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Wan L.,Huazhong Agricultural University | Wan L.,Key Laboratory of Rapeseed Genetic Improvement | Hu Q.,Huazhong Agricultural University | Hong D.,Huazhong Agricultural University | And 3 more authors.
Gene | Year: 2012

In Brassica napus, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes regulating anther cell differentiation and function. Here, we report two floral organ specific genes, BnC15 and BnATA20, derived from a B. napus two-line Rs1046A/B floral subtractive library. Although BnC15 and BnATA20 genes have a different expression pattern in anthers demonstrated by in situ hybridization and real-time PCR analysis, silencing of both genes in B. napus by antisense suppression resulted in pollen abortion after microspore release. Light and electron microscopy observation revealed the lack of plastoglobuli, lipid bodies and sporopollenin secreted from the tapetum leading to aberrations in exine sculpturing and the formation of a pollen coat. In addition, the microspores were squeezed to the irregular shape in the locule in the end. As shown by gene expression analysis in transgenic plants and the comparison of anther development between bnc15 or bnata20 mutants and Rs1046A, BnC15 and BnATA20 were positively regulated downstream of Rf gene controlling the fertility of Rs1046B in the same pathway. The results support the hypothesis that BnC15 and BnATA20 are crucial components of a genetic network that controls tapetum development and exine sculpturing. © 2012 Elsevier B.V. Source


Zhang C.,Huazhong Agricultural University | Zhang C.,Key Laboratory of Rapeseed Genetic Improvement | Zhang C.,University of Nebraska - Lincoln | Zhang W.,Huazhong Agricultural University | And 12 more authors.
Plant Physiology | Year: 2015

Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. © 2015 American Society of Plant Biologists. All rights reserved. Source


Wang Y.,Huazhong Agricultural University | Wang Y.,Key Laboratory of Rapeseed Genetic Improvement | Zhang Y.,Huazhong Agricultural University | Zhang Y.,Key Laboratory of Rapeseed Genetic Improvement | And 6 more authors.
Canadian Journal of Plant Science | Year: 2014

Bacillus thuringiensis (Bt) cry1Ac gene has been transformed into rapeseed to control diamondback moth (DBM, Plutella xylostella), which is one of the major lepidopteran pests of rapeseed (Brassica napus). However, Cry1A-resistant DBMpopulations have already developed in the field. Cry1C* is a new synthetic Bt gene based on the original cry1Ca5 sequence through optimizing its codons as well as removing AT-rich sequences and inverted repeats. In our present study, the cry1C* gene was introduced into rapeseed via Agrobacterium-mediated transformation, and a total of 42 transgenic lines were recovered. The results of polymerase chain reaction (PCR) and Southern blot both confirmed the expression of the cry1C* gene in the genomes of the transformants. We also assessed the expression of this foreign gene at the mRNA level in some selected transgenic lines by real-time reverse transcription (RT) PCR analysis. Enzyme-linked immunosorbent assay (ELISA) showed that the Cry1C* expression at the protein level greatly varied among individual transgenic plants, and transgenic line 1C-8 had the highest protein level of 799.32 ng g-1. The transgenic rapeseed plants expressing cry1C* gene showed a high efficacy against DBM. Taken together, the cry1C*-transgenic rapeseed could be employed as a useful germplasm in pest management and in the broad bioinsecticidal spectrum to prevent and postpone the development of pest resistance. © 2014, Agricultural Institute of Canada. All rights reserved. Source


Chen X.,Huazhong Agricultural University | Chen X.,Key Laboratory of Rapeseed Genetic Improvement | Li X.,Huazhong Agricultural University | Li X.,Key Laboratory of Rapeseed Genetic Improvement | And 19 more authors.
BMC Genomics | Year: 2013

Background: The presence of homoeologous sequences and absence of a reference genome sequence make discovery and genotyping of single nucleotide polymorphisms (SNPs) more challenging in polyploid crops.Results: To address this challenge, we constructed reduced representation libraries (RRLs) for two Brassica napus inbred lines and their 91 doubled haploid (DH) progenies using a modified ddRADseq technique. A bioinformatics pipeline termed RFAPtools was developed to discover and genotype SNPs and presence/absence variations (PAVs). Using this pipeline, a pseudo-reference sequence (PRF) containing 180,991 sequence tags was constructed. By aligning sequence reads to the pseudo-reference sequence, allelic SNPs as well as PAVs were identified and genotyped with RFAPtools. Two parallel linkage maps, one SNP bin map containing 8,780 SNP loci and one PAV linkage map containing 12,423 dominant loci, were constructed. By aligning marker sequences to B. rapa sequence scaffolds, whose genome is available, we assigned 44 unassembled sequence scaffolds comprising 8.15 Mb onto the B. rapa chromosomes, and also identified 14 instances of misassembly and eight instances of mis-ordering sequence scaffolds.Conclusions: These results indicate that the modified ddRADseq approach is a cost-effective and simple method to genotype tens of thousands SNPs and PAV markers in a polyploidy plant species. The results also demonstrated that RFAPtools developed in this study are powerful to mine allelic SNPs from homoeologous sequences in polyploids, therefore they are generally applicable in either diploid or polyploid species with or without a reference genome sequence. © 2013 Chen et al.; licensee BioMed Central Ltd. Source


Li H.,Huazhong Agricultural University | Li H.,Key Laboratory of Rapeseed Genetic Improvement | Younas M.,Huazhong Agricultural University | Younas M.,Key Laboratory of Rapeseed Genetic Improvement | And 28 more authors.
Theoretical and Applied Genetics | Year: 2013

Brassica napus (AACC) is a recent allotetraploid species evolved through hybridization between two diploids, B. rapa (AA) and B. oleracea (CC). Due to extensive genome duplication and homoeology within and between the A and C genomes of B. napus, most SSR markers display multiple fragments or loci, which limit their application in genetics and breeding studies of this economically important crop. In this study, we collected 3,890 SSR markers from previous studies and also developed 5,968 SSR markers from genomic sequences of B. rapa, B. oleracea and B. napus. Of these, 2,701 markers that produced single amplicons were putative single-locus markers in the B. napus genome. Finally, a set of 230 high-quality single-locus SSR markers were established and assigned to the 19 linkage groups of B. napus using a segregating population with 154 DH individuals. A subset of 78 selected single-locus SSR markers was proved to be highly stable and could successfully discriminate each of the 45 inbred lines and hybrids. In addition, most of the 230 SSR markers showed the single-locus nature in at least one of the Brassica species of the U's triangle besides B. napus. These results indicated that this set of single-locus SSR markers has a wide range of coverage with excellent stability and would be useful for gene tagging, sequence scaffold assignment, comparative mapping, diversity analysis, variety identification and association mapping in Brassica species. © 2012 Springer-Verlag Berlin Heidelberg. Source

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