Mapping of QTLs Related to Grain Weight Using Chromosome Single Segment Substitution Lines in Rice

Grain weight, one of the major factors determining rice yield, is a typical quantitative trait control ed by multiple genes. With Guangluai 4 as recipient and Nipponbare as donor, a population of 119 chromosome single segment substitution lines had been developed. Correlation analysis between grain weight and grain shape by SPSS revealed that 1 000-grain weight shared extremely significant posi-tive correlation with grain length and length-width ratio, but no significant correlation with grain width and thickness. The QTL analysis of grain weight was carried out using one-way analysis of variance and Dunnett＇s test. Nineteen stable QTLs re-sponsible for grain weight were identified over two years. Al 19 QTLs were identi-fied on al chromosomes except for chromosome 10 and 12 at a significance level of P≤0.001. Among them, 10 QTLs had a positive effect and were derived from the Nipponbare al ele, the additive effect of these QTLs ranged from 0.49 to 2.74 g, and the contributions of the additive effects ranged from 2.00% to 11.05%. Another 9 QTLs had a negative effect and were al derived from Guangluai 4 al ele, the ad-ditive effect of these QTLs ranged from 0.60 to 2.35 g, and the contributions of the additive effects ranged from 2.40% to 9.84%. The results provide a basis for the fine mapping and gene cloning of novel locus associated with rice grain weight.

QTLs Mapping for Salinity Tolerance at Seedling Stage or Rice(Oryza sativa L.) Using Chromosome Segment Substitution Lines

In this study, a population of chromosome segment substitution lines （CSSLs） derived from the cross between 9311 （indica） and Nipponbare （japonica） was employed to map the quantitative trait loci （QTLs） for salt tolerance under the salt stress simulated with 0.5% NaCI, using survival rate as the index. The data were analyzed by QTL IciMapping v3.1, and the results showed that one QTL （QSsr3） related to salt tolerance was located in the vicinity of the marker RM1350 on chromosome 3, into a genetic interval of 113.2-132.8 cM, with a contribution rate of 17.75%. The additive effect was 10.9, indicating that the QTL derived from the parent Nipponbare improved the salt tolerance of rice at seedling stage. This study will provide a theoretical basis for the selection of salt tolerant rice germplasm.

Mapping QTLs Associated with Sheath Blight Resistance Using Chromosome Segment Substitution Lines of Rice(Oryza sativa L.)

In this study, a population of 119 chromosome segment substitution lines （CSSLs） derived from backcross between indica 9311 and japonica Nipponbare was employed to map quantitative trait loci （QTL） associated with sheath blight resis-tance in rice with toothpick inoculation method. A total of three sheath blight resis-tance-associated QTLs （qsb8-1, qsb8-2 and qsb8-3） were identified, which were lo-cated on adjacent molecular markers RM3262, RM5485 and RM3496 of chromo-some 8; the genetic interval was 81.7cM-91.7cM, 91.7cM-108.1cM and 108.1cM-119.6cM, respectively. The additive effect of qsb8-2 was negative, indicating that sheath blight resistance of susceptible parent harboring qsb8-2 fragment was en-hanced; additive effects of qsb8-1 and qsb8-3 were positive, indicating that sheath blight resistance of susceptible parent harboring qsb8-1 and qsb8-3 fragments was reduced.

Identification of long-grain chromosome segment substitution line Z744 and QTL analysis for agronomic traits in rice

Length of grain affects the appearance, quality, and yield of rice. A rice long-grain chromosome segment substitution line Z744, with Nipponbare as the recipient parent and Xihui 18 as the donor parent, was identified. Z744 contains a total of six substitution segments distributed on chromosomes(Chrs.) 1, 2, 6, 7, and 12, with an average substitution length of 2.72 Mb. The grain length, ratio of length to width, and 1 000-grain weight of Z744 were significantly higher than those in Nipponbare. The plant height, panicle number, and seed-set ratio in Z744 were significantly lower than those in Nipponbare, but they were still 78.7 cm, 13.5 per plant, and 86.49%, respectively. Furthermore, eight QTLs of different traits were identified in the secondary F2 population, constructed by Nipponbare and Z744 hybridization. The grain weight of Z744 was controlled by two synergistic QTLs(qGWT1 and q GWT7) and two subtractive QTLs(qGWT2 and qGWT6), respectively. The increase in the grain weight of Z744 was caused mainly by the increase in grain length. Two QTLs were detected, qGL1 and qGL7-3, which accounted for 25.54 and 15.58% of phenotypic variation, respectively. A Chi-square test showed that the long-grain number and the short-grain number were in accordance with the 3:1 separation ratio, which indicates that the long grain is dominant over the short-grain and Z744 was controlled mainly by the principal effect qGL1. These results offered a good basis for further fine mapping of qGL1 and further dissection of other QTLs into single-segment substitution lines.

Identification of QTL for kernel number-related traits in a rice chromosome segment substitution line and fine mapping of qSP1

A chromosome segment substitution line (CSSL) is a powerful tool for combining quantitative trait locus (QTL) mapping with the pyramiding of desirable alleles. The rice CSSL Z1364 with increased kernel number was identified in a BC3F8 population derived from a cross of Nipponbare as the recipient with Xihui 18 as the donor parent. Z1364 carried three substitution segments distributed on chromosomes 1, 6, and 8. The mean substitution length was 1.19 Mb. Of 17 QTL identified on the substitution segments, qSP1 for spikelets per panicle, qSSD1 for seed-set density, and qNSB1 for number of secondary branches explained respectively 57.34%, 87.7%, and 49.44% of the corresponding phenotypic variance and were all linked to RM6777. Chi-square analysis showed that the increased kernel number in Z1364 was inherited recessively by a single gene. By fine mapping, qSP1 was delimited to a 50-kb region on the short arm of chromosome 1. Based on DNA sequence, a previously uncharacterized rice homolog of Arabidopsis thaliana AT4G32551 was identified as a candidate gene for qSP1 in which mutation increases the number of spikelets and kernels in Z1364. qSP1 was expressed in all tissues, but particularly in 1-cm panicles. The expression levels of OsMADS22, GN1A, and DST were upregulated and those of LAX2, GNP1, and GHD7 were downregulated in Nipponbare. These results provide a foundation for functional research on qSP1.

Detection of QTL for Cold Tolerance at Bud Bursting Stage Using Chromosome Segment Substitution Lines in Rice (Oryza sativa)

Ab The cold tolerance at the bud bursting stage （CTB） was evaluated at 5℃ by using a set of 95 chromosome segment substitution lines （CSSLs） derived from an indica rice 9311 and a japonica rice Nipponbare with a genetic background of 9311. The result showed that six CSSLs had slightly stronger effect on CTB than 9311. Total four quantitative trait loci （QTLs） for CTB were preliminary mapped on chromosomes 5 and 7 by substitution mapping, qCTB-5-1, qCTB-5-2 and qCTB-5-3 were mapped in the region of RM267-RM1237, RM2422-RM6054 and RM3321-RM1054, which were 21.3 cM, 27.4 cM and 12.7 cM in genetic distance on rice chromosome 5, respectively, qCTB-7 was mapped in a 6.8-cM region of RM11-RM2752 on rice chromosome 7.

QTL Mapping for Rice RVA Properties Using High-Throughput Re-sequenced Chromosome Segment Substitution Lines

The rapid visco analyser （RVA） profile is an important factor for evaluation of the cooking and eating quality of rice. To improve rice quality, the identification of new quantitative trait loci （QTLs） for RVA profiling is of great significance. We used a japonica rice cultivar Nipponbare as the recipient and indica rice 9311 as the donor to develop a population containing 38 chromosome segment substitution lines （CSSLs） genotyped by a high-throughput re-sequencing strategy. In this study, the population and the parent lines, which contained similar apparent amylose contents, were used to map the QTLs of RVA properties including peak paste viscosity （PKV）, hot paste viscosity （HPV）, cool paste viscosity （CPV）, breakdown viscosity （BKV）, setback viscosity （SBV）, consistency viscosity （CSV）, peak time （PET） and pasting temperature （PAT）. QTL analysis was carried out using one-way analysis of variance and Dunnett＇s test, and stable QTLs were identified over two years and under two environments. We identified 10 stable QTLs： qPKV2-1, qSBV2-1; qPKV5-1, qHPV5-1, qCPV5-1; qPKV7-1, qHPV7-1, qCPV7-1, qSBV7-1; and qPKV8-1 on chromosomes 2, 5, 7 and 8, respectively, with contributions ranging from -95.6% to 47.1%. Besides, there was pleiotropy in the QTLs on chromosomes 2, 5 and 7.

Evaluation of Agronomic Traits in Chromosome Segment Substitution Lines of KDML105 Containing Drought Tolerance QTL under Drought Stress

Drought is a major abiotic constraint to rice production in rainfed lowland and insufficiently irrigated areas. The improvement of drought tolerant varieties is one of the strategies to reduce the negative effects of drought. Quantitative trait loci （QTLs） for primary and secondary traits related to drought tolerance （DT） on chromosomes 1, 3, 4, 8 and 9 that determined from double haploid lines derived from a cross between CT9993 and IR62266 were introgressed and dissected into small pieces in the genetic background of Khao Dawk Mall 105 （KDML105） to develop chromosome segment substitution line （CSSL） population. The CSSLs were evaluated at the reproductive stage for their agronomic performance and yield components under drought stress, and results were compared with irrigated condition. The flowering of CSSL lines was 6 to 7 d earlier than KDML105. The mean values of grain yields in the CSSLs were higher than KDML105 under drought and irrigated conditions. At irrigated condition, the grain yields of introgression lines carrying DT-QTLs from chromosomes 4 and 8 were higher than that of KDML105, whereas other traits showed little difference with KDML105. Analysis indicated that grain yield has positive correlation with plant height, tiller and panicle number per plant, and total grain weight per plant under drought stress while negatively correlated with days to flowering. As mentioned above, CSSLs showing good adaptation under drought stress can be used as genetic materials to improve drought tolerance in Thai rainfed lowland rice breeding program, and as materials to dissect genes underlying drought tolerance.

Characterization of chromosome segment substitution lines reveals candidate genes associated with the nodule number in soybean

Soybean is one of the most important food crops worldwide.Like other legumes,soybean can form symbiotic relationships with Rhizobium species.Nitrogen fixation of soybean via its symbiosis with Rhizobium is pivotal for sustainable agriculture.Type Ⅲ effectors(T3Es)are essential regulators of the establishment of the symbiosis,and nodule number is a feature of nitrogen-affected nodulation.However,genes encoding T3Es at quantitative trait loci(QTLs)related to nodulation have rarely been identified.Chromosome segment substitution lines(CSSLs)have a common genetic background but only a few loci with heterogeneous genetic information;thus,they are suitable materials for identifying candidate genes at a target locus.In this study,a CSSL population was used to identify the QTLs related to nodule number in soybean.Single nucleotide polymorphism(SNP)markers and candidate genes within the QTLs interval were detected,and it was determined which genes showed differential expression between isolines.Four candidate genes(GmCDPK28,GmNAC1,GmbHLH,and GmERF5)linked to the SNPs were identified as being related to nodule traits and pivotal processes and pathways involved in symbiosis establishment.A candidate gene(GmERF5)encoding a transcription factor that may interact directly with the T3E NopAA was identified.The confirmed CSSLs with important segments and candidate genes identified in this study are valuable resources for further studies on the genetic network and T3Es involved in the signaling pathway that is essential for symbiosis establishment.

A Preliminary Study of Mapping Genes Underlying Complex Traits Based on Chromosome Segment Substitution Lines

Complex traits are the features whose properties are determined by multiple factors, which can be genetic or environmental. Most of economically important characteristics of plants and animals belong to this special catego-

Mapping QTLs for Heat Tolerance in Rice (Oryza sativa L. ) at Heading Stage Using Chromosome Segment Substitution Lines

In the present study, a japonica rice （ Oryza sativa L. ） variety Nipponbare, an indica variety 9311 and a set of chromosome segment substitution lines （CSSLs） which were generated using Nipponbare as the recipient parent and 9311 as the donor parent were used as the experimental materials. The CSSLs were grown in 2012 （normal temperature condition） and 2013 （high temperature condition） in Yangzhou, Jiangsu, and were used to map the quantitative trait loci （QTLs） for heat tolerance, based on the heat tolerance index [ （The seed setting rate under normal temperature condition -The seed setting rate under high temper- ature condition） / The seed setting rate under normal temperature condition]. As a result, three QTLs related to heat tolerance in rice were mapped on chromo- somes 2, 4 and 12, respectively. They had LOD （logarithm of rntds） scores of 2.56, 4.02 and 2.79, and contributian rates of 4.95%, 7.99% and 5.44%. Among them, qHT12.1 showed positive effect, while qHT2.1 and qHT4. t showed negative effect on heat tolerance. The results lay a foundation for the fine mapping and cloning of the QTLs and genes related to heat tolerance, and for the breeding of heat-tolerant rice varieties.

Dynamic Expression Analysis and Introgressive Gene Identification of Fiber Length Using Chromosome Segment Substitution Lines from G.hirsutum×G.barbadense

Fiber length is a critical trait that principally determines cotton spinning quality,while Upland cotton as the most widely cultivated Gossypium species around the world subjects to the relatively ordinary fiber performance.Chromosome segment substitution lines(CSSLs)have been introduced in cotton breeding to take full advantages of superior fiber quality and high yield from Sea Island and Upland cotton,respectively,which serve as ideal materials for elucidating the genetic mechanism of complex quantitative traits.Here,three CSSLs derived from CCRI45(G.hirsutum)×Hai1(G.barbadense),two superior(MBI7561 and MBI7747)and one(MBI7285)with ordinary fiber-quality,were subjected to transcriptome sequencing during fiber elongation together with their recurrent parent CCRI45,and 471.425 million clean reads were obtained with 91.47%average Q30 and 45.23%mean GC content.In total,5,673 differentially expressed genes(DEGs)were identified from multi-sample comparisons,which were mainly involved in the oxidation-reduction process,protein phosphorylation,regulation of transcription,DNA template,and carbohydrate metabolic process.Eight temporal expression patterns were monitored on the DEGs of different lines,of which the significantly enriched profile revealed higher similarities between two superior CSSLs or the ordinary CSSL and CCRI45 with respect to fiber performance.Based on the intersection between the predicted introgressive genes from RNAseq data and the published gene information from the G.barbadense genome,1,535 introgressive genes were identified in three CSSLs.Further analysis of the three common introgressive sections in superior CSSLs revealed eight candidate genes that were identified to be involved in fiber development,namely,O-fucosyltransferase family protein(GB_A02G0240),glutamine synthetase 2(GB_A02G0272),Ankyrin repeat family protein(GB_A02G0264),beta-6 tubulin(GB_D03G1742),WRKY DNA-binding protein 2(GB_D03G1655),quinolinate synthase(GB_D07G0623),nuclear factor Y,subunit B13(GB_D07G0631),and leucine-rich repeat transmembrane protein kinase(GB_D07G0797).Our results provide novel insights into the mechanism underlying fiber formation and lay a solid foundation for further high-efficiency determination of candidate genes by combining RNA-seq data and pivotal chromosome regions.

Multi-Environmental Genetic Analysis of Grain Size Traits Based on Chromosome Segment Substitution Line in Rice(Oryza sativa L.)

Grain size traits are critical agronomic traits which directly determine grain yield,but the genetic bases of these traits are still not well understood.In this study,a total of 154 chromosome segment substitution lines(CSSLs)population derived from a cross between a japonica variety Koshihikari and an indica variety Nona Bokra was used to investigate grain length(GL),grain width(GW),length-width ratio(LWR),grain perimeter(GP),grain area(GA),and thousand grain weight(TGW)under four environments.QTL mapping analysis of six grain size traits was performed by QTL IciMapping 4.2 with an inclusive composite interval mapping(ICIM)model.A total of 64 QTLs were identified for these traits,which mapped to chromosomes 1,2,3,4,6,7,8,10,11,and 12 and accounted for 1.6%–27.1%of the total phenotypic variations.Among these QTLs,thirty-six loci were novel and seven QTLs were identified under four environments.One locus containing the known grain size gene,qGL3/GL3.1/OsPPKL1,also have been found.Moreover,five pairs of digenic epistatic interactions were identified except for GL and GP.These findings will facilitate fine mapping of the candidate gene and QTL pyramiding to genetically improve grain yield in rice.

Development of Chromosomal Segment Substitution Lines from a Backcross Recombinant Inbred Population of Interspecific Rice Cross

A backcross recombinant inbred line population consisting of 202 lines was developed from Xieqingzao B//Xieqingzao B / Dongxiang wild rice. The population was assayed with DNA markers and phenotyped on planthopper resistance and yield traits. A linkage map consisting of 119 DNA markers and spanned for 1188 cM over the 12 rice chromosomes was constructed. Thirty-two chromosomal segment substitution lines were selected based on the percentage of Xieqingzao B allele at marker loci. These lines are of great potential for gene mapping and alien gene introgression.

利用CSSL群体研究稻米加工品质相关QTL表达的稳定性

【目的】研究稻米加工品质的分子遗传基础,为遗传改良和分子标记辅助选择提供有用信息。【方法】利用Asominori为遗传背景具有IR24染色体片段的置换系(CSSL)群体,在4个环境下对稻米糙米率、精米率和整精米率进行QTL定位和稳定性分析。【结果】结果共检测到30个QTL与稻米加工品质相关,其中qMR-6、qMR-8、qHR-3和qHR-6在4个环境中都能被重复检测到,影响精米率的qMR-6和qMR-8的平均贡献率分别为21.1%和22.2%;与整精米率相关的qHR-3和qHR-6,平均贡献率分别为34.6%和22.3%;且qMR-6、qMR-8、qHR-3和qHR-6对应的置换系与背景亲本Asominori在4个环境中相应性状的表现型之间都存在显著差异(P<0.05)。【结论】qMR-8、qBR-8b和qHR-8共定位在第8染色体R727-G1149区间的QTL簇上,在笔者以前的研究中发现该QTL簇包含与稻米蒸煮食味、外观和营养品质等相关的多个主效QTL,这为剖析同一染色体区段内的1个或多个基因协同调控多个品质性状形成的复杂代谢网络提供信息。

利用CSSL群体研究稻米AC和PC相关QTL表达稳定性

利用以Asominori为遗传背景具IR24染色体片段的置换系（CSSL）群体，在“2年4点”8个环境对稻米直链淀粉含量（AC）和蛋白质含量（PC）进行QTL定位和表达稳定性分析。结果共检测到8个AC和PC相关QTL，其中2个QTL在8个环境中都能重复出现，即影响AC的qAC-8和控制PC的qPC-8，平均贡献率分别为21．0％和26．9％。qAC-8和qPC-8对应置换系与背景亲本Asominori在8个环境中相应性状的表现型都达到极显著差异（P〈0．01）；都仅与8个环境中的2个环境之间存在显著的互作效应；说明qAC-8和qPC-8的效应显著且稳定性较高。此外，qAC-8和qPC-8都被定位在第8染色体R727～G1149区间，IR24的等位基因可同时提高AC和PC，这为研究水稻籽粒直链淀粉和蛋白质形成途径之间的相互关系以及碳氮代谢协同调控的遗传机制提供了新的信息。

基于CSSL的水稻穗颈长度QTL的代换作图

水稻穗颈长度是影响杂交水稻制种产量提高的重要农艺性状之一。文章利用94个以籼稻品种9311为遗传背景、粳稻品种日本晴为染色体片段供体的覆盖全基因组的染色体片段置换系(Chromosome segment substitution lines,CSSL)为材料,调查和分析CSSL群体及双亲的穗颈长度。结果表明:在17个置换系中检测到8个控制水稻穗颈长度的数量性状位点(Quantitative trait loci,QTL),分别位于第2、3、7、8、9和第11染色体;利用代换作图法,定位了其中的7个穗颈长度QTL;其加性效应值介于0.10～3.20之间,其中qPE-9和qPE-11的加性效应值较大,平均效应值分别为3.15和2.95,表现为主效基因特征;qPE-2-2、qPE-3-1、qPE-3-2、qPE-7和qPE-8等5个QTL被定位在小于10.0cM的区段内。利用CSSL可以有效地鉴定水稻穗颈长度QTL,这些QTL为分子标记辅助选育穗颈长度适中的水稻品系及其进一步的精细定位奠定了基础。

利用CSSLs群体研究稻米粒型QTL的表达稳定性

利用Asominori×IR2 4的染色体片段置换系 (CSSLs)群体 ,对稻米粒长、粒宽和长宽比进行连续两年及 4个地点的QTL表达稳定性分析。结果表明 :3个性状“两年四点”的表现型都为连续分布 ,均存在超亲遗传类型 ;共检测到 13个粒型相关QTL ,其中在 8个环境中都能被重复检测到的QTL有 6个 ,即影响粒长的 qGL 3、控制粒宽的qGW 5a和 qGW 5b以及共同作用于长宽比的qLWR 3、qLWR 5a和 qLWR 5b。这 6个QTL对应置换系的相应性状与背景亲本Asominori的表现型差异在 8个环境中都达到极显著水平 (P <0 0 0 1) ,且同一QTL对应置换系相应性状的表现型在不同环境间呈显著正相关 (r≥ 0 75 ,r0 0 5=0 6 6 6 ) ,说明这 6个QTL表达稳定性较高。由于 qGL 3和 qLWR 3均位于R19 C16 77标记区间 ,qGW 5a和 qLWR 5a位于C2 6 3标记附近 ,qGW 5b和 qLWR 5b被定位在R5 6 9标记附近 ,因此R19、C16 77、C2 6 3和R5 6 9这 4个RFLP标记对优良水稻外观品质的标记辅助选择 (MAS)

利用染色体片段置换系(CSSLs)群体检测水稻剑叶形态性状QTL

2007和2008年对以粳稻Asominori为受体、籼稻IR24为供体的染色体片段置换系(chromosome segment substitu-tion lines,CSSLs)群体的剑叶形态性状(剑叶长、宽、长宽比及叶面积)进行了相关性分析和QTL检测。结果表明:不同剑叶形态性状间存在极显著的正相关性,剑叶形态性状与单株产量间也存在极显著的正相关性。利用基于完备复合区间作图方法的QTL检测软件(QTL IciMapping V2.0)进行QTL的联合定位分析,两年共定位到17个控制剑叶形态性状的QTL,分布在第1、2、4、6、7和8等多条染色体上,贡献率为5.30%～32.22%。其中位于第2染色体RM262标记位点控制剑叶长宽比性状的qRFLW2的贡献率最大,对改良剑叶长宽比性状具有重要的育种价值,而在第1染色体RM5496标记、第2染色体RM262标记、第7染色体RM248和RM455标记附近以及第8条染色体RM331位置上存在同时控制剑叶长、宽和叶面积的QTL簇,可为协同改良水稻剑叶形态性状的分子育种提供有用信息。

基于CSSL的水稻抽穗期QTL定位及遗传分析

抽穗期是水稻(Oryza sativa)品种的重要农艺性状之一,适宜的抽穗期是获得理想产量的前提。鉴定和定位水稻抽穗期基因/QTL,分析其遗传效应对改良水稻抽穗期至关重要。以籼稻品种9311(Oryzasativa ssp.indica‘Yangdao 6’)为受体,粳稻品种日本晴(Oryza sativa ssp.japonica‘Nipponbare’)为供体构建的94个染色体片段置换系群体为材料,以P≤0.01为阈值,对置换片段上的抽穗期QTL进行了鉴定。采用代换作图法共定位了4个控制水稻抽穗期的QTL,分别位于第3、第4、第5和第8染色体;QTL的加性效应值变化范围为–6.4––2.7,加性效应百分率变化范围为–6.4%––2.7%;qHD-3和qHD-8加性效应值较大,表现主效基因特征。为了进一步定位qHD-3和qHD-8,在目标区域加密16对SSR引物,qHD-3和qHD-8分别被界定在第3染色体RM3166–RM16206之间及第8染色体RM4085–RM8271之间,其遗传距离分别为13.9cM和6.4cM。研究结果为利用分子标记辅助选择改良水稻抽穗期奠定了基础。