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Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

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Journal of Integrative Agriculture 2013年第10期12卷 1799-1806页

作者： LI Jin-ping LI Yan SHI Yan-xia XIE Xue-wen Chai A-li LI Bao-ju institute of vegetables and flowers Chinese Academy of Agricultural Sciences

A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA（rDNA） and internal transcribed spacer（ITS）.A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of *** positive plasmid pB12 was obtained and used as the template to create standard *** specificity,sensitivity,and reproducibility of real-time PCR were evaluated *** and artificially infested soil samples containing different concentrations of *** were *** results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template *** melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of *** genomic DNA was approximately 40 copies per 25 μ*** sensitivity of the assay was at least 100-fold higher than conventional *** DNA from *** could be amplified and detected using this assay,suggesting the highly specific of this *** coefficient of variation was less than 3%,indicating the PCR method revealed high *** detection limit in soil samples corresponded to 1 000 resting spores *** plants were used to validate the real-time PCR *** developed real-time PCR assay allows for fast and sensitive detection of *** in soil and should be useful in disease management and pest interception so as to prevent further spread of ***.

关键词： species-specific rDNA-ITS gene Plasmodiophora brassicae real-time quantitative PCR SYBR Green I dye

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

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Journal of Integrative Agriculture 2017年第1期16卷 57-64页

作者： GUO Xiao YANG Xiao-hui YANG Yu MAO Zhen-chuan LIU Feng MA Wei-qing XIE Bing-yan LI Guang-cun institute of vegetables and flowers Shandong Academy of Agricultural SciencesMolecular Biology Key Laboratory of Shandong Facility Vegetable institute of vegetables and flowers Chinese Academy of Agricultural Sciences

Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

关键词： pepper bacterial artificial chromosome library.(BAC) root-knot nematode Me3 gene contig

Heterotic Group Classification of 63 Inbred Lines and Hybrid Purity Identification by Using SSR Markers in Winter Cabbage(Brassica Oleracea L. var. capitata)

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Horticultural Plant Journal 2018年第4期4卷 158-164页

作者： LI Xing YU Hailong LI Zhiyuan LIU Xiaoping FANG Zhiyuan LIU Yumei YANG Limei ZHUANG Mu LV Honghao ZHANG Yangyong institute of vegetables and flowers Chinese Academy of Agricultural Sciences

Winter cabbage is an important crop cultivated through winter in the region near the Yangtze River, enabling the supply of fresh cabbage there at that time of year. However, a problem has emerged regarding the newly developed parents of winter cabbage, which is completely different from spring and autumn cabbage, namely, how to combine these parents to breed an elite hybrid. To classify the heterotic groups and improve the efficiency of parent selection in winter cabbage breeding, 20 polymorphic SSR markers were selected to screen 63 winter cabbage inbred lines. Seventeen pairs among the 20 SSR markers amplified polymorphic bands. These primers amplified two to six bands,with an average of 2.8 bands per primer, and a total of 47 polymorphic bands were generated in the 63 inbred lines. These lines included flatheaded morphotype and round-headed morphotype, thus they were separately classified into heterotic groups based on the SSR markers. The flat-headed morphotype contained 21 inbred lines and was classified into three heterotic groups, named Hanchun 4, Jiali, and Dongsheng, in accordance with the representative germplasm contained in each group. The round-headed morphotype contained 42 inbred lines and was classified into five heterotic groups, named Parte, Bejo1039, YK-143, SCA002, and Golden B90. Meanwhile, parent analysis of 20 developed elite combinations showed that their parents were all distributed in different heterotic groups, indicating that the group classification was reasonable,which can provide a basis for further parent selection in winter cabbage breeding. Furthermore, polymorphic SSR primers were successfully used to identify the hybrid purity of three elite varieties.

关键词： winter cabbage head morphotype SSR marker heterotic group hybrid purity identification

Research Progress on Agrobacterium tumefaciens-based Transgenic Technology in Brassica rapa

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Horticultural Plant Journal 2018年第3期4卷 126-132页

作者： LI Guoliang YUE Lixin LI Fei ZHANG Shifan ZHANG Hui QIAN Wei FANG Zhiyuan WU Jian WANG Xiaowu ZHANG Shujiang SUN Rifei institute of vegetables and flowers Chinese Academy of Agricultural Sciences

Brassica rapa *** cultivated globally and consumed in many areas *** the transgenic Agrobacterium-mediated transformation method,which is a reproducible and efficient technique,genes can be transferred into various *** *** review summarizes the processes involved in Agrobacterium-mediated transformation of ***,including surface seed sterilization,co-cultivation with ***,induction of callus/shoot/root formation,and confirmation of transgenic *** addition,factors such as the Agrobacterium strain,plant genotype,explant age,transformation efficiency of the hybrid or inbred line,and the concentrations of N6-benzyl amino purine and naphthalene acetic acid,are *** this review shows clearly how to do it,what to do,and what not to do in the transgenic Agrobacterium-mediated in Brassica *** information presented here lays the foundation for a simple and efficient method that resolves existing problems and improves overall transgenic *** production,thereby benefiting both basic and applied research.

关键词： Brassica rapa Agrobacterium tumefaciens transgenic transformation

Genome-wide identification and analysis of sulfatase and sulfatase modifying factor genes in Bemisia tabaci (Hemiptera: Aleyrodidae)

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Insect Science 2021年第6期28卷 1541-1552页

作者： Jun Ren Zheng-Ke Peng Ze-Zhong Yang Li-Xia Tian Shao-Nan Liu Shao-Li Wang Qing-Jun Wu Wen Xie You-Jun Zhang institute of vegetables and flowers Chinese Academy of Agricultural SciencesBeijing100081China

The invasive pest whitefly(Bemisia tabaci)is a complex species,of which Middle East-Minor Asia 1(MEAM1)and Mediterranean(MED)are the two most damaging *** research showed that cabbage is frequently infested with MEAM1 but seldomly with MED,and this difference in performance is associated with glucosi-nolate(GS)*** insects can modify GS using glucosinolate sulfatase(SULF),the activity of which is regulated by sulfatase modifying factor 1(SUMF1);therefore,to increase our understanding of different performances of MEAM1 and MED on cabbage plants,we identified and compared nine putative SULFs and one SUMF in MEAM1 and *** found that the lengths of two genes,BtSulf2 and BtSulf4,differed between MEAM1 and *** messenger RNA levels of BtSulf4 increased more than 20-fold after MEAMl and MED adults were exposed to GS,but BtSulJ2 expression was only induced by GS in *** of BtSulf2 and BtSulf4 in MEAM1 resulted in a substantial increase in the mortality of GS-treated adults but not in *** results indicate that differences in BtSulJ2 and BtSulf4 sequences and/or expression may explain why MEAM1 performs better than MED on *** results provide a basis for future functional research on SULF and SUMF in ***.

关键词： Bemisia tabaci cabbage glucosinolates(GS) sulfatase(SULF) sulfatase modifying factor 1(SUMF 1)

Molecular cloning and functional analysis of the pepper resistance gene Me3 to root-knot nematode

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Horticultural Plant Journal 2023年第1期9卷 133-144页

作者： Yang Liu Hongyi Cao Jian Ling Yuhong Yang Yan Li Bingyan Xie Jianlong Zhao Zhenchuan Mao institute of vegetables and flowers Chinese Academy of Agricultural ScienceBeijing 100081China

Root-knot nematodes(RKNs)cause severe diseases in peppers annually around the *** pepper,the Me3 gene provides a heat-stable and broad-spectrum resistance to *** this study,several simple sequence repeat(SSR)markers and insertion/deletion(In Del)markers were developed to fine map the Me3 *** of 2272 individuals(F2progenies)revealed that Me3 was located in a 45-kb DNA region between markers SSR784 and SSR339,in which there were three candidate *** them,as a novel nucleotide binding site and leucine rich repeat(NBS-LRR)family gene,the DNA sequence of Capana09g000163 of pepper line‘HDA149’was 6348 bp in length,with a 2802-bp open reading frame encoding 933 amino acids,including NB-ARC and LRR *** transient expression assays demonstrated that expression of Capana09g000163 triggered a hypersensitive response(HR)in Nicotiana benthamiana *** localization results showed that the Capana09g000163 protein was localized in the cell *** expression of Capana09g000163 in Arabidopsis significantly increased resistance against Meloidogyne incognita compared with the wild-type(WT)***,*** was almost unable to develop in transgenic Arabidopsis expressing *** together,we cloned the Me3 gene and verified that it induced resistance against *** with the methods of map-based cloning and transgenic technology,which may be of great significance to pepper breeding for resistance against RKNs.

关键词： Capsicum annuum L Meloidogyne incognita Nematode-resistant gene Fine mapping Transgenic technology

Genome-wide association study reveals candidate genes for gummy stem blight resistance in cucumber

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Horticultural Plant Journal 2023年第2期9卷 261-272页

作者： Jianan Han Shaoyun Dong Yanxia Shi Zhuonan Dai Han Miao Baoju Li Xingfang Gu Shengping Zhang institute of vegetables and flowers Chinese Academy of Agricultural SciencesBeijing 100081China

Gummy stem blight(GSB),caused by Didymella bryoniae,is a serious fungal disease that leads to decline in cucumber yield and *** molecular mechanism of GSB resistance in cucumber remains ***,we investigated the GSB resistance of cucumber core germplasms from four geographic groups at the seedling and adult stages.A total of 9 SNPs related to GSB resistance at the seedling stage and 26 SNPs at the adult stage were identified,of which some are co-localized to previously mapped Quantitative trait loci(QTLs)for GSB resistance(gsb3.2/gsb3.3,gsb5.1,and gsb-s6.2).Based on haplotype analysis and expression levels after inoculation,four candidate genes were identified within the region identified by both Genome-wide association study(GWAS)and previous identified QTL mapping,including Csa3G129470 for gsb3.2/gsb3.3,Csa5G606820 and Csa5G606850 for gsb5.1,and Csa6G079730 for *** novel GSB resistant accessions,significant SNPs,and candidate genes facilitate the breeding of GSB resistant cucumber cultivars and provide a novel idea for understanding GSB resistance mechanism in cucumber.

关键词： Cucumber(Cucumis sativus L.) GSB GWAS Quantitative trait loci QTL Candidate genes

Frequencies and mechanisms of pesticide resistance in Tetranychus urticae field populations in China

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Insect Science 2022年第3期29卷 827-839页

作者： Yan Zhang Dandan Xu Youjun Zhang Qingjun Wu Wen Xie Zhaojiang Guo Shaoli Wang institute of vegetables and flowers Chinese Academy of Agricultural SciencesBeijing100081China

The two-spotted spider mite Tetranychus urticate is an important agricultural pest *** is extremely polyphagous and has developed resistance to many ***,we assessed the pesticide resistance of seven field populations of *** in China,their target site mutations and the activities of their detoxification *** results showed that abamectin and the traditional pesticides pyridaben,profenofos and bifenthrin had higher resistance or lower toxicity than more recently developed pesticides including chlorfenapyr,spinetoram,cyflumetofen,cyenopyrafen,bifenazate and *** frequency of point mutations related to abamectin resistance,G314D in the glutamate-gated chloride channel 1(GluCl1)and G326E in GluC13,ranged 47%-70%and 0%-97%,*** frequency of point mutations in A1215D and F1538I of the voltage-gated sodium channel gene(VGSC),which may increase resistance to pyrethroids,ranged 88%-100%and 10%-100%,*** target sites related to organophosphate resistance,mutation frequencies ranged 25%-92%for G119S and 0%-23%for A201S in the acetycholinesterase gene(Ace).Mutation G126S in the bifenazate resistance-related cytochrome b gene(Cytb)was observed in three of the seven *** *** activities of detoxification enzymes(P450,GST,CarEs and UGTs)were observed in two *** populations,with significant difference in the XY-SX *** results provide useful information on the status of pesticide resistance of *** in China and suggest that *** field populations may have multiple resistance mechanisms.

关键词： detoxification enzyme field populations pesticide resistance target site mutations Tetranychus urticae

Comparative Proteomic Analysis of Cucumber Fruits Under Nitrogen Deficiency At the Fruiting Stage

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Horticultural Plant Journal 2021年第1期7卷 59-72页

作者： Jia Hao Qiang Li Hongjun Yu Heng Wang Lin Chai Tiantian Miao Weijie Jiang institute of vegetables and flowers Chinese Academy of Agricultural SciencesBeijing 100081China

Nitrogen deficiency is a major factor that affects the yield and quality of horticultural *** expressed proteins were identified in cucumber fruit under nitrogen deficiency treatment,and are expected to be highly useful for dissecting carbon,nitrogen and ascorbate metabolism in cucumber and for improving nitrogen fertilizer *** plants grown hydroponically in Hoagland solution were transferred to nitrogen-free conditions for 3 or 5 d after the blossom of female *** proteome profiles were assessed using twodimensional *** relative expression of the genes encoding 50 selected differentially expressed proteins and 4 key enzymes in carbon metabolism was analyzed by *** showed that most of the identified differentially expressed proteins were mainly associated with carbon metabolism,amino acid biosynthesis,ascorbate metabolism,and *** phosphorylation was enhanced,while the pentose phosphate pathway was *** metabolism and the synthesis of most amino acids was affected significantly,and ascorbate might be accumulated under nitrogen deficiency in cucumber fruit.

关键词： cucumber fruit nitrogen deficiency proteomics