Novel chromosome segment substitution lines derived from japonica cultivar ‘Yukihikari’ in the genetic background of ‘Joiku462’ cultivar and identification of quantitative trait loci for heading date and grain quality

Quantitative trait loci (QTLs) associated with eating quality, grain appearance quality and yield-related traits were mapped in recombinant inbred lines (RILs) derived from closely related rice (Oryza sativa L. subsp. japonica) cultivars, Yukihikari (good eating quality) and Joiku462 (superior eating quality and high grain appearance quality). Apparent amylose content (AAC), protein content (PC), brown grain length (BGL), brown grain width (BGWI), brown grain thickness (BGT), brown grain weight per plant (BGW) and nine yield-related traits were evaluated in 133 RILs grown in four different environments in Hokkaido, near the northernmost limit for rice paddy cultivation. Using 178 molecular markers, a total of 72 QTLs were detected, including three for AAC, eight for PC, two for BGL, four for BGWI, seven for BGT, and six for BGW, on chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 11 and 12. Fifteen intervals were found to harbor multiple QTLs affecting these different traits, with most of these QTL clusters located on chromosomes 4, 6, 8, 9 and 12. These QTL findings should facilitate gene isolation and breeding application for improvement of eating quality, grain appearance quality and yield of rice cultivars.

Grain yield-related traits and grain quality-related traits are important for rice cultivars. The quantitative trait loci (QTLs) involved in controlling the natural variation in these traits among closely related cultivars are still unclear. The present study describes the development of a novel chromosome segment substitution line (CSSL) population derived from a cross between the temperate japonica cultivars Yukihikari and Kirara397, which are grown in Hokkaido, the northernmost limit for rice cultivation. Days to heading, culm length, panicle length, panicle number, brown grain weight per plant, thousand brown grain weight, brown grain length, brown grain width, brown grain thickness, apparent amylose content, and protein content were evaluated. Panicle length, brown grain length and amylose content differed significantly in the parental cultivars. Thirty-five significant changes in the evaluated traits were identified in the CSSLs. A total of 28 QTLs were located on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 and 12. These findings could be useful for breeding rice cultivars in the northernmost limit for rice cultivation.

Rice grain quality is a determinant of rice breeding success. Although several studies have independently looked at the different traits of quality to infer the phenotype of the grain, few studies have identified the various metabolites that are produced in high quality rice. In recent years, genetic markers have proven to be effective in increasing the efficiency of breeding line selection and therefore shortening the breeding cycle. In complex traits such as rice grain quality, the availability of more specific phenotypes will increase the value of these genetic markers. Many traits of quality in rice are associated with starch; however, lipids interact with starch and have been shown to influence many of the traits of eating quality and aroma. To identify these new phenotypes and understand their genetic basis, using relevant tools that can discriminate between the phenotypes of varieties at the metabolite and genetic levels is necessary.To identify these new phenotypes, in Chapter 3, a set of 40 Cambodian rice varieties were screened for physical and texture-associated traits including apparent amylose content (AAC), gelatinisation temperature (GT), gel consistency (GelCon) and pasting properties and then a subset from this set was screened for volatile compounds using a two-dimensional gas chromatography-time-of-flight-mass spectrometer (GC×GC-TOF-MS) and fatty acids (FA) using gas chromatography-mass spectrometry (GC-MS). The FA analysis showed that unsaturated FA (UFA) oleic (C18:1n-9) and linoleic acid (C18:2n-6) were the most abundant in milled rice grains, followed by palmitic acid (C16:0). These results infer about the lipid origin of odour-active volatile compounds, which were found to be characterising the group of known fragrant, low AAC and soft-textured rice varieties, including the Jasmine-style indica variety Phka Rumduol (PRD).To identify the genetic determinants of these important traits of quality, in Chapters 4 and 5, quantitative trait loci (QTL) mapping was carried out for the texture and aroma traits in PRD using a population of about 300 F6 recombinant-inbred lines (RIL) derived from an intraspecific cross between PRD and Thmar Krem (TMK), both of which were identified as the most discriminating varieties in Chapter 3. This RIL population was genotyped for single nucleotide polymorphisms (SNPs). QTL analysis revealed large-effect QTLs for AAC, GT and all of the pasting properties. Candidate genes for these QTLs relate back to the starch biosynthesis pathways. Major QTLs were colocalised to the Waxy (Wx) gene and starch synthase IIa (SSIIa) on chromosome 6. Minor QTLs on chromosomes 3 and 8 were identified for AAC, SB, PV and HPC, and at chromosomes 4 and 7 for GT and pasting temperature (PTemp). This Chapter has shown that the rice texture is regulated by multiple genes and that although correlations do exist among the routinely measured textural traits, these correlations may not necessarily translate into the already existing molecular markers.In Chapter 5, QTLs for odour-active volatile compounds and FA were carried out on the RIL population. The presence of 2AP and its derivatives significantly characterised the fragrant lines from the non-fragrant ones and this was supported by the identification of large-effect QTLs pointing to the region of the fragrance gene (FGR). Odour-active and low odour threshold volatile compounds that can be derived from the oxidation of UFA were identified as segregating traits in the population as supported by the presence of significant SNPs on rice chromosome 6. These volatile compounds explain the differences in varieties that cannot be differentiated by the presence or absence of 2AP alone. To verify that these volatile compounds can indeed be derived from UFA, the % FA composition in the population was also determined. Here, that C18:1n-9, C18:2n-6 and C16:0 were the identified as the most abundant FA, coinciding with the results observed in Chapter 3. PRD had higher levels of C18:1n-9 and TMK had higher levels of C18:2n-6, a difference which was exhibited in a normal distribution across the population. QTL analyses revealed a common QTL on rice chromosome 7 explaining the variation in % composition of these two UFA. This QTL encompassed the regions where fatty acid biosynthesis genes were located.In order to trace back the origins of the most important FAs to their lipid molecule and to screen the most abundant lipids in the rice samples, in Chapter 6, rice lipidomics was carried out for the first time using a reversed-phase ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results of this experiment showed that glycerolipids in the form of triacylglycerols (TAG) and diacylglycerols (DAG), and glycerophospholipids were the most abundant lipid features in milled rice. Statistical analyses showed strong statistical correlations between traits of quality and several lipid features, indicating a substantial influence that lipid play in determining rice quality.The information in this research will be delivered to rice improvement programs to enable breeders to select more accurately for particular traits of quality, and for novel quality traits identified in this study.

Grain protein content (GPC) is gaining attention due to increasing consumer demand for nutritious foods. The present study carried out at ICAR-IIRR, Hyderabad, focused on the identification of quantitative trait loci (QTLs) linked with GPC and other quality traits. We utilized a population of 188 F2 individuals developed from BPT 5204 (low GPC) X JAK 686 (high GPC) for QTL analysis. QTL analysis yielded four significant QTLs for GPC, three for amylose content, and multiple QTLs for other quality traits. qPC1.2, a major QTL in milled rice, was located in the marker interval RM562-RM11307 on chromosome 1 with an LOD value of 4.4. qPC1.2 explained 15.71% of the phenotypic variance (PVE). Additionally, the Interval Mapping for Epistatic QTLs (IM-EPI) method detected 332 pairs of di-genic epistatic QTLs. Fifteen QTLs exhibited a positive additive effect, indicating that the contributing allele(s) was from JAK 686. Five F2 plants, viz., F2-140, F2-12, F2-7, F2-147, and F2-41, exhibited a high GPC of 14.67%, 14.36%, 14.32%, 13.60%, and 13.36%, respectively. Additionally, these plants also exhibited high per-plant grain yield (~17.0-29.0 g) with desirable agronomic traits. The QTLs identified are valuable resources for developing high-grain-protein varieties with high grain yield and desirable quality traits.

Identification of quantitative trait loci associated with rice eating quality traits using a population of recombinant inbred lines derived from a cross between two temperate japonica cultivars.

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 effective tools used in mapping QTLs. In this study, we constructed 154 CSSLs from backcross progeny (BC(3)F(2)) 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.

A high level of heterosis exists in intersubspecific crosses between indica and japonica rice (Oryza sativa L.) cultivars, and the exploitation of heterosis has long been considered a promising method of increasing rice yield. To analyze the chromosome segments associated with heterosis for yield‐related traits and to dissect the major heterosis quantitative trait loci (QTL) for marker‐assisted selection in intersubspecific crosses to improve rice yield, we introduced a set of 37 chromosome‐segment substitution lines (CSSLs), each of which carried one or a few chromosome segments of the indica rice cultivar Habataki, in the genetic background of the japonica rice cultivar Sasanishiki. The CSSLs were individually crossed with ‘Changhui T025’ to generate a set of corresponding F1 hybrids (chromosome‐segment substitution line hybrids, CSSLHs). These CSSLHs were field‐tested for nine yield‐related traits in two different environments, and 20 heterosis QTL were detected. These QTL were divided into two groups: the positive alleles of one group originated from the Habataki × Changhui T025 hybrid, whereas those of the other group originated from the Sasanishiki × Changhui T025 hybrid. This implied that many different genes that positively contribute to heterosis likely exist in indica and japonica cultivars. Four key heterosis QTL were identified in both environments: qHppp10 for panicle number per plant, qHph1‐1 for plant height, qHtgw4‐1 for 1000‐grain weight, and qHdsp1 for spikelet density per plant. The indica × indica segments at these QTL represent good candidates for marker‐assisted selection for the corresponding traits in intersubspecific crosses to improve yield. Moreover, the CSSLs harboring these QTL represent candidate parents of F1 hybrids along with Changhui T025 or other indica lines.

Bending-type lodging is one of the most important factors affecting the yield and grain quality of rice. This study identified quantitative trait loci (QTLs) for physical strength of the upper culms, and evaluated QTL effects on lodging resistance. In 2010 and 2011, QTLs for breaking strength, length, and diameter of the top three internodes were identified by analyzing chromosomal segment substitution lines (CSSLs) developed from ‘Koshihikari’ and ‘Kasalath’. The QTL analysis indicated that ‘Kasalath’ had two types of QTLs: one to strengthen specific internodes and one to simultaneously improve the physical strengths of plural internodes or the top three internodes. A QTL for breaking strengths of the top three internodes (bsuc11) was detected on chromosome 11 in both years. This QTL did not overlap with that for internode length. To evaluate the effects of bsuc11 on lodging resistance, this study selected three CSSLs with bsuc11 and analyzed the breaking strengths of the top three internodes after heading and the pushing resistance of the lower part. Internodes of ‘Koshihikari’ showed decreased breaking strengths after grain filling, while those of CSSLs with bsuc11 did not show this decrease in breaking strength. The pushing resistance of the lower part at the fully ripe stage was the same in ‘Koshihikari’ and CSSLs with bsuc11. These results suggested that bsuc11 could be a target to improve the physical strength of the upper culms to resist bending-type lodging, and that the physical strengths of upper and lower parts are controlled by different genetic factors in rice.

Cold damage stress significantly affects rice growth (germination and seedling) and causes serious losses in yield in temperate and high-altitude areas around the globe. This study aimed to explore the cold tolerance (CT) locus of rice and create new cold-tolerant germplasm. We constructed a chromosome segment substitution line (CSSL) with strong CT and fine mapped quantitative trait loci (QTLs) associated with CT by performing the whole-genome resequencing of CSSL with phenotypes under cold treatment. A chromosome CSSL, including 271 lines from a cross between the cold-tolerant wild rice Y11 (Oryza rufipogon Griff.) and the cold-sensitive rice variety GH998, was developed to map QTLs conferring CT at the germination stage. The whole-genome resequencing was performed on CSSL for mapping QTLs of associated with CT at the germination stage. A high-density linkage map of the CSSLs was developed using the whole-genome resequencing of 1484 bins. The QTL analysis using 615,466 single-nucleotide polymorphisms (SNPs) led to the identification of 2 QTLs related to germination rate at low-temperature on chromosome 8 (qCTG-8) and chromosome 11 (qCTG-11). The qCTG-8 and qCTG-11 explained 14.55% and 14.31% of the total phenotypic variation, respectively. We narrowed down qCTG-8 and qCTG-11 to 195.5 and 78.83-kb regions, respectively. The expression patterns of important candidate genes in different tissues, and of RNA-sequencing (RNA-seq) in CSSLs, were identified based on gene sequences in qCTG-8 and qCTG-11 cold-induced expression analysis. LOC_Os08g01120 and LOC_Os08g01390 were identified as candidate genes in qCTG-8, and LOC_Os11g32880 was identified as a candidate gene in qCTG-11. This study demonstrated a general method that could be used to identify useful loci and genes in wild rice and aid in the future cloning of candidate genes of qCTG-8 and qCTG-11. The CSSLs with strong CT were supported for breeding cold-tolerant rice varieties.

High grain quality is a key target in wheat breeding and is influenced by genetic and environmental factors. This study evaluated 94 recombinant inbred lines (RILs) from a Pamyati Azieva × Paragon (PA × P) mapping population grown in two regions in Kazakhstan to assess the genetic basis of six grain quality traits: the test weight per liter (TWL, g/L), grain protein content (GPC, %), gluten content (GC, %), gluten deformation index in flour (GDI, unit), sedimentation value in a 2% acetic acid solution (SV, mL), and grain starch content (GSC, %). A correlation analysis revealed a trade-off between protein and starch accumulation and an inverse relationship between grain quality and yield components. Additionally, GPC exhibited a negative correlation with yield per square meter (YM2), underscoring the challenge of simultaneously improving grain quality and yield. With the use of the QTL Cartographer statistical package, 71 quantitative trait loci (QTLs) were identified for the six grain quality traits, including 20 QTLs showing stability across multiple environments. Notable stable QTLs were detected for GPC on chromosomes 4A, 5B, 6A, and 7B and for GC on chromosomes 1D and 6A, highlighting their potential for marker-assisted selection (MAS). A major QTL found on chromosome 1D (QGDI-PA × P.ipbb-1D.1, LOD 19.4) showed a strong association with gluten deformation index, emphasizing its importance in improving flour quality. A survey of published studies on QTL identification in common wheat suggested the likely novelty of 12 QTLs identified for GDI (five QTLs), TWL (three QTLs), SV, and GSC (two QTLs each). These findings underscore the need for balanced breeding strategies that optimize grain composition while maintaining high productivity. With the use of SNP markers associated with the identified QTLs for grain quality traits, the MAS approach can be implemented in wheat breeding programs.

Most agronomic traits of rice (Oryza sativa), such as grain length, are complex traits controlled by multiple genes. Chromosome segment substitution lines (CSSLs) are ideal materials for dissecting these complex traits. We developed the novel rice CSSL ‘Z414’, which has short, wide grains, from progeny of the recipient parent ‘Xihui 18’ (an indica restorer line) and the donor parent ‘Huhan 3’ (a japonica cultivar). Z414 contains four substitution segments with an average length of 3.04 Mb. Z414 displays seven traits that significantly differ from those of Xihui 18, including differences in grain length, width, and weight; degree of chalkiness; and brown rice rate. We identified seven quantitative trait loci (QTL) that are responsible for these differences in an F2 population from a cross between Xihui 18 and Z414. Among these, six QTL (qPL3, qGW5, qGL11, qRLW5, qRLW11, and qGWT5) were detected in newly developed single-segment substitution lines (SSSLs) S1–S6. In addition, four QTL (qGL3, qGL5, qCD3, and qCD5) were detected in S1 and S5. Analysis of these SSSLs attributed the short, wide grain trait of Z414 to qGL11, qGL3, qGL5, and qGW5. Substitution mapping delimited qGL11 within an 810-kb interval on chromosome 11. Sequencing, real time quantitative PCR, and cell morphology analysis revealed that qGL11 might be a novel QTL encoding the cyclin CycT1;3. Finally, pyramiding qGL3 (a = 0.43) and qGL11 (a = − 0.37) led to shorter grains in the dual-segment substitution line D2 and revealed that qGL11 is epistatic to qGL3. In addition, S1 and D2 exhibited different grain sizes and less chalkiness than Z414. In conclusion, the short grain phenotype of the CSSL Z414 is controlled by qGL11, qGL3, and qGL5. qGL11 might be a novel QTL encoding CycT1;3, whose specific role in regulating grain length was previously unknown, and qGL11 is epistatic to qGL3. S1 and D2 could potentially be used in hybrid rice breeding.

BackgroundGenetic populations provide the basis for a wide range of genetic and genomic studies and have been widely used in genetic mapping, gene discovery and genomics-assisted breeding. Chromosome segment substitution lines (CSSLs) are the most powerful tools for the detection and precise mapping of quantitative trait loci (QTLs), for the analysis of complex traits in plant molecular genetics.ResultsIn this study, a wide population consisting of 128 CSSLs was developed, derived from the crossing and back-crossing of two sequenced rice cultivars: 9311, an elite indica cultivar as the recipient and Nipponbare, a japonica cultivar as the donor. First, a physical map of the 128 CSSLs was constructed on the basis of estimates of the lengths and locations of the substituted chromosome segments using 254 PCR-based molecular markers. From this map, the total size of the 142 substituted segments in the population was 882.2 Mb, was 2.37 times that of the rice genome. Second, every CSSL underwent high-throughput genotyping by whole-genome re-sequencing with a 0.13× genome sequence, and an ultrahigh-quality physical map was constructed. This sequencing-based physical map indicated that 117 new segments were detected; almost all were shorter than 3 Mb and were not apparent in the molecular marker map. Furthermore, relative to the molecular marker-based map, the sequencing-based map yielded more precise recombination breakpoint determination and greater accuracy of the lengths of the substituted segments, and provided more accurate background information. Third, using the 128 CSSLs combined with the bin-map converted from the sequencing-based physical map, a multiple linear regression QTL analysis mapped nine QTLs, which explained 89.50% of the phenotypic variance for culm length. A large-effect QTL was located in a 791,655 bp region that contained the rice 'green revolution' gene.ConclusionsThe present results demonstrated that high throughput genotyped CSSLs combine the advantages of an ultrahigh-quality physical map with high mapping accuracy, thus being of great potential value for gene discovery and genetic mapping. These CSSLs may provide powerful tools for future whole genome large-scale gene discovery in rice and offer foundations enabling the development of superior rice varieties.

Construction of chromosome segment substitution lines of Dongxiang common wild rice (Oryza rufipogon Griff.) in the background of the japonica rice cultivar Nipponbare (Oryza sativa L.)

Quantitative trait locus (QTL) mapping and stability analysis were carried out for 16 rice (Oryza sativa L.) quality traits across eight environments, by using a set of chromosome segment substitution lines with 'Asominori' as genetic background. The 16 quality traits include percentage of grain with chalkiness (PGWC), area of chalky endosperm (ACE), amylose content (AC), protein content (PC), peak viscosity, hot paste viscosity, cool paste viscosity, breakdown viscosity (BDV), setback viscosity (SBV), consistency viscosity, cooked-rice luster (LT), scent, tenderness (TD), viscosity, elasticity, and the integrated values of organleptic evaluation (IVOE). A total of 132 additive effect QTLs are detected for the 16 quality straits in the eight environments. Among these QTLs, 56 loci were detected repeatedly in at least three environments. Interestingly, several QTL clusters were observed for multiple quality traits. Especially, one QTL cluster near the G1149 marker on chromosome 8 includes nine QTLs: qPGWC-8, qACE-8, qAC-8, qPC-8a, qBDV-8a, qSBV-8b, qLT-8a, qTD-8a, and qIVOE-8a, which control PGWC, ACE, AC, PC, BDV, SBV, LT, TD, and IVOE, respectively. Moreover, this QTL cluster shows high stability and repeatability in all eight environments. In addition, one QTL cluster was located near the C2340 marker on chromosome 1 and another was detected near the XNpb67 marker on chromosome 2; each cluster contained five loci. Near the C563 marker on chromosome 3, one QTL cluster with four loci was found. Also, there were nine QTL clusters that each had two or three loci; however, their repeatability in different environments was relatively lower, and the genetic contribution rate was relatively smaller. Considering the correlations among all of the 16 quality traits with QTL cluster distributions, we can conclude that the stable and major QTL cluster on chromosome 8 is the main genetic basis for the effect of rice chalkiness, AC, PC, and rapid viscosity analyzer profile characteristics on the eating quality of cooked rice. Consequently, this QTL cluster is a novel gene resource for controlling rice high-quality traits and should be of great significance for research on formation mechanism and molecule improvement of rice quality.

Backcross inbred lines (BILs) derived from elite x wild crosses are very useful for basic studies and breeding. The aim of this study was to map quantitative trait loci (QTLs) associated with yield and related traits and to identify chromosomal segment substitution lines (CSSLs) from unselected BC2F8 BILs of Swarna/Oryza nivara IRGC81848. In all, 94 BILs were field evaluated in 2 years (wet seasons, 2014 and 2015) for nine traits; days to 50% flowering, days to maturity (DM), plant height (PH), number of tillers, number of productive tillers, panicle weight, yield per plant, bulk yield, and biomass. BILs were genotyped using 111 polymorphic simple sequence repeats distributed across the genome. Fifteen QTLs including 10 novel QTLs were identified using composite interval mapping, Inclusive composite interval mapping and multiple interval mapping (MIM). O. nivara alleles were trait-enhancing in 26% of QTLs. Only 3 of 15 QTLs were also reported previously in BC2F2 of the same cross. These three included the two major effect QTLs for DM and PH detected in both years with 13 and 20% phenotypic variance. Further, a set of 74 CSSLs was identified using CSSL Finder and 22 of these showed significantly higher values than Swarna for five yield traits. CSSLs, 220S for panicle weight and 10-2S with consistent high yield in both years are worthy of large scale field evaluation. The major QTLs and 22 significantly different CSSLs are a useful resource for rice improvement and dissecting yield related traits.