[The history and prospect of rice genetic breeding in China].

Rice breeding in China has experienced three major leaps of dwarf breeding, heterosis utilization and green super rice cultivation, accompanied by six important processes: dwarf breeding (the first green revolution), three-line hybrid rice cultivation, two-line hybrid rice cultivation, inter-subspecies heterosis utilization, ideal plant type breeding and green super rice cultivation. The breeding subject ranges from the unique trait of high yield to the complex traits of resistance, high quality and high yield. The breeding concept is gradually upgraded from high yield and quality to the second green revolution concept of "less investment, more output, and better environment". Rice functional genomics achievements have prepared many genes with important utilization values for the second green revolution, and rice breeding is moving towards a new era of design breeding. The genomic selection technology and transgenic technology will help to develop the green super rice for "less pesticides, less fertilizers, water saving and drought tolerance, superior quality and high yield". Here, we summarize the development process of rice genetics and breeding in China, point out advantages and disadvantages of various breeding methods and breeding techniques, systematically introduce the molecular mechanisms on cytoplasmic male sterility, photoperiod-sensitive male genic sterility and indica-japonica hybrid sterility, review the important functional genes related to rice plant architecture, panicle architecture, grain size and nutrient use efficiency, clarify the correlation between yield and heading date, and highlight the important position of China in the rice basic research in the world. In particular, we emphasize the fact that Chinese rice production styles have undergone or are undergoing tremendous changes in recent years, and the breeding concept must also keep pace with the changing production styles. In the future, the hybrid breeding technology should be closely integrated with modern breeding technologies to breed rice varieties that must not only meet the market demand, but also have the natural and healthy characteristics and adapt to the new farming system and methods.

[The application of genome editing in identification of plant gene function and crop breeding].

Plant genome can be modified via current biotechnology with high specificity and excellent efficiency. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system are the key engineered nucleases used in the genome editing. Genome editing techniques enable gene targeted mutagenesis, gene knock-out, gene insertion or replacement at the target sites during the endogenous DNA repair process, including non-homologous end joining (NHEJ) and homologous recombination (HR), triggered by the induction of DNA double-strand break (DSB). Genome editing has been successfully applied in the genome modification of diverse plant species, such as Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. In this review, we summarize the application of genome editing in identification of plant gene function and crop breeding. Moreover, we also discuss the improving points of genome editing in crop precision genetic improvement for further study.

[Ghd7, a pleiotropic gene controlling flag leaf area in rice].

Photosynthesis is the unique source of energy for plant. Flag leaf contributed the majority of photosynthate after rice flowering. Ghd7 is a pleiotropic gene, which can significantly increase rice production. In order to study the genetic effects of Ghd7 on the flag leaf morphology, we made quantitative trait locus (QTL) analysis for flag leaf length (FLL), flag leaf width (FLW), and flag leaf area (FLA) using a Ghd7-BC2F2 population of 190 plants. In the BC2F2 population, the frequency distribution of FLL, FLW, and FLA were bimodal and in agreement with single Mendelian segregation ratio (3:1). FLL, FLW, and FLA were positively correlated with grains per panicle in the population. One QTL was mapped to the in-terval between markers RM3859 and C39 on chromosome 7, which explained 73.3%, 62.3%, and 71.8% of the variations for FLL, FLW, and FLA, and co-segregated with Ghd7. Two near-isogenic lines of NIL (mh7) and NIL (tq7) were devel-oped using Zhenshan 97 as the recurrent parent and Minghui 63 and Teqing as the donor parent, respectively. Both NILs significantly increased the phenotypic values of FLL, FLW, and FLA as compared with Zhenshan 97. FLL, The values of FLW and FLA for Ghd7 over-expression transgenic plants were 8.9 cm, 0.5 cm, and 17.8 cm2 larger than its recipient Heji-ang 19. These results demonstrated that Ghd7 plays an important role in controlling the flag leaf area in rice.

[Discovery of QTLs increasing yield related traits in common wild rice].

Common wild rice (Oryza rufipogon) is an important genetic resource. Discovery of desirable alleles in wild rice will make important contributions to rice genetic improvement. In this study, Zhenshan 97 as the recurrent parent and wild rice as the donor parent were used to develop a BC2F1 population. One plant BC2F1-15 in the population showed distinct phenotype from Zhenshan 97 was selected to produce a population of BC2F5 by continuous self-crossing. The genotype assay of the plant BC2F1-15 with 126 polymorphic SSR markers evenly distributed on 12 chromosomes showed that it was heterozygous at 30% of the control marker loci. Four, 3, 4, 2, and 1 QTLs were detected for heading date, plant height, spikelets per panicle, grain weight, and single plant yield in the BC2F5 population, respectively. One QTL region flanked by the marker interval of RM481-RM2 on chromosome 7 had pleiotropic effects on heading date, spikelets per panicle, and grain yield per plant, and the alleles of wild rice increased phenotypic values. At the other 3 QTLs for spikelets per panicle, common wild rice had positive effects. These results clearly showed that common wild rice carried desirable alleles for yield related traits. The favorable alleles from common wild rice are new valuable genes for rice breeding.

Quantitative trait loci for rice yield-related traits using recombinant inbred lines derived from two diverse cultivars.

The thousand-grain weight and spikelets per panicle directly contribute to rice yield. Heading date and plant height also greatly influence the yield. Dissection of genetic bases of yield-related traits would provide tools for yield improvement. In this study, quantitative trait loci (QTL) mapping for spikelets per panicle, thousand-grain weight, heading date and plant height was performed using recombinant inbred lines derived from a cross between two diverse cultivars, Nanyangzhan and Chuan7. In total, 20 QTLs were identified for four traits. They were located to 11 chromosomes except on chromosome 4. Seven and five QTLs were detected for thousand-grain weight and spikelets per panicle, respectively. Four QTLs were identified for both heading date and plant height. About half the QTLs were commonly detected in both years, 2006 and 2007. Six QTLs are being reported for the first time. Two QTL clusters were identified in regions flanked by RM22065 and RM5720 on chromosome 7 and by RM502 and RM264 on chromosome 8, respectively. The parent, Nanyangzhan with heavy thousand-grain weight, carried alleles with increased effects on all seven thousand-grain weight QTL, which explained why there was no transgressive segregation for thousand-grain weight in the population. In contrast, Chuan7 with more spikelets per panicle carried positive alleles at all five spikelets per panicle QTL except qspp5. Further work on distinction between pleiotropic QTL and linked QTL is needed in two yield-related QTL clusters.

[A new rice dwarf1 mutant caused by a frame-shift mutation].

A dwarf mutant C6PS, which has the similar phenotype as the recessive mutant Dwarf1 (d1), was produced from tissue-cultured plants of Zhonghua 11. In its progeny (T2), the ratio of tall to dwarf plants was in agreement with the expected segregation ratio (3:1) of a single Mendelian inheritance gene, which indicated that the variation of plant height is caused by a single gene. To locate the mutation, C6PS was crossed with Zhenshan 97 and Mudanjiang 8 for producing two F2 populations of F2 (CM) and F2 (CZ), respectively. The plant height in each F2 population also showed the same segregation pattern as that in T2 generation. SSR marker RM430 closely linked to Dwarf1 was preferentially used to genotype the F2 (CZ) population because C6PS showed the similar phenotype to d1 mutant. RM430 was significantly associated with plant height, which indicated that the mutant gene might be D1. Comparative sequencing of D1 between C6PS and Zhonghua 11 showed a 6 bp deletion occurred in the splice site of its ninth exon. The marker C6PS-D1L/R designed on the 6 bp deletion was co-segregated with plant height in T2 generation. The results indicated that C6PS was a new mutant of D1. This mutation led to a 26 bp deletion of the transcript and resulted in a frame-shift mutation and a premature stop codon in C6PS, which could not translate the functional Gα protein. C6PS was weakly sensitive to Brassinolide based on the leaf inclination angle test.

A major QTL, Ghd8, plays pleiotropic roles in regulating grain productivity, plant height, and heading date in rice.

Rice yield and heading date are two distinct traits controlled by quantitative trait loci (QTLs). The dissection of molecular mechanisms underlying rice yield traits is important for developing high-yielding rice varieties. Here, we report the cloning and characterization of Ghd8, a major QTL with pleiotropic effects on grain yield, heading date, and plant height. Two sets of near isogenic line populations were developed for the cloning of Ghd8. Ghd8 was narrowed down to a 20-kb region containing two putative genes, of which one encodes the OsHAP3 subunit of a CCAAT-box binding protein (HAP complex); this gene was regarded as the Ghd8 candidate. A complementary test confirmed the identity and pleiotropic effects of the gene; interestingly, the genetic effect of Ghd8 was dependent on its genetic background. By regulating Ehd1, RFT1, and Hd3a, Ghd8 delayed flowering under long-day conditions, but promoted flowering under short-day conditions. Ghd8 up-regulated MOC1, a key gene controlling tillering and branching; this increased the number of tillers, primary and secondary branches, thus producing 50% more grains per plant. The ectopic expression of Ghd8 in Arabidopsis caused early flowering by 10 d-a situation similar to the one observed by its homolog AtHAP3b, when compared to wild-type under long-day conditions; these findings indicate the conserved function of Ghd8 and AtHAP3b in flowering in Arabidopsis. Our results demonstrated the important roles of Ghd8 in rice yield formation and flowering, as well as its opposite functions in flowering between rice and Arabidopsis under long-day conditions.

QTL analysis for flag leaf characteristics and their relationships with yield and yield traits in rice.

Photosynthesis of carbohydrate is the primary source of grain yield in rice (Oryza sativa L.). It is important to genetically analyze the morphological and the physiological characteristics of functional leaves, especially flag leaf, in rice improvement. In this study, a recombinant inbred population derived from a cross between an indica (O. sativa L. ssp. indica) cultivar and a japonica (O. sativa L. ssp. japonica) cultivar was employed to map quantitative traits loci (QTLs) for the morphological (i.e., leaf length, width, and area) and physiological (i.e., leaf color rating and stay-green) characteristics of flag leaf and their relationships with yield and yield traits in 2003 and 2004. A total of 17 QTLs for morphological traits (flag leaf length, width, and area), 6 QTLs for degree of greenness and 14 QTLs for stay-green-related traits (retention-degrees of greenness, relative retention of greenness, and retention of the green area) were resolved, and 10 QTLs were commonly detected in both the years. Correlation analysis revealed that flag leaf area increased grain yield by increasing spikelet number per panicle. However, the physiological traits including degree of greenness and stay-green traits were not or negatively correlated to grain yield and yield traits, which may arise from the negative relation between degree of greenness and flag leaf size and the partial sterility occurred in a fraction of the lines in this population. The region RM255-RM349 on chromosome 4 controlled the three leaf morphological traits simultaneously and explained a large part of variation, which was very useful for genetic improvement of grain yield. The region RM422-RM565 on chromosome 3 was associated with the three stay-green traits simultaneously, and the use of this region in genetic improvement of grain yield needs to be assessed by constructing near-isogenic lines.

Analysis of digenic epistatic effects and QE interaction effects QTL controlling grain weight in rice.

Immortalized F(2) population of rice (Oryza sativa L.) was developed by randomly mating F(1) among recombinant inbred (RI) lines derived from (Zhenshan 97B x Minghui 63), which allowed replications within and across environments. QTL (quantitative trait loci) mapping analysis on kilo-grain weight of immortalized F(2) population was performed by using newly developed software for QTL mapping, QTLMapper 2.0. Eleven distinctly digenic epistatic loci included a total of 15 QTL were located on eight chromosomes. QTL main effects of additive, dominance, and additive x additive, additive x dominance, and dominance x dominance interactions were estimated. Interaction effects between QTL main effects and environments (QE) were predicted. Less than 40% of single effects, most of which were additive effects, for identified QTL were significant at 5% level. The directional difference for QTL main effects suggested that these QTL were distributed in parents in the repulsion phase. This should make it feasible to improve kilo-grain weight of both parents by selecting appropriate new recombinants. Only few of the QE interaction effects were significant. Application prospect for QTL mapping achievements in genetic breeding was discussed.

[Mapping QTLs for horizontal resistance to sheath blight in an elite rice restorer line, Minghui 63].

This study was conducted with a recombinant inbred line (RILs) population consisting of 240 recombination lines, derived from an elite combination, Zhenshan 97B x Minghui 63. The RILs and their parents were grown in a randomized complete design with two replications in the years of 1999 and 2000. Sheath blight response ratings for the population and their parents were identified by an improved method of inoculation, which was carried out with short woody toothpicks incubated with a Rhizoctonia solani strain, RH-9, and inserted the third sheath in the late tillering/green ring stage of growth. A linkage map was constructed from the RILs. The QTL mapping of sheath blight resistance was carried out by the method of interval QTL mapping. Two QTLs for sheath blight resistance were detected in each year, and were located on chromosome 5 and chromosome 9, respectively. The QTL for sheath blight resistance on chromosome 5 was flanked by markers C624 and C246 on the basis of 1999 data, and by markers C246 and RM26 using 2000 data. The 1-LOD-confidence intervals of QTLs for sheath blight resistance on chromosome 5 detected in two years greatly overlapped with each other, and the peak of the 1-LOD-confidence intervals were approximately the same site. This suggested that the QTL for resistance on chromosome 5 detected in 1999 was probably the same as the QTL detected in 2000. The QTL for sheath blight resistance on chromosome 9 was located on the marker interval of C472-R2638 in term of 1999 data, and on the interval of RM257-RM242 based on 2000 data, and the two intervals were 9.7 cM away from each other. Based on the effect analysis of QTLs for resistance, the genotype of MH63 had negative additive effects or reduced sheath blight rating.

Genetic analysis of the panicle traits related to yield sink size of rice.

In the study, ten panicle traits associated with yield sink size were measured in a recombinant inbred population derived from Zhenshan 97 x Minghui 63. Generally, spikelets per panicle were more closely correlated with number of secondary branch per panicle, spikelets on secondary branch per secondary branch, and spikelet density. A total of 53 QTL were detected for ten traits in two years. Approximately 43.4% QTLs were detected in both two years, suggesting environmental effects on traits. Five chromosomal regions (G359-RG532 and C567-C86-RG236 on chromosome 1, R712-RM29 on chromosome 2, P-RG424 on chromosome 6, C148-RM258 on chromosome 10) were detected to have effects on multiple panicle traits. QTLs for traits, which were correlated, were generally localized in similar chromosomal regions, suggesting that pleiotropy and (or) linkage are the molecular basis of relationship between them. A large number of digenic interactions were detected, 18.2% of which were detected simultaneously in both two years. The proportion of common interactions was trait-depended, ranging 8.7% for spikelets on secondary branch per secondary branch to 32.6% for panicle length. Approximately 26.7% of common two-locus combinations had pleiotropic effects by simultaneously influencing two or more traits. Overall, the results indicate that each panicle trait is controlled by several QTLs, genotype x environment interaction, and a large number of epistatic interactions.