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.

Interspecific Chromosome Substitution Lines as Genetic Resources for Improvement,Trait Analysis and Genomic Inference

The genetic base that cotton breeders commonly use to improve Upland cultivars is very narrow.The AD-genome species Gossypium barbadense,G.tomentosum,and G.mustelinum are part of

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.

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

In the present study,ajaponicarice(Oryza sativa L.)variety Nipponbare,an indicavariety 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 temperature condition)/The seed setting rate under normal temperature condition].As a result,three QTLs related to heat tolerance in rice were mapped on chromosomes 2,4and 12,respectively.They had LOD(logarithm of odds)scores of 2.56,4.02 and 2.79,and contribution rates of 4.95%,7.99%and 5.44%.Among them,qHT12.1showed positive effect,while qHT2.1and qHT4.1showed 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.

Constructing Molecular Marker Linkage Maps of Chromosome 14Sh and 22Sh and QTL Mapping for Major Traits by Use of Substitution Lines of Gossypium hirsutum L.

CSB14Sh,which is isogenic for its recurrent parent TM-1 except for chromosome 14 short arm,was crossed with TM-1,and the F2 population was produced.A total of 3800 SSR primer pairs covering the whole genome were used to screen polymorphism among two parents,TM-1 and CSB14Sh,

QTL Detection for Rice Grain Shape Using Chromosome Single Segment Substitution Lines

Rice grain shape is one of the important factors affecting grain quality and yield,but it is liable to be influenced by genetic backgrounds and environments.The chromosome single segment substitution lines(SSSLs) in rice have been considered as ideal populations to identify the quantitative trait loci(QTLs).In this study,22 QTLs affecting rice grain shape were detected to be distributed on eight chromosomes except chromosomes 6,9,11 and 12 by using SSSLs.Among them,seven QTLs conditioned grain length,six conditioned grain width,five affected grain length-width ratio and four controlled grain thickness.

Development and characterization of wheat–Aegilops kotschyi 1U^(k)(1A)substitution line with positive dough quality parameters

Exploring novel high molecular weight glutenin subunits(HMW-GSs)from wild related species is a strategy to improve wheat processing quality.The objective of the present investigation was to identify the chromosomes of the wheatalien introgression line N124,derived from the hybridization between Triticum aestivum with Aegilops kotschyi,and characterize the effects on quality-related traits.Fluorescence in situ hybridization karyotypes showed that N124 is a disomic 1U^(k)(1A)substitution line.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and reversedphase high-performance liquid chromatography verified N124 expressed two HMW-GSs of the Ae.kotschyi parent.PacBio RNA sequencing and phylogenetic analysis confirmed that the two HMW-GSs were U^(k)x and U^(k)y.Compared to the wheat parent,the substitution line had no obvious agronomic defects except fewer grains per spike but improved several major quality parameters.It can be served as a donor or bridge material for wheat quality improvement.

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.

Identification of Differentially Expressed Genes Associated with Cotton Fiber Development in a Chromosomal Substitution Line(CS-B22sh)

One of the impediments in the genetic improvement of cotton fiber is the paucity of information about genes associated with fiber development.Availability of chromosome arm substitution line CS-

Fine Mapping of QTLs for Stigma Exsertion Rate from Oryza glaberrima by Chromosome Segment Substitution

Stigma exsertion is an important trait for outcrossing ability in rice.Stigma exsertion rate(SER)of male sterile lines(MSLs)is a key factor affecting F1-seed production in hybrid rice.In this study,seven QTLs for SER were detected on five chromosomes using a set of single-segment substitution lines(SSSLs)derived from O.glaberrima.Three of the QTLs were mapped in the estimated intervals of 92.5–333.0 kb.qSER-5 was located in a substitution segment of 92.5 kb.qS ER-1 b and qS ER-8 b were respectively limited to 333.0 kb and 107.5 kb by secondary substitution mapping.qSER-1 b and qSER-3 had bigger additive effects of 11.5%and 11.9%,respectively,while the other five QTLs had smaller additive effects from 5.7%to 8.6%.Open reading frames were identified in the regions of qS ER-5 and qSER-8 b in O.sativa and O.glaberrima genomes.Fine mapping of the QTLs laid a foundation for the cloning of genes,and QTLs for SER will be used to develop MSLs with strong ability of outcrossing.

GL9 from Oryza glumaepatula controls grain size and chalkiness in rice

Grain size is a key factor influencing grain yield and appearance quality in rice.We identified twelve quantitative trait loci(QTL)for grain length(GL),nine for grain width(GW),and nine for 1000-kernel weight(TKW)using GLU-SSSLs,which are single-segment substitution lines with Oryza glumaepatula as donor parent and Huajingxian 74(HJX74)as recipient parent.Among the QTL,qGL1-2,qGL1-4,qGL9-2,qGW2-2,qGW9-1 and qTKW9-2 contributed to high grain yield.GL9 was identified as a candidate gene for qGL9-2 by map-based cloning and sequencing,and is a novel allele of GS9.The kernel of NIL-gl9was slenderer and longer than that of HJX74,and the TKW and grain yield per plant of NIL-gl9 were higher than those of HJX74.The proportion of grain chalkiness of NIL-gl9 was much lower than that of HJX74.Thus,gl9 increased grain yield and appearance quality simultaneously.Three pyramid lines,NIL-gs3/gl9,NIL-GW7/gl9 and NIL-gw8/gl9,were developed and the kernel of each was longer than that of the corresponding recipient parent lines.The gl9 allele may be beneficial for breeding rice varieties with high grain yield and good appearance quality.

Experimental and genomic evidence for the indica-type cytoplasmic effect in Oryza sativa L.ssp.japonica

Cytoplasmic effects are important agronomical phenomena that have generated widespread interest in both theory and application. In the present study, five high yield rice cultivars(Oryza sativa L. ssp. japonica) in large-scale cultivation in northeast China were determined to possess Oryza sativa L. ssp. indica-type cytoplasm using cytoplasmic subspecies-specific molecular markers. This was confirmed by cytoplasmic genome-wide single nucleotide polymorphisms(SNPs) and functional gene sequencing. Two of these five japonica cultivars were core breeding parents with high yield and the other three were super-high-yield varieties registered by the Ministry of Agriculture of China. We constructed nuclear substitution lines to further demonstrate whether and how this indica-type cytoplasm contributed to yield improvement by comparing yield components. The results showed that under the same japonica nuclear background, the lines with indica-type cytoplasm had a significant decrease in tillers in exchange for increased grain number per panicle compared with their recurrent parents. Our results implied that botanical basis of this cytoplasmic effect was to reduce the plant's branching differentiation to produce more floral organs under the constant nutrition. Our findings open another door for the utilization of inter-subspecific hybridization for the improvement of rice cultivar.

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.

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.