Pyramiding of Low Chalkiness QTLs Is an Effective Way to Reduce Rice Chalkiness.

Rice chalkiness is a key limiting factor of high-quality rice. The breeding of low chalkiness varieties has always been a challenging task due to the complexity of chalkiness and its susceptibility to environmental factors. In previous studies, we identified six QTLs for the percentage of grain chalkiness (PGC), named qPGC5, qPGC6, qPGC8.1, qPGC8.2, qPGC9 and qPGC11, using single-segment substitution lines (SSSLs) with genetic background of Huajingxian 74 (HJX74). In this study, we utilized the six low chalkiness QTLs to develop 17 pyramiding lines with 2-4 QTLs. The results showed that the PGC decreased with the increase of QTLs in the pyramiding lines. The pyramiding lines with 4 QTLs significantly reduced the chalkiness of rice and reached the best quality level. Among the six QTLs, qPGC5 and qPGC6 showed greater additive effects and were classified as Group A, while the other four QTLs showed smaller additive effects and were classified as Group B. In pyramiding lines, although the presence of epistasis, additivity remained the main component of QTL effects. qPGC5 and qPGC6 showed stronger ability to reduce rice chalkiness, particularly in the environment of high temperature (HT) in the first cropping season (FCS). Our research demonstrates that by pyramiding low chalkiness QTLs, it is feasible to develop the high-quality rice varieties with low chalkiness at the best quality level even in the HT environment of FCS.

QTL epistasis plays a role of homeostasis on heading date in rice.

If there was no gene interaction, the gene aggregation effect would increase infinitely with the increase of gene number. Epistasis avoids the endless accumulation of gene effects, playing a role of homeostasis. To confirm the role, QTL epistases were analyzed by four single-segment substitution lines with heading date QTLs in this paper. We found that QTLs of three positive effects and one negative effect generated 62.5% negative dual QTL epistatic effects and 57.7% positive triple QTL epistatic effects, forming the relationship "positive QTLs-negative one order interactions-positive two order interactions". In this way, the aggregation effect of QTLs was partially neutralized by the opposite epistatic effect sum. There also were two exceptions, QTL OsMADS50 and gene Hd3a-2 were always with consistent effect directions with their epistases, implying they could be employed in pyramiding breeding with different objectives. This study elucidated the mechanism of epistatic interactions among four QTLs and provided valuable genetic resources for improving heading date in rice.

qGLF5 from Oryza rufipogon Griff. improves kernel shape, plant architecture, and yield in rice.

KEY MESSAGE: A novel QTL qGLF5 from Oryza rufipogon Griff. improves yield per plant and plant architecture in rice. Kernel size and plant architecture are critical agronomic traits that are key targets for improving crop yield. From the single-segment substitution lines of Oryza rufipogon Griff. in the indica cultivar Huajingxian74 (HJX74) background, we identified a novel quantitative trait locus (QTL), named qGLF5, which improves kernel shape, plant architecture, and yield per plant in rice. Compared with the control HJX74, the plant height, panicles per plant, panicle length, primary branches per panicle, secondary branches per panicle, and kernels per plant of the near-isogenic line-qGLF5 (NIL-qGLF5) are significantly increased. NIL-qGLF5 has long and narrow kernels by regulating cell number, cell length and width in the spikelet hulls. Yield per plant of NIL-qGLF5 is increased by 35.02% compared with that of HJX74. In addition, qGLF5 significantly improves yield per plant and plant architecture of NIL-gw5 and NIL-GW7. These results indicate that qGLF5 might be beneficial for improving plant architecture and kernel yield in rice breeding by molecular design.

Breeding of ZhongKeFaZaoGeng1 by molecular design.

Double-cropping early-season rice is one important part of staple crop rice. In recent years, great progress has been made in breeding the double-cropping early-season japonica rice variety, ZhongKeFaZaoGeng1 (ZKFZG1), with high yield, good quality, and high resistance. The breeding of ZKFZG1 aimed at the severe problems of low quality, low income and pre-harvest sprouting in double-cropping early-season rice production, and was achieved through molecular design by selecting three parents with different beneficial genes, KongYu131, NanFangChangLiGeng, and JiGeng88 and screening for key agronomic genes in cross-breeding. ZKFZG1 has a compact plant architecture, a plant height of ~90 cm, a number of ~120 grains per panicle, a setting rate of ~85%, a 1000-grain weight of 26 grams, a yield of 8.25 t/ha, and especially good grain quality. The successful breeding of ZKFZG1 provides a new direction for double-cropping early-season rice production.

GS6.1 controls kernel size and plant architecture in rice.

MAIN CONCLUSION: A novel QTL GS6.1 increases yield per plant by controlling kernel size, plant architecture, and kernel filling in rice. Kernel size and plant architecture are critical agronomic traits that greatly influence kernel yield in rice. Using the single-segment substitution lines (SSSLs) with an indica cultivar Huajingxian74 as a recipient parent and American Jasmine as a donor parent, we identified a novel quantitative trait locus (QTL), named GS6.1. Near isogenic line-GS6.1 (NIL-GS6.1) produces long and narrow kernels by regulating cell length and width in the spikelet hulls, thus increasing the 1000-kernel weight. Compared with the control, the plant height, panicles per plant, panicle length, kernels per plant, secondary branches per panicle, and yield per plant of NIL-GS6.1 are increased. In addition, GS6.1 regulates the kernel filling rate. GS6.1 controls kernel size by modulating the transcription levels of part of EXPANSINs, kernel filling-related genes, and kernel size-related genes. These results indicate that GS6.1 might be beneficial for improving kernel yield and plant architecture in rice breeding by molecular design.

Loss of function of SSIIIa and SSIIIb coordinately confers high RS content in cooked rice.

The sedentary lifestyle and refined food consumption significantly lead to obesity, type 2 diabetes, and related complications, which have become one of the major threats to global health. This incidence could be potentially reduced by daily foods rich in resistant starch (RS). However, it remains a challenge to breed high-RS rice varieties. Here, we reported a high-RS mutant rs4 with an RS content of ~10.8% in cooked rice. The genetic study revealed that the loss-of-function SSIIIb and SSIIIa together with a strong Wx allele in the background collaboratively contributed to the high-RS phenotype of the rs4 mutant. The increased RS contents in ssIIIa and ssIIIa ssIIIb mutants were associated with the increased amylose and lipid contents. SSIIIb and SSIIIa proteins were functionally redundant, whereas SSIIIb mainly functioned in leaves and SSIIIa largely in endosperm owing to their divergent tissue-specific expression patterns. Furthermore, we found that SSIII experienced duplication in different cereals, of which one SSIII paralog was mainly expressed in leaves and another in the endosperm. SSII but not SSIV showed a similar evolutionary pattern to SSIII. The copies of endosperm-expressed SSIII and SSII were associated with high total starch contents and low RS levels in the seeds of tested cereals, compared with low starch contents and high RS levels in tested dicots. These results provided critical genetic resources for breeding high-RS rice cultivars, and the evolutionary features of these genes may facilitate to generate high-RS varieties in different cereals.

Genomic decoding of breeding history to guide breeding-by-design in rice.

Deciphering the intrinsic molecular logic of empirical crop breeding from a genomic perspective is a decisive prerequisite for breeding-by-design (BbD), but remains not well established. Here, we decoded the historical features of past rice breeding by phenotyping and haplotyping 546 accessions covering the majority of cultivars bred in the history of Northeast China (NEC). We revealed that three groups founded the genetic diversities in NEC rice with distinct evolution patterns and traced and verified the breeding footprints to known or genome-wide association study (GWAS)-detected quantitative trait loci (QTLs), or introgressions from indica sub-species with chronological changes in allele frequencies. Then we summarized a rice breeding trend/principle in NEC, and combined with the successful example in breeding and application of Zhongkefa5 to demonstrate the guiding value of our conclusion for BbD in practice. Our study provides a paradigm for decoding the breeding history of a specific crop to guide BbD, which may have implications in different crop breeding.

A Gγ protein regulates alkaline sensitivity in crops.

The use of alkaline salt lands for crop production is hindered by a scarcity of knowledge and breeding efforts for plant alkaline tolerance. Through genome association analysis of sorghum, a naturally high-alkaline-tolerant crop, we detected a major locus, Alkaline Tolerance 1 (AT1), specifically related to alkaline-salinity sensitivity. An at1 allele with a carboxyl-terminal truncation increased sensitivity, whereas knockout of AT1 increased tolerance to alkalinity in sorghum, millet, rice, and maize. AT1 encodes an atypical G protein γ subunit that affects the phosphorylation of aquaporins to modulate the distribution of hydrogen peroxide (H2O2). These processes appear to protect plants against oxidative stress by alkali. Designing knockouts of AT1 homologs or selecting its natural nonfunctional alleles could improve crop productivity in sodic lands.

LAZY3 interacts with LAZY2 to regulate tiller angle by modulating shoot gravity perception in rice.

Starch biosynthesis in gravity-sensing tissues of rice shoot determines the magnitude of rice shoot gravitropism and thus tiller angle. However, the molecular mechanism underlying starch biosynthesis in rice gravity-sensing tissues is still unclear. We characterized a novel tiller angle gene LAZY3 (LA3) in rice through map-based cloning. Biochemical, molecular and genetic studies further demonstrated the essential roles of LA3 in gravity perception of rice shoot and tiller angle control. The shoot gravitropism and lateral auxin transport were defective in la3 mutant upon gravistimulation. We showed that LA3 encodes a chloroplast-localized tryptophan-rich protein associated with starch granules via Tryptophan-rich region (TRR) domain. Moreover, LA3 could interact with the starch biosynthesis regulator LA2, determining starch granule formation in shoot gravity-sensing tissues. LA3 and LA2 negatively regulate tiller angle in the same pathway acting upstream of LA1 to mediate asymmetric distribution of auxin. Our study defined LA3 as an indispensable factor of starch biosynthesis in rice gravity-sensing tissues that greatly broadens current understanding in the molecular mechanisms underlying the starch granule formation in gravity-sensing tissues, and provides new insights into the regulatory mechanism of shoot gravitropism and rice tiller angle.

Grain shape is a factor affecting the stigma exsertion rate in rice.

Stigma exsertion rate (SER) is an index of outcrossing ability in rice and is a key trait of male sterile lines (MSLs) in hybrid rice. In this study, it was found that the maintainer lines carrying gs3 and gs3/gw8 showed higher SER. Single-segment substitution lines (SSSLs) carrying gs3, gw5, GW7 or gw8 genes for grain shape and gene pyramiding lines were used to reveal the relationship between grain shape and SER. The results showed that the grain shape regulatory genes had pleiotropic effects on SER. The SERs were affected by grain shapes including grain length, grain width and the ratio of length to width (RLW) not only in low SER background, but also in high SER background. The coefficients of determination (R2) between grain length and SER, grain width and SER, and grain RLW and SER were 0.78, 0.72, and 0.91 respectively. The grain RLW was the most important parameter affecting SER, and a larger grain RLW was beneficial to stigma exsertion. The pyramiding line PL-gs3/GW7/gw8 showed the largest grain RLW and the highest SER, which will be a fine breeding resource. Further research showed that the grain shape regulatory genes had pleiotropic effects on stigma shape, although the R2 values between grain shape and stigma shape, and stigma shape and SER were lower. Our results demonstrate that grain shape is a factor affecting SER in rice, in part by affecting stigma shape. This finding will be helpful for breeding MSLs with high SER in hybrid rice.

OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice.

Salt stress adversely affects plant growth, development, and crop yield. Rice (Oryza sativa L.) is one of the most salt-sensitive cereal crops, especially at the early seedling stage. Mitogen-activated protein kinase (MAPK/MPK) cascades have been shown to play critical roles in salt response in Arabidopsis. However, the roles of the MPK cascade signaling in rice salt response and substrates of OsMPK remain largely unknown. Here, we report that the salt-induced OsMPK4-Ideal Plant Architecture 1 (IPA1) signaling pathway regulates the salt tolerance in rice. Under salt stress, OsMPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180, leading to degradation of IPA1. Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice, whereas OsMPK4 promotes salt response in an IPA1-dependent manner. Taken together, our results uncover an OsMPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.

Chilling-induced phosphorylation of IPA1 by OsSAPK6 activates chilling tolerance responses in rice.

Chilling is a major abiotic stress harming rice development and productivity. The C-REPEAT BINDING FACTOR (CBF)-dependent transcriptional regulatory pathway plays a central role in cold stress and acclimation in Arabidopsis. In rice, several genes have been reported in conferring chilling tolerance, however, the chilling signaling in rice remains largely unknown. Here, we report the chilling-induced OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 6 (OsSAPK6)-IDEAL PLANT ARCHITECTURE 1 (IPA1)-OsCBF3 signal pathway in rice. Under chilling stress, OsSAPK6 could phosphorylate IPA1 and increase its stability. In turn, IPA1 could directly bind to the GTAC motif on the OsCBF3 promoter to elevate its expression. Genetic evidence showed that OsSAPK6, IPA1 and OsCBF3 were all positive regulators of rice chilling tolerance. The function of OsSAPK6 in chilling tolerance depended on IPA1, and overexpression of OsCBF3 could rescue the chilling-sensitive phenotype of ipa1 loss-of-function mutant. Moreover, the natural gain-of-function allele ipa1-2D could simultaneously enhance seedling chilling tolerance and increase grain yield. Taken together, our results revealed a chilling-induced OsSAPK6-IPA1-OsCBF signal cascade in rice, which shed new lights on chilling stress-tolerant rice breeding.

Fine Mapping of Two Major Quantitative Trait Loci for Rice Chalkiness With High Temperature-Enhanced Additive Effects.

Chalkiness is a crucial determinant of rice quality. During seed filling period, high temperature usually increases grain chalkiness, resulting in poor grain quality. Rice chalkiness was controlled by quantitative trait loci (QTLs) and influenced by environmental conditions. In this study, we identified two single-segment substitution lines (SSSLs) 22-05 and 15-06 with significantly lower percentage of grain chalkiness (PGC) than recipient Huajingxian 74 (HJX74) over 6 cropping seasons. Two major QTLs for chalkiness, qPGC5 and qPGC6, were located by substitution mapping of SSSLs 22-05 and 15-06, respectively. qPGC5 was located in the 876.5 kb interval of chromosome 5 and qPGC6 was located in the 269.1 kb interval of chromosome 6. Interestingly, the PGC of HJX74 was significantly different between the two cropping seasons per year, with 25.8% in the first cropping season (FCS) and 16.6% in the second cropping season (SCS), while the PGC of SSSLs 22-05 and 15-06 did not significantly differ between FCS and SCS. The additive effects of qPGC5 and qPGC6 on chalkiness in the SSSLs were significantly greater in FCS than in SCS. These results showed that qPGC5 and qPGC6 had major effects on chalkiness and the SSSL alleles were more effective in reducing chalkiness under high temperature condition in FCS. The fine-mapping of the two QTLs will facilitate the cloning of genes for chalkiness and provide new genetic resources to develop new cultivars with low chalkiness even under high temperature condition.

Reconstruction of the High Stigma Exsertion Rate Trait in Rice by Pyramiding Multiple QTLs.

Asian cultivated rice is a self-pollinating crop, which has already lost some traits of natural outcrossing in the process of domestication. However, male sterility lines (MSLs) need to have a strong outcrossing ability to produce hybrid seeds by outcrossing with restorer lines of male parents in hybrid rice seed production. Stigma exsertion rate (SER) is a trait related to outcrossing ability. Reconstruction of the high-SER trait is essential in the MSL breeding of rice. In previous studies, we detected eighteen quantitative trait loci (QTLs) for SER from Oryza sativa, Oryza glaberrima, and Oryza glumaepatula using single-segment substitution lines (SSSLs) in the genetic background of Huajingxian 74 (HJX74). In this study, eleven of the QTLs were used to develop pyramiding lines. A total of 29 pyramiding lines with 2-6 QTLs were developed from 10 SSSLs carrying QTLs for SER in the HJX74 genetic background. The results showed that the SER increased with increasing QTLs in the pyramiding lines. The SER in the lines with 5-6 QTLs was as high as wild rice with strong outcrossing ability. The epistasis of additive by additive interaction between QTLs in the pyramiding lines was less-than-additive or negative effect. One QTL, qSER3a-sat, showed minor-effect epistasis and increased higher SER than other QTLs in pyramiding lines. The detection of epistasis of QTLs on SER uncovered the genetic architecture of SER, which provides a basis for using these QTLs to improve SER levels in MSL breeding. The reconstruction of the high-SER trait will help to develop the MSLs with strong outcrossing ability in rice.

An engineered platform for reconstituting functional multisubunit SCF E3 ligase in vitro.

Multisubunit SKP1/Cullin1/F-box (SCF) E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes. Detecting E3 ligase activity in vitro is important for exploring the molecular mechanism of protein ubiquitination. However, in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve, especially in plants, mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs. In this study, we characterized components of the rice SCFDWARF3 (SCFD3) E3 ligase, screened the coordinated E2, and reconstituted active SCFD3 E3 ligase in vitro. We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1 (eSCR) protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53, which is the key transcriptional repressor in strigolactone signaling. Finally, we replaced D3 with other F-box proteins from rice and humans and reconstituted active eSCF E3 ligases, including eSCFGID2, eSCFFBXL18, and eSCFCDC4 E3 ligases. Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins, providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.

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 49 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.

Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size.

Crop genetic improvement requires balancing complex tradeoffs caused by gene pleiotropy and linkage drags, as exemplified by IPA1 (Ideal Plant Architecture 1), a typical pleiotropic gene in rice that increases grains per panicle but reduces tillers. In this study, we identified a 54-base pair cis-regulatory region in IPA1 via a tiling-deletion-based CRISPR-Cas9 screen that, when deleted, resolves the tradeoff between grains per panicle and tiller number, leading to substantially enhanced grain yield per plant. Mechanistic studies revealed that the deleted fragment is a target site for the transcription factor An-1 to repress IPA1 expression in panicles and roots. Targeting gene regulatory regions should help dissect tradeoff effects and provide a rich source of targets for breeding complementary beneficial traits.

Natural variations in grain length 10 (GL10) regulate rice grain size.

Grain size is an important determinant of grain weight and yield in rice. Although several genes related to grain size have been identified, natural variations in these genes that affect grain size are poorly characterized. Here, we describe the grain length QTL GL10, encoding MADS56, which positively regulates grain length and grain weight. A natural allelic variation of NIL-gl10, containing an ∼1.0-kb deletion in the first exon that abolishes its transcription, results in shorter grain length, lower grain weight and delayed flowering in gl10 plants. The knockout of GL10 in the HJX74 background leads to grain phenotypes similar to that of NIL-gl10, while overexpression of GL10 results in increased grain length and weight and earlier heading date. GL10 regulates grain length by promoting greater longitudinal cell growth in the grain glume. Additionally, GL10 participates in the regulation of gibberellic acid (GA) signaling pathway genes in young panicle tissues. Analysis of GL10 haplotypes shows obvious divergence between the japonica and indica lineages. Our findings reveal an allelic variation of GL10 that may explain differences in grain length among modern cultivars and could be used to breed rice varieties with optimized grain shape.

From Green Super Rice to green agriculture: Reaping the promise of functional genomics research.

Producing sufficient food with finite resources to feed the growing global population while having a smaller impact on the environment has always been a great challenge. Here, we review the concept and practices of Green Super Rice (GSR) that have led to a paradigm shift in goals for crop genetic improvement and models of food production for promoting sustainable agriculture. The momentous achievements and global deliveries of GSR have been fueled by the integration of abundant genetic resources, functional gene discoveries, and innovative breeding techniques with precise gene and whole-genome selection and efficient agronomic management to promote resource-saving, environmentally friendly crop production systems. We also provide perspectives on new horizons in genomic breeding technologies geared toward delivering green and nutritious crop varieties to further enhance the development of green agriculture and better nourish the world population.

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.