Innovation and development of the third-generation hybrid rice technology

The breeding and large-scale application of hybrid rice contribute significantly to the food supply worldwide.Currently,hybrid seed production uses cytoplasmic male sterile(CMS)lines or photoperiod/thermo-sensitive genic male sterile(PTGMS)lines as female parent.Despite huge successes,both systems have intrinsic problems.CMS systems are mainly restricted by the narrow restorer resources that make it difficult to breed superior hybrids,while PTGMS systems are limited by conditional sterility of the male sterile lines that makes the propagation of both PTGMS seeds and hybrid seeds vulnerable to unpredictable climate changes.Recessive nuclear male sterile(NMS)lines insensitive to environmental conditions are widely distributed and are ideal for hybrid rice breeding and production,but the lack of effective ways to propagate the pure NMS lines in a large scale renders it impossible to use them for hybrid rice production.The development of"the third-generation hybrid rice technology"enables efficient propagation of the pure NMS lines in commercial scale.This paper discusses the establishment of"the thirdgeneration hybrid rice technology"and further innovations.This new technology breaks the limitations of CMS and PTGMS systems and will bring a big leap forward in hybrid rice production.

Development of herbicide resistance genes and their application in rice

Rice is one of the most important food crops in the world.Weeds seriously affect the rice yield and grain quality.In recent years,there are tremendous progresses in the research and application of herbicideresistant genes in rice worldwide.This article reviews the working mechanisms of six herbicides(glyphosate,glufosinate,acetolactate synthase inhibitor herbicides,acetyl-Co A carboxylase inhibitor herbicides,hydroxyhenylpyruvate dioxygenase(HPPD)inhibitor herbicides and dinitroaniline herbicides),the resistance mutations of the corresponding herbicide-target genes,and the herbicide detoxification mechanisms by non-target genes.Examples are provided on herbicide-resistant rice materials obtained by transformation of exogenous resistance genes,by artificial mutagenesis and mutant screening,and by modifying the target genes through gene editing.This paper also introduces the current application of herbicide-resistant rice,points out problems that may be caused by utilization of herbicide resistant rice and solutions to the problems,and discusses the future prospects for the development of herbicideresistant rice.

Trp_(548)Met mutation of acetolactate synthase in rice confers resistance to a broad spectrum of ALS-inhibiting herbicides

Herbicide resistance in crop plants is valuable for integrated weed management in agriculture. Herbicide resistant rice, in particular, is important to management of weedy rice, a close relative of cultivated rice and a noxious weed prevalent in rice fields that remains challenging to farmers worldwide. Herbicide resistant plants can be obtained through transgenic approach or by mutagenesis of regular plant and screening of mutants with elevated resistance to herbicide. In this study, we conducted ethyl methyl sulfonate mutagenesis(EMS) to elite indica cultivar Huanghuazhan(HHZ) and screened for mutants resistant to imazapic, a herbicide that can inhibit the acetolactate synthase(ALS) in plants. We obtained three mutants of Os ALS gene that have not been reported previously in rice. One of the mutants, with Trp_(548) changed to Met(W_(548)M), was analyzed in more details in this study. This mutation had no negative effect on the plant physiology and morphology as well as rice yield. Compared with the imidazolinone-resistant mutant S_(627)N(Ser_(627) changed to Asn) that has been deployed for Clearfield rice development, W_(548)M mutant showed high levels of resistance to a broad spectrum of five families of ALSinhibiting herbicides, in addition to a higher level of resistance to herbicides of the imidazolinone family.The herbicide-resistance was stably inherited by crossing into other rice lines. Thus, the W_(548)M mutation provides a valuable resource for breeding of herbicide resistant rice and weed management.

A functional characterization of TaMs1 orthologs in Poaceae plants

TaMs1 encodes a non-specific lipid transfer protein(nsLTP) and is required for pollen development in wheat. Although MS1 is a Poaceae-specific gene, the roles of MS1 genes in other Poaceae plants are unknown, especially in rice and maize. Here, we identified one ortholog in rice(OsLTPg29) and two orthologs in maize(ZmLTPg11 and ZmLTPx2). Similar to TaMs1, both OsLTPg29 and ZmLTPg11 genes are specifically expressed in the microsporocytes, and both OsLTPg29 and ZmLTPg11 proteins showed lipid-binding ability to phosphatidic acid and several phosphoinositides. To determine their roles in pollen development, we created osltpg29 mutants and zmltpg11 zmltpx2 double mutants by CRISPR/Cas9.osltpg29, not zmltpg11 zmltpx2, is defective in pollen development, and only OsLTPg29, not ZmLTPg11,can rescue the male sterility of tams1 mutant. Our results demonstrate that the biological function of MS1 in pollen development differs in the evolution of Poaceae plants.

Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis

Alternative splicing can generate multiple mRNAs that differ in their untranslated regions or coding sequences,and these differences might affect mRNA stability or result in different protein isoforms with diverse functions and/or localizations.In this study,we isolated a sterile mutant in rice with abnormal meiosis of microspore mother cells and megaspore mother cells that carried a point mutation in OsRAD1 gene.Cloning of OsRAD1 cDNAs revealed three transcript variants,named as OsRAD1.1,OsRAD1.2 and OsRAD1.3,respectively,which were derived from alternative splicing of the last intron.Proteins derived from the three transcripts were mostly identical except the difference in the very C-terminal domain.The three transcripts exhibited similar expression patterns in various tissues,but the expression level of OsRAD1.1 was the highest.Specific knockout of OsRAD1.1 led to sterility,while knockout of OsRAD1.2 and OsRAD1.3 together did not change the plant fertility.Overexpression of OsRAD1.2 and OsRAD1.3 cDNAs in OsRAD1.1-specific mutant did not complement the plant fertility.Yeast two-hybrid assay showed that OsRAD1.1,but not OsRAD1.2 and OsRAD1.3,interacted with the three other meiosis proteins OsHUS1,OsRAD9 and OsRAD17,suggesting that the C-terminal domain of OsRAD1.1 is critical for the protein function.

A High-Density SNP Genotyping Array for Rice Biology and Molecular Breeding

A high-density single nucleotide polymorphism （SNP） array is critically important for geneticists and molecu- lar breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the devel- opment of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for dif- ferent objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies （GWAS） analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

A Global Identification and Analysis of Small Nucleolar RNAs and Possible Intermediate-Sized Non-Coding RNAs in Oryza sativa

Accumulating evidence suggests that non-coding RNAs （ncRNAs） are both widespread and functionally important in many eukaryotic organisms. In this study, we employed a special size fractionation and cDNA library construction method followed by 454 deep sequencing to systematically profile rice intermediate-size ncRNAs. Our analysis resulted in the identification of 1349 ncRNAs in total, including 754 novel ncRNAs of an unknown functional category. Chromosome distribution of all identified ncRNAs showed no strand bias, and displayed a pattern similar to that observed in protein-coding genes with few chromosome dependencies. More than half of the ncRNAs were centered around the plus-strand of the 5＇ and 3＇ termini of the coding regions. The majority of the novel ncRNAs were rice specific, while 78% of the small nucleolar RNAs （snoRNAs） were conserved. Tandem duplication drove the expansion of over half of the snoRNA gene families. Furthermore, 90% of the snoRNA candidates were shown to produce small RNAs between 20-30 nt, 80% of which were associated with ARGONAUT proteins generally, and AGOlb in particular. Overall, our findings provide a comprehensive view of an intermediate-size non-coding transcriptome in a monocot species, which will serve as a useful platform for an in-depth analysis of ncRNA functions.

Identification of late-stage pollen-specific promoters for construction of pollen-inactivation system in rice

Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant.We attempted to construct this system in rice by using a late-stage pollen-specific(LSP)promoter driving the expression of maizeα-amylase gene ZM-AA1.To obtain such promoters in rice,we conducted comparative RNA-seq analysis of mature pollen with meiosis anther,and compared this with the transcriptomic data of various tissues in the Rice Expression Database,resulting in 269 candidate LSP genes.Initial test of nine LSP genes showed that only the most active OsLSP3 promoter could drive ZM-AA1 to disrupt pollen.We then analyzed an additional 22 LSP genes and found 12 genes stronger than OsLSP3 in late-stage anthers.The promoters of OsLSP5 and OsLSP6 showing higher expression than OsLSP3 at stages 11 and 12 could drive ZM-AA1 to inactivate pollen,while the promoter of OsLSP4 showing higher expression at stage 12 only could not drive ZM-AA1 to disrupt pollen,suggesting that strong promoter activity at stage 11 was critical for pollen inactivation.The strong pollen-specific promoters identified in this study provided valuable tools for genetic engineering of rice male sterile system for hybrid rice production.

GDSL esterase/lipases OsGELP34 and OsGELP110/OsGELP115 are essential for rice pollen development

Pollen exine contains complex biopolymers of aliphatic lipids and phenolics.Abnormal development of pollen exine often leads to plant sterility.Molecular mechanisms regulating exine formation have been studied extensively but remain ambiguous.Here we report the analyses of three GDSL esterase/lipase protein genes,OsGELP34,OsGELP110,and OsGELP115,for rice exine for-mation.OsGELP34 was identified by cloning of a male sterile mutant gene.OsGELP34 encodes an endoplasmic reticulum protein and was mainly expressed in anthers during pollen exine formation.osgelp34 mutant displayed abnormal exine and altered expression of a number of key genes required for pollen development.OsGELP110 was previously identified as a gene differentially expressed in meiotic anthers.OsGELP110 was most homologous to OsGELP115,and the two genes showed similar gene expression patterns.Both OsGELP110 and OsGELP115 proteins were localized in peroxisomes.Individual knockout of OsGELP110 and OsGELP115 did not affect the plant fertility,but double knockout of both genes altered the exine structure and rendered the plant male sterile.OsGELP34 is distant from OsGELP110 and OsGELP115 in sequence,and osgelp34 and osgelp110/osgelp115 mutants were different in anther morphology despite both were male sterile.These results suggested that OsGELP34 and OsGELP110/OsGELP115 catalyze different compounds for pollen exine development.

Lectin receptor kinase OsLecRK-S.7 is required for pollen development and male fertility

Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination.Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture.The mutant gene encodes a novel plasma membrane-localized legume-lectin receptor kinase that we named OsLecRK-S.7.OsLecRK-S.7 was expressed at different levels in all tested tissues and throughout anther development.In vitro kinase assay showed OsLecRK-S.7 capable of autophosporylation.Mutation in s13283(E560K)and mutation of the conserved ATP binding site(K418E)both knocked out the kinase activity.Mass spectrometry showed Thr376,Ser378,Thr386,Thr403,and Thr657 to be the autophosphorylation sites.Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees,but did not abolish the gene function in fertility complementation.oslecrk-s.7 mutant plant overexpressing OsLecRK-S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers,and these phenotypes were abolished by E560K or K418E mutation.The results indicated that OsLecRK-S.7 was a key regulator of pollen development.

The calcium-dependent kinase OsCPK24 functions in cold stress responses in rice

Calcium-dependent protein kinases（CPKs）are serine/threonine protein kinases that function in plant stress responses. Although CPKs are recognized as key messengers in signal transduction, the specific roles of CPKs and the molecular mechanisms underlying their activity remain largely unknown. Here, we characterized the function of Os CPK_（24）, a cytosol-localized calciumdependent protein kinase in rice. Os CPK_（24） was universally and highly expressed in rice plants and was induced by cold treatment. Whereas Os CPK_（24） knockdown plants exhibited increased sensitivity to cold compared to wild type（WT）, Os CPK_（24）-overexpressing plants exhibited increased cold tolerance. Plants overexpressing Os CPK_（24） exhibited increased accumulation of proline（an osmoprotectant） and glutathione（an antioxidant） and maintained a higher GSH/GSSG（reduced glutathione to oxidized glutathione） ratio during cold stress compared to WT. In addition to these effects in response to cold stress, we observed the kinase activity of Os CPK_（24） varied under different calcium concentrations. Further,Os CPK_（24） phosphorylated Os Grx_（10）, a glutathionedependent thioltransferase, at rates modulated by changes in calcium concentration. Together, our results support the hypothesis that Os CPK_（24） functions as a positive regulator of cold stress tolerance in rice, a process mediated by calcium signaling and involving phosphorylation and the inhibition of Os Grx_（10） to sustain higher glutathione levels.