Clubroot Resistance in Brassica rapa: Genetics, Functional Genomics and Marker- Assisted Breeding

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1 Clubroot Resistance in Brassica rapa: Genetics, Functional Genomics and Marker- Assisted Breeding Zhongyun Piao LOGO

plant has clubbed root and wilt in plant.

2 Clubroot disease Clubroot disease is caused by Plasmodiophora brassicae, which specifically infect the crucifer plants The infected plant has clubbed root and wilt in plant. most dangerous disease in Brassica crops worldwide decrease of yield about 10-15% in the world (Dioxin, 2009)

3 Clubroot disease in China It was firstly reported in 1955 In resent years, it became the most serious problem in the cultivation area of Brassica crops, mainly Chinese cabbage, rapeseeds, zha-tsai and others

4 Control of clubroot disease Chemical / microbial fungicide control Fluazinam, Cyazofamid: more expensive for farmers Reduce disease index, less effective Agronomical practice control rotation: need more time, slightly diseased application of lime: less effective

5 Control of clubroot disease Resistant breeding most effective to control, and environmental friendly identification of clubroot resistant (CR) genetic sources characterize CR genes, including gene structure, inheritance and the model of gene action the mechanism of resistance CR breeding

6 Genetics of clubroot resistance in B. rapa CR genetic resources in Brassica crops Number of accessions B. rapa B. juncea B. oleracea B. napus B. carinata Unknown Total: 1,024 lines 0 < Disease reduction (%) Peng et al.

7 The main CR resources in B. rapa Turnip (B. rapa ssp. rapifera) ECD01 (B+C, CRb) ECD02 (A+C, CRa) ECD03 (A+B) ECD04 (A+B+C) Debra (CRc, CRk) Siloga (Crr1, Crr2, Crr4) Milan White (Crr3)

8 CR genes identified and mapped in B. rapa A1 A2 A3 A6 A BRMS096 BSA3 Crr2 BSA4 BSA5 BRMS px155 CRc m6r BRMS OPC11-1S BrSTS61 Crr3 BrSTS54 CRk OPC11-2S BrSTS33 pw125 BRMS BRMS125 Crr4 BRMS BRMS173BRMS297 HC352 BSA2 Crr1 BSA7 BRMS HC352b TCR413 CRb TCR05 TCR01 CRa

9 CR gene identified and mapped in B. rapa CR gene CR origin Chrom Effect Reference Crr1 Siloga A8 Major Suwabe et al. (2006) Crr2 A1 Modifier Crr4 A6 Minor Crr3 Milan White A3 Major Hirai et al. (2003) CRa ECD02 A3 Major Matsumoto et al.(2005) CRb ECD01 A3 Major Piao et al. (2004) CRc Debra A3 Major Sakamoto et al. (2008) CRk Debra A2 Major Sakamoto et al. (2008)

10 Pathotypes of P. brassicae in China Province Race Province Race Yunan 1, 2, 4, 6, 7, 10, 11,12, 13 Shanghai 2, 5, 7 Tibet 2, 4, 5, 7 Sichuan 1, 4, 5, 7, 10, 15 Guizhou 4 Liaoning 2, 4, 11, 14 Anhui 2, 4, 9, 13 Jilin 4 Shandong 2, 4, 7 Hubei 4 Hunan 1, 4, 9, 13 Race 4 distributed in different area are the same pathotype? A new clubroot differentiation set is needed with CR plants which carry known CR gene.

11 QTL mapping for clubroot resistance 59-1 ECD Map construction BC1 BC1F1 Back crossing QTL analysis Phenotyping in BC1F2 families CSSL (including CR lines)

12 Isolate-specific resistance of CR Pb2 Pb4 Pb7 Pb10

13 A1 A3 A PbBa3.1 sau_um438a hri_mbrms173 cnu_490a PbBa8.1 Crr1 nia_095a Crr PbBa1.1 BSA Crr sau_041a CRk BrSTS61 PbBa3.2 Five CR QTLs were identified. Two were novel CRb sau_um398a PbBa3.3 TCR05 Three were located in the respective position of Crr1 Crr2, and Crr3 (CRk) Chen et al. 2013

14 Origin of the CR genes

15 CSSL constructed by integration of chromosome fragment of ECD04 into Chinese cabbage

16 QTL mapping of clubroot resistance In siloga, clubroot resistance is isolate-specific Crr1 region possible also with the aids of CRb (CRa) against race 10 CRb (CRa) to race 4 CRb (CRa) to the mixture of race 4 and race 10 Are there any unknown interaction between different pathotypes?

17 Analysis of epistatic interaction Clubroot resistance is also controlled by epistatic Race4 interaction between CR QTL and non-cr QTL Maker for QTL-a sau_um026 (A3) Epistasis Maker for QTLb BRMS019 (A10) Epistasisvalue H 2 (%) P value AA

18 Isolate-specific resistance of CR genes CR gene Race Crr1, Crr3, CRa, PbBa1.1 + PbBa3.1 2 Crr1+Crr2+Crr4, PbBa8.1 4 CRc CRk K04 2, K04 CRb 2, 4, 8 PbBa1.1 + PbBa3.3 7 PbBa Are these CR genes resistance to other pathotype?

19 Inheritance of CR genes in B. rapa Single gene or QTL, depend on the CR resources Single gene:crr3, CRa, CRb, CRc, CRk QTL(Turnip): Crr1, Crr2, Crr4, CRb (CRa) (Siloga) PbBa1.1, PbBa3.1, PbBa3.2, PbBa3.3 and PbBa8.1(ECD04) Dominant or incomplete dominant Dominant:Crr3, CRa, CRb, CRc, CRk Incomplete dominant:crr1 and Crr2

20 Fine mapping of the CRb gene A total of 2,896 and 1,486 (susceptible plants selected from 5,800) F2 individuals were used for fine mapping of CRb. 67 recombinants were found Zhang et al. in press

cnu_m413 TCR079 CRb TCR108 TCR031 TCR30 TCR37 TCR74 TCR05 TCR17 TCR01 Candidates of the CRb genes 0.04 0.11 0.03 0.

21 cnu_m413 TCR079 CRb TCR108 TCR031 TCR30 TCR37 TCR74 TCR05 TCR17 TCR01 Candidates of the CRb genes B. rapa physical map using BAC clones KBrB085J bp KBrH039L17 KBrE041C10KBrH097C05 KBrB039B11

22 CRa is not identical to CRb TCR79 TCR108 B4732 CRaim-T

23 Conclusion 1. Clubroot resistance is controlled either by single gene or polygenes depend on the CR germplasm. 2. Ten CR genes were identified in B. rapa. 3. CR genes are isolate-specific. 4. Epistatic effects is also present in clubroot resistance. 5. CR genes might be originated by at least 3 common ancestors. 6. CRa and CRb are not the same gene.

24 Functional Genomics of Clubroot Resistance in B. rapa PAMPs: pathogenassociated molecular patterns PRRs:pattern recognition receptors PTI: PAMP-triggered immunity ETI: Pathogen effectorstriggered immunity Boller and Felix, 2009

25 Life cycle of P. brassicae

26 Primary infection of P. brassicae into CR line pzs: primary zoospore; pp: primary plasmodium; z: zoosporangium; sz: secondary zoospore; sp: secondary plasmodium CS NIL CR NIL

27 Statistics of illumina 100 reads and comparison to the B. rapa reference genome Samples Times Total reads Total Mapped reads % (hai) nucleotides Uniq Multiple CR NIL 0 22,595,546 4,563,380, ,182,353 4,681,760, ,372,251 3,912,648, ,484,527 4,743,122, CS NIL 0 21,745,045 4,391,765, ,598,749 4,563,816, ,736,266 4,187,963, ,470,268 5,345,954, Total 180,185, Gb

0 hai 2056 773 403 241 12 hai 1597 96 hai 1452 155 461 135 158 409 57 57 103 119 538 72 hai 1569 Number of

28 0 hai hai hai hai 1569 Number of transcripts in the CR NIL that were differentially expressed (FDR<0.01, Fold change>2.0 or <-2.0) compared to CS NIL.

29 12 hai hai hai hai hai hai hai hai genes were down-regulated 76 genes were up-regulated A total of 165 unique genes related to defense response were differentially expressed in CR NIL at various times after inoculation. Of these, 89 were up-regulated and 76 were down-regulated at 0, 12, 72, and 96 hai in CR NIL. Of these, only 16 genes were differentially regulated at each time point.

30 12 hai hai hai hai hai 23 0 hai hai hai genes down-regulation 76 genes up-regulation A total of 137 unique genes that related to immune response in GO terms were up- or down regulated in CR NIL at various times after inoculation. Of these, 76 were up-regulated and 65 were down-regulated at 0, 12, 72, and/or 96 hai in CR NIL. Of these, 14 genes were differentially regulated at each time point.

31 Conclusion 1. Among the putative Chinese cabbage defence response genes identified (GO: ), 165 exhibited significant differences in expression between the CR and CS NILs. 2. Pathogen-associated molecular pattern (PAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the CR NIL.

32 Breeding of Clubroot resistance in B. rapa Traditional breeding Marker-assisted breeding of single gene Pyramiding CR genes with linked markers

33 Marker-assisted breeding An elite line of Chinese cabbage 91-12A, 91-12B CR Shinki (Rr) F A, 91-12B 2007 (rr) Marker Assisted Selection Genotype of CRb 2008 (Rr) (Rr) BC A, 91-12B (rr) BC A, 91-12B (rr) (rr) (Rr) Genotyping with TCR01 and TCR Resistant test in the field (Rr) BC 3 BC 3 F 2 BC 3 F 3 (RR) (RR, Rr) (rr) (Rr) (rr) (Rr) (RR) Whole genome selectio n

34 CR12 CR17

35 青梗菜 CR 702

Integration of CR genes into B. napus from ECD04 A1 A3 A8 16.5 PbBa3.1 sau_um438a 7.7 8.4 17.

36 Integration of CR genes into B. napus from ECD04 A1 A3 A PbBa3.1 sau_um438a hri_mbrms173 cnu_490a PbBa8.1 Crr1 nia_095a Crr PbBa1.1 BSA Crr sau_041a BrSTS61 PbBa3.2 sau_um398a PbBa3.3 CRb TCR05

37 Some considerations in CR breeding Isolate-specific resistance of CR genes Breeding and cultivation of CR cultivars resistance to specific pathotypes Action of CR genes Dominant genes CMS or SI F1 hybrid between CR parent (CRa and CRb) and non-cr parent Incomplete dominant CMS or SI F1 hybrid between two CR parents with same CR allele (Crr1 and Crr2)

38 Some considerations in CR breeding Gene interaction with additive effects interaction between CR genes (Crr1 and Crr2) interaction between CR gene and non-cr loci Pyramiding CR genes from CR turnip or other CR resource for durable resistance Transfer several genes simultaneously Transfer CR gene to an elite line once a time, then combine them all

39 Acknowledgement Prof. Xiangqun Shen Ph. D students: Jingjing Chen, Teng Zhang, Hong Li, Master students: Pengpeng Li, Sha Yu, Chunsha Zhang, Huazhong Agricultural University Prof. Chunyu Zhang Graduate student: Zhongxiang Zhan Supported by National Natural Science foundation of China

40 Thank you LOGO

Genome-wide identification and characterization of mirnas responsive to Verticillium longisporum infection in Brassica napus by deep sequencing

Genome-wide identification and characterization of mirnas responsive to Verticillium longisporum infection in Brassica napus by deep sequencing Longjiang Fan, Dan Shen, Daguang Cai (Zhejiang University/Kiel