing. Structural differences were detected in the regions surrounding the NOR among homoeologous chromosomes of groups 1 and 6. The adjacent regions distal to the major NORs were expanded compared with their homoeologous counterparts, and the gene density of these expanded regions was relatively low. We provide evidence that these regions are likely to be important for autoregulation of the associated major NORs as well as silencing of minor NORs.

Identification of candidate genes responsible for the susceptibility of apple (Malus × domestica Borkh.) to Alternaria blotch.

BACKGROUND: The mechanism underlying the interaction between host plant and host-selective toxin (HST)-producing Alternaria alternata during infection is of particular interest for sustainable crop production. Alternaria blotch of apple (Malus × domestica Borkh.) caused by A. alternata apple pathotype is a major disease particularly in East Asia, which is the largest producer of apples globally. A single dominant gene, Alt, controls the susceptibility of the apple cultivar 'Delicious' to Alternaria blotch. In this study, we fine mapped the Alt locus and characterized three potential candidate genes. RESULTS: We used 797 F1 individuals derived from 15 crosses between apple accessions susceptible (Alt/alt) and resistant (alt/alt) to Alternaria blotch to construct physical and genetic maps of the Alt locus located on the top of chromosome 11. Susceptible accessions were derived from 'Delicious.' To fine map the Alt locus, we constructed a BAC library of 'Starking Delicious,' a sport of 'Delicious,' and used graphical genotyping to delimit the Alt locus to a region of 43 kb. Three genes predicted within the candidate Alt region were potentially involved in plant defense response, among which the gene encoding a coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) type disease resistance protein was the most promising. Moreover, a 12-bp insertion was uniquely identified in the 5' untranslated region of the Alt-associated allele of this gene, the presence or absence of which co-segregated with the susceptibility or resistance to A. alternata apple pathotype, respectively, among 43 tested cultivars including old ones and founders of modern apple breeding. CONCLUSION: A disease resistance protein has been suggested as a determinant of susceptibility/resistance to HST-producing A. alternata for the first time. Our finding provides new insight into the mechanism of HST-mediated disease control used by A. alternata against host plants.

Scanning RNA-Seq and RAD-Seq approach to develop SNP markers closely linked to MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

BACKGROUND: Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. RESULTS: To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. CONCLUSION: We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Genome-wide analysis of rice cis-natural antisense transcription under cadmium exposure using strand-specific RNA-Seq.

BACKGROUND: The elucidation of novel transcripts and their expression in response to various stress conditions is necessary to understand the transcriptional network of plants as an adaptation to biotic and abiotic stresses. We performed strand-specific RNA-Seq (ssRNA-Seq) on rice exposed to cadmium (Cd) for 24 h and investigated the expression of cis-natural antisense transcripts (cis-NATs), a class of endogenous coding or non-protein-coding RNAs with sequence complementarity to the opposite strands of RAP transcripts. RESULTS: Many RAP transcripts possessed cis-NATs and these cis-NATs were responsive to some extent. Cis-NATs were upregulated from 26, 266 and 409 RAP gene loci, while 2054, 2501 and 2825 RAP transcripts were upregulated from 38,123 RAP loci under high Cd exposure in roots at 1, 12 and 24 h, respectively. In addition, most of the upregulated cis-NATs showed little upregulation under ABA or cold treatment. A number of cis-NATs were upregulated from less than 35 RAP gene loci in different tissue and time-point combinations under low Cd exposure, suggesting that cis-NATs respond to environmental stress. Furthermore, 409 RAP transcripts with upregulated cis-NATs were classified into three groups based on the expression of the RAP transcripts from the opposite DNA strand, including 138 upregulated, 128 invariable, and 143 downregulated transcripts, although the responses of cis-NATs and RAP transcripts were not always correlated. CONCLUSIONS: We have shown that the cis-NATs identified by ssRNA-Seq analysis are novel genes and that some of them are stress-specific and show different responses depending on the degree of stress and tissue. These results improve our understanding of the complete molecular mechanism of plant adaptation to Cd exposure.

Correction to: Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

After publication of the original article [1] the authors noted that the following errors had occurred.

Sequence differences in the seed dormancy gene Qsd1 among various wheat genomes.

BACKGROUND: Pre-harvest sprouting frequently occurs in Triticum aestivum (wheat) and Hordeum vulgare (barley) at the end of the maturity period due to high rainfall, particularly in Asian monsoon areas. Seed dormancy is a major mechanism preventing pre-harvest sprouting in these crops. RESULTS: We identified orthologous sequences of the major Hordeum vulgare (barley) seed dormancy gene Qsd1 in hexaploid wheat cv. Chinese Spring by performing genomic clone sequencing, followed by transcript sequencing. We detected 13 non-synonymous amino acid substitutions among the three sub-genomes of wheat and found that the Qsd1 sequence in the B sub-genome is most similar to that in barley. The Qsd1 sequence in A genome diploid wheat is highly similar to that in the hexaploid A sub-genome. Wheat orthologs of Qsd1 showed closer similarities to barley Qsd1 than did those of other accessions in the DNA database. Like barley Qsd1, all three wheat Qsd1s showed embryo-specific gene expression patterns, indicating that barley and wheat Qsd1 share an orthologous origin. The alignment of four hexaploid wheat cultivars indicated that the amino acid sequences of three spring cultivars, Chinese Spring, Haruyo Koi, and Fielder, are exactly the same in each sub-genome. Only Kitahonami has three amino acid substitutions at the B sub-genome. CONCLUSIONS: Kitahonami has a longer seed dormancy period than does Chinese Spring. Sequence polymorphisms between Chiniese Spring and Kitahonami in the B sub-genome may underlie the phenotypic differences in seed dormancy between these hexaploid wheat cultivars.

TENOR: Database for Comprehensive mRNA-Seq Experiments in Rice.

Here we present TENOR (Transcriptome ENcyclopedia Of Rice, http://tenor.dna.affrc.go.jp), a database that encompasses large-scale mRNA sequencing (mRNA-Seq) data obtained from rice under a wide variety of conditions. Since the elucidation of the ability of plants to adapt to various growing conditions is a key issue in plant sciences, it is of great interest to understand the regulatory networks of genes responsible for environmental changes. We used mRNA-Seq and performed a time-course transcriptome analysis of rice, Oryza sativa L. (cv. Nipponbare), under 10 abiotic stress conditions (high salinity; high and low phosphate; high, low and extremely low cadmium; drought; osmotic; cold; and flood) and two plant hormone treatment conditions (ABA and jasmonic acid). A large number of genes that were responsive to abiotic stresses and plant hormones were detected by differential expression analysis. Furthermore, several responsive genes were found to encode transcription factors that could control the transcriptional network of stress responses, but the timing of the induction of these genes was not uniform across conditions. A significant number of cis-regulatory elements were enriched in the promoter regions of the responsive genes and were shared among conditions. These data suggest that some key components of gene regulation networks are shared between different stress signaling pathways. All the resources (novel genes identified from mRNA-Seq data, expression profiles, co-expressed genes and cis-regulatory elements) can be searched for and are available in TENOR.

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

Characterization of a mini core collection of Japanese wheat varieties using single-nucleotide polymorphisms generated by genotyping-by-sequencing.

A core collection of Japanese wheat varieties (JWC) consisting of 96 accessions was established based on their passport data and breeding pedigrees. To clarify the molecular basis of the JWC collection, genome-wide single-nucleotide polymorphism (SNP) genotyping was performed using the genotyping-by-sequencing (GBS) approach. Phylogenetic tree and population structure analyses using these SNP data revealed the genetic diversity and relationships among the JWC accessions, classifying them into four groups; "varieties in the Hokkaido area", "modern varieties in the northeast part of Japan", "modern varieties in the southwest part of Japan" and "classical varieties including landraces". This clustering closely reflected the history of wheat breeding in Japan. Furthermore, to demonstrate the utility of the JWC collection, we performed a genome-wide association study (GWAS) for three traits, namely, "days to heading in autumn sowing", "days to heading in spring sowing" and "culm length". We found significantly associated SNP markers with each trait, and some of these were closely linked to known major genes for heading date or culm length on the genetic map. Our study indicates that this JWC collection is a useful set of germplasm for basic and applied research aimed at understanding and utilizing the genetic diversity among Japanese wheat varieties.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BACKGROUND: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. RESULTS: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. CONCLUSIONS: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.