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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 crops.Developing cultivars with clubroot resistance(CR)is the most effective control measure.For the two major brassica vegetable species B.rapa and B.oleracea,several commercial cultivars with unclear CR pedigrees have been intensively used as CR donors in breeding.However,the continuous occurrence of CR-breaking makes the CR pedigree underlying these cultivars one of the breeders'most urgent concerns.The complex intraspecific diversity of these two major brassica vegetables has also limited the applicability of CR markers in different breeding programs.Here 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 data.In B.rapa,a major locus CRzi8 underlying the CR of the commercial CR donor‘DegaoCR117’was identified.CRzi8 was further shown to have been introgressed from turnip(B.rapa ssp.rapifera)and that it carried a potential functional allele of Crr1a.The turnip introgression carried CRb^(c),sharing the same coding sequence with the CRb that was also identified from chromosome C07 of B.oleracea CR cultivars with different morphotypes.Within 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 genes.The specificity of these markers was tested in diverse cultivars panels,and each exhibited high reliability in breeding.Our 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

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Turnip mosaic virus pathogenesis and host resistance mechanisms in brassica

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Horticultural Plant Journal 2024年第4期10卷 947-960页

作者： Guanwei Wu Xinxin Fang Tianqi Yu Jianping Chen Fei Yan State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products Institute of Plant Virology Ningbo University Key Laboratory of Biotechnology in Plant Protection of MARA and Zhejiang Province Institute of Plant Virology Ningbo University

Turnip mosaic virus(TuMV) is a devastating potyvirus pathogen that infects a wide variety of both cultivated and wild brassicaceae plants.We urgently need more information and understanding of TuMV pathogenesis and the host responses involved in disease development in cruciferous crops. TuMV displays great versatility in viral pathogenesis, especially in its replication and intercellular movement. Moreover, in the coevolutionary arms races between TuMV and its hosts, the virus has evolved to co-opt host factors to facilitate its infection and counter host defense responses. This review mainly focuses on recent advances in understanding the viral factors that contribute to the TuMV infection cycle and the host resistance mechanism in brassica. Finally, we propose some future research directions on TuMV pathogenesis and control strategies to design durable TuMV-resistant brassica crops.

关键词： Turnip mosaic virus brassica Resistance genes Host factors Infection biology

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Studies on the temporal, structural, and interacting features of the clubroot resistance gene Rcr1 using CRISPR/Cas9-based systems

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Horticultural Plant Journal 2024年第4期10卷 1035-1048页

作者： Hao Hu Fengqun Yu Agriculture and Agri-Food Canada Saskatoon Research and Development Centre107 Science Place

Clubroot disease is a severe threat to brassica crops globally, particularly in western Canada. Genetic resistance, achieved through pyramiding clubroot resistance(CR) genes with different modes of action, is the most important strategy for managing the disease. However,studies on the CR gene functions are quite limited. In this study, we have conducted investigations into the temporal, structural, and interacting features of a newly cloned CR gene, Rcr1, using CRISPR/Cas9 technology. For temporal functionality, we developed a novel CRISPR/Cas9-based binary vector, pHHIGR-Hsp18.2, to deliver Rcr1 into a susceptible canola line(DH12075) and observed that early expression of Rcr1 is critical for conferring resistance. For structural functionality, several independent mutations in specific domains of Rcr1 resulted in loss-offunction, highlighting their importance for CR phenotype. In the study of the interacting features of Rcr1, a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein, resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1. These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response. This study, the first of its kind, provides valuable insights into the functionality of Rcr1. Further, the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits, which should be useful for functional genomics and other similar research in brassica crops.

关键词： Clubroot resistance brassica crops Canola Rcr1 CRISPR/Cas9 system Gene knock-out Timing control Non-synonymous mutation Protein–protein interaction

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Conserved noncoding sequences correlate with distant gene contacts in Arabidopsis and brassica

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Journal of Integrative Plant Biology 2023年第6期65卷 1467-1478页

作者： Lei Zhang Jian Wu Jianli Liang Runmao Lin Chao Sun Qirui Dai Lupeng Zhang Huiling Guo Ranze Zhao Xiaowu Wang State Key Laboratory of Vegetable Biobreeding Institute of Vegetables and FlowersChinese Academy of Agricultural SciencesBeijing 100081China Research Institute of Forestry Chinese Academy of ForestryBeijing 100091China

Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions.To investigate the potential importance of distant contacts,we performed high-throughput chromatin conformation capture(Hi-C)experiments on leaf nuclei isolated from brassica rapa and brassica oleracea.We then combined our results with published Hi-C data from Arabidopsis thaliana.We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene.Genes with higher numbers of conserved noncoding sequences(CNSs)nearby were more likely to have contact with distant genes.With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity.In addition,for the genes we studied,distant contacting genes with CNSs were more likely to be transcriptionally coordinated.These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors,correlating with distant contacts to ensure coordinated expression.This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.

关键词： Arabidopsis brassica conserved noncoding sequences distant gene contacts Hi-C

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The application of CRISPR/Cas technologies to brassica crops:current progress and future perspectives

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aBIOTECH 2022年第2期3卷 146-161页

作者： Jun Li Xiaoxiao Yu Chao Zhang Na Li Jianjun Zhao State Key Laboratory of North China Crop Improvement and Regulation Cllge of Life SciencesHebei Agricultural UniversityBaoding 071001China Key Laboratory of Vegetable Germplasm Innovation and Utilization of Hebei ollaborative Innovation Center of Vegetable Industry in Hebei College of HorticultureHebei Agricultural UniversityBaoding 071001China

brassica species are a global source of nutrients and edible vegetable oil for humans.However,all commercially important brassica crops underwent a whole-genome triplication event,hindering the development of functional genomics and breeding programs.Fortunately,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas)technologies,by allowing multiplex and precise genome engineering,have become valuable genome-editing tools and opened up new avenues for biotechnology.Here,we review current progress in the use of CRISPR/Cas technologies with an emphasis on the latest breakthroughs in precise genome editing.We also summarize the application of CRISPR/Cas technologies to brassica crops for trait improvements.Finally,we discuss the challenges and future directions of these technologies for comprehensive application in brassica crops.Ongoing advancement in CRISPR/Cas technologies,in combination with other achievements,will play a significant role in the genetic improvement and molecular breeding of brassica crops.

关键词： CRISPR Cas9 Cas12 Precise genome editing brassica

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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

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

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Genome-wide identification and characterization of the lateral organ boundaries domain gene family in brassica rapa var.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 species.However,members of the LBD gene family have yet to be identified in brassica rapa var.rapa.In the present study,fifty-nine LBD genes were identified and distributed on 10 chromosomes.The 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 9.68.There were 17 paralogous gene pairs in the BrrLBD family,suggesting that the amplification of the BrrLBD gene family involved largescale gene duplication events.Members 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 motifs.The 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 profiles.However,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

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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.

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Determination of Essential Fatty Acid Composition among Mutant Lines of Canola (brassica napus),through High Pressure Liquid Chromatography

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Journal of Integrative Plant Biology 2009年第12期51卷 1080-1085页

作者： Ghulam Raza Aquil Siddique Imtiaz Ahmad Khan Muhammed Yasin Ashraf Abdullah Khatri Plant Genetic Division Nuclear Institute of Agriculture (NIA) Tando Jam 70060 Pakistan Nuclear Institute for Agriculture and Biology Faisalabad 38000 Pakistan

The present study aimed to quantify the methyl esters of lenoleic acid （LA）, γ-lenolenic acid （LNA） and oleic acid （OL） in the oil of brassica napus mutants. Five stable mutants （ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17） of B. napus cv. ＇Rainbow＇（P） and three mutants （W97-95/16, W97-0.75/11 and W97-.075/13） of B. napus cv. ＇Westar＇（P） at M6 stage, exhibiting better yield and yield components, were analyzed for essential fatty acids. The highest seed yield was observed in the mutant （ROO-100/6） followed by ROO-125/14 of Rainbow, that is, 34% and 32% higher than their parent plants, respectively. Westar mutant W97-75/11 also showed 30% higher seed yield than its parent plant. High performance liquid chromatography analysis of the composition of fatty acids indicated that OL was the most dominant fatty acid, ranging from 39.t to 66.3%; LA was second （15.3-41.6%） and LNA was third （18.1-28.9%）. Mutant ROO-125/14 showed higher OL contents than parent （Rainbow）. These results are expected to support the approval of ROO-125/14 in the National Uniform Varietal Yield Trials （NUVYT） as a new variety based on high oil quality.

关键词： brassica high performance liquid chromatography lenoleic acid lenolenic acid oleic acid.

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A 2-kb Mycovirus Converts a Pathogenic Fungus into a Beneficial Endophyte for brassica Protection and Yield Enhancement

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Molecular Plant 2020年第10期13卷 1420-1433页

作者： Hongxiang Zhang Jiatao Xie Yanping Fu Jiasen Cheng Zheng Qu Zhenzhen Zhao Shufen Cheng Tao Chen Bo Li Qianqian Wang Xinqiang Liu Binnian Tian David B.Collinge Daohong Jiang State Key Laboratory of Agricultural Microbiology College of Plant Science and TechnologyHuazhong Agricultural UniversityWuhanHubei Province 430070China The Provincial Key Lab of Plant Pathology of Hubei Province Huazhong Agricultural UniversityWuhanChina Department of Plant and Environmental Sciences University of Copenhagen1871 Frederiksberg CDenmark

Mycoviruses are viruses that infect fungi,and hypovirulence-associated mycoviruses have the potential to control fungal diseases.However,it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field,and no mycovirus has been applied for field crop protection.In this study,we found that a previously identified small DNA mycovirus(SsHADV-1)can convert its host,Sclerotinia sclerotiorum,from a typical necrotrophic pathogen to a beneficial endophytic fungus.SsHADV-1 downregulates the expression of key pathogenicity factor genes in S.sclerotiorum during infection.When growing in rapeseed,the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense,hormone signaling,and circadian rhythm pathways.As a result,plant growth is promoted and disease resistance is enhanced.Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6%and improve yield by 14.9%.Moreover,we discovered that SsHADV-1 could also infect other S.sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants.These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism.Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

关键词： mycovirus Sclerotinia sclerotiorum endophyte rapeseed stem rot biological control brassica napus

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