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.

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.

Integrative QTL Identification, Fine Mapping and Candidate Gene Analysis of a Major Locus qLTG3a for Seed Low-Temperature Germinability in Rice

Low-temperature germinability (LTG) is an important agronomic trait that can affect the planting time, planting area, and grain yield of staple crops, such as rice. However, the genetic mechanism of LTG is still unclear. In this study, a multi-parental permanent population with 208 single segment substitution lines (SSSLs) was used to conduct a genetic dissection for LTG across four cropping seasons. LTG was a typical quantitative trait with a high combined broad-sense heritability of 0.71. By comparison with the recipient parent, Huajingxian74, 24 SSSLs were identified as carrying LTG QTLs, which were further merged into integrated QTLs with shorter genetic distances by substitution mapping. Finally, 14 LTG QTLs were mapped on ten chromosomes, including seven positive-effect and seven negative-effect QTLs, with additive effect contributions ranging from 19.2 to 39.9%. qLTG3a, a main-effect and novel QTL, was confirmed by bulk segregant analysis using an F2 segregating population, and five key recombinants were selected to develop F3 populations for progeny testing. Marker-trait association analysis fine mapped qLTG3a to a 332.7-kb physical region between markers M6026 and M6341. Within this interval, 40 annotated genes were revealed, and three genes (Os03g0213300, Os03g0214400, and Os03g0214600) were considered as pivotal candidate genes for qLTG3a based on their sequence variations and expression patterns. Besides low temperature, qLTG3a can also enhance seed germination under standard temperature and osmotic stress. In summary, this study identified some genetic factors regulating LTG and opened a new window for breeding elite direct-seeded rice varieties. It will help reduce the climate risk in the production process of rice, which is of great significance to ensuring food security.

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.

Addressing Iron and Zinc Micronutrient Malnutrition Through Nutrigenomics in Pearl Millet: Advances and Prospects

Iron (Fe) and zinc (Zn) micronutrient deficiencies are significant health concerns, particularly among the underprivileged and resource-poor people in the semi-arid tropics globally. Pearl millet is regarded as a climate-smart crop with low water and energy footprints. It thrives well under adverse agro-ecologies such as high temperatures and limited rainfall. Pearl millet is regarded as a nutri-cereal owing to health-promoting traits such as high grain Fe and Zn content, metabolizable energy, high antioxidant and polyphenols, high proportion of slowly digestible starches, dietary fibers, and favorable essential amino acid profile compared to many cereals. Higher genetic variability for grain Fe and Zn content has facilitated considerable progress in mapping and mining QTLs, alleles and genes underlying micronutrient metabolism. This has been made possible by developing efficient genetic and genomic resources in pearl millet over the last decade. These include genetic stocks such as bi-parental RIL mapping populations, association mapping panels, chromosome segment substitution lines (CSSLs) and TILLING populations. On the genomics side, considerable progress has been made in generating genomic markers, such as SSR marker repository development. This was followed by the development of a next-generation sequencing-based genome-wide SNP repository. The circa 1,000 genomes re-sequencing project played a significant role. A high-quality reference genome was made available by re-sequencing of world diversity panel, mapping population parents and hybrid parental lines. This mini-review attempts to provide information on the current developments on mapping Fe and Zn content in pearl millet and future outlook.

Assessing the Weed-Suppressing Potential of Cotton Chromosome Substitution Lines Using the Stair-Step Assay

Palmer amaranth is a problematic common weed species, especially in cotton. With the wide use of chemical herbicide and herbicide-tolerant transgenic cotton lines, Palmer amaranth populations have developed tolerance to commonly used herbicides. It is imperative to develop alternative weed control methods to slow the evolution of herbicide-resistant weed populations and provide new strategies for weed management. Eleven chromosome substitution (CS) cotton lines (CS-B26lo, CS-T17, CS-B16-15, CS-B17-11, CS-B12, CS-T05sh, CS-T26lo, CS-T11sh, CS-M11sh, CS-B22sh, and CS-B22lo) were screened for weed-suppressing abilities in this study. The cotton lines were tested using the established stair-step assay. Height (cm) and chlorophyll concentration (cci) were measured for each plant in the system. The most significant variation in Palmer amaranth height reduction among the CS lines was observed 21 days after establishment. CS-B22sh (76.82%) and T26lo (68.32%) were most effective in reducing Palmer amaranth height. The cluster analysis revealed that CS-B22sh, and CS-T26lo were clustered in one group, suggesting similar genetic potential with reference to Palmer amaranth growth and development. CS-B22sh showed novel genetic potential to control the growth and development of Palmer amaranth, a problematic weed in cotton fields. Future experimentation should implement more parameters and chemical testing to explore allelopathic interactions among CS lines and Palmer amaranth.

Novel QTL For Lodging Resistance, PRL4, Improves Physical Properties With High Non-Structural Carbohydrate Accumulation of Basal Culms in Rice (Oryza Sativa L.)

Resistance to lodging, an important problem in rice production, has three types: low plant height, strong culm, and high strength of the lower part of the plant. The determinants of strength of the lower part remains unclear, compared with plant height and culm strength. This study identified a new genetic factor involved in the strength of the lower part, as assessed by pushing resistance, using chromosomal segment substitution lines (CSSLs) to clarify the determinants of strength of the lower part by functional analysis of the CSSL and the near isogenic line (NIL) harboring the identified quantitative trait locus (QTL). QTL analysis identified the QTL for increasing pushing resistance on chromosome 4, PRL4, which was not related to days to heading. The CSSL with PRL4 showed increased pushing resistance and physical strength of the basal culm, but decreased filled grain ratio and grain weight. The NIL with PRL4, developed by backcrossing this CSSL, improved pushing resistance and the strain of culm until breaking under compression, and did not decrease yield traits. These lines with PRL4 increased the accumulation of non-structural carbohydrate (NSC) in the basal culm at the fully ripe stage. Thus, the genetic control of NSC accumulation in culms by PRL4 may improve the strength of the lower part by enhancing culm toughness with strength and ductility.

Identification and Evaluation of Wheat-Aegilops bicornis Lines with Resistance to Powdery Mildew and Stripe Rust

Wheat pathogens, especially those causing powdery mildew and stripe rust, seriously threaten yield worldwide. Utilizing newly identified disease resistance genes from wheat relatives is an effective strategy to minimize disease damage. In this study, chromosome-specific molecular markers for the 3Sb and 7Sb chromosomes of Aegilops bicornis were developed using PCR-based landmark unique gene (PLUG) primers for screening wheat-Ae. bicornis progenies. Fluorescence in situ hybridization (FISH) was performed to further identify wheat-Ae. bicornis progenies using oligonucleotides probes Oligo-pSc119.2-1, Oligo-pTa535-1, and Oligo-(GAA)8. After establishing Ae. bicornis 3Sb and 7Sb chromosome-specific FISH markers, Holdfast (common wheat)-Ae. bicornis 3Sb addition, 7Sb addition, 3Sb(3A) substitution, 3Sb(3B) substitution, 3Sb(3D) substitution, 7Sb(7A) substitution, and 7Sb(7B) substitution lines were identified by the molecular and cytological markers. Stripe rust and powdery mildew resistance, along with agronomic traits were investigated to evaluate the breeding potential of these lines. Holdfast and Holdfast-Ae. bicornis progenies were all highly resistant to stripe rust, indicating that the stripe rust resistance might derive from Holdfast. However, Holdfast-Ae. bicornis 3Sb addition, 3Sb(3A) substitution, 3Sb(3B) substitution, and 3Sb(3D) substitution lines showed high resistance to powdery mildew while Holdfast was highly susceptible, indicating that chromosome 3Sb of Ae. bicornis carries previously unknown powdery mildew resistance gene(s). Additionally, the transfer of the 3Sb chromosome from Ae. bicornis to wheat significantly increased tiller number, but chromosome 7Sb has a negative effect on agronomic traits. Therefore, wheat germplasm containing Ae. bicornis chromosome 3Sb has potential to contribute to improving powdery mildew resistance and tiller number during wheat breeding.

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.

Assessing the Weed-Suppressing Potential of Cotton Chromosome Substitution Lines Using the Stair-Step Assay

Palmer amaranth (Amaranthus palmeri) is a problematic common weed species, especially in cotton (Gossypium hirsutum). With the wide use of chemical herbicide and herbicide-tolerant transgenic cotton lines, Palmer amaranth populations have developed tolerance to commonly used herbicides. It is imperative to develop alternative weed control methods to slow the evolution of herbicide-resistant weed populations and provide new sources for weed management. Eleven chromosome substitution (CS) cotton lines CS-B26lo, CS-T17, CS-B16-15, CS-B17-11, CS-B12, CS-T05sh, CS-T26lo, CS-T11sh, CS-M11sh, CS-B22sh, and CS-B22lo were screened for weed-suppressing abilities in this study. The cotton lines were tested using the established stair-step structure methodology, which provided scope to study the effect of individual CS lines on the growth and development of Palmer amaranth weed without any interference of other external factors in the greenhouse. Height (cm) and chlorophyll concentration (cci) were measured for each plant in the system. The data were analyzed as a randomized complete block design using LSD mean comparisons of the genotypes at the P≤ .05 level. The 14th day after establishment resulted in the most significant variation in Palmer amaranth height reduction among the CS lines. Results indicated that CS-B22sh had the highest effect in reducing Palmer amaranth height and chlorophyll concentration with the most heightened susceptibility for Palmer amaranth. The cluster analysis revealed that Enlist® cotton, CS-CS-B22sh, and CS-T26lo were clustered in one group suggesting similar genetic potential with reference to Palmer amaranth growth and development. CS-B22sh showed novel genetic potential to control the growth and development of Palmer amaranth, a major weed in cotton fields. In the future, it will be interesting to investigate if CS-B22sh exudates from its root contain allelochemicals able to impede the growth and development of Palmer amaranth.