QTL pyramiding for improving of cold tolerance at fertilization stage in rice

Vigorous cold tolerance at the fertilization stage (CTF) is a very important characteristic for stable rice production in cold temperature conditions. Because CTF is a quantitatively inherited trait, pyramiding quantitative trait loci (QTLs) using marker-assisted selection (MAS) is effective for improving CTF levels in rice breeding programs. We previously identified three QTLs controlling CTF, qCTF7, qCTF8 and qCTF12, using backcrossed inbred lines derived from a cross between rice cultivar Eikei88223 (vigorous CTF) and Suisei (very weak CTF). However, pyramiding of these QTLs for the application of MAS in practical rice breeding programs have not yet been elucidated. In this study, we examined the effect of pyramiding QTLs for improvement of CTF level using eight possible genotype classes from the 152 F₃ population derived from a cross between Eikei88223 and Suisei. Increasing of CTF levels in combinations between qCTF7 and qCTF12 and between qCTF8 and qCTF12 were detected. Furthermore, we compared the haplotype pattern around the QTLs for CTF among the rice cultivars from Hokkaido. These results are useful for improvement of new cultivars with high CTF levels using MAS and identification of genetic resources with the novel QTL(s) for CTF.     

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.     

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.     

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.     

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.     

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.     

Genetic structure and association mapping of cold tolerance in improved japonica rice germplasm at the booting stage

Cold tolerance at booting stage is one of the major determinants for a stable yield of rice (Oryza sativa L.) in many high elevation or high latitude regions. Understanding the genetic basis of cold tolerance is crucial for the improvement of cold tolerance through breeding. In this study, association mapping was performed in 347 rice accessions worldwide with different statistical models in order to identify the genetic marker loci/QTL associated with cold tolerance traits at the booting stage. The evaluation of cold tolerance for all the traits was conducted under natural low temperature in Yunnan and cold water irrigation in Jilin. The 148 SSRs were used for the genotyping. Population structure analysis identified three main subpopulations for the accessions that corresponded to major geographic origins. The relative kinship analysis revealed a weak or no relationship for most of the individual pairs. Model comparisons indicated that the Q+K model controlling both population structure (Q) and the relative kinship (K) was performed better than other models in association mapping. In total, 24 markers were identified that were significantly associated with cold tolerance, including five markers in Yunnan and 19 markers in Jilin. Moreover, RM282, RM252, RM335 and RM6824 were identified in multiple environments or years. Many of these identified markers were located either in or nearby the regions where the QTLs have been reported for cold tolerance at booting stage. These results highlighted the targeted regions for future studies and might be subsequently used in breeding programs to trace and select the useful alleles by MAS.     

Identification of quantitative trait loci for cold tolerance during the germination and seedling stages in rice (Oryza sativa L.)

Low temperature is a serious abiotic stress affecting rice production in subtropical and temperate areas. In this study, cold tolerance of rice at the germination and seedling stages were evaluated using one recombinant inbred line (RIL) population derived from a cross between Daguandao (japonica), with highly cold-tolerant at the seedling stage, and IR28 (indica), with more cold-tolerant at the germination stage, and the quantitative trait loci (QTL) mapping was conducted using the multiple interval mapping (MIM) approach. Continuous segregation in low temperature germinability (LTG) and cold tolerance at the seedling stage (CTS) were observed among the RIL populations. Most RILs were moderately susceptible or tolerant at the germination stage, but were susceptible at the seedling stage. No significant relationship was found in cold tolerance between the germination and seedling stages. A total of seven QTLs were identified with limit of detection (LOD) >3.0 on chromosomes 3, 8, 11 and 12, and the amount of variation (R ²) explained by each QTL ranged from 5.5 to 22.4%. The rice LTG might be regulated by two minor QTLs, with the CTS controlled by one major QTL [qCTS8.1 (LOD = 16.1, R ² = 22.4%)] and several minor loci. Among these loci, one simultaneously controls LTG (qLTG11.1) and CTS (qCTS11.1). Several cold-tolerance-related QTLs identified in previous studies were found to be near the QTLs detected here, and three QTLs are novel alleles. The alleles from Daguandao at six QTLs increased cold tolerance and could be good sources of genes for cold tolerance. In addition, only one digenic interaction was detected for CTS, with a R ² value of 6.4%. Those major or minor QTLs could be used to significantly improve cold tolerance by marker-assisted selection (MAS) in rice.     

Mapping and use of QTLs controlling pod dehiscence in soybean

While the cultivated soybean, Glycine max (L.) Merr., is more recalcitrant to pod dehiscence (shattering-resistant) than wild soybean, Glycine soja Sieb. & Zucc., there is also significant genetic variation in shattering resistance among cultivated soybean cultivars. To reveal the genetic basis and develop DNA markers for pod dehiscence, several research groups have conducted quantitative trait locus (QTL) analysis using segregated populations derived from crosses between G. max accessions or between a G. max and G. soja accession. In the populations of G. max, a major QTL was repeatedly identified near SSR marker Sat_366 on linkage group J (chromosome 16). Minor QTLs were also detected in several studies, although less commonality was found for the magnitudes of effect and location. In G. max × G. soja populations, only QTLs with a relatively small effect were detected. The major QTL found in G. max was further fine-mapped, leading to the development of specific markers for the shattering resistance allele at this locus. The markers were used in a breeding program, resulting in the production of near-isogenic lines with shattering resistance and genetic backgrounds of Japanese elite cultivars. The markers and lines developed will hopefully contribute to the rapid production of a variety of shattering-resistant soybean 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。     

Detection of QTLs for salt tolerance in Asian barley (Hordeum vulgare L.) by association analysis with SNP markers

Two hundred ninety-six Asian barley (Hordeum vulgare L.) accessions were assessed to detect QTLs underlying salt tolerance by association analysis using a 384 single nucleotide polymorphism (SNP) marker system. The experiment was laid out at the seedling stage in a hydroponic solution under control and 250 mM NaCl solution with three replications of four plants each. Salt tolerance was assessed by leaf injury score (LIS) and salt tolerance indices (STIs) of the number of leaves (NL), shoot length (SL), root length (RL), shoot dry weight (SDW) and root dry weight (RDW). LIS was scored from 1 to 5 according to the severity of necrosis and chlorosis observed on leaves. There was a wide variation in salt tolerance among Asian barley accessions. LIS and STI (SDW) were the most suitable traits for screening salt tolerance. Association was estimated between markers and traits to detect QTLs for LIS and STI (SDW). Seven significant QTLs were located on chromosomes 1H (2 QTLs), 2H (2 QTLs), 3H (1 QTL), 4H (1 QTL) and 5H (1 QTL). Five QTLs were associated with LIS and 2 QTLs with STI (SDW). Two QTLs associated with LIS were newly identified on chromosomes 3H and 4H.     

Genetic dissection of seed storability using two different populations with a same parent rice cultivar N22

Seed storability in rice (Oryza sativa L.) is an important agronomic trait. Two segregating populations with N22 (indica) as a common parent, viz. a set of 122 backcross-inbred lines (BILs) derived from the backcross Nanjing35 (japonica)/N22//Nanjing35 and another population comprising 189 recombinant inbred lines (RILs) from the cross of USSR5 (japonica) and N22, were studied to detect quantitative trait loci (QTL) controlling seed storability. Germination percentage (GP) was used to evaluate seed storability after aging treated under three different conditions, viz. natural, artificial and combined aging treatments. A total of seven QTLs were identified on chromosomes 1, 2, 5, 6 and 9. Among them, a major QTL, qSSn-9, was common in the two populations. In contrast, four QTLs (qSSnj-2-1, qSSn-2-2, qSSn-5 and qSSn-6) were detected in BILs and the QTL qSSn-1 was identified in RILs, which was a new QTL for seed storability. The N22-derived alleles increased the seed storability at all the loci except qSSnj-2-1. We also investigated the effect of QTLs using five selected lines with high storability from BILs and verified qSSn-5 with a near-isogenic line (NIL). These results provide an opportunity for pyramiding or map-based cloning major QTLs for seed storability in rice.     

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.     

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.     

Mapping three new interspecific hybrid sterile loci between Oryza sativa and O. glaberrima

Hybrid sterility hinders the transfer of useful traits between Oryza sativa and O. glaberrima. In order to further understand the nature of interspecific hybrid sterility between these two species, a strategy of multi-donors was used to elucidate the range of interspecific hybrid sterility in this study. Fifty-nine accessions of O. glaberrima were used as female parents for hybridization with japonica cultivar Dianjingyou 1, after several backcrossings using Dianjingyou 1 as the recurrent parent and 135 BC₆F₁ sterile plants were selected for genotyping and deducing hybrid sterility QTLs. BC₆F₁ plants containing heterozygous target markers were selected and used to raise BC₇F₁ mapping populations for QTL confirmation and as a result, one locus for gamete elimination on chromosome 1 and two loci for pollen sterility on chromosome 4 and 12, which were distinguished from previous reports, were confirmed and designated as S37(t), S38(t) and S39(t), respectively. These results will be valuable for understanding the range of interspecific hybrid sterility, cloning these genes and improving rice breeding through gene introgression.     

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.     

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。     

Identification and validation of a yield-enhancing QTL cluster in rice (Oryza sativa L.)

Improvement of rice grain yield (YD) is an important goal in rice breeding. YD is determined by its related traits such as spikelet fertility (SF), 1,000-grain weight (TGW), and the number of spikelets per panicle (SPP). We previously mapped quantitative trait loci (QTLs) for SPP and TGW using the recombinant inbred lines (RILs) derived from the crosses between Minghui 63 and Teqing. In this study, four QTLs for SF and four QTLs for YD were detected in the RILs. Comparison of the locations of QTLs for these three yield-related traits identified one QTL cluster in the interval between RM3400 and RM3646 on chromosome 3. The QTL cluster contained three QTLs, SPP3a, SF3 and TGW3a, but no YD QTL was located there. To validate the QTL cluster, a BC₄F₂ population was obtained, in which SPP3a, SF3 and TGW3a were simultaneously mapped to the same region. SPP3a, SF3 and TGW3a explained 36.3, 29.5 and 59.0 % of phenotype variance with additive effect of 16.4 spikelets, 6 % SF and 1.8 g grain weight, respectively. In the BC₄F₂ population, though the region has opposite effects on TGW and SPP/SF, a YD QTL YD3 identified in this cluster region can increase 4.6 g grains per plant, which suggests this QTL cluster is a yield-enhancing QTL cluster and can be targeted to improve rice yield by marker aided selection.     

Genetic studies on saline and sodic tolerances in soybean

Salt-affected soils are generally classified into two main categories: saline and sodic (alkaline). Developing and using soybean (Glycine max (L.) Merr) cultivars with high salt tolerance is an effective way of maintaining sustainable production in areas where soybean growth is threatened by salt stress. Early classical genetics studies revealed that saline tolerance was conditioned by a single dominant gene. Recently, a series of studies consistently revealed a major quantitative trait locus (QTL) for saline tolerance located on linkage group N (chromosome 3) around the SSR markers Satt255 and Sat_091; other minor QTLs were also reported. In the case of sodic tolerance, most studies focused on iron deficiency caused by a high soil pH, and several QTLs associated with iron deficiency were identified. A wild soybean (Glycine soja Sieb. & Zucc.) accession with high sodic tolerance was recently identified, and a significant QTL for sodic tolerance was detected on linkage group D2 (chromosome 17). These studies demonstrated that saline and sodic tolerances were controlled by different genes in soybean. DNA markers closely associated with these QTLs can be used for marker-assisted selection to pyramid tolerance genes in soybean for both saline and sodic stresses.     

Evaluation of the effects of five QTL regions on Fusarium head blight resistance and agronomic traits in spring wheat (Triticum aestivum L.)

Fusarium head blight (FHB) is an important disease of wheat (Triticum aestivum L.). The aim of this study was to determine the effects of quantitative trait locus (QTL) regions for resistance to FHB and estimate their effects on reducing FHB damage to wheat in Hokkaido, northern Japan. We examined 233 F₁-derived doubled-haploid (DH) lines from a cross between ‘Kukeiharu 14’ and ‘Sumai 3’ to determine their reaction to FHB during two seasons under field conditions. The DH lines were genotyped at five known FHB-resistance QTL regions (on chromosomes 3BS, 5AS, 6BS, 2DL and 4BS) by using SSR markers. ‘Sumai 3’ alleles at the QTLs at 3BS and 5AS effectively reduced FHB damage in the environment of Hokkaido, indicating that these QTLs will be useful for breeding spring wheat cultivars suitable for Hokkaido. Some of the QTL regions influenced agronomic traits: ‘Sumai 3’ alleles at the 4BS and 5AS QTLs significantly increased stem length and spike length, that at the 2DL QTL significantly decreased grain weight, and that at the 6BS QTL significantly delayed heading, indicating pleiotropic or linkage effects between these agronomic traits and FHB resistance.