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.

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.

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.

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.

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.

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.

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.

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

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.,

Identification of Rice QTLs for Important Agronomic Traits with Long-Kernel CSSL-Z741 and Three SSSLs

Rice kernel shape affects kernel quality(appearance) and yield(1000-kernel weight) and therefore is an important agronomic trait, but its inheritance is complicated. We identified a long-kernel rice chromosome segment substitution line(CSSL), Z741, derived from Nipponbare as a recipient and Xihui 18 as a donor parent. Z741 has six substitution segments distributed on rice chromosomes 3, 6, 7, 8 and 12 with an average replacement length of 5.82 Mb. Analysis of a secondary F2 population from a cross between Nipponbare and Z741 identified 20 QTLs for important agronomic traits. The kernel length of Z741 is controlled by a major QTL(qKL3) and a minor QTL(qKL7). Candidate gene prediction and sequencing indicated that qKL3 may be an allele of OsPPKL1, which encodes a protein phosphatase implicated in brassinosteroid signaling, and qKL7 is an unreported QTL. Finally, we validated eight QTLs(qKL3, qKL7, qRLW3-1, qRLW7, qPH3-1, qKWT3, qKWT7 and qNPB6) using three selected singlesegment substitution lines(SSSLs), S1, S2 and S3. Also, we detected five QTLs(qKL6, qKW3, qKW7, qKW6 and qRLW6) in S1, S2 and S3, which were not found in the Nipponbare/Z741 F2 population. However, qNPB3, qNPB7 and qPL3 QTLs were not validated by the three SSSLs in 2019, suggesting that minor QTLs are susceptible to environmental factors. These results lay the foundation for studying the biodiversity of kernal length and molecular breeding of different kernel types.

Identification of QTLs for Cooking and Eating Quality of Rice Grain

The BIL （backcross inbred line） population derived from the cross between Koshihikari （good eating and cooking quality, japonica） and Kasalath （poor quality, indica） was used to analyze the QTLs for amylose content （AC）, gelatinization temperature （GT）, gel consistency （GC） and protein content （PC）. Eight main-effect QTLs including 2 for AC, 3 for GT, 2 for GC and 1 for PC were identified, Moreover, 27 epistatic QTL pairs including 7 for AC, 5 for GT, 4 for GC and 11 for PC were also detected while for AC and GT, one main-effect QTL with a major gene was detected, respectively. Therefore, the main-effect QTL might be more responsible for the current variation than the epistetic QTL. The result indicated that the main-effect QTL is the primary genetic basis for those traits. However, for PC, the epistatic QTL explained a much greater portion of the total variation than main-effect QTL, suggesting that epistatic loci are the primary genetic basis for such trait. In the experiment, chromosome segment substitution lines （CSSLs） were used to confirm reliabilities of the main effect QTLs detected in BIL population. Of the 8 main-effect QTLs for 4 traits in BIL analysis, 6 were confirmed end 2 remained unconfirmed by CSSLs analysis.

Confirmation of Novel Quantitative Trait Loci for Seed Dormancy at Different Ripening Stages in Rice

Seed dormancy contributes resistance to pre-harvest sprouting. Effects on respective quantitative trait loci （QTLs） for dormancy should be assessed by using fresh seeds before germinability altered through storage. We investigated QTLs related to seed dormancy using backcross inbred lines derived from a cross between Nipponbare and Kasalath. Four putative QTLs for seed dormancy were detected immediately after harvest using composite interval mapping. These putative QTLs were mapped near C1488 on chromosome 3 （qSD-3.1）, R2171 on chromosome 6 （qSD-6.1）, R1245 on chromosome 7 （qSD-7.1） and C488 on chromosome 10 （qSD-IO.1）. Kasalath alleles promoted dormancy for qSD-3.1, qSD-6.1 and qSD-7.1, and the respective proportions of phenotypic variation explained by each QTL were 12.9%, 9.3% and 8.1%. We evaluated the seed dormancy harvested at different ripening stages during seed development using chromosome segment substitution lines （CSSLs） to confirm gene effects. The germination rates of CSSL27 and CSSL28 substituted with the region including qSD-6.1 were significantly lower than those of Nipponbare and other CSSLs at the late ripening stage. Therefore, qSD-6.1 is considered the most effective novel QTL for pre-harvest sprouting resistance among the QTLs detected in this study.

Analysis of QTLs for Flag Leaf Shape and Its Response to Elevated CO_2 in Rice(Oryza sativa)

To understand the responses of flag leaf shape in rice to elevated CO2 environment and their genetic characteristics, quantitative trait loci （QTLs） for flag leaf shape in rice were mapped onto the molecular marker linkage map of chromosome segment substitution lines （CSSLs） derived from a cross between a japonica variety Asominori and an indica variety IR24 under free air carbon dioxide enrichment （FACE, 200 μmol/mol above current levels） and current CO2 concentration （Ambient, about 370 μmol/mol）. Three flag-leaf traits, flag-leaf length （LL）, width （LW） and the ratio of LL to LW （RLW）, were estimated for each CSSL and their parental varieties. The differences in LL, LW and RLW between parents and in LL and LW within IR24 between FACE and Ambient were significant at 1% level. The continuous distributions and transgressive segregations of LL, LW and RLW were also observed in CSSL population, showing that the three traits were quantitatively inherited under both FACE and Ambient. A total of 16 QTLs for the three traits were detected on chromosomes 1, 2, 3, 4, 6, 8 and 11 with LOD （Log10-1ikelihood ratio） scores ranging from 3.0 to 6.7. Among them, four QTLs （qLL-6＊, qLL-8＊ qLW-4＊ and qRLW-6＊） were commonly detected under both FACE and Ambient. Therefore, based on the different responses to elevated CO2 in comparison with current CO2 level, it can be suggested that the expressions of several QTLs associated with flag-leaf shape in rice could be induced by the high CO2 level.

Genome sequence of Gossypium anomalum facilitates interspecific introgression breeding

Crop wild relatives are an important reservoir of natural biodiversity. However, incorporating wild geneticdiversity into breeding programs is often hampered by reproductive barriers and a lack of accurate genomicinformation. We assembled a high-quality, accurately centromere-anchored genome of Gossypium anomalum, a stress-tolerant wild cotton species. We provided a strategy to discover and transfer agronomicallyvaluable genes from wild diploid species to tetraploid cotton cultivars. With a (Gossypium hirsutum 3 G.anomalum)2 hexaploid as a bridge parent, we developed a set of 74 diploid chromosome segment substitution lines (CSSLs) of the wild cotton species G. anomalum in the G. hirsutum background. This set of CSSLsincluded 70 homozygous substitutions and four heterozygous substitutions, and it collectively containedabout 72.22% of the G. anomalum genome. Twenty-four quantitative trait loci associated with plant height,yield, and fiber qualities were detected on 15 substitution segments. Integrating the reference genome withagronomic trait evaluation of the CSSLs enabled location and cloning of two G. anomalum genes thatencode peroxiredoxin and putative callose synthase 8, respectively, conferring drought tolerance andimproving fiber strength. We have demonstrated the power of a high-quality wild-species reference genomefor identifying agronomically valuable alleles to facilitate interspecific introgression breeding in crops.