Identification of QTLs responsible for culturability, and fine-mapping of QTL qCBT9 related to callus browning derived from Dongxiang common wild rice (Oryza rufipogon Griff.).

Compared to japonica, the lower genetic transformation efficiency of indica is a technical bottleneck for rice molecular breeding. Specifically, callus browning frequently occurs during the culture of the elite indica variety 93-11, leading to poor culturability and lower genetic transformation efficiency. Here, 67 QTLs related to culturability were detected using 97 introgression lines (designated as 9DILs) derived from Dongxiang common wild rice (DXCWR, Oryza rufipogon Griff.) with 93-11 genetic background, explaining 4% ~12% of the phenotypic variations. The QTL qCBT9 on chromosome 9 was a primary QTL for reducing callus browning derived from DXCWR. Five 9DILs with light callus browning and high differentiation were screened. We evaluated the callus browning index (CBI) of 100 F2 population crossed of 93-11 and 9DIL71 and the recombinant plants screened from 3270 individuals. The qCBT9 was delimited to a ~148kb region between the markers X16 and X23. RNA-seq analysis of DEGs between 9DIL71 and 93-11 showed three upregulated DEGs (Os09g0526500, Os09g0527900, Os09g0528200,) and three downregulated DEGs (Os09g0526700, Os09g0526800, Os09g0527700) were located in the candidate region of qCBT9. Furthermore, callus browning may be involved in cell senescence and death caused by oxidative stress. The differentiation of indica and japonica in this region suggested that qCBT9 was possibly a vital QTL contributed to better culturability of japonica. Our results laid a foundation for further cloning of the gene for reduced callus browning in O. rufipogon, and also provided a new genetic resource and material basis for improving the culturability and genetic transformation efficiency of cultivated rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01470-z.

Genetic and Transcriptome Analyses of Callus Browning in Chaling Common Wild Rice (Oryza rufipogon Griff.).

Callus browning during tissue culture of indica rice is genotype dependent, thus limiting the application of genetic transformation for editing-assisted breeding and elucidation of gene function. Here, using 124 introgression lines (HCLs) derived from a cross between the indica rice 9311 and Chaling common wild rice and 2059 SNPs for single-point and interval analysis, we identified two major QTLs, qCBT7 on chromosome 7 and qCBT10 on chromosome 10, related to callus browning, explaining 8-13% of callus browning. Moreover, we performed RNA-seq of two introgression lines with low callus browning, HCL183 and HCL232, with Oryza. rufipogon introgression fragments on chromosomes 10 and 7, respectively. Three candidate genes (Os07g0620700, Os10g0361000, and Os10g0456800) with upregulation were identified by combining interval mapping and weighted gene coexpression network analysis using the DEGs. The qRT-PCR results of the three candidate genes were consistent with those of RNA-seq. The differentiation of indica and japonica subspecies Oryza. sativa and Oryza. rufipogon suggests that these candidate genes are possibly unique in Oryza. rufipogon. GO analyses of hub genes revealed that callus browning may be mainly associated with ethylene and hormone signaling pathways. The results lay a foundation for future cloning of qCBT7 or qCBT10 and will improve genetic transformation efficiency in rice.

OsMADS17 simultaneously increases grain number and grain weight in rice.

During the processes of rice domestication and improvement, a trade-off effect between grain number and grain weight was a major obstacle for increasing yield. Here, we identify a critical gene COG1, encoding the transcription factor OsMADS17, with a 65-bp deletion in the 5' untranslated region (5' UTR) presented in cultivated rice increasing grain number and grain weight simultaneously through decreasing mRNA translation efficiency. OsMADS17 controls grain yield by regulating multiple genes and that the interaction with one of them, OsAP2-39, has been characterized. Besides, the expression of OsMADS17 is regulated by OsMADS1 directly. It indicates that OsMADS1-OsMADS17-OsAP2-39 participates in the regulatory network controlling grain yield, and downregulation of OsMADS17 or OsAP2-39 expression can further improve grain yield by simultaneously increasing grain number and grain weight. Our findings provide insights into understanding the molecular basis co-regulating rice yield-related traits, and offer a strategy for breeding higher-yielding rice varieties.

Mapping quantitative trait loci associated with callus browning in Dongxiang common wild rice (Oryza rufipogon Griff.).

BACKGROUND: Genetic transformation of indica rice (Oryza sativa ssp. indica) is limited by callus browning, which results in poor in vitro tissue culturability. Elucidating the genes in common wild rice (Oryza rufipogon Griff.) that control callus browning is fundamental for improving the tissue culturability of indica rice varieties. METHODS AND RESULTS: We used a population of 129 O. rufipogon (Dongxiang common wild rice; DXCWR) introgression lines in the elite cultivar GC2 (Oryza sativa ssp. indica) background and 159 simple sequence repeat (SSR) markers to identify quantitative trait loci (QTLs) associated with callus browning. We evaluated callus browning based on the indices of callus browning rate (CBR), callus browning index (CBI), and standard callus browning index (SCBI). CONCLUSIONS: We detected 30 QTLs associated with callus browning across all lines, mapping to chromosomes 1, 2, 3, 4, 8, 9, and 12. These genomic regions were repeatedly associated with differences in CBR, CBI, and SCBI. The alleles from DXCWR showed additive effects in reducing callus browning. We identified new QTLs near the markers RM247 and RM7003 on chromosome 12, indicating that these QTLs are unique to DXCWR. Furthermore, we identified six introgression lines with significantly lower callus browning. These lines will be useful germplasms for genetic transformation and fine-mapping of the culturability trait.

The MYB transcription factor Seed Shattering 11 controls seed shattering by repressing lignin synthesis in African rice.

African cultivated rice (Oryza glaberrima Steud.) was domesticated from its wild progenitor species (Oryza barthii) about 3000 years ago. Seed shattering is one of the main constraints on grain production in African cultivated rice, which causes severe grain losses during harvest. By contrast, Asian cultivated rice (Oryza sativa) displays greater resistance to seed shattering, allowing higher grain production. A better understanding in regulation of seed shattering would help to improve harvesting efficiency in African cultivated rice. Here, we report the map-based cloning and characterization of OgSH11, a MYB transcription factor controlling seed shattering in O. glaberrima. OgSH11 represses the expression of lignin biosynthesis genes and lignin deposition by binding to the promoter of GH2. We successfully developed a new O. glaberrima material showing significantly reduced seed shattering by knockout of SH11 in O. glaberrima using CRISPR-Cas9 mediated approach. Identification of SH11 not only supplies a new target for seed shattering improvement in African cultivated rice, but also provides new insights into the molecular mechanism of abscission layer development.

The GZnC1 variant from common wild rice influences grain Zn content.

Zinc (Zn) deficiency, caused by inadequate Zn intake in the human diet, has serious health implications. Rice (Oryza sativa) is the staple food in regions with a high incidence of Zn deficiency, so raising Zn levels in rice grain could help alleviate Zn deficiency. The wild relatives of cultivated rice vary widely in grain Zn content and thus are suitable resources for improving this trait. However, few loci underlying grain Zn content have been identified in wild rice relatives. Here, we identified a major quantitative trait locus for grain Zn content, Grain Zn Content 1 (qGZnC1), from Yuanjiang common wild rice (Oryza rufipogon Griff.) using map-based cloning. Down-regulating GZnC1 expression reduced the grain Zn content, whereas the presence of GZnC1 had the opposite effect, indicating that GZnC1 is involved in grain Zn content in rice. Notably, GZnC1 is identical to a previously reported gene, EMBRYO SAC ABORTION 1 (ESA1), involved in seed setting rate. The mutation in GZnC1/ESA1 at position 1819 (T1819C) causes delayed termination of protein translation. In addition, GZnC1 is specifically expressed in developing panicles. Several genes related to Zn-transporter genes were up-regulated in the presence of GZnC1. Our results suggest that GZnC1 activates Zn transporters to promote Zn distribution in panicles. Our work thus sheds light on the genetic mechanism of Zn accumulation in rice grain and provides a new genetic resource for improving Zn content in rice.

The genetic control of glabrous glume during African rice domestication.

African cultivated rice, Oryza glaberrima, is characterized by its glabrous glumes. During domestication, the pubescent glumes of its wild ancestor, Oryza barthii, lost their trichomes, and in this study, we show that glabrous glume 5 (GLAG5), a WUSCHEL-like homeobox transcription factor gene on chromosome 5, is required for trichome development. DNA methylation associated with an hAT transposable element inserted in the promoter region of GLAG5 is found to reduce its expression, leading to the formation of glabrous glumes and leaves in African cultivated rice. Among 82 African cultivated rice varieties investigated in this study, 59 (approximately 71%) lines exhibit glabrous glumes and harbor the hAT transposon; however, the other 23 varieties (approximately 29%), which exhibit pubescent glumes, lack the hAT transposon, indicating that glag5 had undergone strong artificial selection. The πw/πc ratios also show the hAT transposon insertions influence the genetic diversity of an approximately 150-kb interval encompassing the GLAG5 locus. The identification of the GLAG5 gene provides new insights into the domestication of cultivated rice in Africa. We speculate that the selection of varieties with mutations in their promoter regions is an important aspect of crop domestication.

Polyamine oxidase 3 is involved in salt tolerance at the germination stage in rice.

Soil salinity inhibits seed germination and reduces seedling survival rate, resulting in significant yield reductions in crops. Here, we report the identification of a polyamine oxidase, OsPAO3, conferring salt tolerance at the germination stage in rice (Oryza sativa L.), through map-based cloning approach. OsPAO3 is up-regulated under salt stress at the germination stage and highly expressed in various organs. Overexpression of OsPAO3 increases activity of polyamine oxidases, enhancing the polyamine content in seed coleoptiles. Increased polyamine may lead to the enhance of the activity of ROS-scavenging enzymes to eliminate over-accumulated H2O2 and to reduce Na+ content in seed coleoptiles to maintain ion homeostasis and weaken Na+ damage. These changes resulted in stronger salt tolerance at the germination stage in rice. Our findings not only provide a unique gene for breeding new salt-tolerant rice cultivars but also help to elucidate the mechanism of salt tolerance in rice.

A common wild rice-derived BOC1 allele reduces callus browning in indica rice transformation.

Callus browning, a common trait derived from the indica rice cultivar (Oryza sativa L.), is a challenge to transformation regeneration. Here, we report the map-based cloning of BROWNING OF CALLUS1 (BOC1) using a population derived from crossing Teqing, an elite indica subspecies exhibiting callus browning, and Yuanjiang, a common wild rice accession (Oryza rufipogon Griff.) that is less susceptible to callus browning. We show that BOC1 encodes a SIMILAR TO RADICAL-INDUCED CELL DEATH ONE (SRO) protein. Callus browning can be reduced by appropriate upregulation of BOC1, which consequently improves the genetic transformation efficiency. The presence of a Tourist-like miniature inverted-repeat transposable element (Tourist MITE) specific to wild rice in the promoter of BOC1 increases the expression of BOC1 in callus. BOC1 may decrease cell senescence and death caused by oxidative stress. Our study provides a gene target for improving tissue culturability and genetic transformation.

Natural Variations at TIG1 Encoding a TCP Transcription Factor Contribute to Plant Architecture Domestication in Rice.

The modification of plant architecture is a crucial target in rice domestication and modern genetic improvement. Although several genes regulating rice plant architecture have been characterized, the molecular mechanisms underlying rice plant architecture domestication remain largely unclear. Here we show that the inclined tiller growth in wild rice is controlled by a single dominant gene, TILLER INCLINED GROWTH 1 (TIG1), which is located on chromosome 8 and encodes a TCP transcriptional activator. TIG1 is primarily expressed in the adaxial side of the tiller base, promotes cell elongation, and enlarges the tiller angle in wild rice. Variations in the TIG1 promoter of indica cultivars (tig1 allele) resulted in decreased expression of TIG1 in the adaxial side of tiller base and reduced cell length and tiller angle, leading to the transition from inclined tiller growth in wild rice to erect tiller growth during rice domestication. TIG1 positively regulates the expression of EXPA3, EXPB5, and SAUR39 to promote cell elongation and increase the tiller angle. Selective sweep analysis revealed that the tig1 allele was selected in indica cultivars by human beings. The cloning and characterization of TIG1 supports a new scenario of plant architecture evolution in rice.

ESA1 Is Involved in Embryo Sac Abortion in Interspecific Hybrid Progeny of Rice.

The emergence of sterile individuals in the hybrid backcross progeny of wild and cultivated rice limits the use of wild rice alleles for improving cultivated rice, but the molecular mechanisms underlying this sterility remain unclear. Here, we identified the semisterile introgression line YIL42, derived from a cross between the indica rice variety Teqing (Oryza sativa) and Oryza rufipogon accession YJCWR (Yuanjiang common wild rice), which exhibits semisterility. Using positional cloning, we isolated EMBRYO SAC ABORTION 1 (ESA1), which encodes a nuclear-membrane localized protein containing an armadillo repeat domain. A mutation in ESA1 at position 1819 (T1819C) converts a stop codon into an Arg (R) codon, causing delayed termination of protein translation. Analysis of transgenic lines indicated that the difference in ESA1 protein structure between O. rufipogon-derived ESA1 and Teqing-derived esa1 affects female gamete abortion during early mitosis. Fertility investigation and expression analysis indicated that the interaction between ESA1 T1819 and unknown gene(s) of Teqing affects spikelet fertility of the hybrid backcross progeny. The ESA1 T1819 allele is present in O. rufipogon but absent in O. sativa, suggesting that variation in ESA1 may be associated with interspecific hybrid incompatibility between wild and cultivated rice. Our findings provide insight into the molecular mechanism underlying female sterility, which is useful for improving the panicle seed setting rate of rice and for developing a strategy to overcome interspecific hybrid sterility between cultivated rice and wild rice.

The APETALA2-Like Transcription Factor SUPERNUMERARY BRACT Controls Rice Seed Shattering and Seed Size.

The elimination of seed shattering was a crucial event during crop domestication. Improving and fine-tuning the regulation of this process will further enhance grain yield by avoiding seed losses during crop production. In this work, we identified the loss-of-shattering mutant suppression of shattering1 (ssh1) through a screen of mutagenized wild rice (Oryza rufipogon) introgression lines with naturally high shattering. Using the MutMap approach and transformation experiments, we isolated a genetic factor for seed shattering, SSH1, which is an allele of SUPERNUMERARY BRACT (SNB), a gene encoding a plant-specific APETALA2-like transcription factor. A C-to-A point mutation in the ninth intron of SNB altered the splicing of its messenger RNA, causing the reduced shattering of the ssh1 mutant by altering the development of the abscission layer and vascular bundle at the junction between the seed and the pedicel. Our data suggest that SNB positively regulates the expression of two rice REPLUMLESS orthologs, qSH1 and SH5 In addition, the ssh1 mutant had larger seeds and a higher grain weight, resulting from its increased elongation of the glume longitudinal cells. The further identification of favorable SNB alleles will be valuable for improving rice seed shattering and grain yield using molecular breeding strategies.

Variation in the regulatory region of FZP causes increases in secondary inflorescence branching and grain yield in rice domestication.

Inflorescence branching is a key agronomic trait determining rice yield. The primary branch of the ancestral wild rice (Oryza rufipogon Griff.) bears few grains, due to minimal secondary branching. By contrast, Oryza sativa cultivars have been selected to produce large panicles with more secondary branches. Here we showed that the CONTROL OF SECONDARY BRANCH 1 (COS1) gene, which is identical to FRIZZY PANICLE (FZP), plays an important role in the key transition from few secondary branches in wild rice to more secondary branches in domesticated rice cultivars. A 4-bp tandem repeat deletion approximately 2.7 kb upstream of FZP may affect the binding activities of auxin response factors to the FZP promoter, decrease the expression level of FZP and significantly enhance the number of secondary branches and grain yield in cultivated rice. Functional analyses showed that NARROW LEAF 1 (NAL1), a trypsin-like serine and cysteine protease, interacted with FZP and promoted its degradation. Consistently, downregulating FZP expression or upregulating NAL1 expression in the commercial cultivar Zhonghua 17 increased the number of secondary branches per panicle, grain number per panicle and grain yield per plant. Our findings not only provide insights into the molecular mechanism of increasing grain number and yield during rice domestication, but also offer favorable genes for improving the grain yield of rice.

Genetic control of seed shattering during African rice domestication.

Domestication represents a unique opportunity to study the evolutionary process. The elimination of seed dispersal traits was a key step in the evolution of cereal crops under domestication. Here, we show that ObSH3, a YABBY transcription factor, is required for the development of the seed abscission layer. Moreover, selecting a genomic segment deletion containing SH3 resulted in the loss of seed dispersal in populations of African cultivated rice (Oryza glaberrima Steud.). Functional characterization of SH3 and SH4 (another gene controlling seed shattering on chromosome 4) revealed that multiple genes can lead to a spectrum of non-shattering phenotypes, affecting other traits such as ease of threshing that may be important to tune across different agroecologies and postharvest practices. The molecular evolution analyses of SH3 and SH4 in a panel of 93 landraces provided unprecedented geographical detail of the domestication history of African rice, tracing multiple dispersals from a core heartland and introgression from local wild rice. The cloning of ObSH3 not only provides new insights into a critical crop domestication process but also adds to the body of knowledge on the molecular mechanism of seed dispersal.

The domestication of plant architecture in African rice.

Plant architecture is a key agronomical factor determining crop yield and has been a major target of cereal crop domestication. The transition of plant architecture from the prostrate tiller of typical African wild rice (Oryza barthii) to the erect tiller of African cultivated rice (Oryza glaberrima) was a key step during domestication of African rice. Here we show that PROG7 (PROSTRATE GROWTH 7), a zinc-finger transcription factor gene on chromosome 7, is required for the prostrate growth of African wild rice. Mutations in the promoter region of prog7 reduced the level of gene expression in the tiller base, leading to erect growth in African cultivated rice. Sequence comparison and haplotype analysis show that 90 varieties of cultivated rice from 11 countries carry the same mutations in the prog7 region. A strong signal in a 60-kb genomic region was detected around the prog7 gene, suggesting that the region was under strong positive selection during the domestication process. Identification of the PROG7 gene provides new insights into the molecular basis of plant architecture in crops and facilitates investigation of the history of domestication of African rice.

NOG1 increases grain production in rice.

During rice domestication and improvement, increasing grain yield to meet human needs was the primary objective. Rice grain yield is a quantitative trait determined by multiple genes, but the molecular basis for increased grain yield is still unclear. Here, we show that NUMBER OF GRAINS 1 (NOG1), which encodes an enoyl-CoA hydratase/isomerase, increases the grain yield of rice by enhancing grain number per panicle without a negative effect on the number of panicles per plant or grain weight. NOG1 can significantly increase the grain yield of commercial high-yield varieties: introduction of NOG1 increases the grain yield by 25.8% in the NOG1-deficient rice cultivar Zhonghua 17, and overexpression of NOG1 can further increase the grain yield by 19.5% in the NOG1-containing variety Teqing. Interestingly, NOG1 plays a prominent role in increasing grain number, but does not change heading date or seed-setting rate. Our findings suggest that NOG1 could be used to increase rice production.

GAD1 Encodes a Secreted Peptide That Regulates Grain Number, Grain Length, and Awn Development in Rice Domestication.

Cultivated rice (Oryza sativa) was domesticated from wild rice (Oryza rufipogon), which typically displays fewer grains per panicle and longer grains than cultivated rice. In addition, wild rice has long awns, whereas cultivated rice has short awns or lacks them altogether. These changes represent critical events in rice domestication. Here, we identified a major gene, GRAIN NUMBER, GRAIN LENGTH AND AWN DEVELOPMENT1 (GAD1), that regulates those critical changes during rice domestication. GAD1 is located on chromosome 8 and is predicted to encode a small secretary signal peptide belonging to the EPIDERMAL PATTERNING FACTOR-LIKE family. A frame-shift insertion in gad1 destroyed the conserved cysteine residues of the peptide, resulting in a loss of function, and causing the increased number of grains per panicle, shorter grains, and awnless phenotype characteristic of cultivated rice. Our findings provide a useful paradigm for revealing functions of peptide signal molecules in plant development and helps elucidate the molecular basis of rice domestication.

Genomic structure analysis of a set of Oryza nivara introgression lines and identification of yield-associated QTLs using whole-genome resequencing.

Oryza nivara, an annual wild AA-genome species of rice, is an important gene pool for broadening the genetic diversity of cultivated rice (O. sativa L.). Towards identifying and utilizing favourable alleles from O. nivara, we developed a set of introgression lines (ILs) by introducing O. nivara segments into the elite indica rice variety 93-11 background through advanced backcrossing and repeated selfing. Using whole-genome resequencing, a high-density genetic map containing 1,070 bin-markers was constructed for the 131 ILs, with an average length of 349 kb per bin. The 131 ILs cover 95% of O. nivara genome, providing a relatively complete genomic library for introgressing O. nivara alleles for trait improvement. Using this high-density bin-map, QTL mapping for 13 yield-related traits was performed and a total of 65 QTLs were detected across two environments. At ~36.9% of detected QTLs, the alleles from O. nivara conferred improving effects on yield-associated traits. Six cloned genes, Sh4/SHA1, Bh4, Sd1, TE/TAD1, GS3 and FZP, colocalised in the peak intervals of 9 QTLs. In conclusion, we developed new genetic materials for exploration and use of beneficial alleles from wild rice and provided a basis for future fine mapping and cloning of the favourable O. nivara-derived QTLs.

CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice.

Panicle architecture and seed size are important agronomic traits that directly determine grain yield in rice (Oryza sativa L.). Although a number of key genes controlling panicle architecture and seed size have been cloned and characterized in recent years, their genetic and molecular mechanisms remain unclear. In this study, we identified a mutant that produced panicles with fascicled primary branching and reduced seeds in size. We isolated the underlying CLUSTERED PRIMARY BRANCH 1 (CPB1) gene, a new allele of DWARF11 (D11) encoding a cytochrome P450 protein involved in brassinosteroid (BR) biosynthesis pathway. Genetic transformation experiments confirmed that a His360Leu amino acid substitution residing in the highly conserved region of CPB1/D11 was responsible for the panicle architecture and seed size changes in the cpb1 mutants. Overexpression of CPB1/D11 under the background of cpb1 mutant not only rescued normal panicle architecture and plant height, but also had a larger leaf angle and seed size than the controls. Furthermore, the CPB1/D11 transgenic plants driven by panicle-specific promoters can enlarge seed size and enhance grain yield without affecting other favourable agronomic traits. These results demonstrated that the specific mutation in CPB1/D11 influenced development of panicle architecture and seed size, and manipulation of CPB1/D11 expression using the panicle-specific promoter could be used to increase seed size, leading to grain yield improvement in rice.

LABA1, a Domestication Gene Associated with Long, Barbed Awns in Wild Rice.

Common wild rice (Oryza rufipogon), the wild relative of Asian cultivated rice (Oryza sativa), flaunts long, barbed awns, which are necessary for efficient propagation and dissemination of seeds. By contrast, O. sativa cultivars have been selected to be awnless or to harbor short, barbless awns, which facilitate seed processing and storage. The transition from long, barbed awns to short, barbless awns was a crucial event in rice domestication. Here, we show that the presence of long, barbed awns in wild rice is controlled by a major gene on chromosome 4, LONG AND BARBED AWN1 (LABA1), which encodes a cytokinin-activating enzyme. A frame-shift deletion in LABA1 of cultivated rice reduces the cytokinin concentration in awn primordia, disrupting barb formation and awn elongation. Sequencing analysis demonstrated low nucleotide diversity and a selective sweep encompassing an ∼800-kb region around the derived laba1 allele in cultivated rice. Haplotype analysis revealed that the laba1 allele originated in the japonica subspecies and moved into the indica gene pool via introgression, suggesting that humans selected for this locus in early rice domestication. Identification of LABA1 provides new insights into rice domestication and also sheds light on the molecular mechanism underlying awn development.