Breeding by design for future rice:Genes and genome technologies

1.Introduction Rice is a staple food for 3.2 billion people.The food security threat that shook many Asian countries in 2008 still looms,because farmers are facing the challenge of producing more rice with fewer resources of water,land,and inputs.

Identification of quantitative trait loci for four morphologic traits under water stress in rice (Oryza sativa L.)

Late season drought coinciding with the rice booting to heading stage affects the development of plant height, panicle exserfion, and flag leaf size, and causes significant yield loss. In this study, a recombinant inbred line population derived from a cross between paddy and upland cultivars was used for data collection of the morphologic traits under well water and drought stress conditions. Drought stress was applied at the stage of panicle initiation in the field in 2002 and at the booting stage in PVC pipes in 2003. The data from stress conditions and their ratios （trait measured under stress condition/trait measured under well water condition） or differences （trait measured under stress condition minus trait measured under well water condition） were used for QTL analysis. Totally, 17 and 36 QTLs for these traits were identified in 2002 and 2003, respectively, which explained a range of 2.58%-29.82% of the phenotypic variation. Among them, six QTLs were commonly identified in the two years, suggesting that the drought stress in the two years was different. The genetic basis of these traits will provide useful information for improving rice late season drought resistance, and their application as indirect indices in rice late season drought resistance screening was also discussed.

BSA-seq-based identification of a major additive plant height QTL with an effect equivalent to that of Semi-dwarf 1 in a large rice F_(2) population

Bulked-segregant analysis is a time-and cost-saving strategy for identifying major quantitative trait loci(QTL) in a mapping population. Bulked-segregant analysis combined with whole-genome sequencing(BSA-seq) was performed to rapidly identify QTL for heading date, plant height, and panicle length in a large F_(2) population derived from two landraces: Chuan 7(C7) and Haoboka(HBK). Twenty plants with extremely low or high phenotypic values for the target traits were selected from an F_(2) population of 940 plants to construct low-and high-value bulks. Three pairs of bulks for the three traits were constructed, resulting in six DNA pools. BSA-seq revealed nine QTL: four for heading date, three for plant height, and two for panicle length. These QTL were validated in a random F_(2) population or BC_(4)F_(2) populations. The major novel plant height QTL, qPH8, acting additively with an effect equivalent to that of semi-dwarf 1(sd1), is potentially valuable for hybrid rice breeding. qPH8 controls mainly the elongation of basal internodes. The C7 allele of qPH8 reduces plant height and increases lodging resistance without yield penalty, suggesting a potential role for qPH8 in improving rice plant architecture.

The heading-date gene Ghd7 inhibits seed germination by modulating the balance between abscisic acid and gibberellins

Seed dormancy of cultivated rice was largely weakened during the progress of domestication.Correct timing and uniformity of seed germination are important for rapid seedling establishment and highyield production.In the present study,we found that the heading-date gene Ghd7 acted as a negative regulator of germination.A mutant of ghd7 showed low sensitivity to exogenous ABA treatment during seed germination.Further investigation revealed reduced accumulation of ABA in mature ghd7 seeds as a consequence of dampened expression of OsNCED genes.Moreover,elevated GA_(3) level was detected in seeds of ghd7 mutant during imbibition course,which was attributed to the induction of genes responsible for the synthesis pathways of bioactive GAs.Thus,Ghd7 inhibits seed germination by increasing the ABA/GA_(3) ratio.Besides revealing pleiotropic effects of Ghd7,our results indicate its role in linking seed germination to growth-phase transition in rice,which would enrich the theoretical basis for future breeding practices.

Entropy Production and Fractal Dimensions in Heavy Ion Nuclear Reaction at Intermediate Energies

The characteristics of the nonlinear dynamics in the Heavy Ion Collision (HIC) at intermediate energies have been studied by evaluating the productions of the Generalized Entropy (GE) and the Multifragmentation Entropy (ME) as well as the features of the information and fractal dimensions within the Isospin Quantum Molecular Dynamical Model compensated by the lattice methods. Results demonstrate from various views that the existence of deterministic chaos in the dynamical process of reaction.

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.Mut Map 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 theα2 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 ofα4 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.

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)exhibits 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 Ghd2 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 with Ghd2 overexpressed and 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 and RNA-seq data.Taken together,these results suggest that Ghd2 directly binds to the downstream gene CO3,and the Ghd2eCO3 module constantly delays heading date via the Ehd1-mediated pathway.

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.

Genetic Architecture of Natural Variation in Rice Chlorophyll Content Revealed by a Genome-Wide Association Study

Chlorophyll content is one of the most important physiological traits as it is closely related to leaf photo- synthesis and crop yield potential. So far, few genes have been reported to be involved in natural variation of chlorophyll content in rice （Oryza sativa） and the extent of variations explored is very limited. We con- ducted a genome-wide association study （GWAS） using a diverse worldwide collection of 529 O. sativa accessions. A total of 46 significant association loci were identified. Three F2 mapping populations with parents selected from the association panel were tested for validation of GWAS signals. We clearly demon- strated that Grain number, plant height, andheading date7 （GhdT） was a major locus for natural variation of chlorophyll content at the heading stage by combining evidence from near-isogenic lines and transgenic plants. The enhanced expression of Ghd7 decreased the chlorophyll content, mainly through down- regulating the expression of genes involved in the biosynthesis of chlorophyll and chloroplast. In addition, Narrow leaf1 （NAL1） corresponded to one significant association region repeatedly detected over two years. We revealed a high degree of polymorphism in the 5＇ UTR and four non-synonymous SNPs in the cod- ing region of NAL1, and observed diverse effects of the major haplotypes. The loci or candidate genes iden- tified would help to fine-tune and optimize the antenna size of canopies in rice breeding.

Identification and fine mapping of quantitative trait loci for seed vigor in germination and seedling establishment in rice

Seed vigor is an index of seed quality that is used to describe the rapid and uniform germination and the establish- ment of strong seedlings in any environmental conditions. Strong seed vigor in low-temperature germination conditions is particularly important in direct-sowing rice production systems. However, seed vigor has not been selected as an important breeding trait in traditional breeding programs due to its quantitative inherence. In this study, we identified and mapped eight quantitative trait loci （QTLs） for seed vigor by using a recombinant inbred population from a cross between rice （Oryza sativa L. ssp. indica） cultivars ZS97 and MH63. Conditional QTL analysis identified qSV-1, qSV-Sb, qSV-6a, qSV- 6b, and qSV-11 influenced seedling establishment and that qSV- 5a, qSV-Sc, and qSV-8 influenced only germination. Of these, qSV-1, qSV-Sb, qSV-6a, qSV-6b, and qSV-8 were low-tempera- ture-specific QTLs. Two major-effective QTLs, qSV-1, and qSV-5cwere narrowed down to 1.13-Mbp and 4oo-kbp genomic regions, respectively. The results provide tightly linked DNA markers for the marker-assistant pyramiding of multiple positive alleles for increased low-temperature germination seed vigor in both normal and environments.

Validation and Characterization of Ghd7.1, a Major Quantitative Trait Locus with Pleiotropic Effects on Spikelets per Panicle, Plant Height, and Heading Date in Rice (Oryza sativa L.)

A quantitative trait locus （QTL） that affects heading date （HD） and the number of spikelets per panicle （SPP） was previously identified in a small region on chromosome 7 in rice （Oryza sativa L.）. In order to further characterize the QTL region, near isogenic lines （NILs） were quickly obtained by self-crossing recombinant inbred line 189, which is heterozygous in the vicinity of the target region. The pleiotropic effects of QTL Ghd7.1 on plant height （PH）, SPP, and HD, were validated using an NIL-F2 population. Ghd7.1 explained 50.2%, 45.3%, and 76.9% of phenotypic variation in PH, SPP, and HD, respectively. Ghd7.1 was precisely mapped to a 357-kb region on the basis of analysis of the progeny of the NIL-F2 population. Day-length treatment confirmed that Ghd7.1 is sensitive to photoperiod, with long days delaying heading up to 12.5 d. Identification of panicle initiation and development for the pair of NILs showed that Ghd7.1 elongated the photoperiod-sensitive phase more than 10 d, but did not change the basic vegetative phase and the reproductive growth phase. These findings indicated that Ghd7.1 regulates SPP by controlling the rate of panicle differentiation rather than the duration of panicle development.

Short Panicle 3 controls panicle architecture by upregulating APO2/RFL and increasing cytokinin content in rice

Inflorescence architecture is a major determinant of spikelet numbers per panicle, a key component of grain yield in rice. In this study, Short Panicle 3(SP3) was identified from a short panicle 3(sp3) mutant in which T-DNA was inserted in the promoter of SP3, resulting in a knockdown mutation. SP3 encodes a DNA binding with one finger(Dof) transcriptional activator. Quantitative real time(q RT)-PCR and RNA in situ hybridization assays confirmed that SP3 is preferentially expressed in the young rice inflorescence, specifically in the branch primordial regions. SP3 acts as a negative regulator of inflorescence meristem abortion by upregulating APO2/RFL. SP3 both up-and downregulates expression of genes involved in cytokinin biosynthesis and catabolism, respectively. Consequently, cytokinin concentrations are decreased in young sp3 panicles, thereby leading to small panicles having fewer branches and spikelets. Our findings support a model in which SP3 regulates panicle architecture by modulating cytokinin homeostasis. Potential applications to rice breeding, through gene-editing of the SP3 promoter are assessed.

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 domaincontaining genes involved in regulating heading have been identified, providing diverse, flexible gene combinations for generating rice varieties with a given heading date.

Gene diagnosis and targeted breeding for blast-resistant Kongyu 131without changing regional adaptability

The fungus Magnaporthe oryzae threatens the rice production of Kongyu 131 (KY131),a leading japonica variety in Northeast China.In this study,two rice lines,KP1 and KP2-Hd1,were obtained by introgressing the blast resistance genes Pi1 and Pi2 into KY131,respectively.However,both lines headed later than KY131.RICE60K SNP array analysis showed that Hd1 closely linked to Pi2 was introgressed into KP2-Hd1,and the linkage drag of Hd1 was broken by recombination.On the other hand,no known flowering genes were introgressed into KP1.Gene diagnosis by resequencing six flowering genes showed that KP1 carried functional Hd16 and Ghd8 alleles.Due to its suppression role in heading under long-day conditions,Ghd8 was chosen as the target for gene editing to disrupt its function.Four sgRNAs targeting different sites within Ghd8 were utilized to induce large-deletion mutations,which were easy to detect via agarose gel electrophoresis.All the ghd8-mutated KP1 lines were resistant to rice blast disease and headed earlier than the control KP1,even than KY131,under natural long-day conditions,which ensures its growth in Northeast China.This study confirmed that a combination of gene diagnosis and targeted gene editing is a highly efficient way to quickly eliminate undesired traits in a breeding line.

Two Novel QTLs for Heading Date Are Identified Using a Set of Chromosome Segment Substitution Lines in Rice(Oryza sativa L.)

Heading date in rice is a typical quantitative trait controlled by multiple quantitative trait loci （QTLs）. It is mainly regulated by environmental factors such as photoperiod and temperature （Izawa, 2007）. Many QTLs for heading date have been identified using different mapping populations and methods （http：// www.gramene.org/qtl）. Up to date, several major heading date QTLs have been cloned by map-based cloning strategy （Yano et al., 2000; Takahashi et al., 2001; Kojima et al., 2002; Doi et al., 2004; Xue et al., 2008; Wei et al., 2010; Yan et al.,

Auxin Binding Protein 1 Reinforces Resistance to Sugarcane Mosaic Virus in Maize

Dear Editor,Sugarcane mosaic virus （SCMV） causes severe viral diseases in maize worldwide （Fuchs and Gruntzig, 1995）, resulting in significant losses in grain and forage yield in susceptible cultivars of maize and related crops. The most promising solution is to cultivate resistant varieties, which contribute to sustainable crop production. Two epistatically interacting major SCMV resistance loci （Scmvl and Scmv2） are required to confer complete resistance against SCMV in the resistant nearisogenic line F7RPJRR （the letters left of the slash refer to the genotype at Scmv2 on chromosome 3 and those on the right refer to the genotype at Scmvl on chromosome 6, with R indicating a resistance allele and S a susceptibility allele） （Xing et al., 2006）.

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 q S12 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.

The regulatory network mediated by circadian clock genes is related to heterosis in rice

Exploitation of heterosis in rice（Oryza sativa L.） has contributed greatly to global food security.In this study,we generated three sets of reciprocal F1 hybrids of indica and japonica subspecies to evaluate the relationship between yield heterosis and the circadian clock.There were no differences in trait performance or heterosis between the reciprocal hybrids,indicating no maternal effects on heterosis.The indica-indica and indica-japonica reciprocal F1 hybrids exhibited pronounced heterosis for chlorophyll and starch content in leaves and for grain yield/biomass.In contrast,the japonica-japonica F1 hybrids showed low heterosis.The three circadian clock genes investigated expressed in an above-high-parent pattern（AHP）at seedling stage in all the hybrids.The five genes downstream of the circadian clock,and involved in chlorophyll and starch metabolic pathways,were expressed in AHP in hybrids with strong better-parent heterosis（BPH）.Similarly,three of these Research Arfive genes in the japonica-japonica F1 hybrids showing low BPH were expressed in positive overdominance,but the other two genes were expressed in additive or negative overdominance.These results indicated that the expression patterns of circadian clock genes and their downstream genes are associated with heterosis,which suggests that the circadian rhythm pathway may be related to heterosis in rice.

QTL Scanning for Rice Yield Using a Whole Genome SNP Array

High-throughput SNP genotyping is widely used for plant genetic studies. Recently, a RICE6K SNP array has been developed based on the Illumina Bead Array platform and Infinium SNP assay technology for genome-wide evaluation of allelic variations and breeding applications. In this study, the RICE6K SNP array was used to genotype a recombinant inbred line （RIL） population derived from the cross between the indica variety, Zhenshan 97, and the japonica variety, Xizang 2. A total of 3324 SNP markers of high quality were identified and were grouped into 1495 recombination bins in the RIL population. A high-density linkage map, consisting of the 1495 bins, was developed, covering 1591.2 cM and with average length ofl.1 cM per bin. Segregation distortions were observed in 24 regions of the 11 chromosomes in the RILs. One half of the distorted regions contained fertility genes that had been previously reported. A total of 23 QTLs were identified for yield. Seven QTLs were firstly detected in this study. The positive alleles from about half of the identified QTLs came from Zhenshan 97 and they had lower phenotypic values than Xizang 2. This indicated that favorable alleles for breeding were dispersed in both parents and pyramiding favorable alleles could develop elite lines. The size of the mapping population for QTL analysis using high throughput SNP genotyping platform is also discussed.

Expression of CENH3 alleles in synthesized allopolyploid Oryza species

Synthesized allopolyploids are valuable materials for comparative analyses of two or more distinct genomes, such as the expression changes （activation, inactivation or differential expression） of orthologous genes following allopolyploidization. CENH3 is a centromerespecific histone H3 variant and has been regarded as a central component in kinetochore formation and centromere function. In this study, interspecific hybrids of Oryza genus （AA × CC, AA × CCDD） and their backcross progenies were produced, and the genome constitutions were identified as AC, ACC, ACD, AACD, or AA（CD） by Genomic in situ hybridization （GISH）. We further cloned and sequenced the CENH3 genes from O. sativa （AA）, O. officinalis （CC） and O. latifolia （CCDD）. Sequencing of RT-PCR products revealed that CENH3_C2 and CENH3_D, the two CENH3 alleles from O. latifolia, showed polymophism in several sites, while CENH3_C2 and CENH3_C1 from O. officinalis were different at only two amino acids positions. Moreover, we found that the CENH3 genes from both parents are expressed in interspecific hybrids and their progenies. Specifically, based on our cDNA sequencing data, the ratio of expres- sion level between CENH3_A and CENH3_C1 was approximately 1 in AC and 0.5 in ACC genomes, respectively. As a result, the CENH3 expression patterns shed more light on the inter-coordination between varied centromeric DNA sequences and highly conserved kinetochore protein in synthesized allopolyploids of Oryza genus.