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.

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.

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.

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 and high-throughput genotyping of substitution lines carting the chromosome segments of indica 9311 in the background of japonica Nipponbare

Chromosome segment substitution lines（CSSLs） are useful for the precise mapping of quantitative trait loci（QTLs） and dissection of the genetic basis of complex traits.In this study,two whole-genome sequenced rice cultivars,the japonica Nipponbare and indica 9311 were used as recipient and donor,respectively.A population with 57 CSSLs was developed after crossing and back-crossing assisted by molecular markers, and genotypes were identified using a high-throughput resequencing strategy.Detailed graphical genotypes of 38 lines were constructed based on resequencing data.These CSSLs had a total of 95 substituted segments derived from indica 9311,with an average of about 2.5 segments per CSSL and eight segments per chromosome,and covered about 87.4%of the rice whole genome.A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight.The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice.These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome

Identification of Quantitative Trait Loci for Rice Quality in a Population of Chromosome Segment Substitution Lines

The demand for high quality rice represents a major issue in rice production. The primary components of rice grain quality include appearance, eating, cooking, physico-chemical, milling and nutritional qualities. Most of these traits are complex and controlled by quantitative trait loci （QTLs）, so the genetic characterization of these traits is more difficult than that of traits controlled by a single gene. The detection and genetic identification of QTLs can provide insights into the genetic mechanisms underlying quality traits. Chromosome segment substitution lines （CSSLs） are effectivetools used in mapping QTLs. In this study, we constructed 154 CSSLs from backcross progeny （BC3F2） derived from a cross between ＇Koshihikari＇ （an Oryza sativa L. ssp. japonica variety） as the recurrent parent and ＇Nona Bokra＇ （an O. sativa L. ssp. indica variety） as the donor parent. In this process, we carried out marker-assisted selection by using 102 cleaved amplified polymorphic sequence and simple sequence repeat markers covering most of the rice genome. Finally, this set of CSSLs was used to identify QTLs for rice quality traits. Ten QTLs for rice appearance quality traits were detected and eight QTLs concerned physico-chemical traits. These results supply the foundation for further genetic studies and breeding for the improvement of grain quality.

Study on heterosis of inter-subspecies between indica and japonica rice (Oryza sativa L.) using chromosome segment substitution lines

Heterosis for yield and its component traits between chromosome segments from IR24, an indica variety, and the counterparts from 02428, a japonica rice, was inves- tigated by using a hybrid F1 population composed of 63 com- binations between 02428 and IR24 chromosome segment substitution lines (CSSLs) with the genetic background of Asominori, a japonica variety. Significant differences in het- erosis for yield and yield-component traits were observed among the crosses. Analysis of graphical genotyping showed that 14 substituted segments were responsible for yield het- erosis. All of them were from all the 12 chromosomes of IR24 except chromosomes 8 and 10. Six segments at the intervals of RFLP markers, such as X132—G1340—R459, X48— C393A, R288—R1854, R2918—X52, X257—C1350 and R367 —X189-2—X24-2 on chromosomes 2, 3, 4, 11 and 12 respec- tively, had very significant heterosis for yield at the level of P ≤0.005 based on t-test, individually increasing the hybrid yield by more than 35% compared with the control cross “Asominori×02428”. Most of IR24 chromosome segments were found to have no significant hybrid effect for yield and yield-component traits, and one segment located at R2171 on chromosome 6 possessed significant negative effect with 27% of yield decrease. Advantages of using CSSLs in the heterosis studies were discussed and approaches of the partial and genome-wide exploitation of rice heterosis between indica and japonica by molecular marker-assisted selection were then proposed.

Two Novel QTLs for Heading Date Are Identified Using a Set of Chromosome Segment Substitution Lines in Rice(Oryza sativa L.)

Heading date in rice is a typical quantitative trait controlled by multiple quantitative trait loci （QTLs）. It is mainly regulated by environmental factors such as photoperiod and temperature （Izawa, 2007）. Many QTLs for heading date have been identified using different mapping populations and methods （http：// www.gramene.org/qtl）. Up to date, several major heading date QTLs have been cloned by map-based cloning strategy （Yano et al., 2000; Takahashi et al., 2001; Kojima et al., 2002; Doi et al., 2004; Xue et al., 2008; Wei et al., 2010; Yan et al.,

Development of Gossypium barbadense chromosome segment substitution lines in the genetic standard line TM-1 of Gossypium hirsutum

Chromosome segment substitution lines (CSSL) consist of a battery of nearisogenic lines that have been developed and cover the entire genome of some crops. With the exception of one homozygous chromosome segment transferred from a donor parent, the remaining genome of each CSSL line is the same as the recipient parent. It is an ideal material for genome research and particularly QTL mapping. In the present study, we first developed one set of CSSL lines using G. hirsutum acc. TM-1 (the genetic standard), as the recipient parent and G. barbadense cv. Hai7124 as the donor parent using molecular assisted-selection in BC5S1-3 generations. The CSSL consisted of 330 different lines, in which 1-4 different lines had the same or overlapping substituted segments. The genetic length of the substituted segments covered 5271.9 cM with an average segment distance of 10.9 cM, 1.5 times the total genetic length of Upland cotton (3514.6 cM). The substituted segments of each line varied in length, ranging from 3.5 cM for the shortest segment to 23.2 cM in the longest segment. Our CSSL have not yet to cover the entire tetraploid cotton genome, due to the absence of some donor parent interval segments.

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。

Bin-based model construction and analytical strategies for dissecting complex traits with chromosome segment substitution lines

Chromosome segment substitution lines have been created in several experimental models,including many plant and animal species,and are useful tools for the genetic analysis and mapping of complex traits.The traditional t-test is usually applied to identify a quantitative trait locus (QTL) that is contained within a chromosome segment to estimate the QTL's effect.However,current methods cannot uncover the entire genetic structure of complex traits.For example,current methods cannot distinguish between main effects and epistatic effects.In this paper,a linear epistatic model was constructed to dissect complex traits.First,all the long substituted segments were divided into overlapping small bins,and each small bin was considered a unique independent variable.The genetic model for complex traits was then constructed.When considering all the possible main effects and epistatic effects,the dimensions of the linear model can become extremely high.Therefore,variable selection via stepwise regression (Bin-REG) was proposed for the epistatic QTL analysis in the present study.Furthermore,we tested the feasibility of using the LASSO (least absolute shrinkage and selection operator) algorithm to estimate epistatic effects,examined the fully Bayesian SSVS (stochastic search variable selection) approach,tested the empirical Bayes (E-BAYES) method,and evaluated the penalized likelihood (PENAL) method for mapping epistatic QTLs.Simulation studies suggested that all of the above methods,excluding the LASSO and PENAL approaches,performed satisfactorily.The Bin-REG method appears to outperform all other methods in terms of estimating positions and effects.

基于CSSLs群体定位和图位克隆水稻长芒基因GAD1-2

水稻是世界上最早驯化的重要粮食作物之一。水稻芒可以保护水稻种子不被鸟琢食,是水稻重要的驯化性状之一。芒在野生稻中普遍存在,对野生稻的生存和传播至关重要,然而在驯化和人工选择过程中该性状逐渐被淘汰。定位和克隆水稻长芒相关基因是研究水稻芒驯化遗传机制的基础。本研究以籼稻恢复系东南恢810为受体、漳浦野生稻为供体构建的146个染色体片段置换系(chromosome segment substitution lines, CSSLs)为研究材料,调查了146个CSSLs株系和双亲的芒长,结果表明在4个置换系中检测到1个控制水稻芒长主效基因GAD1-2,位于水稻第8号染色体;利用重叠代换作图法,将GAD1-2定位在Ind8-10和RM4936标记之间,遗传距离约为4.75 Mb。选择分离群体中的显性单株,利用开发的标记,最终将GAD1-2基因定位在两个Indel标记之间,两者间的物理距离约为27kb,该区域内只有两个候选基因Os08g0485500和Os08g0485400。经测序和分析表明,Os08g0485500是GAD1-2的候选基因,GAD1-2在保守的ORF区域存在6个碱基缺失,导致丝氨酸和半胱氨酸这两个氨基酸缺失,从而表现长芒的性状;在Os08g0485500基因位点已克隆了1个控制水稻芒长的GAD1基因,推测GAD1-2与GAD1为等位基因。本研究为进一步理解水稻起源演化和水稻芒长发育基因的遗传机制奠定了基础。