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Utilizing resequencing big data to facilitate brassica vegetable breeding:tracing introgression pedigree and developing highly specific markers for clubroot resistance

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Horticultural Plant Journal 2024年第3期10卷 771-783页

作者： Zhiyong Ren Jinquan Li Xingyu Zhang Xingxu Li Junhong Zhang Zhibiao Ye Yuyang Zhang Qijun Nie Key Laboratory of Vegetable Ecological Cultivation on Highland Ministry of Agriculture and Rural AffairsHubei Key Laboratory of Vegetable Germplasm Enhancement and Genetic ImprovementInstitute of Economic CropsHubei Academy of Agricultural SciencesWuhanHubei 430063China National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops Huazhong Agricultural UniversityWuhanHubei 430070China Vegetable Research Institute Wuhan Academy of Agricultural SciencesWuhanHubei 430345China Hubei Hongshan Laboratory WuhanHubei 430070China Shenzhen Institute of Nutrition and Health Huazhong Agricultural UniversityWuhanHubei 430070China Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of AgricultureAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhenGuangdong 518000China

Clubroot caused by Plasmodiophora brassicae is a devastating disease of Cruciferous *** cultivars with clubroot resistance(CR)is the most effective control *** the two major brassica vegetable species *** and ***,several commercial cultivars with unclear CR pedigrees have been intensively used as CR donors in ***,the continuous occurrence of CR-breaking makes the CR pedigree underlying these cultivars one of the breeders'most urgent *** complex intraspecific diversity of these two major brassica vegetables has also limited the applicability of CR markers in different breeding *** we first traced the pedigree underlying two kinds of CR that have been widely applied in breeding by linkage and introgression analyses based on public resequencing *** ***,a major locus CRzi8 underlying the CR of the commercial CR donor‘DegaoCR117’was ***8 was further shown to have been introgressed from turnip(*** ***)and that it carried a potential functional allele of *** turnip introgression carried CRb^(c),sharing the same coding sequence with the CRb that was also identified from chromosome C07 of *** CR cultivars with different *** natural populations,variation analysis of linkage intervals of CRzi8,PbBa8.1,CRb,and CRb^(c)yielded easily resolved InDel markers(>20 bp)for these fundamental CR *** specificity of these markers was tested in diverse cultivars panels,and each exhibited high reliability in *** research demonstrates the value of the practice of applying resequencing big data to solve urgent concerns in breeding programs.

关键词： brassica Clubroot resistance Resequencing Introgression analysis Molecular marker

Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in brassica

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aBIOTECH 2024年第2期5卷 202-208页

作者： Wenjing Li Xuan Li Chunyang Wang Guanzhong Huo Xinru Zhang Jintai Yu Xiaoxiao Yu Jing Li Chao Zhang Jianjun Zhao Yan Li Jun Li State Key Laboratory of North China Crop Improvement and Regulation College of Life SciencesHebei Agricultural UniversityBaoding 071001China Hebei Key Laboratory of Plant Physiology and Molecular Pathology Hebei Agricultural UniversityBaoding 071001China Key Laboratory of Vegetable Germplasm Innovation and Utilization of Hebei Collaborative Innovation Center of Vegetable Industry in HebeiCollege of HorticultureHebei Agricultural UniversityBaoding 071001China College of Modern Science and Technology Hebei Agricultural UniversityBaoding 071001China

CRISPR/Cas9,presently the most widely used genome editing technology,has provided great potential for functional studies and plant ***,the strict requirement for a protospacer adjacent motif(PAM)has hindered the application of the CRISPR/Cas9 system because the number of targetable genomic sites is ***,the engineered variants Cas9-NG,SpG,and SpRY,which recognize non-canonical PAMs,have been successfully tested in plants(mainly in rice,a monocot).In this study,we evaluated the targeted mutagenesis capabilities of these Cas9 variants in two important brassica vegetables,Chinese cabbage(brassica rapa ***)and cabbage(brassica oleracea ***).Both Cas9-NG and SpG induced efficient mutagenesis at NGN PAMs,while SpG outperformed Cas9-NG at NGC and NGT *** achieved efficient editing at almost all PAMs(NRN>NYN),albeit with some self-targeting activity at transfer(T)-DNA *** SpRY-induced mutants were detected in cabbage plants in a PAM-less ***,an adenine base editor was developed using SpRY and TadA8e deaminase that induced A-to-G conversions within target sites using non-canonical ***,the toolboxes developed here induced successful genome editing in Chinese cabbage and *** work further expands the targeting scope of genome editing and paves the way for future basic research and genetic improvement in brassica.

关键词： brassica Cas9 variants CRISPR Genome editing Non-canonical PAM

Joint enhancement of lead accumulation in brassica plants by EDTA and ammonium sulfate in sand culture

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Journal of Environmental Sciences 2002年第2期14卷 216-220页

作者： XIONG Zhi\|ting, LU Ping (Department of Environmental Sciences, Wuhan University, Wuhan 430072, China. Department of Environmental Sciences Wuhan University Wuhan 430072 Chin

When EDTA was added alone in the Pb\|contaminated sand, the plant biomass and the total Pb amount in plant decreased in both species, brassica pekinensis and B. juncea var. multiceps, though the shoot Pb amount increased. In contrast, when (NH 4) 2SO 4 was added alone in the Pb\|contaminated sand, little effect was observed on the shoot Pb amount, though the root Pb amount was significantly increased in B. juncea var. multiceps. When amending EDTA and (NH 4) 2SO 4 in combination, however, the shoot Pb amount in both species substantially increased, being, on an average, 2 times and 9 times higher than that in EDTA alone or (NH 4) 2SO 4 alone amended treatment, respectively. The two amendments showed antagonism for plant growth, but synergism for Pb bioaccumulation. B. pekinensis showed its highest level of shoot and total Pb amount in the treatment amended with EDTA and (NH 4) 2SO 4 only a half as much as in the other treatments. It is inferred that the mechanisms responsible for the joint\|enhanced Pb accumulation might be concerned with the acidification of the growth medium, cation exchange reaction and relieving EDTA induced toxicity as results by amending ammonium sulfate.

关键词： lead bioaccumulation EDTA ammonium sulfate brassica

Genome-wide association study of heat stresstolerance traits in spring-type brassica napus *** controlled conditions

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The Crop Journal 2018年第2期6卷 115-125页

作者： Mizanur Rahaman Sujan Mamidi Mukhlesur Rahman Department of Plant Sciences North Dakota State UniversityFargoND 58102USA Hudson Alpha Institute for Biotechnology HuntsvilleAL 35806USA

High temperatures have a detrimental effect on growth, development, and yield of brassica napus. Even a short period of heat stress can lead to yield losses of 15%–20%. A collection of spring-type accessions available in Germplasm Resources Information Network(GRIN)were used to assess the effect of short periods of high-temperature stress at the early flowering stage of B. napus. Two sets of accessions with three replications per set were grown in a greenhouse at 22/18 °C day/night temperatures. Plants from the second set at the 6-day flowering stage were exposed to heat-stress conditions(maximum temperature up to 35 °C) in a plant growth chamber for five days. The heat-stressed plants were then allowed to recover in a greenhouse. Pollen sterility, sterile/aborted pods, and number of pods on main raceme were recorded for both control(set 1) and heat stressed(set 2) *** susceptibility indices for all three traits were calculated and an association-mapping study was conducted using 37,539 Single Nucleotide Polymorphisms(SNPs) to identify genomic regions controlling the heat stress traits. A total of 5, 8, and 7 quantitative trait loci(QTL) were associated with pollen sterility, sterile/aborted pods, and number of pods on main raceme, respectively. Together they explained respectively 46.3%, 60.5%, and 60.6% of phenotypic variation. Candidate genes in the QTL regions included genes associated with flowering, male sterility, pollen abortion, embryo abortion reducing pollen development,and pod development.

关键词： brassica napus Heat stress Controlled environment QTL

Physiological and molecular responses to cold stress in rapeseed(brassica napus L.)

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Journal of Integrative Agriculture 2019年第12期18卷 2742-2752页

作者： YAN Lei Tariq Shah CHENG Yong Lü Yan ZHANG Xue-kun ZOU Xi-ling Oil Crops Research Institute Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil CropsMinistry of Agriculture and Rural AffairsWuhan 430062P.R.China College of Plant Science and Technology Huazhong Agricultural UniversityWuhan 430070P.R.China

Low temperature is one of the most important abiotic factors inhibiting growth, productivity, and distribution of rapeseed(brassica napus L.). Therefore, it is important to identify and cultivate cold-tolerant germplasm. The objective of this study was to figure out the mechanism of chilling(4 and 2°C) and freezing(–2 and –4°C) stresses along with a control(22°C) in B. napus cultivars(1801 and C20) under controlled environment(growth chamber). The experiment was arranged in a complete randomized design with three replications. Our results exhibited that under chilling and freezing stresses, the increment of proline accumulation, soluble sugar and protein contents, and antioxidant enzyme activity were enhanced more in 1801 cultivar compared with C20 cultivar. At –2 and –4°C, the seedlings of C20 cultivar died completely compared with 1801 cultivar. Hydrogen peroxide(H2 O2) and malondialdehyde contents(MDA) increased in both cultivars, but when the temperature was decreased up to –2 and –4°C, the MDA and H2 O2 contents continuously dropped in 1801 cultivar. Moreover, we found that leaf abscisic acid(ABA) was enhanced in 1801 cultivar under chilling and freezing stresses. Additionally, the transcriptional regulations of cold-tolerant genes(COLD1, CBF4, COR6.6, COR15, and COR25) were also determined using real-time quantitative PCR(RT-q PCR). RT-q PCR showed that higher expression of these genes were found in 1801 as compared to C20 under cold stress(chilling and freezing stresses). Therefore, it is concluded from this experiment that 1801 cultivar has a higher ability to respond to cold stress(chilling and freezing stresses) by maintaining hormonal, antioxidative, and osmotic activity along with gene transcription process than C20. The result of this study will provide a solid foundation for understanding physiological and molecular mechanisms of cold stress signaling in rapeseed(B. napus).

关键词： brassica napus L. cold stress morphological features molecular regulation physiological indicators

Genome-wide identification and characterization of the lateral organ boundaries domain gene family in brassica rapa ***

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Plant Diversity 2020年第1期42卷 52-60页

作者： Qin Yu Simin Hu Jiancan Du Yongping Yang Xudong Sun Key Laboratory for Plant Diversity and Biogeography of East Asia Kunming Institute of Botany.Chinese Academy of ScienceKunming 650201China Plant Germplasm and Genomics Center The Cermplasm Bank of Wild SpeciesKunming Institute of BotanyChinese Academy of SciencesKunming 650201 China Institute of Tibetan Plateau Research at Kunming.Kunming Institute of Botany Chinese Academy of Sciences.Kunming 650201China University of the Chinese Academy of Sciences Beijing 100049China

The Lateral Organ Boundaries Domain(LBD)genes encode highly conserved plant-specific LOB domain proteins which regulate growth and development in various ***,members of the LBD gene family have yet to be identified in brassica rapa *** the present study,fifty-nine LBD genes were identified and distributed on 10 *** BrrLBD proteins are predicted to encode hydrophobic polypeptides between 118 and 394 amino acids in length and with molecular weights ranging from 13.31 to 44.24 kDa;the theoretical pi for these proteins varies from 4.83 to *** were 17 paralogous gene pairs in the BrrLBD family,suggesting that the amplification of the BrrLBD gene family involved largescale gene duplication *** of the BrrLBD family were divided into 7 subclades(class I a to e,class II a and b).Analysis of gene structure and conserved domains revealed that most BrrLBD genes of the same subclade had similar gene structures and protein *** expression profiles of 59 BrrLBD genes were determined through Quantitative Real-time fluorescent PCR(qRT-PCR).Most BrrLBD genes in the same subclade had similar gene expression ***,the expression patterns of 7 genes differed from their duplicates,indicating that although the gene function of most BrrLBD genes has been conserved,some BrrLBD genes may have undergone evolutionary change.

关键词： LBD Transcription factors LBD gene sequence analysis Expression profiles brassica rapa var.rapa

New Insights into Nested Long Terminal Repeat Retrotransposons in brassica Species

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Molecular Plant 2013年第2期6卷 470-482页

作者： Lijuan Wei Meili Xiaoa Zeshan An Bi Ma Annaliese S. Mason Wei Qian Jiana Li Donghui Fu Chongqing Engineering Research Center for Rapeseed College of Agronomy and Biotechnology Southwest University Chongqing 400716 China School of Agriculture and Food Sciences and centre for Integrative I~egume-Research The University of Queensland 1:he Universit'y of QueenslandBrisbane 4072 Australia Key Laboratory of Crop Physiology Ecology and Genetic Breeding Ministry of Education Agronomy College Jiangxi Agricultural University Nanchang330045 China

Long terminal repeat （LTR） retrotransposons, one of the foremost types of transposons, continually change or modify gene function and reorganize the genome through bursts of dramatic proliferation. Many LTR-TEs preferen-tially insert within other LTR-TEs, but the cause and evolutionary significance of these nested LTR-TEs are not well under-stood. In this study, a total of 1.52 Gb of brassica sequence containing 2020 bacterial artificial chromosomes （BACs） was scanned, and six bacterial artificial chromosome （BAC） clones with extremely nested LTR-TEs （LTR-TEs density： 7.24/kb） were selected for further analysis. The majority of the LTR-TEs in four of the six BACs were found to be derived from the rapid proliferation of retrotransposons originating within the BAC regions, with only a few LTR-TEs originating from the proliferation and insertion of retrotransposons from outside the BAC regions approximately 5-23 Mya. LTR-TEs also pref-erably inserted into TA-rich repeat regions. Gene prediction by Genescan identified 207 genes in the 0.84Mb of total BAC sequences. Only a few genes （3/207） could be matched to the brassica expressed sequence tag （EST） database, indicating that most genes were inactive after retrotransposon insertion. Five of the six BACs were putatively centromeric. Hence, nested LTR-TEs in centromere regions are rapidly duplicated, repeatedly inserted, and act to suppress activity of genes and to reshuffle the structure of the centromeric sequences. Our results suggest that LTR-TEs burst and proliferate on a local scale to create nested LTR-TE regions, and that these nested LTR-TEs play a role in the formation of centromeres.

关键词： LTR retrotransposons brassica centromere retrotransposon-rich transposon burst.

Increased BnaMFT-transcript level is associated with secondary dormancy in oilseed rape (brassica napus L.)

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Journal of Integrative Agriculture 2020年第6期19卷 1565-1576页

作者： LIU Lei FAN Wen-qi LIU Fu-xia YI Xin TANG Tang ZHOU Ying TANG Zi-wei CHEN Guimin ZHAO Xiang-xiang Jiangsu Key Laboratory for Eco-agriculture Biotechnology Around Hongze Lake/Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environment Protection/College of Life Science Huaiyin Normal UniversityHuai'an 223300P.R.China School of Food Science and Engineering Yangzhou UniversityYangzhou 225127P.R.China

brassica napus cultivars have little or no primary dormancy;however,they are prone to sec on dary dorma ncy in ducti *** dormant seeds can produce volunteer plants,which can result in genetic contamination,reduced quality and biosafety ***,informati on regarding the molecular mecha nism un derlyi ng sec on dary dormancy is *** MOTHER OF FT AND TFL1(MFT)gene,which is evolutionarily conserved in the plant kingdom,acts in a complex gene network in the seed dormancy or germination *** this study,we identified four *** genes that share high homology with AtMFT,named as *** of c/s-acting elements showed that BnaMFT promoters contain multiple seed-specific regulatory elements,and various stress-and hormone-responsive *** experiments validated that BnaMFTs were specifically expressed during seed maturation and in the dry seed,with peaks at 35-42 days after *** were not sufficient for primary dormancy;however,they were significantly enhanced by secondary dormancy induction with PEG6000 ***,BnaMFTtranscripts were elevated by treatment with abscisic acid(ABA),which is known to be accumulated duri ng sec on dary dorma *** results collectively suggest that increased BnaMFT tra nscription levels are associated with sec on dary dorma ncy inducti on in an ABA-depe ndent mariner in ***.

关键词： brassica napus DELAY OF GERMINATION 1(DOG7) MFT primary dormancy secondary dormancy

Mapping loci controlling fatty acid profiles, oil and protein content by genome-wide association study in brassica napus

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The Crop Journal 2019年第2期7卷 217-226页

作者： Minqiang Tang Yuanyuan Zhang Yueying Liu Chaobo Tong Xiaohui Cheng Wei Zhu Zaiyun Li Junyan Huang Shengyi Liu The Key Laboratory of Biology and Genetic Improvement of Oil Crops The Ministry of Agriculture Oil Crops Research Institute of Chinese Academy of Agricultural Sciences National Key Laboratory of Crop Genetic Improvement National Center of Crop Molecular Breeding Technology National Center of Oil Crop Improvement (Wuhan) Huazhong Agricultural University Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources Faculty of Life Science Hubei University

Optimizing the profile and quantity of fatty acids in rapeseed(brassica napus L.) is critical for maximizing the value of edible oil and biodiesel. However, selection of these complex seed quality traits is difficult before haplotypes controlling their contents are identified. To efficiently identify genetic loci influencing these traits and underlying candidate genes and networks, we performed a genome-wide association study(GWAS) of eight seed quality traits(oil and protein content, palmitic, stearic, oleic, linoleic, eicosenoic and erucic acids content). The GWAS population comprised 370 diverse accessions, which were phenotyped in five environments and genotyped using 60K SNP arrays. The results indicated that oil and protein contents generally showed negative correlations, while fatty acid contents showed positive or negative correlations,with palmitic and erucic acid contents directly affecting oil content. Seven SNPs on five chromosomes were associated with both seed oil and protein content, and five genes orthologous to genes in Arabidopsis thaliana were predicted as candidates. From resequencing data, besides known haplotypes in Bna ***1.a and Bna ***1.a, three accessions harboring a new haplotype conferring moderate erucic acid content were identified. Interestingly, in a haplotype block, one haplotype was associated with high palmitic acid content and low oil content, while the others showed the reverse effects. This finding was consistent with a negative correlation between palmitic acid and oil contents, suggesting historical selection for high oil content. The identification by this study of genetic variation and complex correlations of eight seed quality traits may be beneficial for crop selection strategies.

关键词： brassica napus Oil content Fatty acid profiles Association study Haplotypes