Aiping Zheng

The evolution and pathogenic mechanisms of the rice sheath blight pathogen

Rhizoctonia solani is a major fungal pathogen of rice (Oryza sativa L.) that causes great yield losses in all rice-growing regions of the world. Here we report the draft genome sequence of the rice sheath blight disease pathogen, R. solani AG1 IA, assembled using next-generation Illumina Genome Analyser sequencing technologies. The genome encodes a large and diverse set of secreted proteins, enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, which probably reflect an exclusive necrotrophic lifestyle. We find few repetitive elements, a closer relationship to Agaricomycotina among Basidiomycetes, and expand protein domains and families. Among the 25 candidate pathogen effectors identified according to their functionality and evolution, we validate 3 that trigger crop defence responses; hence we reveal the exclusive expression patterns of the pathogenic determinants during host infection.

Effects of missing marker and segregation distortion on QTL mapping in F2 populations.

Missing marker and segregation distortion are commonly encountered in actual quantitative trait locus (QTL) mapping populations. Our objective in this study was to investigate the impact of the two factors on QTL mapping through computer simulations. Results indicate that detection power decreases with increasing levels of missing markers, and the false discovery rate increases. Missing markers have greater effects on smaller effect QTL and smaller size populations. The effect of missing markers can be quantified by a population with a reduced size similar to the marker missing rate. As for segregation distortion, if the distorted marker is not closely linked with any QTL, it will not have significant impact on QTL mapping; otherwise, the impact of the distortion will depend on the degree of dominance of QTL, frequencies of the three marker types, the linkage distance between the distorted marker and QTL, and the mapping population size. Sometimes, the distortion can result in a higher genetic variance than that of non-distortion, and therefore benefits the detection of linked QTL. A formula of the ratio of genetic variance explained by QTL under distortion and non-distortion was given in this study, so as to easily determine whether the segregation distortion marker (SDM) increases or decreases the QTL detection power. The effect of SDM decreases rapidly as its linkage relationship with QTL becomes looser. In general, distorted markers will not have a great effect on the position and effect estimations of QTL, and their effects can be ignored in large-size mapping populations.

Characterization of insecticidal crystal proteincry gene ofBacillus thuringiensis from soil of Sichuan Basin, China and cloning of novel haplotypescry gene

Sichuan Basin situated in the west of China, the fourth-largest basin of China, is a special area with complicated geomorphology (mountain, pasture, gorge, virgin forest, highland, hurst, glacier, and plain), and contains a rich and unique biodiversity. In order to describe a systematic study ofcry gene resources fromBacillus thuringiensis (Bt) strains of different ecological regions in Sichuan Basin, a total of 791 Bt strains have been screened from 2650 soil samples. The analysis of thecry genes was based on the method of PCR-restriction fragment length polymorphism (PCR-RFLP).cry1, cry2, cry3, cry4/10, cry9, cry30, andcry40-type genes were found in this basin. Strains containingcry1 genes were the most abundant in our collection (66%), and twenty-one differentcry1-type gene combinations were found. Bt strains harboringcry2 genes were the second most abundant (39.5%), and the strains containingcry3, cry9, cry4/10, cry30, andcry40 genes were found in 2.5, 3.5, 4.2, 4.2, and < 1%, respectively. Furthermore, several novel haplotypescry genes were found, and the full-length sequences of three novelcry genes were obtained, which were designated ascry52Ba1, cry54Aa1, cry30Fa1 by theB. thuringiensis Pesticide Crystal Protein Nomenclature Committee, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay of 80 strains which did not produce any PCR products indicated that these strains may harbour potentially novel Cry proteins. All these researches mentioned above revealed that the diversity and particularity ofcry gene resources fromB. thuringiensis strains in Sichuan Basin.

Characterization of Vegetative Insecticidal Protein vip Genes of Bacillus thuringiensis from Sichuan Basin in China

Vegetative insecticidal proteins (Vip), the second generation of insecticides, are produced during the vegetative growth stage of Bacillus thuringiensis (Bt). To perform a systematic study of vip genes in Bt strains from different ecological regions of Sichuan Basin, 1,789 soil samples were collected from this basin, which is situated in the western region of China. The basin has a complicated geomorphology and contains mountains, forests, highlands, hursts, and plains. A total of 2,134 Bt strains have been screened from the 1,789 soil samples. According to the results, three vip-type genes were found in this basin, namely the vip1, vip2, and vip3-type genes. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vip1-type genes (8.1%). The three types of vip genes were distributed in most of the regions, but E Mei Mountain and the Ba Lang Mountains only contained vip3 genes in environments with high elevation, low temperature, insufficient oxygen, and abundant snow. Moreover, five novel vip3 genes were found, and these Vip proteins were toxic for Chilo suppressalis. All the results mentioned above suggest that Sichuan Basin is a rich resource for vip genes.

The OsmiR396c‐OsGRF4‐OsGIF1 regulatory module determines grain size and yield in rice

Summary Grain weight is the most important component of rice yield and is mainly determined by grain size, which is generally controlled by quantitative trait loci (QTLs). Although numerous QTLs that regulate grain weight have been identified, the genetic network that controls grain size remains unclear. Herein, we report the cloning and functional analysis of a dominant QTL, grain length and width 2 (GLW2), which positively regulates grain weight by simultaneously increasing grain length and width. The GLW2 locus encodes OsGRF4 (growth‐regulating factor 4) and is regulated by the microRNA miR396c in vivo. The mutation in OsGRF4 perturbs the OsmiR396 target regulation of OsGRF4, generating a larger grain size and enhanced grain yield. We also demonstrate that OsGIF1 (GRF‐interacting factors 1) directly interacts with OsGRF4, and increasing its expression improves grain size. Our results suggest that the miR396c‐OsGRF4‐OsGIF1 regulatory module plays an important role in grain size determination and holds implications for rice yield improvement.

Natural variation in PTB1 regulates rice seed setting rate by controlling pollen tube growth

Grain number, panicle seed setting rate, panicle number and grain weight are the most important components of rice grain yield. To date, several genes related to grain weight, grain number and panicle number have been described in rice. However, no genes regulating the panicle seed setting rate have been functionally characterized. Here we show that the domestication-related POLLEN TUBE BLOCKED 1 (PTB1), a RING-type E3 ubiquitin ligase, positively regulates the rice panicle seed setting rate by promoting pollen tube growth. The natural variation in expression of PTB1 which is affected by the promoter haplotype and the environmental temperature, correlates with the rice panicle seed setting rate. Our results support the hypothesis that PTB1 is an important maternal sporophytic factor of pollen tube growth and a key modulator of the rice panicle seed setting rate. This finding has implications for the improvement of rice yield.

Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin, China

Sichuan basin, situated in the west of China, is the fourth biggest basin in China. In order to describe a systematic study of the cry2-type genes resources from Bacillus thuringiensis strains of Sichuan basin, a total of 791 Bacillus thuringiensis strains have been screened from 2650 soil samples in different ecological regions. The method of PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the type of cry2 genes. The results showed that 322 Bacillus thuringiensis strains harbored cry2-type genes and four different RFLP patterns were found. The combination of cry2Aa/cry2Ab genes was the most frequent (90.4%), followed by cry2Aa (6.8%) and cry2Ab alone (2.5%), and only one novel type of cry2 gene was cloned from one isolate (JF19-2). The full-length of this novel gene was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR), which was designated as cry2Ag1 (GenBank No. ACH91610) by the Bt Pesticide Crystal Protein Nomenclature Committee. In addition, the result of scanning electron microscopic (SEM) observation showed that these strains had erose, spherical, bipyramidal, and square crystal. And the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these strains harbored about one to three major proteins. These strains exhibited a wide range of insecticidal spectrum toxic to Aedes aegypti (Diptera) and Pieris rapae Linnaeus, 1758 (Lepidoptera). Particularly, JF19-2 contained cry2Ag gene had the highest insecticidal activity. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bacillus thuringiensis strains in Sichuan basin.

Morphological structure of propagules and electrophoretic karyotype analysis of false smut Villosiclava virens in rice

The target pathogen Villosiclava virens (teleomorph: claviceps oryzae-sativae) was isolated from the infected rice, where it caused false smut. In our study, the forming processes of the chlamydospores, chlamydospore balls, conidiospores, and secondary conidiospores during the asexual reproduction were observed more precisely and in greater detail than previous descriptions. The microstructure of the infected rice kernel showed that the outer dense chlamydospores piled around the false smut balls grown on XBZ medium; moreover the sclerotia consisting of dense mycelium were found. The different morphology was observed across the different growing conditions. In addition, we observed the nuclear numbers of both the conidiospores and hyphae using 4′,6-diamidino-2-phenylindole (DAPI) staining. Because the fungus has small chromosomes and the numbers were not previously known, we analyzed the electrophoretic karyotype using a pulsed field gel electrophoresis (PFGE) technique. The results showed that V. virens has at least 10 chromosomes ranging in size from 0.6 kb to 6 Mb. The V. virens genome size is estimated to be 23 Mb. Here, we report the morphological characteristics of the fungus and the process of asexual spores forming asexual propagules, along with the first analyze the molecular karyotype of V. virens. These results supply a foundation for further study of the pathogenicity and biology of this devastating pathogen.

Identification of Genome-Wide Variations among Three Elite Restorer Lines for Hybrid-Rice

Rice restorer lines play an important role in three-line hybrid rice production. Previous research based on molecular tagging has suggested that the restorer lines used widely today have narrow genetic backgrounds. However, patterns of genetic variation at a genome-wide scale in these restorer lines remain largely unknown. The present study performed re-sequencing and genome-wide variation analysis of three important representative restorer lines, namely, IR24, MH63, and SH527, using the Solexa sequencing technology. With the genomic sequence of the Indica cultivar 9311 as the reference, the following genetic features were identified: 267,383 single-nucleotide polymorphisms (SNPs), 52,847 insertion/deletion polymorphisms (InDels), and 3,286 structural variations (SVs) in the genome of IR24; 288,764 SNPs, 59,658 InDels, and 3,226 SVs in MH63; and 259,862 SNPs, 55,500 InDels, and 3,127 SVs in SH527. Variations between samples were also determined by comparative analysis of authentic collections of SNPs, InDels, and SVs, and were functionally annotated. Furthermore, variations in several important genes were also surveyed by alignment analysis in these lines. Our results suggest that genetic variations among these lines, although far lower than those reported in the landrace population, are greater than expected, indicating a complicated genetic basis for the phenotypic diversity of the restorer lines. Identification of genome-wide variation and pattern analysis among the restorer lines will facilitate future genetic studies and the molecular improvement of hybrid rice.

Complete Genome Sequence of Bacillus thuringiensis Serovar Sichuansis Strain MC28

Bacillus thuringiensis is an important microbial insecticide used in the control of agricultural pests. Here we report the finished, annotated genome sequence of Bacillus thuringiensis serovar Sichuansis strain MC28, which can form parasporal crystals consisting of Cry4Cc1, Cry30Fa1, Cry53Ab1, Cry54Aa1, Cry54Ab1, Cry68Aa1, Cry69Aa1, Cry69Aa2, Cry70Ba1, Cyt1Da1, and Cyt2Aa3. It is also highly toxic to lepidopterous and dipterous insects.