Fine mapping of the panicle length QTL qPL5 in rice.

Panicle length is a crucial trait tightly associated with spikelets per panicle and grain yield in rice. To dissect the genetic basis of panicle length, a population of 161 recombinant inbred lines (RILs) was developed from the cross between an aus variety Chuan 7 (C7) and a tropical Geng variety Haoboka (HBK). C7 has a panicle length of 30 cm, 7 cm longer than that of HBK, and the panicle length was normally distributed in the RIL population. A total of six quantitative trait loci (QTLs) for panicle length were identified, and single QTLs explained the phenotypic variance from 4.9 to 18.1%. Among them, three QTLs were mapped to the regions harbored sd1, DLT, and Ehd1, respectively. To validate the genetic effect of a minor QTL qPL5, a near-isogenic F2 (NIF2) population segregated at qPL5 was developed. Interestingly, panicle length displayed bimodal distribution, and heading date also exhibited significant variation in the NIF2 population. qPL5 accounted for 66.5% of the panicle length variance. The C7 allele at qPL5 increased panicle length by 2.4 cm and promoted heading date by 5 days. Finally, qPL5 was narrowed down to an 80-kb region flanked by markers M2197 and M2205 using a large NIF2 population of 7600 plants. LOC_Os05g37540, encoding a phytochrome signal protein whose homolog in Arabidopsis enlarges panicle length, is regarded as the candidate gene because a single-nucleotide mutation (C1099T) caused a premature stop codon in HBK. The characterization of qPL5 with enlarging panicle length but promoting heading date makes its great value in breeding early mature varieties without yield penalty in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01443-2.

Suppressing a phosphohydrolase of cytokinin nucleotide enhances grain yield in rice.

One-step and two-step pathways are proposed to synthesize cytokinin in plants. The one-step pathway is mediated by LONELY GUY (LOG) proteins. However, the enzyme for the two-step pathway remains to be identified. Here, we show that quantitative trait locus GY3 may boost grain yield by more than 20% through manipulating a two-step pathway. Locus GY3 encodes a LOG protein that acts as a 5'-ribonucleotide phosphohydrolase by excessively consuming the cytokinin precursors, which contrasts with the activity of canonical LOG members as phosphoribohydrolases in a one-step pathway. The residue S41 of GY3 is crucial for the dephosphorylation of iPRMP to produce iPR. A solo-LTR insertion within the promoter of GY3 suppressed its expression and resulted in a higher content of active cytokinins in young panicles. Introgression of GY302428 increased grain yield per plot by 7.4% to 16.3% in all investigated indica backgrounds, which demonstrates the great value of GY302428 in indica rice production.

A Pseudo-near isogenic F2 population strategy for rapid QTL cloning.

Near isogenic F2 (NIF2) population frequently developed by conventional backcross has dramatically contributed to QTL identification in plants. Developing such a NIF2 population is time-consuming. Thus, it is urgent to rapidly produce a NIF2 population for QTL cloning. Here, we proposed a rapid QTL cloning strategy by generating a Pseudo-near isogenic F2 population (Pseudo-NIF2), which segregates at the target QTL but is fixed at other QTLs for the target trait. Nineteen QTLs for GL, GW, and TGW were detected in the F2 population from the cross between Zhenshan 97 and Egy316. To verify the efficiency of Pseudo-NIF2 in QTL quick cloning, the novel moderate QTL qGL10.1 which explained 9.1% and 5.6% of grain length variation in F2 and F2:3 populations was taken as an example. An F2 plant (F2-120), which segregated at qGL10.1 but fixed at other 8 QTLs for grain length, was screened to generate a Pseudo-NIF2 population by selfing cross. In the Pseudo-NIF2 population, the segregation ratio of plants with long grains to short grains fits 3:1, indicating that one gene controlled the variation of grain length. Based on the Pseudo-NIF2 and its progeny, qGL10.1 was fine mapped to a 19.3-kb region, where a gene OsMADS56 was verified as the candidate by functional polymorphism between parental alleles. Pseudo-NIF2 strategy is a rapid way for QTL cloning, which saves 3 to 4 cropping seasons compared to the conventional way. Applying the method for cloning QTL with moderate or major effects is promising. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01408-x.

The CCT transcriptional activator Ghd2 constantly delays the heading date by upregulating CO3 in rice.

CONSTANS, CO-like, and TOC1 (CCT) family genes play important roles in regulating heading date, which exerts a large impact on the regional and seasonal adaptation of rice. Previous studies have shown that Grain number, plant height, and heading date2 (Ghd2) exhibit a negative response to drought stress by directly upregulating Rubisco activase and exerting a negative effect on heading date. However, the target gene of Ghd2 regulating heading date is still unknown. In this study, CO3 is identified by analyzing ChIP-seq data. Ghd2 activates CO3 expression by binding to the CO3 promoter through its CCT domain. EMSA experiments show that the motif CCACTA in the CO3 promoter was recognized by Ghd2. A comparison of the heading dates among plants with CO3 knocked out or overexpressed and double mutants overexpressing Ghd2 with CO3 knocked out shows that CO3 negatively and constantly regulates flowering by repressing the transcription of Ehd1, Hd3a, and RFT1. In addition, the target genes of CO3 are explored via a comprehensive analysis of DAP-seq data and RNA-seq data. Taken together, these results suggest that Ghd2 directly binds to the downstream gene CO3, and the Ghd2-CO3 module constantly delays heading date via the Ehd1-mediated pathway.

The amino acid residue E96 of Ghd8 is crucial for the formation of the flowering repression complex Ghd7-Ghd8-OsHAP5C in rice.

Ghd7 is an important gene involved in the photoperiod flowering pathway in rice. A Ghd7-involved transcriptional regulatory network has been established, but its translational regulatory pathway is poorly understood. The mutant suppressor of overexpression of Ghd7 (sog7) was identified from EMS-induced mutagenesis on the background of ZH11 overexpressing Ghd7. MutMap analysis revealed that SOG7 is allelic to Ghd8 and delayed flowering under long-day (LD) conditions. Biochemical assays showed that Ghd8 interacts with OsHAP5C and Ghd7 both in vivo and in vitro. Surprisingly, a point mutation E96K in the α2 helix of the Ghd8 histone fold domain (HFD) destroyed its ability to interact with Ghd7. The prediction of the structure shows that mutated amino acid is located in the interaction region of CCT/NF-YB/YC complexes, which alter the structure of α4 of Ghd8. This structural difference prevents the formation of complex NF-YB/YC. The triple complex of Ghd8-OsHAP5C-Ghd7 directly bound to the promotor of Hd3a and downregulated the expression of Ehd1, Hd3a and RFT1, and finally resulted in a delayed heading. These findings are helpful in deeply understanding the Ghd7-involved photoperiod flowering pathway and promote the elucidation of rice heading.

Conservation and divergence: Regulatory networks underlying reproductive branching in rice and maize.

BACKGROUND: Cereal crops are a major source of raw food and nutrition for humans worldwide. Inflorescence of cereal crops is their reproductive organ, which also contributes to crop productivity. The branching pattern in flowering plant species not only determines inflorescence architecture but also determines the grain yield. There are good reviews describing the grass inflorescence architecture contributing to the final grain yield. However, very few discuss the aspects of inflorescence branching. AIM OF REVIEW: This review aimed at systematically and comprehensively summarizing the latest progress in the field of conservation and divergence of genetic regulatory network that controls inflorescence branching in maize and rice, provide strategies to efficiently utilize the achievements in reproductive branching for crop yield improvement, and suggest a potential regulatory network underlying the inflorescence branching and vegetative branching system. KEY SCIENTIFIC CONCEPTS OF REVIEW: Inflorescence branching is the consequence of a series of developmental events including the initiation, outgrowth, determinacy, and identity of reproductive axillary meristems, and it is controlled by a complex functional hierarchy of genetic networks. Initially, we compared the inflorescence architecture of maize and rice; then, we reviewed the genetic regulatory pathways controlling the inflorescence meristem size, bud initiation, and outgrowth, and the key transition steps that shape the inflorescence branching in maize and rice; additionally, we summarized strategies to effectively apply the recent advances in inflorescence branching for crop yield improvement. Finally, we discussed how the newly discovered hormones coordinate the regulation of inflorescence branching and yield traits. Furthermore, we discussed the possible reason behind distinct regulatory pathways for vegetative and inflorescence branching.

A Ghd7-centered regulatory network provides a mechanistic approximation to optimal heterosis in an elite rice hybrid.

Heterosis refers to the superior performance of hybrids over their parents, which is a general phenomenon occurring in diverse organisms. Many commercial hybrids produce high yield without delayed flowering, which we refer to as optimal heterosis and is desired in hybrid breeding. Here, we attempted to illustrate the genomic basis of optimal heterosis by reinvestigating the single-locus quantitative trait loci and digenic interactions of two traits, the number of spikelets per panicle (SP) and heading date (HD), using recombinant inbred lines and 'immortalized F2 s' derived from the elite rice (Oryza sativa) hybrid Shanyou 63. Our analysis revealed a regulatory network that may provide an approximation to the genetic constitution of the optimal heterosis observed in this hybrid. In this network, Ghd7 works as the core element, and three other genes, Ghd7.1, Hd1, and Hd3a/RFT1, also have major roles. The effects of positive dominance by Ghd7 and Ghd7.1 and negative dominance by Hd1 and Hd3a/RFT1 in the hybrid background contribute the major part to the high SP without delaying HD; numerous epistatic interactions, most of which involve Ghd7, also play important roles collectively. The results expand our understanding of the genic interaction networks underlying hybrid rice breeding programs, which may be very useful in future crop genetic improvement.

Introgression Lines: Valuable Resources for Functional Genomics Research and Breeding in Rice (Oryza sativa L.).

The narrow base of genetic diversity of modern rice varieties is mainly attributed to the overuse of the common backbone parents that leads to the lack of varied favorable alleles in the process of breeding new varieties. Introgression lines (ILs) developed by a backcross strategy combined with marker-assisted selection (MAS) are powerful prebreeding tools for broadening the genetic base of existing cultivars. They have high power for mapping quantitative trait loci (QTLs) either with major or minor effects, and are used for precisely evaluating the genetic effects of QTLs and detecting the gene-by-gene or gene-by-environment interactions due to their low genetic background noise. ILs developed from multiple donors in a fixed background can be used as an IL platform to identify the best alleles or allele combinations for breeding by design. In the present paper, we reviewed the recent achievements from ILs in rice functional genomics research and breeding, including the genetic dissection of complex traits, identification of elite alleles and background-independent and epistatic QTLs, analysis of genetic interaction, and genetic improvement of single and multiple target traits. We also discussed how to develop ILs for further identification of new elite alleles, and how to utilize IL platforms for rice genetic improvement.

Fixation of hybrid sterility genes and favorable alleles of key yield-related genes with dominance contribute to the high yield of the Yongyou series of intersubspecific hybrid rice.

In rice, the Yongyou series of Xian-Geng intersubspecific hybrids have excellent production performance, as shown by their extremely high yield. However, the mechanisms underlying the success of these rice hybrids are unclear. In this study, three F2 populations are generated from three Yongyou hybrids to determine the genetic basis of the extremely high yield of intersubspecific hybrids. Genome constitution analysis reveals that the female and male parental lines belong to the Geng and Xian subspecies, respectively, although introgression of 20% of the Xian ancestry and 14% of the Geng ancestry are observed. Twenty-five percent of the hybrid genomes carries homozygous Xian or Geng fragments, which harbors hybrid sterility genes such as Sd, Sc, f5, and qS12 and favorable alleles of key yield-related genes, including NAL1, Ghd7, and Ghd8. None of the parents carries the S5+ killer of the S5 killer-protector system. Compatible allele combinations of hybrid sterility genes ensure the fertility of these intersubspecific hybrids and overcome the bottleneck in applying intersubspecific hybrids. Additive effects of favorable alleles of yield-related genes fixed in both parents enhances midparent values. Many QTLs for yield and its key component spikelets per panicle shows dominance and the net positive dominant effects lead to heterosis. These factors result in an extremely high yield of the hybrids. These findings will aid in the development of new intersubspecific rice hybrids with diverse genetic backgrounds.

A long transcript mutant of the rubisco activase gene RCA upregulated by the transcription factor Ghd2 enhances drought tolerance in rice.

The transcription factor Ghd2 increases rice yield potential under normal conditions and accelerates leaf senescence under drought stress. However, its mechanism on the regulation of leaf senescence under drought stress remains unclear. In the present study, to unveil the mechanism, one target of Ghd2, the Rubisco activase gene RCA, was identified through the combined analysis of Ghd2-CRISPR transcriptome data and Ghd2-overexpression microarray data. Ghd2 binds to the 'CACA' motif in the RCA promoter by its CCT domain and upregulates RCA expression. RCA has alternative transcripts, RCAS and RCAL, which are predominantly expressed under normal conditions and drought stress, respectively. Similar to Ghd2-overexpressing plants, RCAL-overexpressing plants were more sensitive to drought stress than the wild-type. However, the plants overexpressing RCAS showed a weak drought-sensitive phenotype. Moreover, RCAL knockdown and knockout plants did not show yield loss under normal conditions, but exhibited enhanced drought tolerance and delayed leaf senescence. The chlorophyll content, the free amino acid content and the expression of senescence-related genes in the RCAL mutant were lower than those in the wild-type plants under drought stress. In summary, Ghd2 induces leaf senescence by upregulating RCAL expression under drought stress, and the RCAL mutant has important values in breeding drought-tolerant varieties.

Assembly of yield heterosis of an elite rice hybrid is promising by manipulating dominant quantitative trait loci.

In the past, rice hybrids with strong heterosis have been obtained empirically, by developing and testing thousands of combinations. Here, we aimed to determine whether heterosis of an elite hybrid could be achieved by manipulating major quantitative trait loci. We used 202 chromosome segment substitution lines from the elite hybrid Shanyou 63 to evaluate single segment heterosis (SSH) of yield per plant and identify heterotic loci. All nine detected heterotic loci acted in a dominant fashion, and no SSH exhibited overdominance. Functional alleles of key yield-related genes Ghd7, Ghd7.1, Hd1, and GS3 were dispersed in both parents. No functional alleles of three investigated genes were expressed at higher levels in the hybrids than in the more desirable parents. A hybrid pyramiding eight heterotic loci in the female parent Zhenshan 97 background had a comparable yield to Shanyou 63 and much higher yield than Zhenshan 97. Five hybrids pyramiding eight or nine heterotic loci in the combined parental genome background showed similar yield performance to that of Shanyou 63. These results suggest that dominance underlying functional complementation is an important contributor to yield heterosis and that heterosis assembly might be successfully promised by manipulating several major dominant heterotic loci.

Rice functional genomics: decades' efforts and roads ahead.

Rice (Oryza sativa L.) is one of the most important crops in the world. Since the completion of rice reference genome sequences, tremendous progress has been achieved in understanding the molecular mechanisms on various rice traits and dissecting the underlying regulatory networks. In this review, we summarize the research progress of rice biology over past decades, including omics, genome-wide association study, phytohormone action, nutrient use, biotic and abiotic responses, photoperiodic flowering, and reproductive development (fertility and sterility). For the roads ahead, cutting-edge technologies such as new genomics methods, high-throughput phenotyping platforms, precise genome-editing tools, environmental microbiome optimization, and synthetic methods will further extend our understanding of unsolved molecular biology questions in rice, and facilitate integrations of the knowledge for agricultural applications.

Five plants per RIL for phenotyping traits of high or moderate heritability ensure the power of QTL mapping in a rice MAGIC population.

Currently, the power of QTL mapping is mainly dependent on the quality of phenotypic data in a given population, regardless of the statistical method, as the quality of genotypic data is easily guaranteed in the laboratory. Increasing the sample size per line used for phenotyping is a good way to improve the quality of phenotypic data. However, accommodating a large-scale mapping population takes a large area of rice field, which frequently results in high costs and extra environmental noises. To acquire a reasonable small sample size without a penalty in mapping power, we conducted three experiments with a 4-way MAGIC population and measured phenotypes of 5, 10, and 20 plants per RIL. Three traits including heading date, plant height, and tillers per plant were focused. With SNP- and bin-based QTL mapping, 3 major and 3 minor QTLs for heading date with high heritability and 2 major QTLs for plant height with moderate heritability were commonly detected across the three experiments, but no QTL for tillers per plant with low heritability were commonly identified. In addition, bin-based QTL mapping was more powerful than SNP-based mapping and able to rank the genetic effects of parental alleles. Thus, 5 plants per RIL for phenotyping ensure the power of QTL mapping for traits of high or moderate heritability, and bin-based QTL mapping is recommended for multiparent populations.

Root-to-Shoot Long-Distance Mobile miRNAs Identified from Nicotiana Rootstocks.

Root-derived mobile signals play critical roles in coordinating a shoot's response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in which these two taxonomically distant species with clear genetic backgrounds had sufficient diversity in differentiating miRNA sources. Small RNA deep sequencing analysis revealed that 82 miRNAs from the Arabidopsis scion could travel through the graft union to reach the rootstock, whereas only a very small subset of miRNA (6 miRNAs) preferred the root-to-shoot movement. We demonstrated in an ex vivo RNA imaging experiment that the root-to-shoot mobile Nb-miR164, Nb-miR395 and Nb-miR397 were targeted to plasmodesmata using the bacteriophage coat protein MS2 system. Furthermore, the Nb-miR164 was shown to move from the roots to the shoots to induce phenotypic changes when its overexpressing line was used as rootstock, strongly supporting that root-derived Nb-miR164 was able to modify the scion trait via its long-distance movement.

Combinations of Ghd7, Ghd8, and Hd1 determine strong heterosis of commercial rice hybrids in diverse ecological regions.

Heterosis of grain yield is closely associated with heading date in crops. Gene combinations of the major heading date genes Ghd7, Ghd8, and Hd1 play important roles in enhancing grain yield and adaptation to ecological regions in rice. However, the predominant three-gene combinations for a specific ecological region remain unclear in both three-line and two-line hybrids. In this study, we sequenced these three genes of 50 cytoplasmic male sterile/maintainer lines, 31 photo-thermo-sensitive genic male sterile lines, and 109 restorer lines. Sequence analysis showed that hybrids carrying strong functional alleles of Ghd7 and Hd1 and non-functional Ghd8 are predominant in three-line hybrids and are recommended for rice production in the subtropics around 30°N/S. Hybrids carrying strong functional Ghd7 and Ghd8 and non-functional Hd1 are predominant in two-line hybrids and are recommended for low latitude areas around 23.5°N/S rich in photothermal resources. Hybrids carrying strong functional Ghd7 and Ghd8 and functional Hd1 were not identified in commercial hybrids in the middle and lower reaches of the Yangtze River, but they have high yield potential in tropical regions because they have the strongest photoperiod sensitivity. Based on these findings, two genic sterile lines, Xiangling 628S and C815S, whose hybrids often head very late, were diagnosed with these three genes, and Hd1 was targeted to be knocked out in Xiangling 628S and replaced with hd1 in C815S. The hybrids developed from both modified sterile lines in turn had appropriate heading dates and significantly improved grain yield. This study provides new insights for breeding design to develop hybrids for various regions.

The chromosome-scale reference genome of safflower (Carthamus tinctorius) provides insights into linoleic acid and flavonoid biosynthesis.

Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high-quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single-molecule real-time sequencing, Hi-C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2-12 was specifically expressed in seeds and was vital for high-LA content in seeds, while tandemly duplicated CarFAD2 genes were up-regulated in ovaries compared to CarFAD2-12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem-duplicated CarCHS5˜CarCHS6, which were up-regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high-quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower.

OsPRR37 Alternatively Promotes Heading Date Through Suppressing the Expression of Ghd7 in the Japonica Variety Zhonghua 11 under Natural Long-Day Conditions.

Heading date is an important agronomic trait of rice (Oryza sativa L.) and is regulated by numerous genes, some of which exhibit functional divergence in a genetic background-dependent manner. Here, we identified a late heading date 7 (lhd7) mutant that flowered later than wild-type Zhonghua 11 (ZH11) under natural long-day (NLD) conditions. Map-based cloning facilitated by the MutMap strategy revealed that LHD7 was on the same locus as OsPRR37 but exhibited a novel function as a promoter of heading date. A single-nucleotide mutation of G-to-A in the coding region caused a substitution of aspartic acid for glycine at site 159 within the pseudo-receiver (PR) domain of OsPRR37. Transcriptional analysis revealed that OsPRR37 suppressed Ghd7 expression in both ZH11 background under NLD conditions and the Zhenshan 97 background under natural short-day conditions. Consistently, the expression of Ehd1, Hd3a and RFT1 was enhanced by OsPRR37 in the ZH11 background. Genetic analysis indicated that the promotion of heading date and reduction in grain yield by OsPRR37 were partially dependent on Ghd7. Further investigation showed that the alternative function of OsPRR37 required an intact Ghd7-related regulatory pathway involving not only its upstream regulators OsGI and PhyB but also its interacting partner Hd1. Our study revealed the distinct role of OsPRR37 in the ZH11 background, which provides a more comprehensive understanding of OsPRR37 function and enriches the theoretical bases for improvement of rice heading date in the future.

Global analysis of CCT family knockout mutants identifies four genes involved in regulating heading date in rice.

Many genes encoding CCT domain-containing proteins regulate flowering time. In rice (Oryza sativa), 41 such genes have been identified, but only a few have been shown to regulate heading date. Here, to test whether and how additional CCT family genes regulate heading date in rice, we classified these genes into five groups based on their diurnal expression patterns. The expression patterns of genes in the same subfamily or in close phylogenetic clades tended to be similar. We generated knockout mutants of the entire gene family via CRISPR/Cas9. The heading dates of knockout mutants of only 4 of 14 genes previously shown to regulate heading date were altered, pointing to functional redundancy of CCT family genes in regulating this trait. Analysis of mutants of four other genes showed that OsCCT22, OsCCT38, and OsCCT41 suppress heading under long-day conditions and promote heading under short-day conditions. OsCCT03 promotes heading under both conditions and upregulates the expression of Hd1 and Ehd1, a phenomenon not previously reported for other such genes. To date, at least 18 CCT domain-containing genes involved in regulating heading have been identified, providing diverse, flexible gene combinations for generating rice varieties with a given heading date.