Introgression of Bacterial Blight Resistance Genes in the Rice Cultivar Ciherang: Response against Xanthomonas oryzae pv. oryzae in the F6 Generation.

Bacterial blight (BB) is caused by Xanthomonas oryzae pv. oryzae and is one of the most important diseases in rice. It results in significantly reduced productivity throughout all rice-growing regions of the world. Four BB resistance genes have been reported; however, introgression of a single gene into rice has not been able to sufficiently protect rice against BB infection. Pyramiding of effective BB resistance genes (i.e., Xa genes) into background varieties is a potential approach to controlling BB infection. In this study, combinations of four BB resistance genes, Xa4, xa5, xa13, and Xa21, were pyramided into populations. The populations were derived from crossing Ciherang (a widespread Indonesian rice variety) with IRBB60 (resistance to BB). Promising recombinants from the F6 generation were identified by scoring the phenotype against three virulent bacterial strains, C5, P6, and V, which cause widespread BB infection in most rice-growing countries. Pyramiding of genes for BB resistance in 265 recombinant introgressed lines (RILs) were confirmed through marker-assisted selection (MAS) of the F5 and F6 generations using gene-specific primers. Of these 265 RILs, 11, 34 and 45 lines had four, three, or two BB resistance genes, respectively. The RILs had pyramiding of two or three resistance genes, with the Xa4 resistance gene showing broad spectrum resistance against Xoo races with higher agronomic performance compared to their donor and recipients parents. The developed BB-resistant RILs have high yield potential to be further developed for cultivation or as sources of BB resistance donor material for varietal improvement in other rice lines.

Harnessing the hidden genetic diversity for improving multiple abiotic stress tolerance in rice (Oryza sativa L.).

To develop superior rice varieties with improved yield in most rainfed areas of Asia/Africa, we started an introgression-breeding program for simultaneously improving yield and tolerances of multiple abiotic stresses. Using eight BC1 populations derived from a widely adaptable recipient and eight donors plus three rounds of phenotypic selection, we developed 496 introgression lines (ILs) with significantly higher yield under drought, salt and/or non-stress conditions in 5 years. Six new varieties were released in the Philippines and Pakistan and many more are being evaluated in multi-location yield trials for releasing in several countries. Marker-facilitated genetic characterization revealed three interesting aspects of the breeding procedure: (1) the donor introgression pattern in specific BC populations was characteristic; (2) introgression frequency in different genomic regions varied considerably, resulting primarily from strong selection for the target traits; and (3) significantly lower heterozygosity was observed in BC progenies selected for drought and salinity tolerance. Applying strong phenotypic selection under abiotic stresses in early segregating generations has major advantages for not only improving multiple abiotic stress tolerance but also achieving quicker homozygosity in early generations. This breeding procedure can be easily adopted by small breeding programs in developing countries to develop high-yielding varieties tolerant of abiotic stresses. The large set of trait-specific ILs can be used for genetic mapping of genes/QTL that affect target and non-target traits and for efficient varietal development by designed QTL pyramiding and genomics-based recurrent selection in our Green Super Rice breeding technology.

Genome-wide response to selection and genetic basis of cold tolerance in rice (Oryza sativa L.).

BACKGROUND: Cold stress is an important factor limiting rice yield in many areas of high latitude and altitude. Considerable efforts have been taken to genetically dissect cold tolerance (CT) in rice using DNA markers. Because of possible epistasis and gene × environment interactions associated with identified quantitative trait loci, the results of these genetic studies have unfortunately not been directly applicable to marker-assisted selection for improved rice CT. In this study, we demonstrated the utility of a selective introgression strategy for simultaneous improvement and genetic dissection of rice seedling CT. RESULTS: A set of japonica introgression lines (ILs) with significantly improved seedling CT were developed from four backcross populations based on two rounds of selection. Genetic characterization of these cold-tolerant ILs revealed two important aspects of genome-wide responses to strong phenotypic selection for rice CT: (1) significant over-introgression of donor alleles at 57 loci in 29 functional genetic units (FGUs) across the rice genome and (2) pronounced non-random associations between or among alleles at many unlinked CT loci. Linkage disequilibrium analyses of the detected CT loci allowed us to construct putative genetic networks (multi-locus structures) underlying the seedling CT of rice. Each network consisted of a single FGU, with high introgression as the putative regulator plus two to three groups of highly associated downstream FGUs. A bioinformatics search of rice genomic regions harboring these putative regulators identified a small set of candidate regulatory genes that are known to be involved in plant stress response. CONCLUSIONS: Our results suggest that CT in rice is controlled by multiple pathways. Genetic complementarity between parental-derived functional alleles at many loci within a given pathway provides an appropriate explanation for the commonly observed hidden diversity and transgressive segregation of CT and other complex traits in rice.

Three genetic systems controlling growth, development and productivity of rice (Oryza sativa L.): a reevaluation of the 'Green Revolution'.

The Green Revolution (GR-I) included worldwide adoption of semi-dwarf rice cultivars (SRCs) with mutant alleles at GA20ox2 or SD1 encoding gibberellin 20-oxidase. Two series of experiments were conducted to characterize the pleiotropic effects of SD1 and its relationships with large numbers of QTLs affecting rice growth, development and productivity. The pleiotropic effects of SD1 in the IR64 genetic background for increased height, root length/mass and grain weight, and for reduced spikelet fertility and delayed heading were first demonstrated using large populations derived from near isogenic IR64 lines of SD1. In the second set of experiments, QTLs controlling nine growth and yield traits were characterized using a new molecular quantitative genetics model and the phenotypic data of the well-known IR64/Azucena DH population evaluated across 11 environments, which revealed three genetic systems: the SD1-mediated, SD1-repressed and SD1-independent pathways that control rice growth, development and productivity. The SD1-mediated system comprised 43 functional genetic units (FGUs) controlled by GA. The SD1-repressed system was the alternative one comprising 38 FGUs that were only expressed in the mutant sd1 backgrounds. The SD1-independent one comprised 64 FGUs that were independent of SD1. GR-I resulted from the overall differences between the former two systems in the three aspects: (1) trait/environment-specific contributions; (2) distribution of favorable alleles for increased productivity in the parents; and (3) different responses to (fertilizer) inputs. Our results suggest that at 71.4 % of the detected loci, a QTL resulted from the difference between a functional allele and a loss-of-function mutant, whereas at the remaining 28.6 % of loci, from two functional alleles with differentiated effects. Our results suggest two general strategies to achieve GR-II (1) by further exploiting the genetic potential of the SD1-repressed and SD1-independent pathways and (2) by restoring the SD1-mediated pathways, or 'back to the nature' to fully exploit the genetic diversity of those loci in the SD1-mediated pathways which are virtually inaccessible to most rice-breeding programs worldwide that are exclusively based on sd1.

Development and application of a set of breeder-friendly SNP markers for genetic analyses and molecular breeding of rice (Oryza sativa L.).

Single nucleotide polymorphisms (SNPs) are the most abundant DNA markers in plant genomes. In this study, based on 54,465 SNPs between the genomes of two Indica varieties, Minghui 63 (MH63) and Zhenshan 97 (ZS97) and additional 20,705 SNPs between the MH63 and Nipponbare genomes, we identified and confirmed 1,633 well-distributed SNPs by PCR and Sanger sequencing. From these, a set of 372 SNPs were further selected to analyze the patterns of genetic diversity in 300 representative rice inbred lines from 22 rice growing countries worldwide. Using this set of SNPs, we were able to uncover the well-known Indica-Japonica subspecific differentiation and geographic differentiations within Indica and Japonica. Furthermore, our SNP results revealed some common and contrasting patterns of the haplotype diversity along different rice chromosomes in the Indica and Japonica accessions, which suggest different evolutionary forces possibly acting in specific regions of the rice genome during domestication and evolution of rice. Our results demonstrated that this set of SNPs can be used as anchor SNPs for large scale genotyping in rice molecular breeding research involving Indica-Japonica and Indica-Indica crosses.

Dissecting genetic networks underlying complex phenotypes: the theoretical framework.

Great progress has been made in genetic dissection of quantitative trait variation during the past two decades, but many studies still reveal only a small fraction of quantitative trait loci (QTLs), and epistasis remains elusive. We integrate contemporary knowledge of signal transduction pathways with principles of quantitative and population genetics to characterize genetic networks underlying complex traits, using a model founded upon one-way functional dependency of downstream genes on upstream regulators (the principle of hierarchy) and mutual functional dependency among related genes (functional genetic units, FGU). Both simulated and real data suggest that complementary epistasis contributes greatly to quantitative trait variation, and obscures the phenotypic effects of many 'downstream' loci in pathways. The mathematical relationships between the main effects and epistatic effects of genes acting at different levels of signaling pathways were established using the quantitative and population genetic parameters. Both loss of function and "co-adapted" gene complexes formed by multiple alleles with differentiated functions (effects) are predicted to be frequent types of allelic diversity at loci that contribute to the genetic variation of complex traits in populations. Downstream FGUs appear to be more vulnerable to loss of function than their upstream regulators, but this vulnerability is apparently compensated by different FGUs of similar functions. Other predictions from the model may account for puzzling results regarding responses to selection, genotype by environment interaction, and the genetic basis of heterosis.

[Meta-analysis of relationship of vitamin D receptor gene polymorphism and tuberculosis susceptibility].

OBJECTIVE: To provide a quantitative summary in estimating the association between polymorphisms of 2 loci of FokI and TaqI in vitamin D receptor gene and susceptibility to tuberculosis by means of meta-analysis. METHODS: Databases CNKI, CBM, WanFang DATA, MEDLINE and PubMed were searched using "VDR", "polymorphisms" or "alleles", in combination with "tuberculosis", and a manual search of citations from relevant original studies and literature was also performed between January 1980 and November 2007. RevMan 4.2 was applied in the process of analyzing the data of 14 case-control studies. RESULTS: The summary ORs for studies with polymorphisms of FokI, TaqI loci of the VDR gene were 1.12 (95%CI: 0.98 - 1.28) in Ff vs FF, 1.66 (95%CI: 1.12 - 2.46) in ff vs FF, 0.97 (95%CI: 0.82 - 1.14) in Tt vs TT, 1.27 (95%CI: 0.96 - 1.69) in tt vs TT. The fail-safe number was 109.69 for FokI and 75.24 for TaqI. The analysis result was reliable. CONCLUSION: Polymorphisms at FokI loci (VDR-ff) showed statistically significant association between the VDR variants and susceptibility to tuberculosis, and variant in the TaqI locus failed to show statistically significant association.

Identification of functional candidate genes for drought tolerance in rice.

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs.

Mapping QTLs with digenic epistasis under multiple environments and predicting heterosis based on QTL effects.

Mixed linear model approach was proposed for mapping QTLs with the digenic epistasis and QTL by environment (QE) interaction as well as additive and dominant effects. Monte Carlo simulations indicated that the proposed method could provide unbiased estimations for both positions and genetic main effects of QTLs, as well as unbiased predictions for QE interaction effects. A method was suggested for predicting heterosis based on individual QTL effects. The immortalized F(2) (IF(2)) population constructed by random mating among RI or DH lines is appropriate for mapping QTLs with epistasis and their QE interaction. Based on the models and methodology proposed, we developed a QTL mapping software, QTLMapper 2.0 on the basis of QTLmapper 1.0, which is suitable for analyzing populations of DH, RIL, F(2) and IF(2). Data of thousand grain weight of IF(2) population with 240 lines derived from elite hybrid rice Shanyou 63 were analyzed as a worked example.

Genome-wide introgression lines and their use in genetic and molecular dissection of complex phenotypes in rice (Oryza sativa L.).

Tremendous efforts have been taken worldwide to develop genome-wide genetic stocks for rice functional genomic (FG) research since the rice genome was completely sequenced. To facilitate FG research of complex polygenic phenotypes in rice, we report the development of over 20,000 introgression lines (ILs) in three elite rice genetic backgrounds for a wide range of complex traits, including resistances/tolerances to many biotic and abiotic stresses, morpho-agronomic traits, physiological traits, etc., by selective introgression. ILs within each genetic background are phenotypically similar to their recurrent parent but each carries one or a few traits introgressed from a known donor. Together, these ILs contain a significant portion of loci affecting the selected complex phenotypes at which allelic diversity exists in the primary gene pool of rice. A forward genetics strategy was proposed and demonstrated with examples on how to use these ILs for large-scale FG research. Complementary to the genome-wide insertional mutants, these ILs opens a new way for highly efficient discovery, candidate gene identification and cloning of important QTLs for specific phenotypes based on convergent evidence from QTL position, expression profiling, functional and molecular diversity analyses of candidate genes, highlights the importance of genetic networks underlying complex phenotypes in rice that may ultimately lead to more complete understanding of the genetic and molecular bases of quantitative trait variation in rice.

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.

[Studies of marker screening efficiency and corresponding influencing factors in QTL composite interval mapping].

Screening markers efficiently is the foundation of mapping QTLs by composite interval mapping. Main and interaction markers distinguished, besides using background control for genetic variation, could also be used to construct intervals of two-way searching for mapping QTLs with epistasis, which can save a lot of calculation time. Therefore, the efficiency of marker screening would affect power and precision of QTL mapping. A doubled haploid population with 200 individuals and 5 chromosomes was constructed, with 50 markers evenly distributed at 10 cM space. Among a total of 6 QTLs, one was placed on chromosome I, two linked on chromosome II, and the other three linked on chromosome IV. QTL setting included additive effects and epistatic effects of additive x additive, the corresponding QTL interaction effects were set if data were collected under multiple environments. The heritability was assumed to be 0.5 if no special declaration. The power of marker screening by stepwise regression, forward regression, and three methods for random effect prediction, e.g. best linear unbiased prediction (BLUP), linear unbiased prediction (LUP) and adjusted unbiased prediction (AUP), was studied and compared through 100 Monte Carlo simulations. The results indicated that the marker screening power by stepwise regression at 0.1, 0.05 and 0.01 significant level changed from 2% to 68%, the power changed from 2% to 72% by forward regression. The larger the QTL effects, the higher the marker screening power. While the power of marker screening by three random effect prediction was very low, the maximum was only 13%. That suggested that regression methods were much better than those by using the approaches of random effect prediction to identify efficient markers flanking QTLs, and forward selection method was more simple and efficient. The results of simulation study on heritability showed that heightening of both general heritability and interaction heritability of genotype x environments could enhance marker screening power, the former had a greater influence on QTLs with larger main and/or epistatic effects, while the later on QTLs with small main and/or epistatic effects. The simulation of 100 times was also conducted to study the influence of different marker number and density on marker screening power. It is indicated that the marker screening power would decrease if there were too many markers, especially with high density in a mapping population, which suggested that a mapping population with definite individuals could only hold limited markers. According to the simulation study, the reasonable number of markers should not be more than individuals. The simulation study of marker screening under multiple environments showed high total power of marker screening. In order to relieve the problem that marker screening power restricted the efficiency of QTL mapping, markers identified in multiple environments could be used to construct two search intervals.