Shinya Yoshida

Genome-wide association study using whole-genome sequencing rapidly identifies new genes influencing agronomic traits in rice

A genome-wide association study (GWAS) can be a powerful tool for the identification of genes associated with agronomic traits in crop species, but it is often hindered by population structure and the large extent of linkage disequilibrium. In this study, we identified agronomically important genes in rice using GWAS based on whole-genome sequencing, followed by the screening of candidate genes based on the estimated effect of nucleotide polymorphisms. Using this approach, we identified four new genes associated with agronomic traits. Some genes were undetectable by standard SNP analysis, but we detected them using gene-based association analysis. This study provides fundamental insights relevant to the rapid identification of genes associated with agronomic traits using GWAS and will accelerate future efforts aimed at crop improvement.

Pathogenic diversity of Phytophthora sojae and breeding strategies to develop Phytophthora-resistant soybeans

Phytophthora stem and root rot, caused by Phytophthora sojae, is one of the most destructive diseases of soybean [Glycine max (L.) Merr.], and the incidence of this disease has been increasing in several soybean-producing areas around the world. This presents serious limitations for soybean production, with yield losses from 4 to 100%. The most effective method to reduce damage would be to grow Phytophthora-resistant soybean cultivars, and two types of host resistance have been described. Race-specific resistance conditioned by single dominant Rps (“resistance to Phytophthora sojae”) genes and quantitatively inherited partial resistance conferred by multiple genes could both provide protection from the pathogen. Molecular markers linked to Rps genes or quantitative trait loci (QTLs) underlying partial resistance have been identified on several molecular linkage groups corresponding to chromosomes. These markers can be used to screen for Phytophthora-resistant plants rapidly and efficiently, and to combine multiple resistance genes in the same background. This paper reviews what is currently known about pathogenic races of P. sojae in the USA and Japan, selection of sources of Rps genes or minor genes providing partial resistance, and the current state and future scope of breeding Phytophthora-resistant soybean cultivars.

Genetic Analysis of Long Chain Synthesis in Rice Amylopectin

The amount of long chains (LC) of amylopectin in high-amylose rice is thought to be one of the important determinants of its quality when cooked. A wide range of differences in LC content have been reported in rice varieties, which can be clearly divided into four classes based on LC and apparent amylose content: namely, amylose and LC-free, low or medium-amylose and low-LC, high-amylose and medium-LC, high-amylose and high-LC. However, genetic factors controlling LC content have not been fully understood. Here, we performed quantitative trait loci (QTL) analysis of LC content using 157 recombinant inbred lines (RILs) derived from a cross of a low-LC cultivar, Hyogokitanishiki, and a high-LC line, Hokuriku 142. By analyzing randomly selected 15 RILs, it was shown that high LC content (≥11%) was associated with high setback viscosity (≥200 RVU), and that low LC (≤ 3%) was associated with low setback viscosity (≤ 130 RVU), as measured by a Rapid Visco Analyzer. With setback viscosity as an indicator for LC content, QTL analysis was conducted using 60 DNA markers including a CAPS marker that distinguished Wxa and Wxb alleles coding for granule-bound starch synthase I (GBSSI or Wx protein), the enzyme working for amylose biosynthesis. Only one QTL with a peak log of likelihood score at the wx locus was detected, and no line showing setback viscosity corresponding to the medium-LC class appeared. The fact that wx mutants of Hokuriku 142 lacked LC in their rice starch supports the view that the functional Wx allele is indispensable for LC synthesis in addition to amylose synthesis in rice endosperm. We suggest three possible reasons why no line with medium-LC content was observed. First, the locus (loci) responsible for generation of medium-LC may be located very close to the wx locus and not able to be dissected by the population and DNA markers we used. Second, there may be special QTLs for medium-LC cultivars that do not exist in low- or high-LC cultivars. Third, medium-LC cultivars may have an as-yet unidentified Wx allele with lower capability in LC synthesis compared to the Wx allele in high-LC cultivars.

A simple, rapid and reliable bioassay for evaluating seedling vigor under submergence in indica and japonica rice (Oryza sativa L.)

Submergence is a major stress causing yield losses particularly in the direct-seeded rice cultivation system and necessitates the development of a simple, rapid and reliable bioassay for a large scale screening of rice germplasms with tolerance against submergence stress. We developed two new bioassay methods that were based primarily on the seedling vigor evaluated by the ability of fast shoot elongation under submerged conditions, and compared their effectiveness with two other available methods. All four bioassay methods using cultivars of 7 indica and 6 japonica types revealed significant and consistent cultivar differences in seedling vigor under submergence and/or submergence tolerance. Japonica cultivars were more vigorous than indica cultivars, with Nipponbare being the most vigorous. The simplest test tube method showed the highest correlations to all other methods. Our results suggest that seedling vigor serves as a submergence avoidance mechanism and confers tolerance on rice seedlings to flooding during early crop establishment. A possible relationship is discussed between seedling vigor based on fast shoot elongation and submergence tolerance defined by recovery from submergence stress.

Race distribution of Phytophthora sojae on soybean in Hyogo, Japan

Since 1987, Phytophthora root and stem rot of soybean [Glycine max (L.) Merr. cv. Tanbakuro], caused by Phytophthora sojae Kaufman and Gerdemann, has been increasing in the Sasayama, Nishiwaki, and Kasai regions in Hyogo, the most famous soybean (cv. Tanbakuro)-producing areas in Japan. In 2002 to 2004, 51 isolates (one from each field) of P. sojae were recovered from 51 fields in Hyogo. These isolates were tested for virulence on six Japanese differential soybean cultivars used for race determination in Japan, and three additional ones containing four Rps genes used in Indiana, USA. Race E was the most prevalent from 2002 to 2004, followed by races A, C, D, and four new races (proposed as races K, L, M, and N). Interestingly, none of the new races had high virulence on the Japanese differential cultivars, compared with other races in each area. One (race N) was avirulent on all six soybean differentials. There was a difference in race distribution on each of three individual areas; race E seemed to be a major component of the P. sojae population in Sasayama, whereas race A and the new race M were the most prevalent in Nishiwaki and Kasai, respectively. Rps6 (cv. Altona) and Rps1a + Rps7 (cv. Harosoy 63) were infected by 90.2% and 33.3% of all isolates, respectively. However, Rps1d (cv. PI103091) was not susceptible to any of the 51 isolates, nor was cv. Gedenshirazu-1. These two soybean cultivars were considered to be potential sources of resistance to breed new resistant cultivars with the desirable characteristics of cv. Tanbakuro for this region.

Mapping QTLs for cold tolerance at germination and the early seedling stage in rice (Oryza sativa L.)

Cold tolerance is an important breeding target in rice production. We studied quantitative trait loci (QTLs) controlling cold tolerance at germination (CTG) and early seedling (CTS) stages, using recombinant inbred lines derived from a japonica × indica cross. CTG was evaluated based on the percentage rate of germination at 15 °C for 12 days after pre-incubation of imbibed seeds at 20 °C for 2 days. For CTS, seven-day-old seedlings grown at 25 °C were subjected to two consecutive periods of three-day cold stress at 4 °C with an intervening eight-day recovery at 25 °C. CTS evaluation was according to an arbitrary five-point rating system at the fifth day of recovery after each stress period. No correlations were found between CTG and CTS, while a weak correlation was detected between CTS after the first and second stress. By the composite interval mapping, five QTLs for CTG explaining 5.7%–9.3% of the total phenotypic variance (PVE) and nine for CTS with PVE of 5.8%–35.6% were detected. Only one of these QTLs was common, indicating growth-stage specificity of cold tolerance. Four of the five QTLs after the second cold stress were different from the ones after the first cold stress. Analysis of variance test showed significant interactions between alleles at the QTL sites and the two stress conditions with respect to the mean CTS scores. A possible involvement of cold acclimation and usefulness of japonica germplasms in breeding for cold tolerance in indica rice was discussed.

Diversity and population structure of black soybean landraces originating from Tanba and neighboring regions

Black soybean landraces that had been cultivated in Tanba region and the neighboring regions and conserved black soybean landraces, including those from other regions in Japan, were used in this study. The polymorphisms of 78 SSR markers in nuclear DNA and 6 SSRs in chloroplast DNA were analyzed in the black soybean landrace populations. The result of phylogenic analysis revealed that the black soybeans can be classified into six clades. The landraces originating from Tanba region were classed into first and second clades, and two chloroplast genotypes were found in the population of black soybeans from the Tanba region. Genotype A chloroplast was predominantly identified in major populations of the Tanba, while genotype B was widely distributed in the black soybean population. Population structure analysis in the Japanese black soybean accessions inferred there are six groups. The black soybean landrace from the Tanba region was classified into three groups, mainly corresponding to the distance-based phylogenic results. The two groups were probably derived from different ancestors with Type A and B chloroplast genomes, respectively, whereas the other group showed both types of chloroplast genome. The admixture situations suggested that the landraces in the main group have been widely cultivated in Tanba region, while the landraces that belong to other groups were cultivated in localized area. Several phenotypes were compared among genotype groups, dividing into two sub-groups: founder sub-group and admixed sub-group. Phenotypic differences were observed between founder landraces in group 1 and group 3. On the other hand, landraces in admixture landraces in group 1 and group 2 segregated for several traits, while founder landraces in group 1 were stabled for each trait. These observations suggest that gene flow events have occurred between different founder landraces.

MAPPING OF QUANTITATIVE TRAIT LOCI ASSOCIATED WITH COLD TOLERANCE AT THE POST-GERMINATION STAGE IN RICE

ABSTRACT Cold tolerance at the post-germination stage in rice was studied using gained shoot length (GSL) as a new parameter, which was measured as increased shoot length during cold stress at 4°C followed by a 4-day recovery period at 35°C. GSL decreased until 4th day in both japonica and indica cultivar, while a marked increase occurred in a japonica cultivar after 5 days of cold stress, indicating its higher ability of cold acclimation. Quantitative trait loci (QTLs) associated with cold tolerance were identified using recombinant inbred lines derived from these parents on a linkage map constructed with 102 simple sequence repeat (SSR) markers. By the interval analysis, one and five putative QTLs were identified after 5 and 11 days of cold stress, respectively. The single point analysis indicated that these QTLs explained 16% and 54% of the phenotypic variance after 5 and 11 days of cold stress, respectively. A cold tolerant japonica parent contributed all QTLs, among which one on the long arm of chromosome 7 was common for the two cold stress periods. Based on gained root length one QTL was identified on chromosome 12 explaining 11% of the variance.

Non-random gametoclonal variation in rice regenerants from callus subcultured for a prolonged period under high osmotic stress

Gametoclonal variations were investigated in rice plants regenerated from anther-derived callus after subculture for two different periods (4–6 and 20 weeks) in medium with two levels (292 and 438 mM) of mannitol. A high osmotic stress by mannitol in subculture medium contributed to the maintenance of high regeneration ability. The frequency of diploid regenerants (R0 plants) increased and that of haploids decreased from callus subcultured for the longer period in medium with the higher concentration of mannitol. R1 regenerant lines displayed variations in all six agronomical traits studied at the frequencies of 63% and 88% after subculture for 4–6 weeks and 20 weeks, respectively. High homozygosity was suggested in most of the R1 lines for these traits by the similar levels of variance to those in the control variety. A principal component analysis showed a skewed distribution of variants towards dwarfness and less panicle weight in the regenerants from callus subcultured for 20 weeks with 438 mM mannitol. Two short culmn variants were shown to be due to single or double recessive mutations. The results suggested that prolonged subculture under high osmotic stress causes non-random selection of homozygous gametoclonal variations in rice anther culture.

Submergence-Induced ADH and ALDH Gene Expression in Japonica and Indica Rice with Contrasting Levels of Seedling Vigor under Submergence Stress

ABSTRACT ADH and ALDH gene expression was studied by reverse transcriptase polymerase chain reaction (RT-PCR) using selected indica and japonica rice cultivars showing vigorous and non-vigorous post-germination seedling growth under submergence stress. Transcript amount of ADH1, ADH2 and ALDH2a increased dramatically under submergence irrespective of dark/light conditions in both indica and japonica rice, whereas ALDH2b gene expression was greatly suppressed. Submergence-induced increases of the transcript levels also occurred in floating rice as well as in selected chromosome segment substitution lines that were derived from crosses between ‘Nipponbare’ and ‘Kasalath’. However, no cultivar and line differences were observed in their transcript levels under submergence. Our results suggest that the expression of ADH1, ADH2 and ALDH2a genes is highly responsive to submergence stress but is not a major determinant of the cultivar/genotype-specific level of seedling vigor under submergence.