Kiyosumi Hori

Comprehensive Sequence Analysis of 24,783 Barley Full-Length cDNAs Derived from 12 Clone Libraries

Full-length cDNA (FLcDNA) libraries consisting of 172,000 clones were constructed from a two-row malting barley cultivar (Hordeum vulgare ‘Haruna Nijo’) under normal and stressed conditions. After sequencing the clones from both ends and clustering the sequences, a total of 24,783 complete sequences were produced. By removing duplicates between these and publicly available sequences, 22,651 representative sequences were obtained: 17,773 were novel barley FLcDNAs, and 1,699 were barley specific. Highly conserved genes were found in the barley FLcDNA sequences for 721 of 881 rice (Oryza sativa) trait genes with 50% or greater identity. These FLcDNA resources from our Haruna Nijo cDNA libraries and the full-length sequences of representative clones will improve our understanding of the biological functions of genes in barley, which is the cereal crop with the fourth highest production in the world, and will provide a powerful tool for annotating the barley genome sequences that will become available in the near future.

Natural Variation in Hd17, a Homolog of Arabidopsis ELF3 That is Involved in Rice Photoperiodic Flowering

Flowering time of rice depends strongly on photoperiodic responses. We previously identified a quantitative trait locus, Heading date 17 (Hd17), that is associated with a difference in flowering time between Japanese rice (Oryza sativa L.) cultivars. Here, we show that the difference may result from a single nucleotide polymorphism within a putative gene that encodes a homolog of the Arabidopsis EARLY FLOWERING 3 protein, which plays important roles in maintaining circadian rhythms. Our results demonstrate that natural variation in Hd17 may change the transcription level of a flowering repressor, Grain number, plant height and heading date 7 (Ghd7), suggesting that Hd17 is part of rices photoperiodic flowering pathway.

Efficient construction of high-density linkage map and its application to QTL analysis in barley

Abstract. Using a High Efficiency Genome Scanning (HEGS) system and recombinant inbred (RI) lines derived from the cross of Russia 6 and H.E.S. 4, a high-density genetic map was constructed in barley. The resulting 1,595.7-cM map encompassed 1,172 loci distributed on the seven linkage groups comprising 1,134 AFLP, 34 SSR, three STS and vrs1 (kernel row type) loci. Including PCR reactions, gel electrophoresis and data processing, 6 months of work by a single person was sufficient for the whole mapping procedure under a reasonable cost. To make an appraisal of the resolution of genetic analysis for the 95 RI lines based on the constructed linkage map, we measured three agronomic traits: plant height, spike exsertion length and 1,000-kernel weight, and the analyzed quantitative trait loci (QTLs) associated with these traits. The results were compared on the number of detected QTLs and their effects between a high-density map and a skeleton map constructed by selected AFLP and anchor markers. The composite interval mapping on the high-density map detected more QTLs than the other analyses. Closely linked markers with QTLs on the high-density map could be powerful tools for marker-assisted selection in barley breeding programs and further genetic analyses including an advanced backcross analysis or a map-based cloning of QTL.

Q-TARO: QTL Annotation Rice Online Database

Over the past two decades, genetic dissection of complex phenotypes of economic and biological interest has revealed the chromosomal locations of many quantitative trait loci (QTLs) in rice and their contributions to phenotypic variation. Mapping resolution has varied considerably among QTL studies owing to differences in population size and number of DNA markers used. Additionally, the same QTLs have often been reported with different locus designations. This situation has made it difficult to determine allelic relationships among QTLs and to compare their positions. To facilitate reliable comparisons of rice QTLs, we extracted QTL information from published research papers and constructed a database of 1,051 representative QTLs, which we classified into 21 trait categories. This database (QTL Annotation Rice Online database; Q-TARO, http://qtaro.abr.affrc.go.jp/) consists of two web interfaces. One interface is a table containing information on the mapping of each QTL and its genetic parameters. The other interface is a genome viewer for viewing genomic locations of the QTLs. Q-TARO clearly displays the co-localization of QTLs and distribution of QTL clusters on the rice genome.

SmartGrain: High-throughput phenotyping software for measuring seed shape through image analysis

Seed shape and size are among the most important agronomic traits because they affect yield and market price. To obtain accurate seed size data, a large number of measurements are needed because there is little difference in size among seeds from one plant. To promote genetic analysis and selection for seed shape in plant breeding, efficient, reliable, high-throughput seed phenotyping methods are required. We developed SmartGrain software for high-throughput measurement of seed shape. This software uses a new image analysis method to reduce the time taken in the preparation of seeds and in image capture. Outlines of seeds are automatically recognized from digital images, and several shape parameters, such as seed length, width, area, and perimeter length, are calculated. To validate the software, we performed a quantitative trait locus (QTL) analysis for rice (Oryza sativa) seed shape using backcrossed inbred lines derived from a cross between japonica cultivars Koshihikari and Nipponbare, which showed small differences in seed shape. SmartGrain removed areas of awns and pedicels automatically, and several QTLs were detected for six shape parameters. The allelic effect of a QTL for seed length detected on chromosome 11 was confirmed in advanced backcross progeny; the cv Nipponbare allele increased seed length and, thus, seed weight. High-throughput measurement with SmartGrain reduced sampling error and made it possible to distinguish between lines with small differences in seed shape. SmartGrain could accurately recognize seed not only of rice but also of several other species, including Arabidopsis (Arabidopsis thaliana). The software is free to researchers.

Hd16, a gene for casein kinase I, is involved in the control of rice flowering time by modulating the day‐length response

The alteration of photoperiod sensitivity has let breeders diversify flowering time in Oryza sativa (rice) and develop cultivars adjusted to a range of growing season periods. Map-based cloning revealed that the rice flowering-time quantitative trait locus (QTL) Heading date 16 (Hd16) encodes a casein kinase-I protein. One non-synonymous substitution in Hd16 resulted in decreased photoperiod sensitivity in rice, and this substitution occurred naturally in an old rice cultivar. By using near-isogenic lines with functional or deficient alleles of several rice flowering-time genes, we observed significant digenetic interactions between Hd16 and four other flowering-time genes (Ghd7, Hd1, DTH8 and Hd2). In a near-isogenic line with the weak-photoperiod-sensitivity allele of Hd16, transcription levels of Ehd1, Hd3a, and RFT1 increased under long-day conditions, and transcription levels of Hd3a and RFT1 decreased under short-day conditions. Expression analysis under continuous light and dark conditions showed that Hd16 was not likely to be associated with circadian clock regulation. Biochemical characterization indicated that the functional Hd16 recombinant protein specifically phosphorylated Ghd7. These results demonstrate that Hd16 acts as an inhibitor in the rice flowering pathway by enhancing the photoperiod response as a result of the phosphorylation of Ghd7.

Novel QTLs for photoperiodic flowering revealed by using reciprocal backcross inbred lines from crosses between japonica rice cultivars

The rice japonica cultivars Nipponbare and Koshihikari differ in heading date and response of heading to photoperiod (photoperiod sensitivity). Using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers, we conducted quantitative trait locus (QTL) analyses for heading date in a set of reciprocal backcross inbred lines (BILs) from crosses between Nipponbare and Koshihikari. Under natural-day conditions, transgressive segregation in days to heading (DTH) toward both early and late heading was observed in both BIL populations. QTL analyses revealed that two QTLs—on chromosomes 3 and 6—were involved in the difference in heading date between the parental cultivars. The Nipponbare allele at the QTLs on chromosomes 3 and 6 showed, respectively, increasing and decreasing effects on DTH in both BIL populations. The transgressive segregation observed in the BILs could be accounted for mainly by the complementary action of a set of alleles with opposing effects. Both QTLs were finely mapped as single Mendelian factors in secondary mapping populations (BC2F2 plants/BC2F3 lines). The QTL on chromosome 3 was mapped in the 1,140-kb interval between 94O03-4 (SSR) and OJ21G19-4 (SNP) and was designated Hd16. The QTL on chromosome 6 was mapped in the 328-kb interval between P548D347 (SSR) and 0007O20 (SSR) and was designated Hd17. Both Hd16 and Hd17 were involved in photoperiod sensitivity, as revealed by observation of the DTH of nearly isogenic lines of Nipponbare under short- and long-day conditions, suggesting that allelic differences in both Hd16 and Hd17 account for most of the difference in photoperiod sensitivity between the parental cultivars.

Application of genomics-assisted breeding for generation of climate resilient crops: progress and prospects

Climate change affects agricultural productivity worldwide. Increased prices of food commodities are the initial indication of drastic edible yield loss, which is expected to increase further due to global warming. This situation has compelled plant scientists to develop climate change-resilient crops, which can withstand broad-spectrum stresses such as drought, heat, cold, salinity, flood, submergence and pests, thus helping to deliver increased productivity. Genomics appears to be a promising tool for deciphering the stress responsiveness of crop species with adaptation traits or in wild relatives toward identifying underlying genes, alleles or quantitative trait loci. Molecular breeding approaches have proven helpful in enhancing the stress adaptation of crop plants, and recent advances in high-throughput sequencing and phenotyping platforms have transformed molecular breeding to genomics-assisted breeding (GAB). In view of this, the present review elaborates the progress and prospects of GAB for improving climate change resilience in crops, which is likely to play an ever increasing role in the effort to ensure global food security.

Uncovering of major genetic factors generating naturally occurring variation in heading date among Asian rice cultivars.

To dissect the genetic factors controlling naturally occurring variation of heading date in Asian rice cultivars, we performed QTL analyses using F2 populations derived from crosses between a japonica cultivar, Koshihikari, and each of 12 cultivars originating from various regions in Asia. These 12 diverse cultivars varied in heading date under natural field conditions in Tsukuba, Japan. Transgressive segregation was observed in 10 F2 combinations. QTL analyses using multiple crosses revealed a comprehensive series of loci involved in natural variation in flowering time. One to four QTLs were detected in each cross combination, and some QTLs were shared among combinations. The chromosomal locations of these QTLs corresponded well with those detected in other studies. The allelic effects of the QTLs varied among the cross combinations. Sequence analysis of several previously cloned genes controlling heading date, including Hd1, Hd3a, Hd6, RFT1, and Ghd7, identified several functional polymorphisms, indicating that allelic variation at these loci probably contributes to variation in heading date. Taken together, the QTL and sequencing results indicate that a large portion of the phenotypic variation in heading date in Asian rice cultivars could be generated by combinations of different alleles (possibly both loss- and gain-of-function) of the QTLs detected in this study.

Detection of quantitative trait loci controlling pre-harvest sprouting resistance by using backcrossed populations of japonica rice cultivars

Backcrossed inbred lines (BILs) and a set of reciprocal chromosome segment substitution lines (CSSLs) derived from crosses between japonica rice cultivars Nipponbare and Koshihikari were used to detect quantitative trait loci (QTLs) for pre-harvest sprouting resistance. In the BILs, we detected one QTL on chromosome 3 and one QTL on chromosome 12. The QTL on the short arm of chromosome 3 accounted for 45.0% of the phenotypic variance and the Nipponbare allele of the QTL increased germination percentage by 21.3%. In the CSSLs, we detected seven QTLs, which were located on chromosomes 2, 3 (two), 5, 8 and 11 (two). All Nipponbare alleles of the QTLs were associated with an increased rate of germination. The major QTL for pre-harvest sprouting resistance on the short arm of chromosome 3 was localized to a 474-kbp region in the Nipponbare genome by the SSR markers RM14240 and RM14275 by using 11 substitution lines to replace the different short chromosome segments on chromosome 3. This QTL co-localized with the low-temperature germinability gene qLTG3-1. The level of germinability under low temperature strongly correlated with the level of pre-harvest sprouting resistance in the substitution lines. Sequence analyses revealed a novel functional allele of qLTG3-1 in Nipponbare and a loss-of-function allele in Koshihikari. The allelic difference in qLTG3-1 between Nipponbare and Koshihikari is likely to be associated with differences in both pre-harvest sprouting resistance and low-temperature germinability.