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.

水稻CSSL-Z481代换片段携带的穗部性状QTL分析及次级代换系培育

【背景】粮食安全是保障国家安全的重要基础,水稻是人民赖以生存的主要粮食作物,提高其产量是重要的育种目标。水稻产量由每株有效穗数、每穗实粒数和粒重等性状构成,其中,粒重与籽粒形状、充实程度等密切相关。但这些性状都是由多基因控制,遗传基础复杂。染色体片段代换系(CSSL)可将这些复杂性状的QTL较准确地分解为单个孟得尔因子研究,且与育种工作紧密衔接,因而是理想的遗传研究和育种材料。【目的】前期以4代换片段的水稻染色体片段代换系Z481精细定位了一个易落粒基因SH6,但Z481与受体日本晴间还存在多个显著差异的穗部性状。明晰控制这些差异性状的QTL在代换片段上如何分布,并分解为单片段代换系,对目标QTL的图位克隆及应用于水稻分子设计育种有重要应用价值。【方法】利用受体亲本日本晴与Z481杂交构建的次级F2分离群体以SAS9.3统计软件的混合线性模型(mixed linear model,MLM)法进行穗部性状QTL定位(P<0.05),然后,根据基因型和表型,从F2选择42个单株在F3株系利用MAS法培育单片段及双片段代换系,并利用IBM SPSS Statistics 25.0的ONE-WAY ANOVA和TWO-WAY ANOVA分析及LSD和Duncans多重比较(P<0.05)分析这些单片段代换系(SSSL)和双片段代换系(DSSL)的QTL加性和上位性效应。【结果】以日本晴/Z481构建的次级F2群体共定位出12个控制水稻穗部性状的QTL,并培育出相应QTL的11个单片段代换系(S1—S11)和3个双片段代换系(D1—D3)。其中,有8个QTL(qGL1、qGL3、qGL6、qGW1、qGW3、qRLW1、qRLW3和qRLW6)可被单片段代换系所验证,表明这些QTL遗传稳定。此外,利用单片段代换系鉴定到qGL1-2、qGL1-3、qGL3-2等33个QTL。其中,qNSB1-1等15个可能为新鉴定的QTL。且利用3个DSSL分析了非等位QTL间的上位性效应,结果表明,不同QTL聚合会产生不同的上位性效应,如qGL3(a=1.26)和qGL6-2(a=0.86)聚合产生了-0.77的上位性效应,据DSSL遗传模型,D2的粒长遗传效应(1.35)产生了更长的粒长表型;qGWT3-2(a=3.18)和qGWT6-2(a=3.39)聚合产生了-5.46的上位性效应,则D2的千粒重遗传效应(1.11)产生了更小的籽粒。【结论】Z481的4个代换片段上共携带45个水稻穗部性状的QTL,并进一步分解到11个次级单片段代换系上,单片段代换系具有比F2群体更高的QTL检测效率。利用SSSL和DSSL解析的水稻穗部性状QTL的加性效应和上位性效应,有助于根据这些遗传信息预测设计基因型的表型,从而选择合适的SSSL进行育种设计。

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.