Fine mapping and candidate gene analysis of QS_B3.1 conferring clubroot resistance in Brassica rapa

doi:10.21203/rs.3.rs-1738495/v1

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Fine mapping and candidate gene analysis of QS_B3.1 conferring clubroot resistance in Brassica rapa

https://doi.org/10.21203/rs.3.rs-1738495/v1

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Chinese cabbage is an important leafy vegetable rich in many nutrients widely grown in China. Clubroot disease caused by an obligate biotrophic pathogen Plasmodiophora brassicae was rapidly spread and challenged to Chinese cabbage production. A clubroot resistance (CR) gene, QS_B3.1, was mapped on chromosome A03 of B. rapa. A Chinese cabbage line ‘CR510’, which harbor homozygous resistance locus QS_B3.1 was selected from a BC₄F₃ family. ‘CR510’ was crossed with a clubroot susceptible Chinese cabbage inbred line ‘59-1’. Total 51 recombinant plants were identified from an F₂ population including 3000 individuals. These recombinants were selfed and the clubroot resistance of F_2/3 families were evaluated. Finally, QS_B3.1 was fine mapped to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008. According to the reference genome, total 54 genes including five encoding the TIR-NBS-LRR proteins was annotated in the fine mapped region. Further nine candidate’s gene expression in parental lines at 7, 14 and 21 days after inoculation of P.brassicae were evaluated. Bra019376, Bra019401, Bra019403 and Bra019410 are highly expressed in ‘CR510’ than ‘59-1’. Gene sequence of Bra019410 from ‘CR510’ was cloned and identified different from CRa. Therefore, Bra019376, Bra019401, Bra019403 and Bra019410 are the most likely candidates of QS_B3.1. Our research provides a valuable germplasm resource against P. brassicae and QS_B3.1 closely linked marker for marker-assisted selection in CR cultivars breeding.

Chinese cabbage

clubroot resistance

Plasmodiophora brassicae

QS_B3.1

TIR-NBS-LRR proteins

In this study, we fine mapped a clubroot resistance gene QS_B3.1 in Chinese cabbage and developed its closely linked marker syau-InDel3008 for marker-assisted selection in CR cultivars breeding.

Clubroot disease caused by soil-borne obligate plant pathogen P. brassicae Woronin lead to heavy yield losses in Chinese cabbage production in China, Korea and Japan (Song et al., 2021). Growing clubroot-resistant (CR) cultivars is the most effective and environment-friendly strategy for clubroot management (Pang et al., 2020). Therefore, many CR gene/genes were identified and their linked molecular markers were developed for breeding of CR varieties in Chinese cabbage. In the previously research, CR loci such as CRa (Ueno et al., 2012), CRb (Piao et al., 2004), CRd (Pang et al., 2018), CRk and Crr3 (Sakamoto et al., 2008) were mapped on A3, while, Crr1 (Suwabe et al., 2003), CRc (Sakamoto et al. 2008), Crr2 and Crr4 (Suwabe et al., 2006) mapped on chromosome A8, A2, A1 and A6 in Brassica rapa, respectively. PbBa3.1and PbBa3.3 (Chen et al., 2013) were identified as two novel QTLs for clubroot resistance. Three more QTLs QS_B1.1, QS_B3.1, and QS_B8.1 (Pang et al., 2014) were detected using an F₂ population by crossing a clubroot resistant inbred line ‘Siloga’ and susceptible Chinese cabbage ‘BJN3’. Most of the CR loci were detected from European fodder turnip ‘Gelria R’, ‘Siloga’, ‘Milan White’, and ‘Debra’. Of the detected CR genes, CRb^Kato and CRa were identified as same clubroot resistant gene (Hatakeyama et al., 2017). Whether the other loci detected are identical remains to be clarify. Therefore, fine mapping and gene functional verification are necessary for the CR gene identification.

In breeding programs, CR genes from the European fodder turnip have been introduced into Chinese cabbage and several resistant commercial cultivars have been released (Diederichsen et al., 2009; Drury et al., 2021). However, most of the commercial resistant cultivars with CR genes from turnip showed susceptible in field of Japan, whereas CR turnips still showed resistance (Kuginuki et al., 1999). Discover the mechanism which caused the accumulated CR genes lost their resistance to P. brassicae in commercial cultivars is still a challenge to the scientists and breeders. CR genes were appeared to be isolate-specific resistance to P. brassicae in B. rapa (Chen et al. 2013), B. oleracea (Rocherieux et al., 2004)d napus (Werner et al., 2008). Genetic variance of pathogen lead to the pathotype was changed to more virulent. It has been reported that the pathotypes of P. brassicae include 2, 4, 7 and 11 according the Williams’ classification system (Williams 1966) in China (Pang et al. 2020). Further study shows 16 pathotypes were identified from 132 field isolates using a new developed Sinitic clubroot differential (SCD) set in different regions of China and Korea (Pang et al., 2020). Therefore, more CR genes identification are required for breeding cultivars of Brassica crops with broad-spectrum resistance to multiple P. brassicae pathotypes (Lv et al., 2020).

In the previous study, we reported three CR loci QS_B1.1, QS_B3.1 and QS_B8.1 (Pang et al., 2014) in ‘Siloga’. Near-isogenic lines of Chinese cabbage containing homozygous QS_B3.1 were developed through backcross and marker assisted selection. QS_B3.1 was validated and narrowed to a range of 2.89 Mb in chromosome A03’ (Ning et al., 2015). In this study, we aim to fine map the CR locus QS_B3.1 using large F_2/3 mapping population and develop closely linked molecular marker for CR cultivars breeding in Chinese cabbage. Moreover, candidate’s gene will be predicated and analyzed in the fine mapped QTL region.

Plant materials

A Chinese cabbage line ‘CR510’, which harbor homozygous resistance locus QS_B3.1 was selected from BC₄F₃ family through marker-assisted backcrossing approach (Ning et al., 2015). ‘CR510’ was crossed with a susceptible Chinese cabbage inbred line ‘59 − 1’ to produce F₁ hybrid in year 2016. The F₁ was selfed to obtain F₂ which 3000 F₂ individuals were used for recombinant individuals’ selection. Total 180 individuals were random selected from the large F₂ population and used for genetic map construction.

Indel Marker Development And Genetic Map Construction

‘CR510’ and ‘59 − 1’ were sent for re-sequencing using 6-fold Illumina sequence coverage. To call InDel markers, we aligned the clean short reads of two parental lines which were fileted with NGSQC Toolkit to the reference genome of Chiifu-401-42, using BWA software (Li and Dubin, 2009). Alignment files were converted to SAM/BAM files using SAMtools (Li et al., 2009), and applied to the InDel -calling filter with VarScan 2 (Koboldt et al., 2012). Flanking sequences of the InDel site were used to design makers with Primer 3. Genetic map was constructed using JoinMap version 4 and the details have been described by pang et al. (2014) (Stam, 1993; Van Ooijen and Voorrips, 2001).

Recombinant’s selection and P. brassicae resistance evaluation

Three molecular markers cnu_ssr316, syau_InDel3008 and sau_um026 closely linked to QS_B3.1 were used for recombinant individuals’ selection. The selected recombinant individuals were selfed to develop F₃ family. Parental lines and F₃ plants were inoculated with P. brassicae isolate ‘LNND-2’ which has been identified as Pb3 according to SCD (Pang et al., 2020). Three replicates were performed with twelve individuals of each replicate. The inoculation process and disease index evaluation were according to Pang et al. (2020).

Fine mapping of QS_B3.1 and candidate genes expression analysis

The disease index of recombinant individuals’ F₃ family and genotype of recombinant individuals were combined to fine map the clubroot resistant gene QS_B3.1. The sequences of parental lines were compared to reference genome (Chiifu-401-42) and the fundamental functional annotation of genes located in the QS_B3.1 region was analyzed. Candidate genes expression of were analyzed from infected and uninfected parental lines.

Roots of parental lines were sampled at 7, 14 and 21 days after inoculation with 1 ml 1×10⁷ spores of P. brassicae or 1 ml distilled water as control. Three biological replicates were performed for the RNA isolation and quantitative RT-PCR analysis. Total RNA isolation and PCR conditions were according to Ma et al (2022). The expression patterns of candidate genes in root tissue of ‘CR510’ and ‘59 − 1’ were determined in a quantitative RT-PCR assay using GAPDH as an internal control.

CRa was cloned from the cDNA of ‘CR510’ and ‘59 − 1’. The primers for gene cloning and expression analysis were designed using Primer 3. PCR products were detected and purified using a QIA quick Gel Extraction Kit (Qiagen, Germany). The purified fragments were ligated to a pGEM-T Easy vector and transformed into E. coli DH5α. The positive colonies were screened on LB medium containing ampicillin (Amp +) and verified by PCR. The positive clones were sequenced by Biorun Biotechnology Co., (Wuhan, China). SeqMan (SeqMan NGen®. Version 12.0. DNASTAR. Madison, WI.) was used for splicing the candidate genes sequences.

Molecular marker development and genetic map construction

‘CR510’ and ‘59 − 1’ were sequenced and the total amount of clean data was 3.5Gb (Clean Q30 bases rate, 93.14%) and 3.5Gb (Clean Q30 bases rate, 93.22%), respectively (SRA accession: SAMN28869001 and SAMN28869002). Based on the resequencing result of ‘CR510’ and ‘59 − 1’, twenty-six InDel markers were developed in the confidence interval of the QS_B3.1 (Table S1). Out of the 26 InDel markers 20 showed polymorphisms between ‘CR510’ and ‘59 − 1’. Total 180 out of 3000 F₂ individuals were randomly selected and used for genetic map construction in year 2018. A partial genetic map including 11 SSR and InDel markers from 20 polymorphisms markers was constructed (Fig. 1A). The genetic map covers 15.9 CM corresponding to a physical length of about 2.76 Mb.

Recombinant Individuals’ Selection And Disease Resistant Evaluation

Total 51 recombinants were identified from 3000 F₂ individuals using QS_B3.1 linked molecular cnu_ssr316, syau_InDel3008 and sau_um026. The genotyping of selected recombinants was performed using the above 11 molecular markers which mapped on the confidence interval of the QS_B3.1 (Fig. 1B). Forty five out of 51 recombinants were succussed obtain F_2/3 families which were used for disease resistant test in year 2018. Parental lines and 45 F_2/3 families were inoculated with P. brassicae isolate ‘LNND-2’ in year 2019. Eight and six teen F_2/3 families showed highly resistant and susceptible to ‘LNND-2’, respectively. The other 21 F_2/3 families disease index ranged from 8.3 to 41 (Fig. 1C).

Fine mapping of QS_B3.1 and candidate genes prediction

QS_B3.1 was fine mapped to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008 according to the genotype and disease evaluation of 45 identified recombinants F_2/3 families. Total 54 genes (Table S2) were annotated within 4 genes (Bra019409, Bra019410, Bra019412 and Bra019413) identified as encoding disease resistance protein (TIR-NBS-LRR). Five function unknown genes Bra019376, Bra019381, Bra019401, Bra019403 and Bra019404 were found in the fine mapped region. In addition, each 3 genes belong to the multi antimicrobial extrusion (MATE) family, F-box and RNA binding, respectively. Eight enzyme genes belong to glycosyl hydrolase 1 family, peptidase, calmodulin-domain protein kinase, phospholipase or reductase. The other 28 genes belonged to other families such as alkaline ceramidase 3-like, calmodulin-domain protein kinase 9, golgin family A protein, cellular response to nitrogen starvation and zinc finger family protein (Table S2).

Candidate gene expression and CRa sequence comparison

The expression patterns of 4 resistance genes (Bra019409, Bra019410, Bra019412, and Bra019413) and 5 unknown genes (Bra019401, Bra019403, Bra019404, Bra019376, and Bra019381) were analyzed in ‘CR510’ and ‘59 − 1’ using a real time quantitative PCR assay. Bra019409 and Bra019410 are expressed as significant differences in ‘CR510’ and ‘59 − 1’ after P. brassicae inoculation at 7, 14 and 14 DAI. These genes expression showed significantly higher in P. brassicae treatment than distilled water in ‘CR510’. Bra019404 is highly expressed in ‘59 − 1’ but not ‘CR510’, however, there is no difference between P. brassicae and distilled water treatments. Bra019403 and Bra019376 is highly expressed in P. brassicae inoculated than distilled water treatments at 7 and 14 DAI ‘CR510 in ‘CR510’. Bra019401 and Bra019413 is highly expressed in ‘CR510’ in P. brassicae treatment at 14 DAI. Bra019412 showed negative regulation with the P. brassicae treatment in both ‘CR510’ and ‘59 − 1’ at 7 DAI. Bra019381 has no differences in ‘CR510’ and ‘59 − 1’ at 7 DAI, However, highly expressed was observed in P. brassicae treatment at 14 DAI in ‘CR510’.

To better distinguish the differences between CRa and QS_B3.1. Bra019410 from ‘CR510’ and ‘59 − 1’ was cloned. The gene sequence of Bra019410 from ‘59 − 1’ was identical to the reference genome (Chiifu-401-42), however, Bra019410 from ‘CR510’ has a 27bp insertion and SNPs compared to the reference genome (Fig S1). In addition, sequence alignment analysis showed that there are only 79% similarity between resistance gene CRa (from DH line T136-8) and candidate gene Bra019410 from ‘CR510’, indicating a possible novel candidate resistance gene was identified in B. rapa (Fig S2).

P.brassicae could infect most cruciferous crops and Chinese cabbage is reported highly susceptible to clubroot disease (Dixon, 2009). Mapping of clubroot resistant genes and breeding of clubroot-resistant (CR) cultivars is the most effective and environment-friendly strategy for clubroot management (Pang et al., 2020). Total 26 clubroot resistant genes/loci have been reported from European turnips such as Siloga, ECD1, ECD2, ECD3, ECD4 etc. in Brassica rapa (Suwabe et al., 2003; Chen et al., 2013; Hirani et al., 2018; Wang et al., 2022). However, only CRa and Crr1a have been cloned and these two genes all encode Toll-interleukin-1 receptor-like domain-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) proteins (Ueno et al., 2012; Hatakeyama et al., 2013). CR genes appeared to confer isolate-specific resistance to P. brassicae (Chen et al., 2013). In addition, resistant genes encoding TIR-NBS-LRR proteins are known as race-specific resistance in higher plants (Larkan et al. 2020). The pathogenic variability of P. brassicae leading to a complex pathotypes classification (Pang et al., 2020) and causing the breakdown of the resistance of CR cultivars in Chinese cabbage and canola (Kuginuki et al., 1999; Strelkov et al., 2016). Therefore, continues exploring isolate specific clubroot resistance gene are important to the breeding of CR cultivars in Brassica.

In the previously study, we identified a clubroot resistant gene QS_B3.1 which was detected from a European turnip ‘Siloga’ (Pang et al., 2014). QS_B3.1 is a major clubroot resistant locus which explained 70.55% of the phenotypic variation. Then, near-isogenic lines (NILs) of Chinese cabbage containing homozygous QS_B3.1 were selected from BC₄F₃ population and used for the validation of QS_B3.1 (Ning et al., 2015). In this study, we fine mapped the CR gene QS_B3.1 to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008 in chromosome A03 using a large F₂ population. QS_B3.1 was mapped in an R gene hot zone of A03 where five major CR genes have been reported including CRa, CRb, Rcr1, Rcr2 and Rcr4. CRa has been successfully cloned, encoding TIR-NBS-LRR protein and CRb was identified that is the same gene as CRa (Hatakeyama et al., 2017). While, the other CR genes Rcr1, Rcr2 and Rcr4 require confirmation. Four resistance genes including Bra019409, Bra019410, Bra019412 and Bra019413 which encode the TIR-NBS-LRR proteins were identified from 54 genes in this fine mapped region. Moreover, five unknown genes were detected in the candidate genes. Gene expression data indicates Bra019376, Bra019401, Bra019403 and Bra019410 are contribute to the clubroot resistance (Fig. 2). Sequence variation between the CRa and candidate gene Bra019410 revealed QS_B3.1 is distinct from CRa. Therefore, Bra019376, Bra019401, Bra019403 and Bra019410 are candidates for further gene function study.

The genetic variation within the pathogen and Brassica species is the main challenge in clubroot resistance breeding. Breeding for broaden clubroot resistance cultivars are by far the best effective and economic strategy for dealing with this devastating disease in Brassica crops (Pang et al., 2018). Many CR cultivars have been released in Japan, China, Korea and Canada including Chinese cabbage and canola (Diederichsen et al., 2009; Drury et al., 2021). CRa is a single dominant resistance gene against several pathotypes of P. brassicae (Ueno et al., 2012; Pang et al., 2020). Therefore, CRa was the most widely distributed in CR cultivars of Chinese cabbage in Japan (Aruga et al., 2013). During previous work, CR genes and their closely linked markers exploration have been greatly improved the pyramiding multiple CR genes through marker-assisted selection in CR breading. Different CR genes rotation or pyramiding could confer great resistance to multiple pathotypes of P. brassicae in field (Dolatabadian et al., 2021). In the present work, molecular cnu_ssr316, syau_InDel3008 and sau_um026 showed excellent clubroot resistant individuals selection ability in F₂ population which indicates that cnu_ssr316, syau_InDel3008 and sau_um026 are closely linked to clubroot resistance gene. The genetic map shows syau_InDel3008 is the closest molecular market to QS_B3.1. Our research provides a valuable germplasm resource against P. brassicae and a co-segregate molecular marker in future breeding programs for Chinese cabbage and oil seed rape.

In conclusion, the major QTL locus QS_B3.1 conferring to clubroot resistance was fine mapped to an interval of 386 kb on chromosome A3 in B. rapa, and 9 putative candidate genes expression pattern were analyzed. Gene expression results indicated that Bra019376, Bra019401, Bra019403 and Bra019410 are highly possible to be the candidate genes for QS_B3.1. In addition, the candidate gene Bra019410 from ‘CR510’ showed sequence variation from CRa that indicated QS_B3.1 is possible to be a novel resistance gene to clubroot. Further candidate gene’s function analysis will be necessary for QS_B3.1 in brasscia rapa. Our research provides a valuable germplasm resource against P. brassicae and QS_B3.1 closely linked marker syau-InDel3008 for marker-assisted selection in the CR cultivars breeding.

Acknowledgment

The authors thank Prof. Yue Liang for the construction suggestions on the writing of this paper, as well as the laboratory members for assistance with the plant materials growing and surveys of clubroot resistance tests.

Author’s contributions

XZ, WXP and YW carried out experiments, generated data. WXP analyzed the data and wrote the original manuscript. YM and ZZ participated in data analysis. WXP and ZYP conceived the study, participated in its coordination. All authors have read and approved the final manuscript.

Funding

This study received funds from the Liaoning Natural Science Foundation (2021-MS-229), the China Postdoctoral Science Foundation (2016M600214).

Availability of data and material

The datasets generated and analyzed during this study are available on reasonable requests from the corresponding authors. The raw data of whole genome re-sequencing have been deposited in the NCBI Sequence Read Archive (SRA) repository under the accession numbers SAMN28869001 and SAMN28869002.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Supplementarydata.pdf
Table S1 Primers used in this study.Table S2 Candidate genes information in the fine mapped region of clubroot resistance gene QS_B3.1.Figure S1 Amino acid sequence alignment analysis of Bra019410 from ‘CR510’ and Chinese cabbage reference genome Chiifu-401-42.Figure S2 Amino acid sequence alignment analysis of Bra019410 from ‘CR510’ and CRa (from DH line T136-8).

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Fine mapping and candidate gene analysis of QS_B3.1 conferring clubroot resistance in Brassica rapa

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Abstract

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Key Message

Introduction

Materials And Methods

Plant materials

Indel Marker Development And Genetic Map Construction

Results

Molecular marker development and genetic map construction

Recombinant Individuals’ Selection And Disease Resistant Evaluation

Discussion

Declarations

References

Supplementary Files

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