水稻育性调控的分子遗传研究进展

杂交水稻为世界粮食安全做出了重要贡献。细胞质雄性不育和光/温敏不育分别是三系和两系杂交稻生产利用的遗传基础,而(亚)种间杂种不育则是杂交稻生产中要克服的主要技术瓶颈。因此,水稻育性调控是杂交水稻生产技术的关键,也是研究植物核质互作和物种生殖隔离等基础科学问题的重要模型。我国植物遗传学家在阐明杂交水稻育性调控的分子遗传基础领域做出了重要贡献。本文回顾了我国杂交水稻的发展历程,系统总结了杂交水稻生产涉及的细胞质雄性不育与恢复、光/温敏不育与育性转换、杂种不育与亲和性的遗传基础和分子作用机制,探讨了我国杂交水稻生产存在的问题,指明了水稻杂种优势利用的发展方向。

Ectopic expression of a male fertility gene,LOGL8,represses LOG and hinders panicle and ovule development

Grain number and seed-setting rate are components of crop yield.Cytokinin influences grain yield.However,emerging studies suggest that high cytokinin signals often lead to reduced branching or seed-setting rate,leading to reduced grain yield,although the mechanisms remain unclear.In this study,we identified and characterized the rice(Oryza sativa L.)gene LONELY GUY-LIKE 8(LOGL8),based on analysis of the LOGL8-pm(promoter mutant of LOGL8)mutant,which harbors a T-DNA insertion in the promoter of this gene.The mutation in LOGL8-pm causes ectopic hyperexpression of LOGL8 in inflorescence organs,resulting in plants with smaller panicles and defective ovules lacking archesporial cells and integuments.Knockout of LOGL8 caused pollen abortion,leading to a reduced seed-setting rate.LOGL8 encodes a putative cytokinin-activating enzyme.Our results showed that LOGL8 directly catalyzes the biosynthesis of bioactive cytokinins.Therefore,we propose that the ectopic expression of LOGL8 disrupts cytokinin spatiotemporal distribution and causes inhibition of LONELY GUY(LOG),which affects panicle branching and female organ development.These findings reveal the important role of LOGL8 in male development,and highlight the delicate balance of local cytokinin levels during panicle branching and female organ development.

Overexpression of maize GOLDEN2 in rice and maize calli improves regeneration by activating chloroplast development

Golden2(G2), a member of the GARP transcription factor superfamily, regulates several biological processes and phytohormone signaling pathways in plants. In this study, we used a rice codon-optimized maize G2 gene(rZmG2) to improve the regeneration efficiency of rice and maize calli for genetic transformation. We isolated a promoter driving strong and callus-specific expression from rice to drive rZmG2 transcription from a transgene after transformation of two indica and two japonica rice cultivars. The resulting rZmG2 transgenic calli turned green in advance at the differentiation stage, thus significantly raising the regeneration rates of the transgenic indica and japonica rice plants relative to control transformations. Similar effect of this gene on improving maize transformation was also observed. Transcriptome sequencing and RT-qPCR analyses showed that many rice genes related to chloroplast development and phytohormones are upregulated in rZmG2-transgenic calli. These results demonstrate that rZmG2 can promote embryogenic callus differentiation and improve regeneration efficiency by activating chloroplast development and phytohormone pathways. We also established a heat-inducible Cre/loxP-based gene-excision system to remove rZmG2 and the antibiotic selectable gene after obtaining the transgenic plants. This study provides a useful tool for functional genomics work and biotechnology in plants.

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high- efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/ Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edi- ted 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homol- ogous end-joining mechanism followed by homologous recombination-based repair. We also obtained uni- form biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mu- tations in To rice and T1Arabidopsis plants by simultaneous targeting of multiple （up to eight） members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

From Golden Rice to aSTARice:Bioengineering Astaxanthin Biosynthesis in Rice Endosperm

Carotenoids are important phytonutrients with antioxidant properties,and are widely used in foods and feedstuffs as Supplements.Astaxanthin,a red-colored ketocarotenoid,has strong antioxidant activity and thus can benefit human health.However,astaxanthin is not produced in most higher plants.Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes,sZmPSY1,sPaCrtl,sCrBKT,and sHpBHY,which encode the enzymes phytoene synthase,phytoene desaturase,β-carotene ketolase,and β-carotene hydroxylase,respectively.Transgneic overexpression of two (sZmPSY1 and sPaCrtl),three (sZmPSY1,sPaCrtl and sCrBKT),and all these four genes driven by rice endosperm-specific promoters established the Carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm,from the yellow-grained β-caro- tene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice,respectively. Grains Of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity.These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in therice endosperm.This work provides a Successful example for synthetic biology in plants and biofortification in crops;the biofortified rice products generated by this study could be consumed as health-promoting foods and processed tO produce dietary supplements.

Development of ＂Purple Endosperm Rice＂ by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice （Oryza sativa L.）, could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes （two regulatory genes from maize and six structural genes from Coleus） driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm ＂Purple Endosperm Rice＂ （called ＂Zijingmi＂ in Chinese）, which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation （or enhancement） of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.

DRISPR/Cas9 Platforms for Genome Editing in Plants： Developments and Applications

The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap- tion of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/ Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac- tors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.

Molecular Control of Male Reproductive Development and Pollen Fertility in Rice

Anther development and male fertility are essential biological pro- cesses for flowering plants and are important for crop seed produc- tion. Genetic manipulation of male fertility/sterility is critical for crop hybrid breeding. Rice （Oryza sativa L.） male sterility phenotypes, including genic male sterility, hybrid male sterility, and cytoplasmic male sterility, are generally caused by mutations of fertility-related genes, by incompatible interactions between divergent allelic or non-allelic genes, or by genetic incompatibilities between cytoplas-mic and nuclear genomes. Here, we review the recent advances in the molecular basis of anther development and male fertility-sterility conversion in specific genetic backgrounds, and the interactions with certain environmental factors. The highlighted findings in this review have significant implications in both basic studies and rice genetic improvement.

The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice （Oryza sativa L.） male sterile mutant. Based on map- based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate （ATP）-binding cassette （ABC） transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcgl5. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 （meiosis II stage） to stage 10 （late microspore stage）. Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcgl5 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther Iocules. Consistently, histological analysis showed that osabcgl5 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.

Molecular mechanisms of hybrid sterility in rice

Hybrid sterility presents a major bottleneck in hybrid crop breeding and causes postzygotic reproductive isolation in speciation.Here, we summarize the current understanding of the genetics of rice hybrid sterility and highlight new advances in deciphering the molecular basis of the major genetic loci for hybrid sterility in rice. We also discuss practical strategies for overcoming reproductive barriers to utilize hybrid vigor in inter-specific and inter-subspecific hybrid rice breeding.

Targeted Genome Editing in Genes and cis- Regulatory Regions Improves Qualitative and Quantitative Traits in Crops

The Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）-associated protein 9 （CRISPR/Cas9）-based genomeediting system is a revolutionary technology for targeted muta- genesis in molecular biology research and genetic improvement of traits in crops （Cong et al., 2013; Ma et al., 2015, 2016）. Agronomic traits of crops are controlled by major genes and quantitative trait loci （QTL）. Therefore, the CRISPR/Cas9 system can be used to effectively and rapidly produce mutant traits by different strategies （Figure 1A-1C）. The most common application of the targeted editing system in genetic improvement is to knock out completely the functions of target genes, usually by editing site（s） in the coding sequences （CDS） to produce null-allele mutants （Figure 1A）.

Genome-editing technologies: the gap between application and policy

Genome editing is a new technology for manipulating genomic DNA sequences at specific sites,and researchers worldwide are rapidly developing new variants of genomeediting applications that target mRNAs,viruses,chromatin structure,and other cellular processes.

A modified high-efficiency thermal asymmetric interlaced PCR method for amplifying long unknown flanking sequences

Rapid and efficient isolation of unknown flanking DNA sequences adjacent to known regions is important for molecular biology research.For this purpose,several PCR-based methods have been reported,including inverse PCR(Uchiyama and Watanabe,2006),ligation-mediated PCR(Yan et al.,2003;Ballester et al.,2005;Wang et al.,2007;Trinh et al.,2012)and randomly primed PCR(Liu and Whittier,1995;Liu et al.,1995;Antal et al.

Characterization of a novel DUF1618 gene family in rice

Domain of unknown function （DUF） proteins represent a number of gene families that encode functionally uncharacterized proteins in eukaryotes. For example, DUF1618 family members in plants possess a 56-199-amino acid conserved domain and this family has not been described previously. Here, we report the characterization of 121 DUF1618 genes identified in the rice genome. Based on phylogenetic analysis, the rice DUF1618 family was divided into two major groups, each group consisting of two clades. Most DUF1618 genes with close phylogenetic relationships are located in gene clusters on the chromosomes, indicating that gene duplications increased the number of DUF1618 genes. A search for DUF1618 genes in genomic and/or expressed sequence tag databases for 35 other plant species showed that DUF1618 genes are only present in several monocot plants, suggesting that DUF1618 is a new gene family that originated after the dicot-monocot divergence. Based on public microarray databases, most rice DUF1618 genes are expressed at relatively low levels. Further experimental analysis showed that the transcriptional levels of some DUF1618 genes varied in different cultivars, and some responded to stress and hormone conditions, suggesting their important roles for development and fitness in rice （Oryza sativa L.）.

PhieCBEs:Plant High-Efficiency Cytidine Base Editors with Expanded Target Range

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base editors offer the ability to induce precise nucleotide substitutions(Komor et al.,2016).Cytidine base editors(CBEs)consist of a cytidine deaminase fused with a Cas9-nickase variant(Cas9n,with a D10A substitu-tion)and can achieve site-specific C-to-T substitution.Similarly,adenine base editors use an adenine deaminase forA-to-G substi-tution.These systems have been used in various organisms(Mishra et al.,2019).However,the Cas9 complex requires target sites containing NGG protospacer adjacent motifs(PAMs),thus restricting selection of potential targets.A number of CBEs have been developed using Cas9 variants(mostly Cas9n),cytidine deaminases(such as rAPOBEC1 and PmCDA1),and uracil glycosylase inhibitor(UGI)domains.These CBEs of the first generation(BE1,rAPOBEC1-dCas9),second generation(BE2,rAPOBEC1-dCas9-UGI),and third generation(BE3,rAPOBEC1-Cas9n-UGI)have moderate editing efficiencies in mammalians(Komor etal.,2016).

An effective strategy to establish a male sterility mutant mini-library by CRISPR/Cas9-mediated knockout of anther-specific genes in rice

Male sterility(MS),characterized by functional defects in male organs or gametes,is an important agronomic trait for hybrid seed production,especially for self-pollinated crops such as rice(Oryza sativa L)(Chen and Liu,2014).Spontaneous MS mutants are rare and difficult to maintain in nature,thus limiting basicresearch and breeding.Artificial mutants are typically generated by physical,chemical,or biological mutagenesis(Wei et al,2013).Recently developed genome editing systems such as CRISPR/Cas9 allow efficient and timesaving knockout of endogenous genes at specific sites(Smith et al,2000;Moscou and Bogdanove,2009;Gasiunas et al.,2012).

Hd1,Ghd7,and DTH8 synergistically determine the rice heading date and yield-related agronomic traits

Heading date determines the seasonal and regional adaptation of rice(Oryza sativa L.)varieties and is mainly controlled by photoperiod sensitivity(PS).The core heading date genes Hd1,Ghd7,DTH8,and PRR37 act synergistically in regulating the PS.In this study,we systematically analyze the heading date,PS,and agronomic traits of eight homozygous lines with various combinations of Hd1,Ghd7,and DTH8 alleles in the prr37 background under long-day(LD)and short-day(SD)conditions,respectively.We find that Hd1 alone promotes heading,regardless of the day length.However,under LDs,Hd1 suppresses flowering,in coordination with functional Ghd7 or with Ghd7 and DTH8.These loci cooperate to negatively regulate the Ehd1-Hd3 a/RFT1 pathway and delay heading.Under SDs,Hd1 competes with various heading suppressors to promote heading.Therefore,the dual function of Hd1 is vital for PS.The lines carrying Hd1 alone show reduced plant height with fewer primary and secondary branches in panicles.Lines carrying Ghd7 and DTH8(with hd1)show delayed heading and improve agronomic traits.Overall,our results reveal the regulation of rice PS flowering by the core heading date genes and their effects on agronomic traits,providing valuable information for the selection of rice varieties for adaptation to different light and temperature conditions.

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice

Oryza rufipogon Griff.is a wild progenitor of the Asian cultivated rice Oryza sativa.To better understand the genomic diversity of the wild rice,high-quality reference genomes of O.rufipogon populations are needed,which also facilitate utilization of the wild genetic resources in rice breeding.In this study,we generated a chromosome-level genome assembly of O.rufipogon using a combination of short-read sequencing,single-molecule sequencing,BioNano and Hi-C platforms.The genome sequence(399.8 Mb)was assembled into 46 scaffolds on the 12 chromosomes,with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb,respectively.The genome contains 36,520 protein-coding genes,and 49.37% of the genome consists of repetitive elements.The genome has strong synteny with those of the O.sativa subspecies indica and japonica,but containing some large structural variations.Evolutionary analysis unveiled the polyphyletic origins of O.sativa,in which the japonica and indica genome formations involved different divergent O.rufipogon(including O.nivara)lineages,accompanied by introgression of genomic regions between japonica and indica.This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O.sativa subspecies,and valuable information for basic research and rice breeding.

A novel CCCH-type zinc finger protein SAW1 activates OsGA20ox3 to regulate gibberellin homeostasis and anther development in rice

Male sterility is a prerequisite for hybrid seed production.The phytohormone gibberellin(GA)is in-volved in regulating male reproductive development,but the mechanism underlying GA homeostasis in anther development remains less understood.Here,we report the isolation and characterization of a new positive regulator of GA homeostasis,swollen anther wall 1(SAW1),for anther development in rice(Oryza sativa L.).Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRIPSR-associated protein 9-mediated knockout of SAW1 in rice generated similar male sterile plants.SAW1 encodes a novel nucleus-localizing CCCH-tandem zinc finger protein,and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther-specific expression.In the saw1 anther,the significantly decreased OsGA20ox3 expression resulted in lower bio-active GA content,which in turn caused the lower expression of the GA-inducible anther-regulator gene OsGAMYB.Thus,our results disclose the mechanism of the SAW1-GA20ox3-GAMYB pathway in controlling rice anther development,and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.

DELLA-GRF4-mediated coordination of growth and nitrogen metabolism paves the way for a new Green Revolution

The Green Revolution (GR) based on semi-dwarf breeding has greatly increased cereal crop yields since the 1960’s.Owing to appropriate plant height, the semi-dwarf crop plants are beneficial to increase light utilization and improve the lodging-resistance, and eventually increase yield potential (Hedden, 2003). During the past 50 years, the worldwide application of semi-dwarf crop breeding has produced plenty of elite varieties. The characters of Green Revolution varieties (GRVs) are caused by the mutant growth-repressing genes, such as reduced height (Rht) in wheat (Peng et al.,