Identification of quantitative trait loci for seedling cold tolerance using RILs derived from a cross between japonica and tropical japonica rice cultivars

Cold tolerance at the seedling stage of rice is an important phenotypic trait that causes normal plant growth and stable rice production in temperate regions as well as tropical high-lands in Asia and Africa. In order to find quantitative trait loci (QTLs)/genes associated with cold tolerance, we constructed a linkage map using 153 recombinant inbred lines (RILs) derived from a cross between a cold-tolerant temperate japonica cultivar, Geumobyeo, and a cold-sensitive tropical japonica breeding line, IR66160-121-4-4-2. The RILs were phenotyped for cold tolerance or sensitivity based on the degrees of cold tolerance as cold tolerance indices at the seedling stage. The seedlings for cold-tolerance/-sensitive traits were scored on the 7th day of the recovery period at 25°C after cold treatment at 10°C. Two QTLs (qCTS4a and qCTS4b) associated with cold tolerance at the seedling stage were identified on the long and short arms of chromosome 4 with an LOD score of 2.89 and 2.75, respectively, using composite interval mapping. The QTLs were flanked by simple sequence repeat (SSR) markers RM3648-RM2799 and RM3375a-RM558 that explained 8.3 and 7.8% of the total phenotypic variation, respectively. Seven of the selected RILs expressed cold tolerance at both the seedling and reproductive stages. The SSR markers associated with the QTLs will be useful for tracking favorable QTLs/genes into cold-sensitive elite cultivars and may have potential for pyramiding different QTLs for the improvement of cold tolerance in rice.     

Identification of QTLs controlling low-temperature germinability and cold tolerance at the seedling stage in rice (<Emphasis Type="Italic">Oryza Sativa</Emphasis> L.)

Both low-temperature germinability (LTG) and cold tolerance at the seedling stage (CTS) are important traits for rice. In this study, a rice population of recombinant inbred lines (RILs), derived from the backcross population of a cross between Dongnong422 and Kongyu131, was developed to detect quantitative trait loci (QTL) affecting LTG and CTS by using seed of different storage times. Correlation analysis indicated that there was no significant relationship between LTG and CTS, suggesting that cold tolerance might be genetic differences for LTG and CTS. In total, Twelve and twenty-three major QTLs were detected for LTG and CTS, respectively, which could explain greater than 10% of the phenotypical variation. Eight (qCG12-1, qGI12-1, qGV9-1, qMLIT12-1, qPV6-1, qMDG12-1, qLDWcold10-1, qLFWcold10-1) significant QTLs were mapped for different storage time, it concluded that such QTLs were not affected by environment (storage time) and were closely related QTLs to cold tolerance. One or more QTLs were identified for each trait with some of these QTLs co-locating, qMLIT7-1, qCG7-1, and qGI7-1 for LTG, qLFWcold10-1, and qLDWcold10-1 for CTS with contributions over 15% were mapped common marker interval, respectively, co-location of QTLs for different traits can be an indication that a locus has pleiotropic effects on multiple traits due to a common mechanistic basis. Two lines, RIL128 and RIL73, might be valuable to improve the LTG and CTS through a combination of crosses. The identified QTLs might be applicable to improve the rice cold tolerance by the marker-assisted selection approach.     

Analysis of decision-making coefficients of the lint yield of upland cotton (Gossypium hirsutum L.)

Cold water irrigation and growth in low temperature phytotron are two commonly used methods to evaluate cold tolerance of rice at the seedling stage and the cold sensitive seedlings exhibit different injury symptoms, respectively. However, so far no one has systematically dissected the differences of cold tolerance at seedling stage in rice under the two cold environments. We used a recombinant inbred line (RIL) population derived from a cross of a cold-tolerant japonica cultivar, Lijiangxintuanheigu and a cold sensitive indica cultivar, Sanhuangzhan-2 for this study. The cold sensitive seedlings exhibited leaf yellowing after cold water irrigation and leaf rolling during growth in the low temperature phytotron. Leaf yellowing and leaf rolling in RILs was significantly correlated, but the correlation coefficient was low. A total of four quantitative trait locus (QTLs) on chromosomes 1, 6, 9 and 12 were detected using leaf yellowing and percent seedling survival as indicators of cold tolerance after cold water irrigation, while five QTLs on chromosomes 7, 8, 9, 11 and 12 were detected using leaf rolling and percent seedling survival as indicators of cold tolerance during growth in the low temperature phytotron. The two QTLs, qCTS-9 and qCTS-12 were detected using different evaluation indicators under the two cold environments. Our results suggest that rice cold tolerance mechanisms at the seedling stage differ between the two environments, but the detection of common QTL implies the existence of overlap in the metabolic pathways for cold tolerance. The two common QTLs have potential value in rice breeding.     

Deployment of cold tolerance loci from Oryza sativa ssp. Japonica cv. ‘Nipponbare’ in a high‐yielding Indica rice cultivar ‘93‐11’

Cold tolerance is a complex trait, and QTL pyramiding is required for rice breeding. In this study, a total of seven QTLs for cold tolerance in the Japonica rice variety ‘Nipponbare’ were identified in an F_2:3 population. A stably inherited major QTL, called qCTS11, was detected in the region adjacent to the centromere of chromosome 11. In a near‐isogenic line population, the QTL was further dissected into two linked loci, qCTS11.1 and qCTS11.2. Both of the homozygous alleles of qCTS11.1 and qCTS11.2 from ‘Nipponbare’ showed major positive effects on cold tolerance. Through pyramiding the linked QTLs in the cold‐sensitive Indica rice cultivar ‘93‐11’, we have developed a new elite, high‐yielding Indica variety with cold tolerance.     

水稻芽性状耐冷性的QTL分析

低温发芽和芽期耐冷性是影响水稻芽生长发育的两个重要因素.本试验利用182个越光(粳型)/Kasalath(籼型)//越光回交重组自交株系(backcross recombinant inbred lines,BILs),对2个芽性状耐冷性(低温发芽和芽期耐冷性)进行QTL(quantitative trait loci)定位和相关性分析.结果表明,BIL群体中这2个芽性状耐冷性均呈连续分布,属于数量性状遗传,两性状间的相关性不显著;控制低温发芽的4个QTL分布于第1、7、9、11染色体上,其贡献率为6.72～12.78%;芽期耐冷性相关的4个QTL分布在第4、6(2个QTL)和11染色体上,贡献率为6.61%～14.93%;其中第11染色体上的2个QTL位于相同区域内,并且其增强耐冷性等位基因均来自耐冷性较差亲本Kasalath.本研究结果及其中检测到的QTLs两侧的连锁分子标记可为水稻芽性状耐冷性分子育种提供理论依据.     

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.     

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.     

一个水稻苗期耐冷性的主效QTL精细定位研究

苗期耐冷性是影响水稻生长发育的重要因素之一。此实验以低温导致叶片卷曲的卷曲度作为水稻苗期耐冷性指标,采用182个越光（粳型）/kasalath（籼型）//越光回交重组自交系（backcross recombinant inbred lines,BILs）和162个RFLP分子标记,对苗期耐冷性进行QTL（quantitative trait loci,QTL）定位分析。结果表明,BIL群体中苗期耐冷性均呈连续分布,属于数量性状遗传,并检测到4个控制苗期耐冷性的QTL,分布在第1、3、11、12染色体上,其贡献率为7.4%～21.9%,所有能增强耐冷性等位基因均来自越光;并在其目标区域内进一步设计分子引物把位于第12染色体上的主效qCTS-12定位在约77kb区域内。此研究结果及其检测到的QTLs两侧的连锁分子标记可为水稻苗期耐冷性分子育种以及相关基因克隆提供理论依据。     

水稻耐冷性遗传及基因定位研究进展

综述了水稻芽期、苗期、孕穗期和开花灌浆期耐冷性的遗传研究概况和耐冷性基因定位的研究进展,并对今后水稻耐冷性研究提出了建议和展望。     

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.     

Quantitative trait loci controlling rice seed germination under salt stress

Salt tolerance of rice (Oryza sativa L.) at the seed germination stage is one of the major determinants for the stable stand establishment in salinity soil. One population of recombinant inbred lines (RILs, F_2:9), derived from a cross between a japonica rice landrace tolerant to salt stress and a sensitive indica rice variety, was used to determine the germination traits including imbibition rate and germination percentage under control (water) and salt stress (100 mM NaCl) for 10 days at 30 °C. The multiple interval mapping (MIM) were applied to conduct QTL for the traits. The results showed that seed germination was a quantitative trait controlled by several genes, and strongly affected by salt stress. A total of 16 QTLs were detected in this study, and each QTL could explain 4.6–43.7% of the total phenotypic variance. The expression of these QTLs might be developmentally regulated and growth stage-specific. In addition, only one digenic interaction was detected under salt stress, showing small effect on germination percentage with R² 2.7%. Among sixteen QTLs detected in this study, four were major QTLs with R² > 30%, and some novel alleles of salt tolerance genes in rice. The results demonstrated that the japonica rice Jiucaiqing is a good source of gene(s) for salt tolerance and the major or minor QTLs identified could be used to improve the salt tolerance by marker-assisted selection (MAS) in rice.     

High resolution genotyping by restriction enzyme-phased sequencing of advanced backcross lines of rice exhibiting differential cold stress recovery

Advanced backcross rice lines MIb 4853-9 and 6885-2 harbor major seedling cold tolerance QTL qCTS4 and qCTS12 from the temperate japonica M202 in the genetic background of the indica IR50. Previous studies have shown that these lines exhibit the same tolerance, based on visual ratings, under constant and intermittent exposure to cold stress. However, some differences between the MIb lines with regard to physiological stress indicators are observed under constant exposure and a clear difference is detected using a cold stress recovery assay. Genotyping with over 120 SSR markers did not detect any polymorphisms. Here we report using a reduced representation sequencing method called Restriction Enzyme Site Comparative Analysis to identify M202 introgressions unique to each MIb line as candidate loci for the cold stress recovery trait.     

Identification of QTLs associated with heat tolerance at the heading and flowering stage in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The ongoing rise in temperatures caused by global climate change is a critical climatic risk factor for rice production, and enhancing rice heat tolerance is an area of particular research interest. A recombinant inbred line (RIL) mapping population was developed from heat sensitive, rice cultivar IAPAR-9 crossed with heat tolerant, Liaoyan241. RIL and parental lines were exposed to high temperature at the heating and flowering stage in experiments in 2014 and 2015. As indicators of heat tolerance, the seed setting rate under natural (NS) and heat stress (HTS) conditions were measured, and the reduction rate of seed set (RRS) was calculated. Quantitative trait loci (QTL) analysis revealed eleven heat tolerance QTLs located on chromosomes 1, 3, 4, 5, and 6. Single QTL contribution rates were 4.75–13.81% and effect values were ? 5.98 to 5.00. Four major QTLs (qNS1, qNS4, qNS6, and qRRS1) were stable detected in different environments in both years. Thirteen QTLs with epistatic interactions and nine QTLs with environmental interactions were also detected. Major QTLs were all involved in epistatic and environmental interactions. Three QTLs from the SSR marker interval RM471 to RM177 region of chromosome 4 (qNS4, qHTS4, and qRRS4) were all involved in epistatic and environmental interactions and contributed to phenotypic variation, indicating that this region constituted a major QTL hotspot. The major QTL for heat tolerance identified in this study will aid in breeding tolerant cultivars and facilitating investigation of the molecular underpinnings of heat tolerance in rice.     

Identification of Quantitative Trait Loci Associated with Aluminum Tolerance in Rice (Oryza Sativa L.)

Summary Quantitative trait loci (QTL) analysis for Al tolerance was performed in rice using a mapping population of 98 BC₁F₁₀ lines (backcross inbred lines: BILs), derived from a cross of Al-tolerant cultivar of rice (Oryza sativa L. cv. Nipponbare) and Al-sensitive cultivar (cv. Kasalath). Three characters related to Al tolerance, including root elongation under non-stress conditions (CRE), root elongation under Al stress (SRE) and the relative root elongation (RRE) under Al stress versus non-stress conditions, were evaluated for the BILs and the parents at seedling stage. A total of seven QTLs for the three traits were identified. Among them, three putative QTLs for CRE (qCRE-6, qCRE-8 and qCRE-9) were mapped on chromosomes 6, 8 and 9, respectively. One QTL for SRE (qSRE-4) was identified on chromosome 4. Three QTLs (qRRE-5, qRRE-9 and qRRE-10) for RRE were detected on chromosomes 5, 9, 10 and accounted for 9.7–11.8% of total phenotypic variation. Interestingly, the QTL qRRE-5 appears to be syntenic with the genomic region carrying a major Al tolerance gene on chromosome 6 of maize. Another QTL, qRRE-9, appears to be similar among different rice populations, while qRRE-10 is unique in the BIL population. The common QTLs for CRE and RRE indicate that candidate genes conferring Al tolerance in the rice chromosome 9 may be associated with root growth rates. The existence of QTLs for Al tolerance was confirmed in substitution lines for corresponding chromosomal segments. These results also provide the possibilities of enhancing Al tolerance in rice through using marker-assisted selection (MAS) and pyramiding QTLs.     

Mapping of QTLs associated with cadmium tolerance and accumulation during seedling stage in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Cadmium (Cd) is a non-essential element and toxic to plants. To investigate the genetics of Cd tolerance and accumulation in rice, quantitative trait loci (QTL) associated with Cd tolerance and accumulation at the seedling stage were mapped using a doubled haploid (DH) population derived from a cross between a japonica JX17 and an indica ZYQ8. A total of 22 QTLs were found to be associated with shoot height (SH), root length (RL), shoot dry weight (SDW), root dry weight (RDW), total dry weight (TDW) and chlorophyll content (CC), and 10 and 12 QTLs were identified under the control and Cd stress conditions, respectively. For Cd tolerant coefficient (CTC), 6 QTLs were detected on chromosomes 1, 3, 5, 8 and 10. Under Cd stress, 3 QTLs controlling root and shoot Cd concentrations were mapped on chromosome 6 and 7. One QTL for shoot/root rate of Cd concentration was identified on chromosome 3. The results indicated that Cd tolerance and accumulation were quantitatively inherited, and the detected QTLs may be useful for marker-assistant selection (MAS) and identification of the genes controlling Cd tolerance and accumulation in rice.     

Mapping quantitative trait loci for salt tolerance at germination and the seedling stage in barley (Hordeum vulgare L.)

Quantitative trait loci (QTLs) controlling salt tolerance at germination and the seedling stage in barley (Hordeum vulgare L.) were identified by interval mapping analysis using marker information from two doubled haploid (DH) populations derived from the crosses, Steptoe/Morex and Harrington/TR306. Interval mapping analysis revealed that the QTLs for salt tolerance at germination in the DH lines of Steptoe/Morex were located on chromosomes 4 (4H), 6(6H), and 7(5H), and in the DH lines of Harrington/TR306 on chromosomes 5(1H) and 7(5H). In both DH populations, the most effective QTLs were found at different loci on chromosome 7(5H). Genetic linkage between salt tolerance at germination and abscisic acid (ABA) response was found from QTL mapping. The QTLs for the most effective ABA response at germination were located very close to those for salt tolerance on chromosome 7 (5H) in both crosses. The QTLs for salt tolerance at the seedling stage were located on chromosomes 2(2H), 5(1H), 6(6H), and 7(5H) in the DH lines of Steptoe/Morex, and on chromosome 7(5H) in the DH lines of Harrington/TR 306. Their positions were different from those of QTLs controlling salt tolerance at germination, indicating that salt tolerance at germination and at the seedling stage were controlled by different loci. This revised version was published online in July 2006 with corrections to the Cover Date.     

水稻芽期与幼苗前期耐碱性状QTL定位

利用包含120个株系的籼粳交来源(春江06/TN1)的加倍单倍体群体, 在Na₂CO₃胁迫下, 以发芽期和幼苗前期的相对发芽势等10个性状作为耐碱性评价指标, 进行水稻耐碱性的QTL定位。相关性分析表明, 相对发芽势和相对发芽率显著正相关, 相对苗高、相对根数和相对根长之间显著正相关。采用QTLNetwork统计软件共定位到14个加性QTL和13个上位性QTL。在第3染色体RM251~RM3280间有2个QTL, 在第7染色体RM3286~RM1279区域有3个QTL; 在第1、2和7染色体同一位置同时检测到2个上位性QTL, 在第12染色体RM1246~RM5199之间集中了4个上位性QTL, 耐碱数量基因表现出一因多效或紧密连锁现象。耐碱性盐QTL可能包括两类, 一类与K+、Na+等离子胁迫有关, 另一类与高pH胁迫有关。不同类型的水稻品种都具有一些耐碱基因, 可以通过有性杂交和分子标记辅助选择的方法选育优良的耐碱品种。     

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。     

Identification of new QTL for salt tolerance from rice variety Pokkali

Salt stress is an ever-present threat to rice production worldwide. Rice salinity tolerance is complex, both genetically and physiologically. The success and effectiveness in selecting salt-tolerant rice variety require the identification of QTL for the tolerance and closely linked molecular markers. In the present study, a RIL population consisting of 148 lines, derived from a cross between IR29 (salt-sensitive) and Pokkali (salt-tolerant), was used to identify new QTL for salt tolerance and investigate the relationships between salt stress caused injury and the changes in different physiological and morphological traits at the seedling stage. 14,470 high-quality SNP markers generated by the Rice 56K SNP array were converted to 1,467 bin markers for linkage mapping. A high-density genetic linkage map covering 1,680.9 cM was constructed, with the physical to genetic distance ratio being 222 Kb/cM. In total, 23 QTL for different salt tolerance indices were identified, including the previously reported Saltol which is currently used in breeding programmes. Three QTL for salt injury score (SIS) were located on chromosomes 1, 4 and 12, all being closely related to the long-distant Na⁺ transport from roots to shoots. These QTL showed additive effects, thus can be effectively used in breeding programme to pyramid various tolerance genes.     

超级杂交稻‘Y两优2号’耐冷性QTL定位与杂种优势分析

挖掘耐冷基因并提高耐冷性对于保证水稻在气候变化条件下的高产稳产具有至关重要的意义。本研究利用‘远恢2号’和‘Y58S’杂交而成的超级杂交稻‘Y两优2号’的高世代重组自交系(RIL F14)276个家系作为作图群体,以SNP为分子标记构建了高密度遗传图谱,对水稻的芽期耐冷性(cold tolerance at the bud bursting period,CTBP)性状进行数量性状位点(quantitative trait locus,QTL)定位分析;同时对全世界范围内收集的水稻自然变异微核心(Minicore)种质群体进行芽期耐冷性全基因组关联分析(genome-wide association study,GWAS)。结果表明,水稻芽期耐冷性在水稻群体内呈连续分布,是由多基因控制的数量性状。同时,在RIL群体的第9号染色体上定位到1个与耐冷性性状相关的QTL,位于区间Block73479和Block72824之间,对表型变异的解释率为9.65%。进一步分析表明该QTL对水稻芽期耐冷性为负显性。结果还显示‘Y两优2号’耐冷性显著强于亲本,具有杂种优势。