Genetical and morphological characterization of cold tolerance at fertilization stage in rice

Cold temperature during the reproductive phase leads to seed sterility, which reduces yield and decreases the grain quality of rice. The fertilization stage, ranging from pollen maturation to the completion of fertilization, is sensitive to unsuitable temperature. Improving cold tolerance at the fertilization stage (CTF) is an important objective of rice breeding program in cold temperature areas. In this study, we characterized fertilization behavior under cold temperature to define the phenotype of CTF and identified quantitative trait loci (QTLs) for CTF. A wide variation in CTF levels has been identified among local cultivars in Hokkaido, which is one of the most northern regions for rice cultivation in the world. Clear varietal differences in pollen germination, and pollen tube elongation due to cold temperature have been observed. These differences may confer a degree of CTF among this population. We conducted QTL analysis for CTF using 120 backcrossed inbred lines derived from a cross between Eikei88223 (vigorous CTF) and Suisei (very weak CTF). Three QTLs for CTF were identified. A clear effect by QTL, qCTF7, for increasing the level of CTF was validated using advanced progeny. These results will facilitate marker-assist selection for desirable QTLs for CTF in rice breeding program.     

Quantitative trait loci for whiteness of cooked rice detected in improved rice cultivars in Hokkaido

Improving the eating quality of cooked rice has been one of the most important objectives in rice breeding programs. Eating quality of cooked rice is a complex trait including several components, such as external appearance, taste, aroma, and texture. Therefore, dissection of these components followed by marker-assisted selection of detected QTL(s) may be a useful approach for achieving desirable eating quality in rice breeding. Whiteness of cooked rice (WCR) is an important factor related to the external appearance of cooked rice. WCR is known to be associated with the amylose and protein contents of the endosperm. However, the genetic basis of WCR remains unclear. In this study, we evaluated phenotypic variation in WCR among recently developed rice cultivars from Hokkaido, Japan. Then, we developed doubled haploid lines (DHLs) derived from a cross between two cultivars from Hokkaido, Joiku No. 462 (high WCR) and Jokei06214 (low WCR). Using the DHLs, we detected two QTLs for WCR, qWCR3 and qWCR11, on chromosomes 3 and 11, respectively. We also examined the dosage effect of the two QTLs based on both the categorized segregants in the DHLs and the relationship between the WCR phenotype and inheritance around the QTL regions in cultivars from Hokkaido.     

Characterization and identification of cold tolerant near-isogenic lines in rice

To exploit the genetic mechanism of cold tolerance in rice, cold tolerant near-isogenic lines (NILs) were developed by backcrossing Kunmingxiaobaigu (KMXBG), reported to be the most cold-tolerant variety at the booting stage, as donor, with the cold sensitive Japanese commercial japonica variety, Towada. Comparisons of cold tolerance-related traits between five BC₆F₅ NILs and recurrent parent Towada under cold treatment and normal temperatures at the booting stage showed that the differences between the NILs and Towada were significant only for spikelet fertility-related traits. Analyses of cold tolerance in the NILs at the budding (germination), seedling and booting stages indicated both correlated effects and differences. Lines 1913-4 and 1916-1 showed strong and stable tolerance at all three stages. Whole genome marker screening showed that the proportion of genetic background recovery was more than 98%. Seventeen markers from KMXBG were introgressed in two or more NILs, and cold tolerance genes were possibly present in these marker regions. The NILs should be excellent materials for both rice improvement and map-based cloning of cold tolerance QTLs.     

Comparison of phenotypic versus marker-assisted background selection for the SUB1 QTL during backcrossing in rice

Marker assisted backcrossing has been used effectively to transfer the submergence tolerance gene SUB1 into popular rice varieties, but the approach can be costly. The selection strategy comprising foreground marker and phenotypic selection was investigated as an alternative. The non-significant correlation coefficients between ranking of phenotypic selection and ranking of background marker selection in BC₂F₁, BC₃F₁ and BC₃F₂ generations indicated inefficiency of phenotypic selection compared to marker-assisted background selection with respect to recovery of the recipient genome. In addition, the introgression size of the chromosome fragment containing SUB1 was approximately 17 Mb, showing the effects of linkage drag. The significant correlation coefficient between rankings of phenotypic selection with the percentage of recipient alleles in the BC₁F₁ generation suggested that background selection could be avoided in this generation to minimize the genotyping cost. The phenotypically selected best plant of the BC₃F₁ generation was selfed and backcross recombinant lines were selected in the resulting BC₃F₄ generation. The selection strategy could be appropriate for the introgression of SUB1 QTL in countries that lack access to high-throughput genotyping facilities.     

Genome-wide association mapping focusing on a rice population derived from rice breeding programs in a region

Plant breeding programs in local regions may generate genetic variations that are desirable to local populations and shape adaptability during the establishment of local populations. To elucidate genetic bases for this process, we proposed a new approach for identifying the genetic bases for the traits improved during rice breeding programs; association mapping focusing on a local population. In the present study, we performed association mapping focusing on a local rice population, consisting of 63 varieties, in Hokkaido, the northernmost region of Japan and one of the northern limits of rice cultivation worldwide. Six and seventeen QTLs were identified for heading date and low temperature germinability, respectively. Of these, 13 were novel QTLs in this population and 10 corresponded to the QTLs previously reported based on QTL mapping. The identification of QTLs for traits in local populations including elite varieties may lead to a better understanding of the genetic bases of elite traits. This is of direct relevance for plant breeding programs in local regions.     

Confirming a major QTL and finding additional loci responsible for field resistance to brown spot (Bipolaris oryzae) in rice

Brown spot is a devastating rice disease. Quantitative resistance has been observed in local varieties (e.g., ‘Tadukan’), but no economically useful resistant variety has been bred. Using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) from ‘Tadukan’ (resistant) × ‘Hinohikari’ (susceptible), we previously found three QTLs (qBS2, qBS9, and qBS11) that conferred resistance in seedlings in a greenhouse. To confirm their effect, the parents and later generations of RILs were transplanted into paddy fields where brown spot severely occurred. Three new resistance QTLs (qBSfR1, qBSfR4, and qBSfR11) were detected on chromosomes 1, 4, and 11, respectively. The ‘Tadukan’ alleles at qBSfR1 and qBSfR11 and the ‘Hinohikari’ allele at qBSfR4 increased resistance. The major QTL qBSfR11 coincided with qBS11 from the previous study, whereas qBSfR1 and qBSfR4 were new but neither qBS2 nor qBS9 were detected. To verify the qBSfR1 and qBSfR11 ‘Tadukan’ resistance alleles, near-isogenic lines (NILs) with one or both QTLs in a susceptible background (‘Koshihikari’) were evaluated under field conditions. NILs with qBSfR11 acquired significant field resistance; those with qBSfR1 did not. This confirms the effectiveness of qBSfR11. Genetic markers flanking qBSfR11 will be powerful tools for marker-assisted selection to improve brown spot resistance.     

QTL analysis on rice grain appearance quality,as exemplifying the typical events of transgenic or backcrossing breeding

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F_9–10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.     

Identification of QTLs controlling rice drought tolerance at seedling stage in hydroponic culture

‘Drought avoidance’ and ‘drought tolerance’ are two mechanisms by which plants adapt under water stress. These mechanisms are difficult to evaluate separately in field experiments. Using hydroponic culture, we studied the genetic control of drought tolerance in rice (Oryza sativa L.) without the effect of drought avoidance. A backcross inbred population of ‘Akihikari’ (lowland cultivar) × ‘IRAT109’ (upland cultivar) with 106 lines was cultured with (stressed condition) and without (non-stressed condition) polyethylene glycol (PEG) at seedling stage. The relative growth rate (RGR), specific water use (SWU), and water use efficiency (WUE) showed significant genotype × environment interactions with or without PEG, indicating that each line responded differently to water stress. A quantitative trait locus (QTL) analysis revealed that these interactions were QTL specific. A total of three QTLs on chromosomes 2, 4, and 7 were detected for RGR. The QTL on chromosome 7 had a constant effect across environments, while the QTL on chromosome 4 had an effect only under non-stressed condition and that on chromosome 2 only under stressed condition. The stress-specific QTL on chromosome 2 was not co-located with any QTLs for root system depth previously reported from the same mapping population. However, this QTL was co-located with a stress-specific QTL for SWU, suggesting that the control of transpiration was relevant to dry matter production under drought. We concluded that PEG-treated hydroponic culture is very effective for use in genetic analyses of drought tolerance at seedling stage.     

Cold tolerance at the early growth stage in wild and cultivated rice

The present study was conducted to understand the pattern of variation and the genetic bases for cold tolerance at the early growth stage in Asian rice. The genetic variation was investigated at the germination, plumule and seedling stages among 57 strains including cultivated rice (Oryza sativa ssp. indica and ssp. japonica) and its wild progenitor (Oryza rufipogon). The significant differentiation of cold tolerance was observed among the taxonomically divided groups. At the germination stage, both indica and japonica subspecies tended to be more tolerant than O. rufipogon, whereas at the plumule and seedling stages, ssp. japonica tended to be more tolerant than ssp. indica and O. rufipogon. Furthermore, in cold tolerance at the plumule stage, the clinal variation across the latitude of origins was observed within O. rufipogon and ssp. japonica, suggesting that the current pattern of variation seems to have been shaped by both their phylogenetic histories and on-going adaptation to the local environments. QTL analysis between O. sativa ssp. japonica (tolerant) and O. rufipogon (susceptible) revealed five putative QTLs for cold tolerance at the plumule and seedling stages but not at the germination stage. Substitution mapping was also carried out to precisely locate the two major QTLs for cold tolerance at the plumule stage, which could be used for improvement of tolerance to cold stress in ssp. indica.     

Mapping and validation of QTLs for rice sheath blight resistance

Sheath blight, caused by Rhizoctonia solani, is one of the most serious diseases of rice. Among 33 rice accessions, mainly from National Institute of Agrobiological Sciences (NIAS) Core Collection, we found three landraces from the Himalayas—Jarjan, Nepal 555 and Nepal 8—with resistance to sheath blight in 3 years’ field testing. Backcrossed inbred lines (BILs) derived from a cross between Jarjan and the leading Japanese cultivar Koshihikari were used in QTL analyses. Since later-heading lines show fewer lesions, we used only earlier-heading BILs to avoid association with heading date. We detected eight QTLs; the Jarjan allele of three of these increased resistance. Only one QTL, on chromosome 9 (between markers Nag08KK18184 and Nag08KK18871), was detected in all 3 years. Chromosome segment substitution lines (CSSLs) carrying it showed resistance in field tests. Thirty F₂ lines derived from a cross between Koshihikari and one CSSL supported the QTL.     

Feasibility of selection for traits associated with cold tolerance in rice under rapid generation advance method

Summary Variability of the hybrid population can be preserved through generations during the process of rapid generation advance (RGA). Effective selection can be done in an early generation for those traits having high heritability value like growth duration and plant height. But selection is not advantageous for traits having low heritability. Thus, selection for panicle exsertion, cold tolerance at seedling stage and panicle length would not be wise during the early generations. However, in the low temperature areas growth duration and plant height are two important factors which determine varietal suitability. As selection of these two characters are possible through RGA, it should be a standard parctice during RGA to eliminate the plants with long growth duration and select plants with optimum plant height.     

A major QTL associated with cold tolerance at seedling stage in rice (Oryza sativa L.)

Chilling injury is one of the most important limiting factors affecting rice production in temperate and high-elevation areas. In this study, 146 microsatellite markers were employed to identify quantitative trait loci (QTL) conferring cold tolerance at seedling stage (CTS) .The mapping population consisted of 193 doubled haploid (DH) lines, which derived from a cross between a cold-tolerant japonica variety (AAV002863) and a cold-sensitive indica cultivar (Zhenshan97B). Tolerance to cold was assessed by the survival percentage of seedlings after cold treatment. In a climate chamber, after treatment at 6°C/10°C for 7 d, the measurement was taken on the sixth day of the recovery stage at room temperature. The phenotypic distribution of the DH population approximately fitted normality with skewness and kurtosis less than 0.3, and the difference among the three repetitions was not significant. Five main effect QTLs were identified with LOD > 4.0 on chromosomes 1, 2, 8 using a composite interval mapping approach. The accumulated contribution of the five QTLs was 62.28%, and a major QTL (LOD = 15.09) was identified on chromosome 2 flanked by RM561 and RM341, which explained 27.42% of the total phenotypic variation. Four significant epistatic interactions were also detected with a total contribution of 20.14%. Liang Chen and Qiaojun Lou had made the equal contribution for the research.     

Variation in cooking and eating quality traits in Japanese rice germplasm accessions

The eating quality of cooked rice is important and determines its market price and consumer acceptance. To comprehensively describe the variation of eating quality in 183 rice germplasm accessions, we evaluated 33 eating-quality traits including amylose and protein contents, pasting properties of rice flour, and texture of cooked rice grains. All eating-quality traits varied widely in the germplasm accessions. Principal-components analysis (PCA) revealed that allelic differences in the Wx gene explained the largest proportion of phenotypic variation of the eating-quality traits. In 146 accessions of non-glutinous temperate japonica rice, PCA revealed that protein content and surface texture of the cooked rice grains significantly explained phenotypic variations of the eating-quality traits. An allelic difference based on simple sequence repeats, which was located near a quantitative trait locus (QTL) on the short arm of chromosome 3, was associated with differences in the eating quality of non-glutinous temperate japonica rice. These results suggest that eating quality is controlled by genetic factors, including the Wx gene and the QTL on chromosome 3, in Japanese rice accessions. These genetic factors have been consciously selected for eating quality during rice breeding programs in Japan.     

Cytoplasmically controlled cold tolerance in a cytoplasmic-genetic male sterile line of rice

Summary The cytoplasmic-genetic male sterile line, Krishna-A with Kalinga-I cytoplasm was developed in rice through repeated backcrossing the completely pollen sterile F₁ of the cross Kalinga-I/Krishna with the recurrent male parent cv. Krishna. The germination percentage and root-shoot length in Krishna-A at 12°C and 9°C indicated a higher degree of tolerance to low temperature than the cold tolerant female parent Kalinga-I. The high yielding cold tolerant variety Kalinga-I was developed from a cross involving the cold tolerant variety Dunghansali and the high yielding variety IR 8. It is inferred that tolerance to low temperature during germination in Krishna-A was inherited from the cytoplasm of cv. Dunghansali through Kalinga-I besides male sterility. The cytoplasmic control of cold tolerance is reported here for the first time in rice literature. The male sterile line Krishna-A because of its tolerance to low temperature would be suitable for the development of hybrid rice especially for areas where low temperature is a problem during germination.     

Identification of qRL7, a major quantitative trait locus associated with rice root length in hydroponic conditions

Root system development is an important target for improving yield in rice. Active roots that can take up nutrients more efficiently are essential for improving grain yield. In this study, we performed quantitative trait locus (QTL) analyses using 215 recombinant inbred lines derived from a cross between Xieqingzao B (XB), a maintainer line with short roots and R9308, a restorer line with long roots. Only a QTLs associated with root length were mapped on chromosomes 7. The QTL, named qRL7, was located between markers RM3859 and RM214 on chromosome 7 and explained 18.14–18.36% of the total phenotypic variance evaluated across two years. Fine mapping of qRL7 using eight BC₃F₃ recombinant lines mapped the QTL to between markers InDel11 and InDel17, which delimit a 657.35 kb interval in the reference cultivar Nipponbare. To determine the genotype classes for the target QTL in these BC₃F₃ recombinants, the root lengths of their BC₃F₄ progeny were investigated, and the result showed that qRL7 plays a crucial role in root length. The results of this study will increase our understanding of the genetic factors controlling root architecture, which will help rice breeders to breed varieties with deep, strong and vigorous root systems.     

Variation in heading date conceals quantitative trait loci for other traits of importance in breeding selection of rice

To identify quantitative trait loci (QTLs) associated with the primary target traits for selection in practical rice breeding programs, backcross inbred lines (BILs) derived from crosses between temperate japonica rice cultivars Nipponbare and Koshihikari were evaluated for 50 agronomic traits at six experimental fields located throughout Japan. Thirty-three of the 50 traits were significantly correlated with heading date. Using a linkage map including 647 single-nucleotide polymorphisms (SNPs), a total of 122 QTLs for 38 traits were mapped on all rice chromosomes except chromosomes 5 and 9. Fifty-eight of the 122 QTLs were detected near the heading date QTLs Hd16 and Hd17 and the remaining 64 QTLs were found in other chromosome regions. QTL analysis of 51 BILs having homozygous for the Koshihikari chromosome segments around Hd16 and Hd17 allowed us to detect 40 QTLs associated with 27 traits; 23 of these QTLs had not been detected in the original analysis. Among the 97 QTLs for the 30 traits measured in multiple environments, the genotype-by-environment interaction was significant for 44 QTLs and not significant for 53 QTLs. These results led us to propose a new selection strategy to improve agronomic performance in temperate japonica rice cultivars.     

基于高密度SNP遗传图谱的粳稻芽期耐低温QTL鉴定

水稻直播由于省时、省工和节约成本而备受农户关注。然而,芽期耐冷性不强致使现行推广的许多优良水稻品种不适于直播生产。因此,挖掘鉴定芽期耐冷位点,为后续的辅助育种提供基因资源就日益受到重视。本研究利用丽江新团黑谷和沈农265构建的重组自交系群体及其重测序构建的包含2818个bin标记的遗传图谱对水稻芽期的耐冷性进行QTL定位分析。共检测到5个芽期耐冷QTL,分布在水稻的1号、3号、9号和11号染色体上,增效等位基因均来自耐冷亲本丽江新团黑谷。这些QTL的LOD值的范围从3.05到24.01,表型贡献率为8.0%～53.5%。其中,表型贡献率最大的主效QTL是qCTB11b,位于11号染色体长臂端的21.24 Mb～22.03 Mb之间,物理图谱区间为790 kb。随后利用"选择作图"的策略进行了QTL验证和累加效应分析,明确了可以通过QTL的累加聚合实现芽期耐冷能力的遗传改良,聚合的增效QTL越多,耐冷能力提升越明显。上述研究结果不仅可以增强人们对芽期水稻耐冷能力遗传基础的认识和理解,也可以为后续直播品种的遗传改良提供理论依据和技术指导。     

Combination of stem elongation ability with submergence tolerance in rice

Summary A study was conducted with six F₂ populations to test the possibility of combining submergence tolerance and stem elongation ability into a single genotype of rice (Oryza sativa L.). Submergence tolerance and stem elongation ability could be combined in the same genotype if strongly submergence tolerant genes are present in submergence tolerant parents.