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

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.

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

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.

Comprehensive analysis of the full-length transcripts and alternative splicing involved in clubroot resistance in Chinese cabbage

Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology,although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames(ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs(lncRNAs),56 transcription factor(TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing(AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes(one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.

Pyramiding of multiple genes generates rapeseed introgression lines with clubroot and herbicide resistance, high oleic acid content, and early maturity

Clubroot and herbicide resistance,high oleic acid(OA)content,and early maturity are targets of rapeseed(Brassica napus L.)breeding.The objective of this study was to develop new male-fertility restorer lines by pyramiding favorable genes to improve these traits simultaneously.Seven elite alleles for the four traits were introduced into the restorer line 621R by speed breeding with marker-assisted and phenotypic selection.Six introgression lines(ILs)were developed with four-to seven-gene combinations and crossed with two elite parents to develop hybrids.All ILs and their corresponding hybrids displayed high resistance to both clubroot pathotype 4 and sulfonylurea herbicides.Three ILs and their hybrids showed large increases in OA contents and four showed earlier maturity.These new ILs may be useful in rapeseed hybrid breeding for the target traits.

Genetic Inheritance and Molecular Marker of Clubroot Resistance Genes in Brassica campestris ssp. chinensis

Objective] This study was conducted to investigate the genetic inheritance of clubroot resistance in Chinese non-heading cabbage （Brassica campestris ssp. chinensis）. [Method] The clubroot resistance gene was introduced from a Brassica campestris ssp. pekinensis cultivar to non-heading Chinese cabbage, and the inheri-tance and molecular markers of clubroot resistance gene in parental lines, F1, F2 and BC1 of non-heading Chinese cabbage were studied through pathogen inoculation at seedling stage and ISSR-PCR. [Result] Clubroot resistance in non-heading Chi-nese cabbage was control ed by a single dominant gene. ISSR molecular markers with Bulk segregant analysis （BSA） found that primer-873 was linked to resistance gene, named CR-873, and the genetic distance between the marker and the resis-tance gene was 9.72 cM. [Conclusion] The results provide references for the molecular marker assisted breeding of non-heading Chinese cabbage.

Influence of 6-BA on Incidence Rate of Chinese Cabbage Clubroot (Plasmodiophora brassicae Woron)

[Objective] The paper was to study the relationship between cytokinin and Chinese cabbage clubroot, and to explore the incidence mechanism of clubroot. [Method]The spores were extracted from the tumors of diseased plant, and their DNA was extracted for PCR test. The diagnosed spores were inoculated to the culture soils in erlenmeyer flask at seeding stage, at seed germination and 21 d after sowing, and 0.08 μmol/L 6-BA was added to the soil at germination. Spores were extracted from the root tumors developed in inoculated plants and examined by scanning electronic microscope （ SEM）, and the incidence rate of clubroot in 6-BA treatment and control was recorded. [ Result] Plasmodiophora brassicae Woron could cause the clubroot incidence of Chinese cabbage planted in erlenmeyer flasks, the incidence rate of chibroot in the treatment adding with 0.08 μmol/L 6-BA was 100%, while that in the treatment without 6-BA was 57%, and the volume of former tumor was much larger than the latter. SEM showed that the size of resting spore of P. brassicae was 1.5 -4.3μm. [ Conduslon] 6-BA applied at germination could significantly promote the formation of tumor of Chinese cabbage clubroot.

Effects of different rotation patterns on the occurrence of clubroot disease and diversity of rhizosphere microbes

Clubroot disease, caused by Plasmodiophora brassicae, is one of the most destructive soil-borne diseases in cruciferous crops worldwide. New strategies are urgently needed to control this disease, as no effective disease-resistant varieties or chemical control agents exist. Previously, we found that the incidence rate and disease index of clubroot in oilseed rape decreased by 50 and 40%, respectively, when oilseed rape was planted after soybean. In order to understand how different rotation patterns affect the occurrence of clubroot in oilseed rape, high-throughput sequencing was used to analyze the rhizosphere microbial community of oilseed rape planted after leguminous (soybean, clover), gramineous (rice, maize) and cruciferous (oilseed rape, Chinese cabbage) crops. Results showed that planting soybeans before oilseed rape significantly increased the population density of microbes that could inhibit P. brassicae (e.g., Sphingomonas, Bacillus, Streptomyces and Trichoderma). Conversely, consecutive cultivation of cruciferous crops significantly accumulated plant pathogens, including P. brassicae, Olpidium and Colletotrichum (P<0.05). These results will help to develop the most effective rotation pattern for reducing clubroot damage.

Breeding of a New Clubroot-resistant Chinese Cabbage Variety, CCR11242

In this study, using Japanese radish cytoplasmic sterile line ＂Half Green Cabbage＂ （HGC） and clubfoot-resistant South Korean cabbage variety ＂ CR Cabbage King＂ （CCK） as experimental materials, serf-incompatible DH lines were obtained by mierosporo culture. Through five gnerations of backeross, CCR11239, a clubroot-resistant radish cytoplasmic sterile line, was obtained. Through six generations of serf-crossing, CCR11240, the maintainer line of CCR11239, was bred. After the cross of CCK and Luchunbai 1 （83 - 1 ） and five generations of self-crossing, serf-compatible line CCRl1241 was obtained. Through crossing CCR11240 with CCRl1241, the new cabbage variety CCRl1242 was obtained. After variety comparison test, provincial regional test and production demonstration test, the new variety was registered in 2012. The average yield of CCR11242 reached 60 975 kg/hm2, which was improved by 142% compared with Luchunbai 1 （83 - 1 ）. The disease index of CCR11242 was 5.63, which was 88.13 lower than the control, indicating high resistance （HR） to clubfoot.

Identification of Genes Associated with Clubroot Resistance in Chinese Cabbage

The transcriptome-wide gene expression was compared between susceptible and resistant Chinese cabbage cultivars to identify genes that contributed to clubroot resistance. A higher number of differentially expressed genes were detected in susceptible cultivars than in resistant cultivars. Fifty-six genes involved in cell wall modification, hormone signaling, root marphogenesis, nematodes response and cell proliferation were uniquely expressed in the resistant cultivars. Among them, 27 genes were involved in cell wall modification and hormone signaling, indicating that genes in these two types might play a vital role in the defense response to pathogen infection.

Inheritance of Clubroot Resistance of Miniature Chinese Baby Cabbage and Molecular Markers-assisted Selection

[Objective] The paper was to analyze the inheritance of clubroot resistance of miniature Chinese baby cabbage,to shorten identification time and to improve breeding efficacy of new disease-resistant varieties. [Method] Taking clubroot-resistant Chinese cabbage CCR001,disease-susceptible baby cabbage CM002,and their F_1,F_2 and BC_1 offspring as the research objects,the inheritance of clubroot resistance of baby cabbage was studied. [Result]The clubroot-resistance of baby cabbage was controlled by a single dominant gene,which conformed to Mendel＇s Laws of inheritance. The molecular markers-assisted selection combing with bacterial soil inoculation confirmed that the disease-resistance indeed passed on from the parents,and was inherited in F_1 and F_2. [Conclusion]It is feasible to breed clubroot-resistant baby cabbage by using molecular markers-assisted selection.

Clubroot resistance introgression in interspecific hybrids between Raphanus sativus and Brassica napus

Clubroot is a prevailing soil-borne disease affecting rapeseed production worldwide.However,few clubroot resistant rapeseed accessions were available for breeding.Identification and introgression of new clubroot resistant genes from closely related species by distant hybridization is an effective strategy.In the present study,9 radish(Raphanus sativus L.,2n=18,RR)lines resistant to Plasmodiophora brassicae pathotype 4 were used as donors to transfer clubroot resistance into a susceptible rapeseed(Brassica napus L.,2n=38,AACC)line by distant hybridization combined with embryo rescue.Nine intergeneric crosses were made but only 1(411×93039)produced F1 plants both from embryo rescue and natural seed-setting.Authenticity of triploid F1 hybrids(2n=28,ACR)were verified by flower color,cytological observation and molecular marker analysis,and 2 genuine F1 hybrids were identified.After chromosome doubling,these synthetic allohexaploid plants(2n=56,AACCRR)became partially fertile(pollen viability rate=35%)and were backcrossed with rapeseed parent to generate a BC1 population(2n=47,AACCR).Totally 178 BC1 plants were obtained,of which the majority(96.1%)were resistant to clubroot.These backcrossing progenies could be used for the breeding of new rapeseed varieties resistant to clubroot.

Two adjacent NLR genes conferring quantitative resistance to clubroot disease in Arabidopsis are regulated by a stably inherited epiallelic variation

Clubroot caused by the protist Plasmodiophora brassicae is a major disease affecting cultivated Brassica-ceae.Using a combination of quantitative trait locus(QTL)fine mapping,CRISPR-Cas9 validation,and extensive analyses of DNA sequence and methylation patterns,we revealed that the two adjacent neigh-boring NLR(nucleotide-binding and leucine-rich repeat)genes AT5G47260 and AT5G47280 cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P.brassicae in Arabidopsis and that they are epigenetically regulated.The variation in DNA methylation is not associated with any nucleotide variation or any transposable element presence/absence variants and is stably inherited.Vari-ations in DNA methylation at the Pb-At5.2 QTL are widespread across Arabidopsis accessions and corre-late negatively with variations in expression of the two genes.Our study demonstrates that natural,stable,and transgenerationally inherited epigenetic variations can play an important role in shaping resistance to plant pathogens by modulating the expression of immune receptors.

油菜抗根肿病品种在云南省西南发病区的田间评价

通过明确‘华双5R’和‘华油杂62R’2个抗根肿病油菜品种的田间抗性、农艺性状和产量情况,为油菜根肿病重发区筛选出优良的抗病品种。本研究在云南省西南地区的腾冲市、梁河县和临翔县3个油菜根肿病病区进行田间试验,采用本课题组研发品种和当地主推品种作为对照。田间试验结果显示,‘华双5R’和‘华油杂62R’在3个试验点均能正常成熟安全越冬,生育期176~199d,各生育期表现整齐一致;‘华双5R’和‘华油杂62R’在腾冲和梁河点对油菜根肿病表现为高度抗病,在临翔点表现为抗病;‘华双5R’和‘华油杂62R’在3个试验点的农艺性状良好;‘华双5R’的产量在3个试验点与7个对照品种相比,比梁河点的‘云油杂12号’减产2.71%,与腾冲点的‘云油杂12号’和‘杂1256’、梁河点的‘云油杂15’和‘花油8号’、临翔点的‘云油杂12号’和‘德油1号’相比增产幅度为7.49%~56.83%;‘华油杂62R’的产量在3个试验点与7个对照品种相比,比梁河点的‘云油杂12号’减产5.76%,比临翔点的‘德油1号’减产4.61%,与腾冲点的‘云油杂12号’和‘杂1256’、梁河点的‘云油杂15’和‘花油8号’、临翔点的‘云油杂12号’相比增产幅度为4.12%~51.90%。‘华双5R’和‘华油杂62R’在3个试验点均能正常成熟,生育期176~199d,对根肿病表现出较强抗性,同时农艺性状良好、产量较高,可在云南省西南地区油菜根肿病病区推广种植。

Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection.Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease.The traditional way of R gene functional validation requires stable transformation that is both time-and labor-consuming.In this study,a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed.The transformation positive rate was over 80%in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation.The system was applicable to different B.napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea.In particular,two known CR genes,CRA3.7.1 and CRA8.2.4 were used respectively,as example to show that the system works well for CR gene study combined with subsequent P.brassicae infection in B.napus.Most importantly,it works both in over-expression that led to disease resistance,as well as in RNAi which led to disease susceptible phenotype.Therefore,this system can be used in batch-wise identification of CR genes,and also offered the possibility of manipulating key genes within the P.brassicae genome that could improve our knowledge on host-pathogen interaction.

土壤根肿菌定量检测方法的建立及常德市油菜根肿病调查

根肿病严重危害油菜及其他十字花科蔬菜,通过检测土壤根肿菌孢子浓度,能够评估田块中根肿病发生的风险程度,为防治根肿病提供参考。本研究利用荧光定量PCR(qPCR)技术,建立了油菜土壤根肿病菌qPCR定量检测技术。以来自湖南省不同地区的发病油菜植株和带病土壤对该技术进行验证,结果表明该技术拥有较高的灵敏性和特异性。该技术判定田间根肿病发病的根肿菌孢子阈值浓度为1.34×10^(3)个/克土壤,当土壤中孢子浓度高于阈值浓度时,油菜发生根肿病风险较大。对常德市各区县油菜种植地区进行了实地调查,并随机采集481块田的土壤样本,利用本研究建立的定量检测技术测定了土壤中根肿病菌孢子浓度,发现根肿病在常德地区已呈多点发展,并有加速扩散的趋势,如不及时采取有效防治措施,油菜根肿病在常德地区将大面积爆发,严重危害该地区的油菜发展。对于常德市油菜种植区,根肿病菌孢子浓度小于1.34×10^(3)个/克土壤的田块,根肿病发病的概率较低,可种植普通非抗性油菜品种,但需加强根肿病病情监控。孢子浓度高于1.34×10^(3)个/克土壤的田块需及时进行化学防治,或者种植根肿病抗性油菜品种。

木醋液对大白菜根肿病的防治效果

以寒玉快菜为试材,采用盆栽方式,研究不同浓度木醋液(稀释600、400、200倍)对大白菜根肿病的防治效果;同时采用Hochest33342-PI双荧光复染法,测定不同浓度木醋液处理下芸薹根肿菌孢子活性,并分析木醋液主要抑菌成分。结果表明:3种浓度木醋液处理均可显著提高大白菜的株高和地上部鲜质量;并显著降低根肿病的发病率和病情指数,且随着木醋液浓度的增加根肿病的发病率和病情指数呈显著下降趋势,其中稀释200倍的木醋液处理的根肿病发病率仅为14.42%,防治效果达到68.72%。木醋液可使芸薹根肿菌孢子失活,稀释200倍的木醋液处理的孢子存活率仅为24.03%。采用GC-MS技术初步测定出供试木醋液含有24种成分,其中有17种成分对芸薹根肿菌孢子具有一定抑制作用,以俞创木酚抑制效果最好,EC50值为113.01μg·mL^(-1)。综上,本试验条件下,稀释200倍的木醋液用于大白菜根肿病防治效果最佳。

大白菜抗根肿病DH系的创制与鉴定

为了快速获得纯合稳定的抗根肿病大白菜DH系,为大白菜抗根肿病育种提供基础材料,以5个大白菜抗根肿病基因型为试材进行游离小孢子培养,并结合分子标记、形态观察和根肿菌人工接种对获得的小孢子再生植株进行了表型和抗性鉴定。结果表明,5个基因型均诱导出胚,出胚率变异在0.02~1.72个胚/蕾,3个基因型20aCR12、21aCR6、21aCR12获得再生植株,再生植株率分别为27.09%,1.45%,26.07%。抗性标记鉴定表明,与CRa、CRb^(kato)连锁的分子标记在获得的50个小孢子再生植株均扩增出了抗性条带。表型调查表明,50个小孢子株系生殖期基生叶片形状、颜色、抽薹早晚、育性等差异很大,自交获得的29个DH系营养期株型、叶片性状及结球性状表现出多样性。抗性接种表明,11份DH系对根肿菌4号和1号生理小种侵染病情指数均小于33.33,表现为抗病或耐病,其中2种菌侵染病情指数均小于5.0的高抗DH系有3份,分别为20aCR12-23、20aCR12-29和21aCR12-39。研究表明,单倍体培养结合分子标记辅助鉴定可快速获得纯合的抗根肿病DH系。

十字花科根肿病拮抗菌贝莱斯芽胞杆菌ZF72分离鉴定及生防效果研究

针对十字花科根肿病日益严重且防治困难的问题,采用平板对峙法,以尖孢镰孢菌Fusarium oxysporum和辣椒疫霉菌Phytophthora capsici为指示菌进行初筛,通过盆栽试验复筛,得到十字花科根肿病高效生防菌株ZF72,盆栽防效74.6%。根据形态学特征、生理生化特性及16S rDNA,gyr B和atp D多基因系统发育分析,鉴定菌株ZF72为贝莱斯芽胞杆菌Bacillus velezensis。采用单因素试验对菌株ZF72摇瓶发酵工艺进行优化,确定最佳配方为山梨醇3 g/L、麸皮8 g/L、CaCl_(2)1 g/L和MgCl_(2)·6H_(2)O 3 g/L,最适发酵条件为温度28℃、pH 6.0、装液量50 mL/250 mL、转速240 r/min、种子液接种量1%(v/v)。田间试验结果显示,菌株ZF72处理根肿病症状明显减轻,田间防效可达63.8%,为根肿病生物防治提供了新的微生物资源。

大白菜根肿病抗感品种间的侵染过程及生理生化差异分析

根肿病是由芸薹根肿菌专性侵染十字花科蔬菜作物引起的一种世界性土传病害,对大白菜等蔬菜造成了严重的经济损失。以抗根肿病大白菜DH系(DH40R)和感病大白菜DH系(DH199S)为试验材料,通过观察根肿菌侵染的差异、防御酶活性、MDA和可溶性蛋白含量的测定研究大白菜根肿病抗性机制。结果表明,DH40R和DH199S的根毛侵染发生在接种后第2天,随着侵染时间的延长,出现皮层侵染,但DH40R的皮层侵染在接菌第8天后被阻断,对根肿病具有完全的抗性,而DH199S在接菌8 d后,根部有肿瘤产生。在接菌后2、8和13 d,分别对抗、感材料接菌和未接菌根部的防御酶活性、MDA和可溶性蛋白含量进行测定,发现DH40R在这3个时间点接菌后SOD的活性均显著高于未接菌对照组,在第8天达到最高峰值,显著高于DH199S;DH40R接菌后的CAT活性在第8天达到最高峰值1572.68μmol·mL^(-1)·min^(-1),显著高于DH40R未接菌对照组和接菌及未接菌处理的DH199S;在接菌后8和13 d,抗根肿病大白菜DH系DH40R的MDA含量显著低于接菌后的感病DH系DH199S;接菌后DH40R的可溶性蛋白含量在接菌后8和13 d均显著高于DH199S。研究结果可为后期解析大白菜根肿病抗性相关机制的研究奠定基础。

云南白菜根肿病拮抗菌的筛选及鉴定

【目的】获得对云南白菜根肿病具有高效拮抗作用的微生物菌株,为云南白菜根肿病的生物防治提供潜在的菌种资源。【方法】从云南省曲靖市麒麟区三宝乡大棚白菜根肿病发病严重的地块,采集正常生长或发病较轻的白菜植株及其根际土壤,分离筛选抑菌效果较好的菌株,并对其进行形态特征鉴定、生理生化检测及分子序列分析。【结果】经初步筛选共获得46株细菌。土壤样品中共分离出30株细菌,其中抑制效果最好的是TB⁃7菌株,该菌株对白菜根肿病菌休眠孢子萌发抑制率为76.88%;白菜植株内共分离出16株内生细菌,其中抑制效果最好的是EB⁃15菌株,该菌株对白菜根肿病菌休眠孢子萌发抑制率为67.38%。通过对比分析选择抑菌效果最好的TB⁃7菌株作为目标菌株,经鉴定,TB⁃7菌株为革兰氏阳性芽孢杆菌,菌体菌落较小,颜色淡黄色,表面光滑且扁平,菌体湿润,不透明,边缘规则;生理生化指标检测结果显示,TB⁃7菌株是好氧菌,V⁃P、淀粉水解、pH 5.7,生长3项检测指标均呈阳性,柠檬酸盐、丙酸盐、D⁃木糖、L⁃阿拉伯糖、D⁃甘露醇、明胶液化、7%NaCl生长、硝酸盐还原8项检测指标均呈阴性;16S rDNA扩增产物约为1500 bp,测序结果表明,TB⁃7菌株与坚强芽孢杆菌(Cytobacillus firmus)同源性达到100%,鉴定该菌株为芽孢杆菌属的坚强芽孢杆菌。【结论】通过初步筛选及鉴定,获得1株对白菜根肿病菌休眠孢子抑制率达到76.88%的拮抗菌株,该菌株的获得可为解决云南本地日益严重的白菜根肿病的生物防治奠定基础。