Planta (2022) 256: 66

https://doi.org/10.1007/s00425-022-03981-5

ORIGINAL ARTICLE

Mapping and identification of a new potential dominant resistance

gene to turnip mosaic virus in Brassica rapa
Xinxin Lu1   · Ze Li1 · Wenyue Huang1 · Shaoxing Wang1 · Shifan Zhang1 · Fei Li1 · Hui Zhang1 · Rifei Sun1 ·
Guoliang Li1 · Shujiang Zhang1

Received: 23 June 2022 / Accepted: 22 August 2022 / Published online: 29 August 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Main conclusion  By constructing an ­F2 population, a new potential dominant resistance gene to TuMV in Brassica
rapa was mapped and identified.

Abstract  Brassica rapa is the most widely grown vegetable crop in China, and turnip mosaic virus (TuMV) is a great threat
to its production. Hence, it is a very important work to excavate more and novel resistance genes in B. rapa. In this study, the
resistant line B80124 and the susceptible line B80450 were used to construct the F
­ 2 populations, and through genetic analysis,
the resistance to TuMV was found to be controlled by a dominant gene. Bulked segregant analysis sequence (BSA-seq) was
used for the primary mapping, and an intersection (22.25–25.03 Mb) was obtained. After fine mapping using single nucleo-
tide polymorphisms (SNP) markers, the candidate region was narrowed to 330 kb between the SNP markers A06S11 and
A06S14, including eight genes relating to disease resistance. Using the transcriptome analysis and sequence identification,
BraA06g035130.3C was screened as the final candidate gene, and it contained two deletion mutations, leading to frameshift
in the susceptible line B80450. In addition, the phylogenetic analysis, hydrophilia and hydrophobicity analysis, subcellular
location prediction analysis, amino acid bias analysis, and 3D modeling structures of BraA06g035130.3C were conducted
to predict its functions. This study was conducive to the identification of a new TuMV resistance gene in B. rapa, which
is of important scientific significance and application value for the improvement of TuMV resistance traits and molecular
design breeding for Brassica crops.

Keywords  Brassica rapa · TuMV · Resistance · Dominant · R gene

Abbreviations eIF(iso)4E Eukaryotic translation initiation factor 4E

TuMV Turnip mosaic virus isoform
BSA-seq Bulked segregant analysis sequence VPg Virus protein of genome-linked
SNP Single nucleotide polymorphisms
DAS-ELISA Double antibody sandwich assay enzyme
linked immunosorbent assay Introduction
KASP Kompetitive allele-specific PCR
eIF4E Eukaryotic translation initiation factor 4E Brassica crops are the very important vegetables and oil
crops in the world. These include three diploid crops—
Brassica rapa (B. rapa, AA), Brassica nigra (B. nigra,
BB), and Brassica oleracea (B. oleracea, CC)—and three
Communicated by Anastasios Melis.
amphidiploids synthesized by three diploid crops above-
* Guoliang Li Brassica juncea (B. juncea, AABB), Brassica carinata (B.
[email protected] carinata, BBCC), and Brassica napus (B. napus, AACC)
* Shujiang Zhang (Cheng et al. 2017). Brassica rapa is the most widely grown
[email protected] vegetable crop in China, with 1,800,000 hectares of Chinese
cabbage playing a significant role in balancing the market
1
Institute of Vegetables and Flowers, Chinese Academy supply and stabilizing vegetable prices (FAO). However, the
of Agricultural Sciences, Beijing 100081, China

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turnip mosaic virus (TuMV), which belongs to the Potyvirus with pathogens, which may accelerate their co-evolution,
genus, is a serious disease of B. rapa. Currently, TuMV has and the protein–protein interactions, through the yeast two
been the only potyvirus reported to infect Brassica crops hybrid (Y2H), co-immunoprecipitation (CoIP), and bi-
(Li et al. 2018). The genome of TuMV is about 10 kb, and molecular fluorescence complement (BiFC), can indicate
contains a positive single-stranded RNA encoding ten pro- their interaction relationships. The interactions between
teins (P1, CH-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb, and Brassica crops and TuMV are related to the cytoplasmic
CP) (Walsh and Jenner 2002). After infection with TuMV, inclusion (CI) protein, which has been demonstrated in
B. rapa shows various symptoms, including leaf shrinking, TuRB01, TuRB01b and TuRB04 genes; However, P3 also
the mosaic leaves, mottling or spotting, which lead to non- plays an important role in resistance to TuMV, as the viral
heading or loose heading (Walsh et al. 2002). TuMV has a factor in TuRB03 and TuRB05 genes (Jenner et al. 2000,
serious impact on B. rapa production, and in many areas of 2002, 2003; Walsh et  al. 2002). From the resistant line
China, B. rapa production has resulted in total crop failure ‘RLR22’ and the susceptible line ‘R-o-18’, the ConTR01
due to TuMV damage. gene was mapped, and four amino acids were found between
TuMV can be transmitted among plants by at least 89 the two lines, which may be responsible for resistance in B.
aphid species, therefore, it is difficult to control TuMV. To rapa (Rusholme et al. 2007). Compared with the dominant
better control TuMV, more resistance genes should be exca- genes, recessive genes show different mechanisms, which
vated in Brassica crops. To date, 20 genes/loci, comprising include nucleotide loss, deletion, or mutation (Dinkova
16 dominant genes/loci and four recessive genes/loci, have et al. 2016). Many recessive genes, such as retr01, retr02,
been mapped and cloned in Brassica crops for resistance and trs, are eukaryotic initiation factor 4E (eIF4E) family
to TuMV. Using restriction fragment length polymorphism genes that interact with the potyvirus VPgs and promote
markers (RFLPs), a B. rapa genetic map was constructed the replication and transmission of the virus in host plants
in BC populations, and the ConTR01 gene was mapped to (Rusholme et al. 2007; Qian et al. 2012; Kim et al. 2013). In
a resistance TuMV CDN1 isolate on A08 (Rusholme et al. our previous studies, the retr02 was cloned as an eIF(iso)4E
2007). Similarly, the amplified fragment length polymor- that has broad-spectrum resistance to TuMV due to a natu-
phisms (AFLPs), random amplified polymorphic DNA ral mutation, resulting in the mis-splicing of the eIF(iso)4E
(RAPDs), and simple sequence repeat markers (SSRs) have gene (Qian et al. 2012; Nellist et al. 2014). In addition, the
been used to establish a linked map in 100 DH line popu- key amino acids of the eIF(iso)4E protein have been exca-
lations, and four quantitative trait loci (QTLs) have been vated by Y2H (Kim et al. 2014). Similarly, the interactions
identified for TuMV Tu1-4 isolates (Zhang et al. 2008). In between different copies of TuMV C4/CDN1/UK1/CHN2/
addition, 183 DH lines were used to identify three QTLs CHN3 VPgs and eIF4E/eIF(iso)4E have been resolved by
on A03, A04, and A06 after screening with the TuMV C4 Y2H and BiFC assays (Li et al. 2018, 2021). Furthermore,
isolate (Zhang et al. 2009). In non-heading Chinese cab- some single nucleotide polymorphisms (SNPs) have also
bage, an R gene (BcTuR3) has been cloned (Ma et al. 2010). been screened, which may play an important role in the
Furthermore, in recent years, TuMV-R (Chung et al. 2013), interaction between eIF(iso)4E and VPg, such as SNP T ­ 106C
TuRB07 (Jin et al. 2014), and TuRBCS01 (Gao et al. 2016)— in BraA.eIF(iso)4E.c and SNP ­A154C in VPg (Li et al. 2018,
have been identified using the ­F2, BC, or DH populations in 2021). These results may lay the foundation for gene edit-
recent years in B. rapa. In addition to the dominant genes/ ing (key amino acids) to accelerate the resistance of TuMV
loci, four recessive genes/loci have been mapped. Moreover, breeding in B. rapa.
retr01 on A04, combined with the dominant gene ConTR01, Further excavating TuMV resistance genes in B. rapa is
is resistant to TuMV CDN1 (Rusholme et al. 2007); rnt1-2 important work that requires the long-term persistence of
combined with rnt1-3 is responsible for TuMV UK1 isolate breeders. At present, more than 20 resistance genes have
resistance in B. rapa (Fujiwara et al. 2011). The trs gene has been identified in Brassica crops, but these genes cannot be
been mapped to A04 in B. rapa and encodes the eIF(iso)4E effectively used for antiviral breeding. Therefore, it is neces-
protein, which confers resistance to TuMV CHN2-5 isolates sary to excavate new resistance genes and conduct polymeri-
(Kim et al. 2013). In our previous study, the insertion-dele- zation breeding of multiple resistance genes in the future. In
tion (InDels) and SSR markers were used to screen a reces- this study, a new dominant gene for resistance to the TuMV
sive gene, retr02, which could also encode the eIF(iso)4E C4 isolate was mapped and cloned by BSA-seq in B. rapa.
protein for the broad-spectrum resistance (Qian et al. 2012). The candidate gene BraA06g035130.3C was an R gene con-
Many resistant genes have been mapped and cloned for taining a CC-NBS-LRR domain. In addition, phylogenetic
TuMV in B. rapa, which could facilitate their application analysis, hydrophilia, hydrophobicity analysis, subcellular
in breeding. location prediction analysis, amino acid bias analysis, and
With many resistant genes cloned, the resistance mecha- 3D modeling structures of BraA06g035130.3C were con-
nisms have also been revealed. Host plants could interact ducted to predict its functions. This study was conducive to

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the identification of new TuMV resistance genes in B. rapa, was performed according to the manufacturer’s instructions.
which are of important scientific significance and applica- Detailed steps for TuMV DAS-ELISA testing were per-
tion value for the improvement of TuMV resistance traits formed according to the DAS-ELISA instructions, and the
and molecular design breeding for Brassica crops. resistant index was divided into positive (susceptible) and
negative (resistant). Samples with an OD value two times
higher than the healthy average were positive, and samples
Materials and methods with an OD value two times lower than the healthy average
were negative (OD value = the O
­ D405 value of the sample/the
Plant materials ­OD405 value of the healthy average) (Lu et al. 2022).

Two high-generation inbred lines of B. rapa, ­P1 (B80124, Resistance mapping by bulk segregant analysis
TuMV-resistant) and ­P2 (B80450, TuMV-susceptible), were (BSA)
used as parental lines to generate F ­ 1 and F
­ 2 populations,
respectively, for inheritance analysis and gene mapping. According to the phenotypic investigation and the OD value
Both ­P1 and ­P2 are head Chinese cabbage lines, which were evaluation of the ­F2 population, 40 individuals with extreme
collected from the rural local varieties, and their maturity is TuMV resistance were selected to construct a resistant DNA
about 70 and 60 days, respectively. They were collected from pool (R-pool), and 40 individuals with extreme TuMV sus-
local rural varieties. All the materials were planted in a 7 × 7 ceptibility were selected to construct a susceptible DNA
nutrient bowl in an insect-proof net in an artificial room in pool (S-pool). DNA extraction from a single plant of the P ­1
the Institute of Vegetables and Flowers, Chinese Academy and ­P2 was used to construct the parent pools ­P1 (B80124)
of Agricultural Sciences. The temperature during the day and ­P2 (B80450), respectively. Genomic DNA was extracted
was 20–25 °C, and the temperature at night was 15–19 °C. using the modified cetyltrimethylammonium bromide
The humidity was about 60%. The plants were thoroughly method (CTAB) (Murray and Thompson 1980). The DNA
watered, and the aphids were controlled regularly. concentration and quality were assessed using an ND-2000
spectrophotometer (Thermo Fisher Scientific, Wilmington,
TuMV C4 isolate inoculation DE, USA) and 1.0% agarose gel electrophoresis, respec-
tively. The parent pool sequencing depth was 10 × , and the
The TuMV C4 isolate (GenBank ID: HQ46217), found R- and S-pool sequencing depth was 40 × by Illumina Hiseq
in the Shunyi base of Beijing, China in 2010, was used in (San Diego, CA, USA) with 150 bp paired-end sequencing
this study and maintained on susceptible line ‘pengbo 116’ reads from the Biomarker Biotech Co., Ltd. (Beijing, China).
(fresh leaves including TuMV stored at − 80 °C). Plants Brassica rapa genome V3.0 was used to align the data using
were inoculated with TuMV following our previous study Burrows-Wheeler Aligner (BWA) software (Li 2013; Chen
(Li et al. 2016). When the third true leaf of the plant was et al. 2021).
fully expanded, a thin layer of emery was evenly sprayed
on the front of the second and third leaves of the expanded SNP marker and linkage map development
plant; 1 g of the fresh leaves infected with TuMV-C4 was
ground in a high-temperature sterilized mortar, and 4 mL of According to the sequencing results of SNP sites, selected
phosphate buffer (0.05 mol/L, pH = 7.0) was added. Evenly the SNP sites with polymorphism between parents, and the
ground samples were immediately used for inoculation. The sites with a sequencing depth greater than 10 were selected.
TuMV homogenate was gently applied in the direction of the Further, 100 bp upstream and downstream sequences of
leaf veins. After inoculation, the leaves were immediately the variation sites (there were not four consecutive varia-
rinsed with clean water and shaded for 24 h. Inoculation tion sites in the sequence) were extracted, and SNP mark-
was repeated 1 day after the first inoculation. TuMV resist- ers were designed. The SNP markers were designed by the
ance identification was conducted 3 weeks after the second Laboratory of the Government Chemist based on the B. rapa
inoculation. genome V3.0 (Li 2013). The developed SNP markers were
first validated in the B80124, B80450 and F ­ 1 lines to screen
Double antibody sandwich assay enzyme linked polymorphic markers using the Kompetitive allele-specific
immunosorbent assay (DAS‑ELISA) test for TuMV PCR (KASP) technique. Then, the polymorphic markers
were employed to genotype in the ­F2 population. The P ­ 1 gen-
The TuMV DAS-ELISA reagent test kit and positive con- otype was marked as ‘2’, and the P ­ 2 genotype was marked as
trol were purchased from Agdia Inc. (Elkhart, IN, USA), ‘0.’ The F ­ 1 heterozygous genotype was marked as ‘1’, and
and an ELx808 microplate reader (BioTek, Winusky, VT, deletion was marked as ‘− 1’ to construct the linkage map
USA) was used to measure absorbance at 405 nm. The test from the F ­ 2 population. All primer pairs were synthesized by

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Sangon Biological and Engineering Co (Shanghai, China). Weblogo (http://​weblo​go.​three​pluso​ne.​com/) (Crooks et al.
Linkage analysis was performed using JoinMap 4.0 and 2004).
Mapchart 2.3.2 software (Voorrips 2002; Van Ooijen 2006).

Statistical analysis
RNA extraction and quantitative real‑time PCR
(qRT‑PCR)
Genetic analysis is based on the combination of phenotypic
investigation results and ELISA OD value. There were three
The TuMV-inoculated and non-inoculated leaves from
biological and three technical replicates for other experi-
B80124 and B80450 were placed in a sterilized 2 ml cen-
ments and/or measurements unless it is specified for a spe-
trifuge tube, quickly frozen in liquid nitrogen, and stored
cific experiment. The statistical analysis was performed
at − 80 °C. Total RNA was extracted using the Plant Total
using the Statistical Package for Social Sciences (SPSS,
RNA Extraction Kit (GeneBetter, Beijing, China). Reverse
2013; TBtools, 2018). All genes mentioned in this article
transcription was conducted using the E ­ asyScriptR One-
can be found in the BRAD with gene ID (http://​brass​icadb.​
STEP gDNA Removal and cDNA Synthesis SuperMix Kit
cn/).
(TransGen, Beijing, China). Real-time PCR was performed
Δ(SNP/InDel_index) is calculated as follows:
using ChamQ Universal SYBR qPCR Master Mix (Vazyme
Biotech Co., Ltd., Nanjing, China) on the CFX96™ Real- Maa
SNP/InDel_index(aa)= . (1)
Time System (Bio-Red, Hercules, California, USA). Rela- Maa + Paa
tive expression levels were calculated using the ­2−ΔΔCT
In Eq. 1, Maa represents the depth of aa pool from female
method (Livak and Schmittgen 2001). The primers were
parent, Paa represents the depth of aa pool from male parent.
designed for qRT-PCR are listed in Table S4.
Mab
SNP/InDel_index(ab)= . (2)
Transcriptome analysis for screening candidate Mab + Pab
genes In Eq. 1, Mab represents the depth of ab pool from female
parent, Pab represents the depth of ab pool from male parent.
The TuMV-resistant line ‘B80124’ and the TuMV-suscep-
tible line ‘B80461’ were used for transcriptome analysis. Δ(SNP/InDel_index) = SNP/InDel_index
(3)
B80124CK and B80461CK were not inoculated with the (aa) − SNP/InDel_index(ab).
TuMV C4 isolate, while B80124 and B80461 were inocu-
lated with the TuMV C4 isolate. The TRIzol reagent method
was used to extract the total RNA, and the quality of the
RNA was detected using Agilent 2010. RNA sequencing
was processed by combinatorial Probe-Anchor Synthesis Results
(cPAS), and a 150 bp sequencing read length was obtained.
The quality of the sequencing data was filtered using the Phenotypic investigation and genetic analysis
FASTQ Trimmer. Alignment of the clean reads and the B.
rapa reference genome V3.0 was conducted using HISAT2, F1 was obtained from the resistant parent P ­ 1 (B80124) × the
as described in our previous study (Lu et al. 2022). susceptible parent ­P2 (B80450), and F ­ 1 was selfed to obtain
­F2 populations. The ­P1, ­P2, ­F1 and the ­F2 populations were
inoculated with the TuMV C4 isolate at the four true leaf
Characteristic analysis of the candidate gene
stage, and the resistance/susceptibility was detected in com-
bination with the phenotypic investigation and DAS-ELISA
Several characteristics were detected about the candi-
test. Compared with the non-inoculated control, the phe-
date gene in this study, such as the phylogenetic analysis
notype of the TuMV-inoculated lines, the resistant parent
by MEGA 6.0 (Tamura et al. 2013), the hydrophilia and
(B80124), did not show the obvious symptoms (Fig. 1A),
hydrophobicity analysis by Protscale (https://​web.​expasy.​
and the susceptible parent (B80450) showed the typical
org/​prots​cale/) (Gasteiger et al. 2005), the subcellular loca-
TuMV infection symptoms (leaf shrinking and mosaic
tion analysis by Plant-mPLoc (http://w ​ ww.c​ sbio.s​ jtu.e​ du.c​ n/​
leaves) (Fig. 1B). The OD values of the B80124 were lower
bioinf/​plant-​multi/) (Chou and Shen 2010), the amino acid
than the average value of the blank control (Table S1), indi-
composition analysis by PredictProtein (https://​predi​ctpro​
cating that the results from the phenotypic investigation
tein.​org/) (Bernhofer et al. 2021), the protein second and 3D
were consistent with those from the DAS-ELISA test and
structures by Phyre2 (http://​www.​sbg.​bio.​ic.​ac.​uk/​phyre2/)
that B80124 was resistant to the TuMV C4 isolate. The OD
(Kelley et al. 2015), and the sequence logos analysis by

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Fig. 1  Phenotypic investigation of TuMV (pathotype C4)-challenged (b), there were 20 plants in each treatment; and F
­ 1 line (­F1CK, non-
Brassica rapa. The resistant parent P ­ 1 (B80124CK, non-inoculated inoculated TuMV; F ­ 1, inoculated TuMV), there were 48 plants in this
TuMV; B80124, inoculated TuMV) (a), the susceptible parent ­ P2 treatment (c)
(B80450CK, non-inoculated TuMV; B80450, in-occulated TuMV)

Table 1  Genetic analysis of the Materials Numbers Expectation ratio Chi square value
two parent lines, F
­ 1, and ­F2
Resistant Susceptible Total

B80124 17 0 17 3:1 𝜒 2 = 1.175 < 𝜒0.05

2
 = 3.841
B80450 0 17 17
F1 45 0 45
F2 362 102 464

Table 2  Mapping of the sequencing data by BSA-seq and the refer- Resistant gene primary mapping by bulk
ence genome segregation analysis sequencing (BSA‑seq)
Pool ID Total reads Mapped (%) Cover degree (%)
The clean reads were obtained by filtering the raw reads,
B80124 51,617,994 98.26 91.92
and the sequencing output data was evaluated (Table S2).
B80450 54,511,794 98.28 92.65
Brassica rapa reference genome V3.0 was used to align the
R-pool 136,512,246 98.14 92.55
clean reads, and the coverage degrees were all above 90%
S-pool 137,471,012 97.96 92.12
(Table 2), indicating that the sequencing data were reli-
BSA-seq bulk segregant analysis sequencing, Pool ID names of test able. In addition, the sequencing depths of the data were
samples, Total reads total number of clean reads, two terminal sep- analyzed (Table S3), showing an average coverage depth
arate statistics, that is, read1 and read2 are recorded as two reads, of about 29 × and the genome coverage of about 94%. The
Mapped (%) percentage of clean reads mapped to the reference
genome in all clean reads, Cover degree (%) two terminal sequences candidate regions were evaluated using BSA-seq. SNPs and
were located on the reference genome, and the distance agreed with Indels were filtered, and respectively 771,931 and 271,923
the length distribution of the sequencing fragments high-quality credible SNPs and Indels, respectively, were
obtained. Correlation analysis was performed according to
SNP-index and Indel-index methods, and Δ(SNP-index)
values of the B80450 were much higher than those of the statistics showed that when the confidence was 0.99. One
blank control (Table S1), implying susceptibility to B80450. region was obtained at 22.25–25.23 Mb on A06, including
Similarly, there were no obvious TuMV-infected symptoms 543 genes (267 genes of non-synonymous mutation sites)
­ 1 lines after inoculation with the TuMV C4 isolate
in the F (Fig. 2A). Identically, Δ(Indel-index) statistics showed that
(Fig. 1C, Table S1), suggesting that the resistant gene was one region was also obtained at 22.07–25.03 Mb on A06,
dominant in this study. Combined with the phenotypic inves- including 541 genes (54 genes with frameshift mutation
tigation and DAS-ELISA test of the ­F2 populations (464 sites) (Fig. 2B). The intersection of the candidate regions
individuals), 362 individuals were resistant, and 102 indi- corresponding to SNPs and Indels revealed an intersection
viduals were susceptible, which showed that the separation at 22.25–25.03 Mb.
ratio of resistant and susceptible in ­F2 populations was 3:1
(Table 1). Fine mapping of the resistance gene

Based on BSA-seq analysis, 26 pairs of SNP markers were

designed based on the genome sequences of the B80124

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Fig. 2  The Δ(SNP-index) graph

(a) and the Δ(Indel-index)
graph (b) of two extreme pools.
The black line is the average
value of Δ(SNP/Indel-index)
in a sliding window of 1 Mb.
The red/blue lines represent
statistical confidence intervals
p < 0.01/0.05. Candidate region
on A06 chromosome (red
box) was identified in Δ(SNP-
index) methods (top panel) at
22.25–25.23 Mb and Δ(Indel-
index) method (lower panel) at
22.07–25.03 Mb

Fig. 3  Fine mapping of the dominant resistance gene to TuMV in score > 3 indicates linkage (b); Fine mapping of the resistant gene.
329 individuals of ­F2 population (B80124 × B80450). Numbers in left The resistant gene was delimited to an interval between the A06S11–
show the genetic distances between adjacent markers (cM), the right A06S14 markers, with an estimated length of 330 kb. 8 R-genes were
represents the physical location on chromosome A06 (bp)  (a); The annotated in this region based on the reference genome sequence (c)
two markers labelled in red denoted the resistant gene position, LOD

and B80450 lines. However, only 15 pairs of SNP mark- SNP markers were further genotyped in 329 F
­ 2 individuals
ers showed polymorphisms among the parent lines and for linkage analysis (SNP marker sequence in Table S5).
­F 1 (Table S4). The SNP markers were selected with no Genotyping of the 15 SNP markers in 329 individuals of
segregation distortion, and markers with more than 25% ­F2 population is shown in Table S6. The dominant resist-
missing data were also excluded. Then, the 15 pairs of ance gene was mapped to the 23.72–24.05  Mb region

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Table 3  Genes related to disease resistance in the candidate region

Gene IDs Egg annotations Nr annotations

BraA06g035110.3C Signal transduction mechanisms LRR receptor-like serine/threonine-protein kinase RCH1

BraA06g035130.3C Signal transduction mechanisms Disease resistance protein (CC-NBS-LRR class) family
BraA06g035180.3C Signal transduction mechanisms Disease resistance protein (CC-NBS-LRR class) family
BraA06g035230.3C Signal transduction mechanisms Disease resistance protein (CC-NBS-LRR class) family
BraA06g035250.3C Putative defensin-like protein Putative defensin-like protein 165
BraA06g035340.3C Signal transduction mechanisms Probable LRR receptor-like serine/threonine-protein
kinase At5g48740
BraA06g035410.3C Defense mechanisms B-cell receptor-associated protein 31-like protein
BraA06g035420.3C Cleavage site for pathogenic type III effector Defense protein-like protein
avirulence factor Avr

between the SNP marker markers A06S11 and A06S14

(Fig. 3a, b), with a likelihood of odd (LOD) > 20. The
candidate region was narrowed to 330 kb. In addition,
another 20 pairs of SNP markers were also designed in
the 330 kb, unfortunately, no recombinant single plant was
found. There were 67 genes in the 330 kb candidate region,
and the homologous genes between Arabidopsis thaliana
and B. rapa were searched, including eight genes related
to disease resistance (Table 3), which were considered as
the candidate genes for TuMV resistance.

Transcriptome analysis for further screening

the candidate genes

The TuMV-resistant line ‘B80124’ and the TuMV-suscep-

tible line ‘B80461’ were used for transcriptome sequenc-
ing in our previous study to analyze the influence of
TuMV infection on volatile organic compounds, which
could help us to further screen the candidate genes in this
study. The expression level analysis of the eight candidate
genes showed that the two genes (BraA06g035110.3C and
BraA06g035250.3C) were not expressed, which was veri-
fied by the qRT-PCR analysis (data not shown). Expres-
sion-level analysis of the other six candidate genes was
used for the heatmap analysis after inoculating with the Fig. 4  The relative expression levels analysis of the six candidate
TuMV C4 isolate (Fig. 4). Compared with B80124CK, the genes by transcriptome data. The resistance lines B80124 (inoculated
TuMV) and B80124CK (non-inoculated TuMV), and the susceptible
relative expression levels of two genes (BraA06g035180.3C lines B80461 (inoculated TuMV) and B80461CK (non-inoculated
and BraA06g035410.3C) were not show the change TuMV), the color indicates the expression amount of this gene in dif-
obviously; the relative expression levels of two genes ferent experimental samples
(BraA06g035130.3C, BraA06g035230.3C) were upregu-
lated, and those of the other two genes (BraA06g035340.3C and BraA06g035410.3C) was downregulated in B80461
and BraA06g035420.3C) were downregulated in B80124 (Fig. 4). Compared with non-inoculated B. rapa, one gene
(Fig. 4). Furthermore, the susceptible line (B80461) was (BraA06g035130.3C) was upregulated after inoculat-
also analyzed before and after inoculation TuMV C4 isolate. ing the resistant line (B80124) with TuMV; however, this
Compared with B80461CK, the relative expression levels of gene was downregulated in the susceptible line (B80461).
three genes (BraA06g035180.3C, BraA06g035230.3C, and Nevertheless, the BraA06g035420.3C gene was opposed
BraA06g035420.3C) were upregulated, and those of the to BraA06g035130.3C, which was downregulated in the
other three genes (BraA06g035130.3C, BraA06g035340.3C resistant line (B80124) and susceptible line (B80461) after

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inoculating with TuMV. The relative expression levels of lines. In addition, there were two deletion mutations, that
the other genes (BraA06g035230.3C, BraA06g035340.3C, could have led to the frameshift in the ‘B80450’ line, located
and BraA06g035410.3C) were consistent in the non-inoc- 864 (deleting 8 nucleotides, AGC​CTA​AG) and 947 (delet-
ulated and inoculated plants in both the resistant and sus- ing two nucleotides, AA), and these mutations may directly
ceptible lines; thus, these genes were not considered to be result in the loss of the TuMV resistance in B. rapa. Finally,
related to TuMV resistance. However, BraA06g035180.3C there was an insertion in the ‘B80450’ line, located at 685
was more highly expressed in B80461 than in B80124. (inserting 12 nucleotides, CTC​CTG​ATG​TGG​), which only
As shown in Table 3, three genes (BraA06g035130.3C, added four amino acids, and rarely changed the functions.
BraA06g035180.3C, and BraA06g035420.3C) were R genes; In conclusion, the frameshift mutations were the main cause
based on the relative expression levels of the three genes, for the loss of resistance in the ‘B80450’ line, and the non-
BraA06g035130.3C was identified as the final candidate synonymous mutations may also play an important role. The
gene. amino acid sequence alignment is shown in Fig. S2.

Characteristics analysis of BraA06g035130.3C

Sequence identification of BraA06g035130.3C
in lines ‘B80124’ and ‘B80450’ Eleven homologous genes were identified in six spe-
cies (three from B. rapa, two from B. oleracea, one from
BraA06g035130.3C from lines ‘B80124’ and ‘B80450’ B. juncea, one from B. napus, one from Raphanus sati-
lines was cloned to analyze the variation (Figs. 5, S1). This vus, and three from Arabidopsis thaliana) for phyloge-
gene contained two exons (first exon, 1071 nucleotides, and netic analysis by MEGA 6.0 (Fig.  6). The three genes
second exon, 138 nucleotides), and one intron (155 nucleo- (AT5G35450, AT5G43470, and AT5G48620) gathered in
tides). Compared with the ‘B80124’ line, there were two a branch that was relatively far away from the candidate
synonymous mutations in the ‘B80450’ line, i.e., ­C771A and gene. BraA06g035130.3C was clustered in a branch with
­A822C. There were also two non-synonymous mutations (the the other gene (BraA06g035180.3C) from B. rapa, which
first G859A, from GTA [aa, Val] to ATA [aa, Ile]; the second was not clearly correlated with the genes from B. oleracea,
A1089T, from AAA [aa, Lys] to ATA [aa, Ile]) between B. juncea, and B. napus (Fig. 6), indicating that the candi-
BraA06g035130.3C from lines the ‘B80124’ and ‘B80450’ date should not be particularly conserved in Brassica crops.

Fig. 5  The alignment analysis of the BraA06g035130.3C cDNA nucleotide sequences partly from the resistance line B80124 and the susceptible
line B80450. The dark part indicated the same sequence, and the light blue part indicates that the sequence was different

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Planta (2022) 256: 66 Page 9 of 13  66

resistance rpp13-like protein structure, which belonged to

the protein kinase superfamily protein, the 3D structure
of BraA06g035130.3C was constructed, and from the 3D
structure of BraA06g035130.3C (Fig. 7A, B), six β-strands
formed a barrel structure, which could help capture patho-
gens when the host is under biotic stress. In addition, the
coiled-coil domain also contributed to resistosome pen-
tamerization by forming an α-helical barrel that interacted
with the leucine-rich repeat and winged-helix domains.
Furthermore, structural remodeling and fold switching dur-
ing activation released the very N-terminal amphipathic α
helix to form a funnel-shaped structure that was required
for the plasma membrane association, cell death trigger-
ing, and disease resistance, offering clues to the biochemi-
cal function of a plant resistosome. In addition to protein
Fig. 6  The phylogenetic analysis of BraA06g035130.3C, eleven secondary and 3D structure analyses, the protein sequence
homologous genes were identified in six species logos were also analyzed to identify the amino acid bias
of the candidate gene. The BraA06g035130.3C protein
The Protscale software was used to predict the amino acid sequence acted as the query, and 273 to 341 amino acids
hydrophilia and hydrophobicity of the BraA06g035130.3C of the BraA06g035130.3C homology proteins from six
protein (Fig. S3). Moreover, the subcellular location predic- species were analyzed (Fig. 7C). It encoded a conserved
tion analysis of BraA06g035130.3C was conducted using hydrophobic core structure and contained the missing key
Plant-mPLoc software (Fig. S3), indicating that this gene domains. The 274/285/288/302/303 sites showed obvious
was expressed in the cell membrane and nucleus. The cell amino acid bias, and the 293–295/316/340 sites did not. In
membrane is selectively permeable, separating the cyto- the co-evolution process of resistance genes from Brassica
plasm from its surroundings, and is known as the cell inner crops, some amino acids were selected to transition to the
membrane in prokaryotes with two membranes. In addition, key amino acids for function, and other amino acids were
the nucleus is the most obvious organelle in any eukary- not selected for amino acid bias. Analyzing the resistance
otic cell and is a membrane-bound organelle surrounded by protein sequence logos helped screen the key amino acids
double membranes, containing most of the cell’s genetic for resistance functions.
material and communicating with the surrounding cytosol
via numerous nuclear pores. To determine the amino acid
composition of the BraA06g035130.3C protein, Predict- Discussion
Protein software was used for the analysis (Fig. S3). The
BraA06g035130.3C protein contained 426 amino acids (20 Turnip mosaic virus (TuMV) can infect the Brassica crops,
kinds), and the top six amino acids were Gly, Leu, Ser, Pro, and cause some obvious symptoms, including plant dwarf-
Ala, and Arg, comprising half of the total amino acids. ism, leaf wrinkle, and mosaic Compared with chemical
control, screening for resistance genes is environmentally
Three‑dimensional (3D) structure and sequence friendly, direct, and efficient and can not only ensure the bal-
logo analysis of the candidate gene ance of the country’s supply but also ensure the safety of the
vegetables. Therefore, excavating new resistance genes for
The protein secondar y and 3D str uctures of TuMV resistance in B. rapa is of important scientific signifi-
BraA06g035130.3C were predicted by Phyre2 (Fig.  7), cance and application value for the improvement of TuMV
including 8 β-strands, 12 α-helices, and 12 extended resistance traits and molecular design breeding for Brassica
loops, and all 3D structure predictions indicated that crops. This study identified a new potential dominant resist-
BraA06g035130.3C encoded a defense response pro- ance gene on chromosome A06 to the TuMV strain C4 iso-
tein, which is involved in disease resistance signal- late in ­F2 populations using KASP technology.
ing (Zhang et  al. 2010). Two deletion mutations could There are two main resistance regulation mechanisms
lead to a frameshift compared to the 3D structure of (passive and active) for plants to fight viruses. The resistance
BraA06g035130.3C from the resistance line (A). The was inherited dominantly. Different inheritance mechanisms
3D structure of BraA06g035130.3C from the suscepti- of resistance to TuMV have been reported, depending on the
ble line had some great changes, including the loss of strains tested, the resistant and susceptible parents used, and
four β-strands and one α-helix (B). Based on the disease the sensitivity of the virus resistance tests (Walsh and Jenner

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Page 10 of 13 Planta (2022) 256: 66

Fig. 7  The 3D structure analysis of the BraA06g035130.3C protein and 1 α-helices lost (b). The sequence logos analysis were conducted
in the resistance line B80124 and the susceptible line B80450. Com- from 273 to 341 amino acids of the BraA06g035130.3C proteins in
pared the 3D structure of BraA06g035130.3C from the resistance line the homologous genes, where it contained the missing key domains
(a), the 3D structure has some great changes, including 4 β-strands (c)

2002). In recent years, there have been many dominant linkage map (Wang et al. 2011a, b). TuRB07 mapped on
genes mapped and cloned for TuMV resistance in Brassica the telomere of the short arm of chromosome A06 (1.5 Mb)
crops, such as TuRB01 to TuRB05 in B. napus (Walsh et al. (Jin et al. 2014). Therefore, BraA06g035130.3C is a new
1999; Jenner et al. 2000, 2002, 2003; Hughes et al. 2002), potential dominant resistance gene to TuMV C4 in B. rapa.
ConTR01 in B. rapa (Rusholme et al. 2007), TuRBCH01 In general, the dominant active resistance mechanism is
in B. rapa (Wang et al. 2011a, b), TuRB07 in B. rapa (Jin mediated by gene silencing and R genes, and R genes con-
et  al. 2014), TuRB01b in B. rapa (Lydiate et  al. 2014), tain some special domains, such as NBS-LRR or CC-NBS-
TuRBCS01 in B. rapa (Li et al. 2015), and TuRBJU01 in B. LRR (Li et al. 2018). Potyviruses can use the replication
juncea (Nyalugwe et al. 2015). Some single dominant genes and translation factors from host plants for reproduction,
have been mapped in Brassica crops; TuRB01, TuRB03, and and the mutation of these factors may result in resistance to
TuRB01b have been mapped on chromosome A06 in a simi- the virus (Wang and Krishnaswamy 2012). In this study, the
lar interval, and two SSR markers flanking TuRB03 have candidate gene was located between SNP markers A06S11
been mapped at 105.5 and 116.2 cM in the BnaDYDH A06 and A06S14 by constructing a gene linkage map. There were

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Planta (2022) 256: 66 Page 11 of 13  66

67 genes in the 330 Kb candidate region, and eight genes mutations may directly result in the loss of TuMV resistance.
related to disease resistance were found. By analyzing the In conclusion, BraA06g035130.3C is a new potential domi-
expression of eight candidate genes through transcriptome nant resistance gene to TuMV in B. rapa. Further functional
data, BraA06g035130.3C was finally determined as the analysis of these candidate genes could help elucidate the
potential gene and was determined to be an R gene with a regulatory mechanism of TuMV resistance in B. rapa. As an
CC-NBS-LRR structure. Many studies have demonstrated increasing number of resistance genes are being identified
the role of NBS-LRR genes in resistance to diverse patho- in Brassica crops, we need to systematically determine and
gens (Kachroo et al. 2006). The C-terminal LRR domain is identify the details of each resistance gene so that researchers
thought to be involved in the specific recognition of patho- can better use these genes. The following measures should be
gen effector molecules and to regulate signal transduction taken. First, the functional markers should be developed based
positively or negatively (Rairdan and Moffett 2006; Weaver on the resistance genes for molecular marker-assisted breed-
et al. 2006). Moreover, the subcellular location prediction ing. Whether these resistance genes have functional mark-
analysis of BraA06g035130.3C was conducted using Plant- ers should be systematically summarized, and if so, whether
mPLoc software, indicating that this gene was expressed in they are good should be determined; if not, functional mark-
the cell membrane and nucleus. Mechanisms induced by ers need to be developed according to gene characteristics
plant R genes, most of which encode NBS-LRR proteins, are (Walsh et al. 1999; Cheng et al. 2016). Second, resistance
initiated by the recognition of pathogen-associated molecu- genes should be pyramided for durable resistance. According
lar patterns in plant transmembrane receptors, thereby induc- to the resistance of different genes to various TuMV isolates,
ing active defense responses (DeYoung and Innes 2006; multiple resistance genes can be aggregated into one line by
Marone et al. 2013). cross-breeding and backcrossing. Gene aggregation would be
In addition to the dominant resistance genes, the reces- much easier if these genes had functional markers to assist in
sive genes also play an important role in defending against screening (Jenner and Walsh 1996; Hughes et al. 2002; Lind-
viruses, and the eukaryotic initiation factor 4E (eIF4E) hout 2002; Gładysz and Hanus-Fajerska 2009). Third, TuMV
family genes are significant genes, including eIF4E, eIF4G, isolates with serious production hazards should be systemati-
eIF(iso)4E, and eIF(iso)4G, that have spectrum resistance to cally identified. In the main producing areas of vegetables, the
potyviral infection (Dinkova et al. 2016). Generally, reces- hazard of TuMV was extensively investigated, and samples
sive gene resistance results from natural mutation and the infected with TuMV were collected for the identification and
long co-existence of host plants and potyviruses may pro- analysis of TuMV isolates, which could enable comprehen-
mote the emergence of virulent isolates in Brassica crops sive strategies for TuMV prevention and control, including
(Sanfaçon 2015). Hence, excavating new resistance genes targeted TuMV-resistant varieties and chemical control. In
is important. Until now, four eIF family genes have been further studies, we can focus on the different varieties of B.
mapped and cloned in Brassica crops, including ConTR01 rapa to take full advantage of the bolting genetic information
[eIF(iso)4E.c] (Rusholme et  al. 2007), retr01/retr02 for breeding.
[eIF(iso)4E. a] (Rusholme et al. 2007; Kim et al. 2013), and
retr03 (eIF2Bβ) (Shopan et al. 2017). After functional inter- Author contributions statement  Data curation, XL, ZL, SW,
action analysis, the viral protein of genome-linked (VPg) and WH; Formal analysis, XL, ZL, SW, and WH; Investi-
from TuMV interacted with the eIF, which was helpful in gation, SZ (Shifan Zhang), FL, and HZ; Methodology, RS;
accelerating virus reproduction in host plants (Michon et al. Supervision, GL; Writing-original draft, XL and SZ (Shu-
2006). In addition, the functions of the eIF-VPg interaction jiang Zhang); Writing-review and editing, GL, JZ, and SZ
were summarized in three points: (1) TuMV VPg is a cap (Shujiang Zhang). All authors read and approved the final
protein that plays an important role in viral RNA translation manuscript.
and accelerates protein translation in the host (Contreras-
Paredes et al. 2013); (2) the interactions between eIF4E and
VPg facilitate virus movement from one tissue to other tis- Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00425-0​ 22-0​ 3981-5.
sues, resulting in whole plant infection (Wang and Krish-
naswamy 2012); and (3) the interactions of VPgs and eIFs Acknowledgements  We thank LetPub (www.​letpub.​com) for its lin-
restrain specific or entire cellular translation and boost viral guistic assistance during the preparation of this manuscript.
RNA translation (Leonard et al. 2000).
The CDs of BraA06g035130.3C were cloned and aligned Funding  This work was funded by the State Key Laboratory of North
between the B80124 line and the B80450 line. There were China Crop Improvement and Regulation, the Beijing Natural Science
Foundation (6212030), the National Natural Science Foundation of
two deletion mutations that led to a frameshift in line China (32102373), and China Agriculture Research System (CARS-
‘B80450,’ located 864 (deleting eight nucleotides, AGC​ 23-A-14). This work was performed at the Key Laboratory of Biology
CTA​AG) and 947 (deleting two nucleotides, AA), and these

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and Genetic Improvement of Horticultural Crops, Ministry of Agri- Gao H, Zeng Q, Zhang Z, Zhao Z, Pei Y, Liu S, Li Y, Liu X, Song X, Li
culture, Beijing, China. Q (2016) The development of molecular markers closely linked to
TuMV resistance gene TuRBCS01 in Chinese cabbage (Brassica
Data availability statement  The date presented in this study are openly campestris ssp. pekinensis). J Agric Biotechnol 24(2):196–205
available in NCBI with BioProject number is PRJNA764554 (https://​ Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch
www.​ncbi.​nlm.​nih.​gov/​search/​all/?​term=​PRJNA​764554). A (2005) Protein identification and analysis tools on the ExPASy
server. In: The proteomics protocols handbook, pp 571–607
Gładysz K, Hanus-Fajerska E (2009) Evaluation of the infectivity of
Declarations  selected Turnip mosaic virus isolates towards white cabbage cul-
tivars. Folia Hortic 21(1):129–138
Conflicts of interest  All the authors declare that they have no conflicts Hughes SL, Green SK, Lydiate DJ, Walsh JA (2002) Resistance to Tur-
of interests. nip mosaic virus in Brassica rapa and B. napus and the analysis of
genetic inheritance in selected lines. Plant Pathol 51(5):567–573.
https://​doi.​org/​10.​1046/j.​1365-​3059.​2002.​00755.x
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