Whole-Genome Sequencing of 117 Chromosome Segment Substitution Lines for Genetic Analyses of Complex Traits in Rice

Rice is one of the most important food crops in Asia. Genetic analyses of complex traits and molecular breeding studies in rice greatly rely on the construction of various genetic populations. Chromosome segment substitution lines (CSSLs) serve as a powerful genetic population for quantitative trait locus (QTL) mapping in rice. Moreover, CSSLs containing target genomic regions can be used as improved varieties in rice breeding. In this study, we developed a set of CSSLs consisting of 117 lines derived from the recipient ‘Huanghuazhan’ (HHZ) and the donor ‘Basmati Surkb 89–15’ (BAS). The 117 lines were extensively genotyped by whole-genome resequencing, and a high-density genotype map was constructed for the CSSL population. The 117 CSSLs covered 99.78% of the BAS genome. Each line contained a single segment, and the average segment length was 6.02 Mb. Using the CSSL population, we investigated three agronomic traits in Shanghai and Hangzhou, China, and a total of 25 QTLs were detected in both environments. Among those QTLs, we found that RFT1 was the causal gene for heading date variance between HHZ and BAS. RFT1 from BAS was found to contain a loss-of-function allele based on yeast two-hybrid assay, and its causal variation was a P to S change in the 94th amino acid of the RFT1 protein. The combination of high-throughput genotyping and marker-assisted selection (MAS) is a highly efficient way to construct CSSLs in rice, and extensively genotyped CSSLs will be a powerful tool for the genetic mapping of agronomic traits and molecular breeding for target QTLs/genes.

Identification of Candidate Cotton Genes Associated With Fiber Length Through Quantitative Trait Loci Mapping and RNA-Sequencing Using a Chromosome Segment Substitution Line

Fiber length is an important determinant of fiber quality, and it is a quantitative multi-genic trait. Identifying genes associated with fiber length is of great importance for efforts to improve fiber quality in the context of cotton breeding. Integrating transcriptomic information and details regarding candidate gene regions can aid in candidate gene identification. In the present study, the CCRI45 line and a chromosome segment substitution line (CSSL) with a significantly higher fiber length (MBI7747) were utilized to establish F2 and F2:3 populations. Using a high-density genetic map published previously, six quantitative trait loci (QTLs) associated with fiber length and two QTLs associated with fiber strength were identified on four chromosomes. Within these QTLs, qFL-A07-1, qFL-A12-2, qFL-A12-5, and qFL-D02-1 were identified in two or three environments and confirmed by a meta-analysis. By integrating transcriptomic data from the two parental lines and through qPCR analyses, four genes associated with these QTLs including Cellulose synthase-like protein D3 (CSLD3, GH_A12G2259 for qFL-A12-2), expansin-A1 (EXPA1, GH_A12G1972 for qFL-A12-5), plasmodesmata callose-binding protein 3 (PDCB3, GH_A12G2014 for qFL-A12-5), and Polygalacturonase (At1g48100, GH_D02G0616 for qFL-D02-1) were identified as promising candidate genes associated with fiber length. Overall, these results offer a robust foundation for further studies regarding the molecular basis for fiber length and for efforts to improve cotton fiber quality.

QTL Analysis Based on A Rice Short-Wide Grain CSSL-Z414 and Substitution Mapping of qGL11 and qGW5

Background: Most of rice agronomic traits as grain length etc. are complex traits controlled by multiple genes. Chromosome segment substitution lines (CSSLs) are ideal materials for dissecting and studying of these complex traits. Results: We developed a novel rice short-wide grain CSSL, Z414, deriving from progeny of the recipient parent Xihui 18 (an indica restorer line) and the donor parent Huhan 3 (a japonica cultivar). Z414 contained 4 substitution segments (average length was 3.04 Mb). Compared with Xihui 18, Z414 displayed seven significantly different traits as grain length, width and weight, chalkiness degree, brown rice rate etc. Then, 8 quantitative trait loci (QTLs) were found responding these difference traits by F2 population from Xihui 18/Z414. Among them, 6 QTLs (qPL3, qGW5, qGL11, qRLW5, qRLW11, qGWT5) could be verified by novel developed single segment substitution lines (SSSLs, S1-S6). In addition, 4 QTLs (qGL3, qGL5, qCD3 and qCD5) were novel detected by S1 and S5. Thus, the short–wide grain of Z414 was responded by qGL11, qGL3, qGL5, and qGW5. Then, qGL11 and qGW5 were delimited within intervals of 0.405 and 1.14 Mb on chromosomes 11 and 5, respectively, by substitution mapping. Again by sequencing, qRT-PCR and cell morphology analysis, qGW5 should be a novel allele of GS5 and qGL11 is novel QTL encoding CycT1;3, whose specific function of regulating grain length was still unknown. Finally, pyramid of qGL3 (a=0.22) and qGL11 (a=-0.19) displayed qGL11 epistatic to qGL3. In addition, novel S1 and D2 exhibited different grain sizes and lower chalkiness degree. They are potential to be directly used in breeding hybrid rice varieties.Conclusions: We constructed a novel rice short–wide grain CSSL-Z414 with 4 substitution segments based on the genetic backgrounds of Xihui 18. The broad grain of Z414 was controlled by qGW5, which should be a novel allele of GS5. The short grain of Z414 was controlled by qGL11, qGL3, and qGL5, and qGL11 is a novel QTL encoding CycT1;3, whose specific function of regulating grain length was still unknown, and qGL11 is epistatic to qGL3. Novel S1 and D2 are potential in hybrid rice varieties.

Identification of Heterotic Loci with Desirable Allelic Interaction to Increase Yield in Rice

Heterosis denotes the superiority of a hybrid plant over its parents. The use of heterosis has contributed significantly to yield improvement in crops. However, the genetic and molecular bases on heterosis are not fully understood. A large number of heterotic loci were identified for 12 yield-related traits in one parental population of chromosome segment substitution lines (CSSLs) and two test populations, which were interconnected by CSSLs derived from two rice genome-sequenced cultivars, Nipponbare and Zhenshan 97. Seventy-five heterotic loci were identified in both homozygous background of Zhenshan 97 and heterogeneous background of an elite hybrid cultivar Shanyou 63. Among the detected loci, at least 11 were colocalized in the same regions encompassing previously reported heterosis-associated genes. Furthermore, a heterotic locus Ghd8NIP for yield advantage was verified using transgenic experiments. Various allelic interaction at Ghd8 exhibited different heterosis levels in hetero-allelic combinations of five near-isogenic lines that contain a particular allele. The significant overdominance effects from some hetero-allelic combinations were found to improve yield heterosis in hybrid cultivars. Our findings support the role of allelic interaction at heterotic loci in the improvement of yield potential, which will be helpful for dissecting the genetic basis of heterosis and provide an optional strategy for the allele replacement in molecular breeding programs in hybrid rice.

Transformation and Overexpression of Primary Cell Wall Synthesis-Related Zinc Finger Gene Gh_A07G1537 to Improve Fiber Length in Cotton

Molecular interventions have helped to explore the genes involved in fiber length, fiber strength, and other quality parameters with improved characteristics, particularly in cotton. The current study is an extension and functional validation of previous findings that Gh_A07G1537 influences fiber length in cotton using a chromosomal segment substitution line MBI7747 through RNA-seq data. The recombinant Gh_A07G1537 derived from the MBI7747 line was over-expressed in CCRI24, a genotype with a low profile of fiber quality parameters. Putative transformants were selected on MS medium containing hygromycin (25mg/ml), acclimatized, and shifted to a greenhouse for further growth and proliferation. Transgene integration was validated through PCR and Southern Blot analysis. Stable integration of the transgene (ΔGh_A07G1537) was validated by tracking its expression in different generations (T0, T1, and T2) of transformed cotton plants. It was found to be 2.97-, 2.86-, and 2.92-folds higher expression in T0, T1, and T2 plants, respectively, of transgenic compared with non-transgenic cotton plants. Fiber quality parameters were also observed to be improved in the engineered cotton line. Genetic modifications of Gh_A07G1537 support the improvement in fiber quality parameters and should be appreciated for the textile industry.

Dynamic Analysis of QTLs on Plant Height with Single Segment Substitution Lines in Rice

Dynamic regulation of QTLs remains mysterious. Single segment substitution lines (SSSLs) and conditional QTL mapping and functional QTL mappings are ideal materials and methods to explore dynamics of QTLs for complex traits. This paper analyzed the dynamics of QTLs on plant height with SSSLs in rice. Five SSSLs were verified with plant height QTLs first. All five QTLs had significant positive effects at one or more developmental stages except QTL1. They interacted each other, with negative effects before 72 d after transplanting and positive effects since then. The five QTLs selectively expressed in specific periods, mainly in the periods from 35 to 42 d and from 49 to 56 d after transplanting. Expressions of epistasis were dispersedly in various periods, negative effects appearing mainly before 35 d. The five QTLs brought the inflexion point ahead of schedule, accelerated growth and degradation, and changed the peak plant height, while their interactions had the opposite effects. The information will be helpful to understand the genetic mechanism for developmental traits.

Comparative transcriptomic analysis of maize ear heterosis during the inflorescence meristem differentiation stage

Heterosis is widely used in many crops; however, its genetic mechanisms are only partly understood. Here, we sampled inflorescence meristem (IM) ears from the single-segment substitution maize (Zea mays) line lx9801hlEW2b, containing a heterotic locus hlEW2b associated with ear width, the receptor parent lx9801, the test parent Zheng58, and their corresponding hybrids. After transcriptomic analysis, 1638 genes were identified in at least one hybrid with nonadditively expressed patterns and different expression levels between the two hybrids. In particular, 2263 (12.89%) and 2352 (14.65%) genes showed allele-specific expression (ASE) in Zheng58 × lx9801 and Zheng58 × lx9801hlEW2b, respectively. A functional analysis showed that these genes were enriched in development-related processes and biosynthesis and catabolism processes, which are potentially associated with heterosis. Additionally, nonadditive expression and ASE may fine-tune the expression levels of crucial genes (such as WUS and KNOX that control IM development) controlling auxin metabolism and ear development to optimal states, and transcriptional variation may play important roles in maize ear heterosis. The results provide new information that increases our understanding of the relationship between transcriptional variation and heterosis formation during maize ear development, which may be helpful in clarifying the genetic and molecular mechanisms of heterosis.

Identification and Pyramid of QTLs Based on Rice Short-wide Grain CSSL-Z414, SSSL, DSSL and Candidate Gene Analysis of qGL11 and qGW5

BackgroundMost of rice agronomic traits as grain length etc. are complex traits controlled by multiple genes. Chromosome segment substitution lines (CSSLs) are ideal materials for dissecting and studying of these complex traits. ResultsA rice short-wide grain CSSL Z414 was identified among progeny of the recipient parent Xihui 18 (an indica restorer line) and the donor parent Huhan 3 (a japonica cultivar). Z414 carried 4 substitution segments (average length was 3.04 Mb), and displayed shorter panicle length and less number of primary branches, shorter, wider and larger grain, higher brown rice rate and chalkiness degree when compared with Xihui 18. Then, 9 quantitative trait loci (QTLs) for associated traits were identified using the secondary F2 population from Xihui 18 / Z414. Among them, 6 QTLs (qPL3, qGW5, qGL11, qRLW5, qRLW11, qGWT5) could be verified by corresponding single segment substitution lines (SSSLs, S1-S6). In addition, 4 QTLs (qGL3, qGL5, qCD3 and qCD5) were detected by S1 and S5, which was not detected by the F2 population. Thus, the grain length of Z414 was controlled by qGL11, qGL3 and qGL5, and the grain width of Z414 was answered by qGW5. Then by substitution mapping, qGL11 and qGW5 were delimited within the estimated substitution length of 1.42 and 1.14 Mb on chromosomes 11 and 5, and 4 and 2 candidate genes were found respectively for qGL11 and qGW5 by sequencing. However, only two had expression differences by qRT-PCR analysis. Finally, Analysis of QTL epistatic effects revealed that pyramid of qGL3 (a= 0.22) and qGL11 (a=-0.19) caused grain length of double segment substitution line (DSSL, D2) shorter than that of S5 (qGL11).ConclusionsWe developed a rice short –wide grain CSSL with 4 substitution segments from Huhan 3 based on the genetic backgrounds of Xihui 18. The grain width of Z414 was controlled by qGW5, and GS5 should be the candidate gene for qGW5 by sequencing and qRT-PCR analysis. The grain length of Z414 was controlled by qGL11, qGL3, and qGL5, and CycT1;3 should be the best candidate gene of qGL11, whose specific function of regulating grain length was still unknown, and qGL11 is epistatic to qGL3.

Combining Genome and Gene Co-expression Network Analyses for the Identification of Genes Potentially Regulating Salt Tolerance in Rice

Salinity stress tolerance is a complex polygenic trait involving multi-molecular pathways. This study aims to demonstrate an effective transcriptomic approach for identifying genes regulating salt tolerance in rice. The chromosome segment substitution lines (CSSLs) of “Khao Dawk Mali 105 (KDML105)” rice containing various regions of DH212 between markers RM1003 and RM3362 displayed differential salt tolerance at the booting stage. CSSL16 and its nearly isogenic parent, KDML105, were used for transcriptome analysis. Differentially expressed genes in the leaves of seedlings, flag leaves, and second leaves of CSSL16 and KDML105 under normal and salt stress conditions were subjected to analyses based on gene co-expression network (GCN), on two-state co-expression with clustering coefficient (CC), and on weighted gene co-expression network (WGCN). GCN identified 57 genes, while 30 and 59 genes were identified using CC and WGCN, respectively. With the three methods, some of the identified genes overlapped, bringing the maximum number of predicted salt tolerance genes to 92. Among the 92 genes, nine genes, OsNodulin, OsBTBZ1, OsPSB28, OsERD, OsSub34, peroxidase precursor genes, and three expressed protein genes, displayed SNPs between CSSL16 and KDML105. The nine genes were differentially expressed in CSSL16 and KDML105 under normal and salt stress conditions. OsBTBZ1 and OsERD were identified by the three methods. These results suggest that the transcriptomic approach described here effectively identified the genes regulating salt tolerance in rice and support the identification of appropriate QTL for salt tolerance improvement.