construction of a sequence-based bin map and mapping of...

Construction of a sequence-based bin map and mappingof QTLs for downy mildew resistance at four developmentalstages in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Shuancang Yu . Tongbing Su . Shenghua Zhi . Fenglan Zhang .

Weihong Wang . Deshuang Zhang . Xiuyun Zhao . Yangjun Yu

Received: 8 September 2015 / Accepted: 23 March 2016 / Published online: 31 March 2016

� Springer Science+Business Media Dordrecht 2016

Abstract Specific-locus amplified fragment

sequencing is a high-resolution method for genetic

mapping, genotyping, and single nucleotide polymor-

phism (SNP) marker discovery. Previously, a major

QTL for downy mildew resistance, BraDM, was

mapped to linkage group A08 in a doubled-haploid

population derived from Chinese cabbage lines

91–112 and T12–19. The aim of the present study

was to improve the linkage map and identify the

genetic factors involved in downy mildew resistance.

We detected 53,692 high quality SLAFs, of which

7230 were polymorphic, and 3482 of the polymorphic

markers were used in genetic map construction. The

final map included 1064 bins on ten linkage groups

and was 858.98 cM in length, with an average inter-

locus distance of 0.81 cM.We identified six QTLs that

are involved in downy mildew resistance. The four

major QTLs, sBrDM8, yBrDM8, rBrDM8, and

hBrDM8, for resistance at the seedling, young plant,

rosette, and heading stages were mapped to A08, and

are identical to BraDM. The two minor resistance

QTLs, rBrDM6 (A06) and hBrDM4 (A04), were

active at the rosette and heading stages. The major

QTL sBrDM8 defined a physical interval of*228 Kbon A08, and a serine/threonine kinase family gene,

Bra016457, was identified as the possible candidate

gene. We report here the first high-density bin map for

Chinese cabbage, which will facilitate mapping QTLs

for economically important traits and SNP marker

development. Our results also expand knowledge of

downy mildew resistance in Chinese cabbage and

provide three SNP markers (A08-709, A08-028, and

A08-018) that we showed to be effective when used in

MAS to breed for downy mildew resistance in B. rapa.

Shuancang Yu and Tongbing Su have been contributed equally

to this work.

Electronic supplementary material The online version ofthis article (doi:10.1007/s11032-016-0467-x) contains supple-mentary material, which is available to authorized users.

S. Yu � T. Su � S. Zhi � F. Zhang (&) �W. Wang � D. Zhang � X. Zhao � Y. YuBeijing Vegetable Research Center (BVRC), Beijing

Academy of Agriculture and Forestry Science (BAAFS),

Beijing 100097, People’s Republic of China

e-mail: [email protected]

S. Yu � T. Su � S. Zhi � F. Zhang � W. Wang �D. Zhang � X. Zhao � Y. YuKey Laboratory of Biology and Genetic Improvement of

Horticultural Crops (North China), Ministry of

Agriculture, Beijing, People’s Republic of China

S. Yu � T. Su � S. Zhi � F. Zhang � W. Wang �D. Zhang � X. Zhao � Y. YuBeijing Key Laboratory of Vegetable Germplasm

Improvement, Beijing 100097, People’s Republic of

China

123

Mol Breeding (2016) 36:44

DOI 10.1007/s11032-016-0467-x

http://dx.doi.org/10.1007/s11032-016-0467-xhttp://crossmark.crossref.org/dialog/?doi=10.1007/s11032-016-0467-x&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1007/s11032-016-0467-x&domain=pdf

Keywords Brassica rapa ssp. pekinensis � Bin map �Downy mildew � Developmental stage � Quantitativetrait loci � SNP

Introduction

Chinese cabbage is a relatively new crop, first

recorded in China in the eighteenth century, that is

now widely cultivated in Asia (Hirai and Matsumoto

2007). In China, cultivation of Chinese cabbage

accounts for *15 % of the total area devoted tovegetable production, ranking first in terms of area and

yield for cultivated vegetables. Chinese cabbage is

also one of the most important vegetable crops in

Japan and Korea. The importance of Chinese cabbage

in agriculture has driven efforts to develop tools for

genetic and genomic studies in B. rapa ssp. pekinensis.

Genetic linkage maps are very useful tools for

studying genome structure and evolution, identifying

introgression between different genomes, and localiz-

ing genes of interest in plants (Quijada et al. 2007).Over

the past 25 years, many genetic maps have been

constructed for B. rapa, and have incorporated various

types of molecular markers; these include random

amplified polymorphic DNA (RAPDs), restriction

fragment length polymorphisms (RFLPs), amplified

fragment length polymorphisms (AFLPs), simple

sequence repeats (SSRs), and sequence-tagged sites

(STS). More recently, markers based on insertions and

deletions (InDels) and SNPs (single nucleotide poly-

morphisms) have also been used to generate genetic

linkage maps (Song et al. 1991; Chyi et al. 1992;

Teutonico and Osborn 1994; Nozaki et al. 1997; Lu

et al. 2002; Lowe et al. 2004; Kim et al. 2006; Suwabe

et al. 2006; Soengas et al. 2007; Choi et al. 2007; Li

et al. 2009; Kapoor et al. 2009; Yu et al. 2009; Xu et al.

2010; Wang et al. 2011a, b, 2014; Chung et al. 2014).

Evenwith such a large number of genetic linkagemaps,

there are few that mainly consist of sequence-tagged

markers.

Recently, the construction of genetic maps has

benefited from the development of new types of

molecular markers that rely on automated sequencing

and genotyping technologies. Due to the availability of

the B. rapa reference genome sequence (Wang et al.

2011a, b), it is possible to carry out high-throughput

sequencing and subsequently check the genome-wide

distribution of DNA polymorphisms across theB. rapa

chromosomes. Specific-locus amplified fragment

sequencing (SLAF-seq) was developed based on

high-throughput, next-generation sequencing technol-

ogy (Sun et al. 2013). This method has several obvious

advantages, such as high-throughput, high accuracy,

low cost, and short cycle times, which allow the

sequencing results to be used directly for molecular

marker development (Sun et al. 2013). To date, this

technology has been used in haplotype mapping,

genetic mapping, linkagemapping, and polymorphism

mapping (Wang et al. 2012; Chen et al. 2013; Zhang

et al. 2013).

Breeding for disease resistance is one of the primary

objectives in most crop-breeding programs. Downy

mildew, caused by Hyaloperonospora parasitica, is a

destructive disease of crops in the family Brassicaceae.

In China, H. parasitica causes one of the most severe

foliar diseases of Chinese cabbage. However, relatively

few studies on resistance to downy mildew infection

have focused on Chinese cabbage. Diverse accessions

of Chinese cabbage have been screened to identify

sources of downy mildew resistance (Yuen 1991; Silue

et al. 1996), and resistance was found to be determined

by a single dominant gene (Niu et al. 1983). Based on a

genetic map derived from a doubled-haploid (DH)

population, a major QTL, BraDM, controlling seedling

resistance, was identified and localized to the A08

linkage group of B. rapa (Yu et al. 2009). In order to

establish marker-assisted selection (MAS) for BraDM,

the sequence-characterized amplified region marker

SCK14-825, and SSR markers kbrb058m10-1 and

kbrb006c05-2, were designed and found to be effective

in MAS (Yu et al. 2011). For adult-plant resistance to

downy mildew, a single dominant locus, designated

BrRHP1, was mapped to linkage group A01 indepen-

dent of BraDM. Using the same population described

by Yu et al. (2009), downy mildew resistance was

investigated at four developmental stages, which

demonstrated that the inheritance of downy mildew

resistance varied slightly during plant development

(Zhang et al. 2012). We hypothesize that the genetic

systems controlling resistance to downy mildew are

different in seedlings and adult plants.

In this study, we used the recently-developed

SLAF-seq approach for large-scale discovery of

DNA sequence polymorphism in B. rapa. We con-

structed a high-density bin map that offers abundant

resources for QTL mapping and SNP marker

44 Page 2 of 12 Mol Breeding (2016) 36:44

123

development. To further investigate the genetic factors

involved in downymildew resistance, the high-density

bin map was used to locate QTLs for resistance to

downy mildew at various stages of growth, and SNP

markers based on the KASPTM SNP genotyping

system were developed for high-throughput molecular

marker-assisted breeding.

Materials and methods

Plant materials

The mapping population DH100 was comprised of

100 microspore culture derived DH lines (DH100-1 to

DH100-100) from a cross between two Chinese

cabbage lines 91–112 and T12–19. The maternal

parent (91–112), an inbred line self-pollinated for nine

generations, is susceptible to downy mildew infection.

The paternal parent (T12–19) is a DH line derived by

microspore culture from an F1 cross between ‘Orange

Queen’ and Beijing local variety (‘Dabaikou’), and is

resistant to downymildew. A BC2 population from the

cross of downy mildew-susceptible DH line DH100-

60 (as the recurrent parent) and resistant DH line

DH100-88, consisting of 164 individuals, was used to

validate the SNP markers linked to the major QTL.

Young leaves from the DH100 population, the BC2population, and lines 91–112 and T12–19 were

collected from 2-week-old seedlings. Plant material

was flash frozen in liquid nitrogen and stored at

-80 �C until DNA extraction was performed.

Downy mildew resistance phenotypic evaluation

The pathogen isolate used in this study was collected

from the experimental field at theBeijingVegetableRe-

search Center, Beijing, China. The inoculum was

maintained on the susceptible host ‘A8 Jian’, a Chinese

cabbage inbred line that is highly susceptible to downy

mildew.

As previously described (Yu et al. 2009), seedling

inoculation tests were carried out in the greenhouse.

Two-week-old seedlings were inoculated by spraying

with a conidial suspension of the P. parasitica isolate.

Three inoculation replicates were conducted, with ten

plants per replicate (n = 30). Phenotypic data was

collected, and disease indexes (DI) for each line were

calculated as described in Yu et al. (2009).

For downy mildew resistance at the young plant,

rosette, and heading stages, plants were grown in an

open field, and the resistance was evaluated using the

leaf disk test as described by Zhang et al. (2012). The

third pair of fully expanded leaves were removed from

the plants for downy mildew inoculation. Ten 20-mm

diameter leaf disks were cut and placed in clear plastic

plates (45 cm 9 30 cm 9 4 cm) with the adaxial disk

surface in direct contact with 1 % agar containing

50 ppm enzimidazole. A spraying process was used to

infect the leaf disks with downy mildew. At least ten

leaf disks per accession were tested, and three

replicates were conducted for each treatment. Pheno-

typic data were collected according to the following

rating system: 0—no host response, no sporulation

observed. 1—light necrotic flecking, no sporulation

observed. 3—heavy necrotic flecking, no sporulation

observed. 5—any host response, sparse sporulation

observed. 7—any host response, moderate sporulation

covering 1/3–2/3 of the disk. 9—any host response,

moderate to heavy sporulation covering the entire

disk. The formula for disease index (DI) calculation

was the same as described for the greenhouse exper-

iment. Results of disease testing to evaluate the

reaction of Chinese cabbage plants to downy mildew

infection at different developmental stages are shown

in Fig. 1.

SLAF library construction and high-throughput

DNA sequencing

An improved SLAF-seq strategy was utilized in our

study. We first used the reference genome of Brassica.

rapa (http://brassicadb.org/brad/index.php) (Cheng

et al. 2011) to design marker discovery experiments

by simulating in silico the number of markers pro-

duced by different restriction enzymes. A SLAF pilot

experiment was then performed, and the SLAF library

was constructed in accordance using the predesigned

scheme. The restriction enzyme RsaI (New England

Biolabs, NEB, USA) was used to digest the genomic

DNA. A single nucleotide (A) overhang was subse-

quently added to the digested fragments using Klenow

Fragment (30 ? 50 exo-) (NEB) and dATP at 37 �C.Duplex tag-labeled sequencing adapters (PAGE-pu-

rified, Life Technologies, USA) were then ligated to

the A-tailed fragments using T4 DNA ligase. Poly-

merase chain reaction (PCR) amplification was per-

formed using diluted restriction-ligation DNA

Mol Breeding (2016) 36:44 Page 3 of 12 44

123

http://brassicadb.org/brad/index.php

samples, dNTPs, Q5� High-Fidelity DNA Poly-

merase, and PCR primers (Forward primer: 50-AAT-GATACGGCGACCACCGA-30, reverse primer: 50-CAAGCAGAAGACGGCATACG-30; PAGE-puri-fied, Life Technologies). PCR products were then

purified using Agencourt AMPure XP beads (Beck-

man Coulter, High Wycombe, UK) and pooled.

Pooled samples were separated by electrophoresis on a

2 % agarose gel. Fragments ranging from 300 to 500

base pairs (with indexes and adaptors) in size were

excised and purified using a QIAquick gel extraction

kit (Qiagen, Hilden, Germany). Gel-purified products

were then diluted, and paired-end sequencing

(2 9 125 b) was performed on an Illumina HiSeq

2500 system (Illumina, Inc; San Diego, CA, USA)

according to the manufacturer’s recommendations.

Linkage map construction

All SLAF pair-end reads with clear index information

were clustered based on sequence similarity as

detected by BLAT (-tileSize = 10, -stepSize = 5).

Sequences with over 90 % identity were grouped into

a single SLAF locus as described by Sun et al. (2013).

Alleles were defined in each SLAF using the minor

allele frequency (MAF) evaluation. Because Chinese

cabbage is a diploid species, one locus contains at

most four SLAF tags, so groups containing more than

four tags were filtered out as repetitive SLAFs. In this

study, SLAFs with a sequence depth of less than ten

were defined as low-depth SLAFs and were filtered

out.

Polymorphic markers were classified into eight

segregation patterns (ab 9 cd, ef 9 e.g., hk 9 hk,

lm 9 ll, nn 9 np, aa 9 bb, ab 9 cc and cc 9 ab).

For the DH population, SLAF markers in which

segregation patterns were aa 9 bb were used for

genetic map construction. For map construction,

segregation data for SLAFs developed in this study

and 211 previously-published SSR and InDel markers

were integrated. The recombination rates between

markers were calculated using HighMap software

(Wenzel 2006) and the genetic map was constructed

using a logarithm of odds difference (LOD) threshold

C4.0 and a maximum recombination fraction of 0.4.

Map distances in centiMorgans were calculated using

the Kosambi mapping function (Kosambi 1944). Due

to the large number of markers, we obtained a very

long map with a total recombinational length of

3324.11 cM.

Fig. 1 Disease testing to evaluate the reaction to downymildew infection in Chinese cabbage plants at different

developmental stages. a Two-week old seedlings for greenhouseinoculation. b–d Chinese cabbage plants grown in the open fieldat the young plant, rosette, and heading stages, respectively.

e Example of infected susceptible plant 7 days post inoculation.f Example of leaf disk test 7 days post inoculation. g Example ofinfected leaf disk 7 days post inoculation. h Example of leafdisk from a resistant leaf 7 days after inoculation with downy

mildew sporangia


123

To improve the map, a bin map was constructed. As

described by Van Os et al. (2006), co-segregating

markers were manually identified using this display

and defined as a ‘‘filled bin’’. A bin signature

comprises the consensus segregation pattern of marker

loci which do not recombine and were thus incorpo-

rated into individual bins. The consensus genotype

data for bins were then used to calculate genetic

distances using JoinMap 4.0 software (Van Ooijen and

Voorrips 2001).

QTL analysis

LOD thresholds for determining significant QTLs

were estimated from 1000 permutation tests

(P\ 0.05; Churchill and Doerge 1994). The gen-ome-wide threshold for downy mildew resistance was

3.2, and interval mapping was used to identify putative

QTLs (Lander and Botstein 1989; Young 1996).

Subsequently, molecular markers coinciding with, or

closely flanking, the maximum QTL LOD were used

as co-factors in multiple QTL analysis. All calcula-

tions employed MapQTL 3.0 software (Van Ooijen

et al. 2002). In order to provide visual images of

marker loci genomic positions, integrated markers and

QTL were plotted using Mapchart 2.2 (Voorrips

2002).

Molecular marker design and SNP assays

SNPs were identified on the basis of alignment of

SLAF-sequenced reads generated from 91 to 112 and

T12–19 against the reference sequence of Chiifu-401-

42 (v1.5). Based on the alignment results, variants in

intervals of the target QTL were identified. Significant

variants were then selected for the Competitive Allele

Specific PCR (KASP) assay.

For each SNP, two allele-specific forward primers

and one common reverse primer were designed and

tested by the LGC (Laboratory of the Government

Chemist). By using these primers, KASP assays were

performed as described by Smith and Maughan

(2015). Fluorescent detection of the reaction products

was performed using an Omega Fluorostar scanner

(BMG LABTECH GmbH, Offenburg, Germany), and

the data were analyzed using KlusterCaller 1.1

software (KBioscience). A total of three SNP markers

were screened to combine with and improve the

resolution of the current bin map.

Results

Analysis of SLAF-seq data and SLAF markers

For map construction, the DH100 population and the

two parental lines (91–112 and T12–19) were sub-

jected to SLAF sequencing. After SLAF library

construction and high-throughput sequencing,

139.46 M paired-end reads were obtained, with each

read being 80 bp in length. The Q30 ratio was 86.45 %

and the mean guanine-cytosine (GC) content was

38.72 %.

The numbers of SLAFs in the male (T12–19)

and female (91–112) parents were 41,969 and

43,495, respectively. The average coverage for

each marker was 19.48-fold in the male parent

and 24.43-fold in the female parent. In the DH

population, the average coverage of whole reads for

the number of SLAF markers was 42,599,158, and

the number for SLAFs was 3,825,246, with an

average coverage of 11.14.

Among the 53,692 developed SLAFs that were

detected, 7230 were polymorphic, with a polymor-

phism rate of 13.47 %. As shown in Supplementary

Fig. 1, the polymorphic SLAF loci are distributed

evenly on the ten chromosomes of Chinese cabbage.

Based on segregation patterns, 5833 SLAF loci could

be used to construct a genetic linkage map. Among

these markers, 3482 high quality markers that had

[10-fold parental sequencing depth, had [80 %integrity of the SLAF tags, and harbored more than

four SNPs, were used for the genetic map

construction.

Construction of a high-density bin map

The combination of newly developed SLAFs with

previously-published SSR and InDel markers resulted

in the construction of a composite B. rapa map for the

DH100 population. A total of 3688 polymorphic loci,

including 206 previously reported molecular markers

and 3482 new SLAF markers, were assembled into 10

linkage groups with LOD values of 5.0–8.0. For bin

mapping, the co-segregating markers were manually

determined and defined as a bin, and then were

submitted to Joinmap. The final bin map included

1064 bins on the ten linkage groups (Fig. 2), and was

858.98 cM in length with an average distance of

0.81 cM between adjacent markers. To our


123

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A02Bin71 70.937A02Bin72 71.013A02Bin73 73.023A02Bin74 73.424A02Bin75 74.192A02Bin76 74.955A02Bin77 75.092A02Bin78 76.138A02Bin79 76.325A02Bin80 76.600A02Bin81 77.453A02Bin82 78.140A02Bin83 78.907A02Bin84 79.864A02Bin85 80.319A02Bin86 81.494A02Bin87 82.626A02Bin88 82.786A02Bin89 83.626A02Bin90 84.563A02Bin91 86.700A02Bin92 87.078A02Bin93 89.627A02Bin94 90.717

A02A03BIn01 0.000A03BIn02 0.184A03BIn03 0.439A03BIn04 1.255A03BIn05 1.680A03BIn06 1.947A03BIn07 2.116A03BIn08 3.157A03BIn09 3.651A03BIn10 4.102A03BIn11 4.274A03BIn12 4.578A03BIn13 4.891A03BIn14 6.273A03BIn15 6.451A03BIn16 6.803A03BIn17 7.043A03BIn18 7.293A03BIn19 8.390A03BIn20 8.872A03BIn21 9.043A03BIn22 10.310A03BIn23 10.466A03BIn24 10.613A03BIn25 10.809A03BIn26 11.504A03BIn27 11.823A03BIn28 12.335A03BIn29 12.364A03BIn30 13.404A03BIn31 13.828A03BIn32 13.977A03BIn33 14.179A03BIn34 14.403A03BIn35 14.932A03BIn36 15.161A03BIn37 16.702A03BIn38 17.361A03BIn39 17.585A03BIn40 18.657A03BIn41 19.432A03BIn42 19.921A03BIn43 20.860A03BIn44 22.210A03BIn45 22.285A03BIn46 22.780A03BIn47 23.273A03BIn48 23.355A03BIn49 23.854A03BIn50 24.639A03BIn51 24.987A03BIn52 25.269A03BIn53 25.298A03BIn54 26.202A03BIn55 26.216A03BIn56 26.382A03BIn57 26.392A03BIn58 26.623A03BIn59 26.953A03BIn60 27.307A03BIn61 27.772A03BIn62 27.822A03BIn63 28.347A03BIn64 29.309A03BIn65 29.667A03BIn66 29.724A03BIn67 29.917A03BIn68 30.134A03BIn69 30.277A03BIn70 30.872A03BIn71 31.855A03BIn72 33.211A03BIn73 33.622A03BIn74 34.414A03BIn75 35.018A03BIn76 35.226A03BIn77 36.488A03BIn78 37.255A03BIn79 39.689A03BIn80 40.405A03BIn81 40.698A03BIn82 41.349A03BIn83 42.133A03BIn84 42.450A03BIn85 43.148A03BIn86 43.866A03BIn87 44.081A03BIn88 45.092A03BIn89 45.458A03BIn90 45.862A03BIn91 46.157A03BIn92 46.578A03BIn93 46.836A03BIn94 48.456A03BIn95 48.698A03BIn96 48.741A03BIn97 48.877A03BIn98 50.638A03BIn99 50.905

A03BIn100 51.428A03BIn101 51.883A03BIn102 52.123A03BIn103 52.671A03BIn104 53.387A03BIn105 54.015A03BIn106 54.073A03BIn107 54.394A03BIn108 55.527A03BIn109 55.785A03BIn110 56.309A03BIn111 56.414A03BIn112 57.115A03BIn113 58.964A03BIn114 58.993A03BIn115 59.679A03BIn116 60.008A03BIn117 60.521A03BIn118 60.536A03BIn119 60.854A03BIn120 60.901A03BIn121 61.463A03BIn122 61.486A03BIn123 61.884A03BIn124 62.496A03BIn125 63.212A03BIn126 66.050A03BIn127 66.344A03BIn128 66.913A03BIn129 67.551A03BIn130 68.264A03BIn131 68.765

A03

A04Bin01 0.000A04Bin02 0.544A04Bin03 1.162A04Bin04 2.901A04Bin05 4.523A04Bin06 4.984A04Bin07 5.965A04Bin08 6.241A04Bin09 7.022A04Bin10 7.454A04Bin11 8.372A04Bin12 9.799A04Bin13 10.044A04Bin14 10.471A04Bin15 10.603A04Bin16 11.317A04Bin17 11.726A04Bin18 12.449A04Bin19 13.197A04Bin20 13.884A04Bin21 13.978A04Bin22 14.680A04Bin23 17.646A04Bin24 19.946A04Bin25 21.145

A04Bin26 26.932A04Bin27 27.799A04Bin28 28.584A04Bin29 29.359A04Bin30 29.866A04Bin31 30.186A04Bin32 32.544A04Bin33 33.759A04Bin34 34.262A04Bin35 34.918A04Bin36 35.521A04Bin37 36.762A04Bin38 37.429A04Bin39 37.725A04Bin40 38.617A04Bin41 39.642

A04Bin42 44.419A04Bin43 45.153A04Bin44 45.649A04Bin45 46.513A04Bin46 46.771A04Bin47 47.892A04Bin48 49.270A04Bin49 50.745A04Bin50 51.852A04Bin51 53.159A04Bin52 54.455A04Bin53 54.895A04Bin54 55.790

A04Bin55 60.640

A04A05Bin01 0.000A05Bin02 0.573A05Bin03 1.640A05Bin04 1.841A05Bin05 2.854A05Bin06 3.315A05Bin07 4.414A05Bin08 6.197A05Bin09 6.655A05Bin10 7.363A05Bin11 8.122A05Bin12 8.655A05Bin13 9.431A05Bin14 9.829A05Bin15 10.113A05Bin16 13.201A05Bin17 14.084A05Bin18 14.783A05Bin19 15.007A05Bin20 15.957A05Bin21 16.218A05Bin22 17.435A05Bin23 17.748A05Bin24 18.760A05Bin25 20.764A05Bin26 22.502A05Bin27 23.528A05Bin28 23.846A05Bin29 24.891A05Bin30 26.178A05Bin31 27.989A05Bin32 29.776A05Bin33 31.033A05Bin34 31.229A05Bin35 32.432A05Bin36 33.566A05Bin37 34.110A05Bin38 35.206A05Bin39 36.302A05Bin40 36.551A05Bin41 36.782A05Bin42 37.104A05Bin43 37.673A05Bin44 39.295A05Bin45 39.516A05Bin46 39.707A05Bin47 40.818A05Bin48 41.537A05Bin49 42.789A05Bin50 43.684A05Bin51 44.090A05Bin52 45.282A05Bin53 45.547A05Bin54 45.937A05Bin55 46.829A05Bin56 47.069A05Bin57 47.930A05Bin58 48.286A05Bin59 50.281A05Bin60 50.865A05Bin61 51.900A05Bin62 52.201A05Bin63 52.385A05Bin64 53.017A05Bin65 53.490A05Bin66 55.012A05Bin67 56.225A05Bin68 57.146A05Bin69 60.326A05Bin70 61.442A05Bin71 61.687A05Bin72 61.906A05Bin73 62.949A05Bin74 63.193A05Bin75 64.519A05Bin76 65.686A05Bin77 66.675A05Bin78 66.969A05Bin79 68.099A05Bin80 68.323A05Bin81 69.354A05Bin82 69.618A05Bin83 70.140A05Bin84 73.758A05Bin85 74.740A05Bin86 75.928A05Bin87 77.244A05Bin88 78.313A05Bin89 81.517A05Bin90 82.196A05Bin91 83.754A05Bin92 84.386A05Bin93 84.921A05Bin94 85.477


A05Bin100 94.504

A05

A06Bin01 0.000A06Bin02 0.286

A06Bin03 2.531A06Bin04 3.522A06Bin05 5.406A06Bin06 5.732A06Bin07 6.335A06Bin08 7.720A06Bin09 8.939A06Bin10 11.088A06Bin11 11.328A06Bin12 11.585A06Bin13 11.775

A06Bin14 14.602A06Bin15 17.910A06Bin16 18.337A06Bin17 18.815A06Bin18 21.235A06Bin19 21.590A06Bin20 22.457A06Bin21 23.446A06Bin22 23.998A06Bin23 24.660A06Bin24 25.916A06Bin25 26.425A06Bin26 27.467A06Bin27 31.156A06Bin28 32.060A06Bin29 34.350A06Bin30 34.818A06Bin31 35.148A06Bin32 35.525A06Bin33 37.656A06Bin34 38.814A06Bin35 39.068A06Bin36 40.295A06Bin37 41.394A06Bin38 42.904A06Bin39 43.498A06Bin40 44.654A06Bin41 45.685A06Bin42 46.394A06Bin43 48.648A06Bin44 49.004A06Bin45 50.444A06Bin46 50.882A06Bin47 51.623A06Bin48 51.755A06Bin49 52.456A06Bin50 52.846A06Bin51 53.215A06Bin52 53.378A06Bin53 54.506A06Bin54 54.600A06Bin55 54.698A06Bin56 54.991A06Bin57 55.779A06Bin58 56.128A06Bin59 56.232A06Bin60 56.715A06Bin61 57.764A06Bin62 57.948A06Bin63 58.136A06Bin64 58.346A06Bin65 58.436A06Bin66 58.944A06Bin67 59.495A06Bin68 59.647A06Bin69 60.551A06Bin70 60.740A06Bin71 60.938A06Bin72 61.027A06Bin73 62.234A06Bin74 63.091A06Bin75 63.256A06Bin76 63.434A06Bin77 64.312A06Bin78 64.437A06Bin79 64.626A06Bin80 65.574A06Bin81 65.788A06Bin82 65.876A06Bin83 66.100A06Bin84 66.383A06Bin85 67.188A06Bin86 68.299A06Bin87 68.819A06Bin88 69.614A06Bin89 69.811A06Bin90 71.186A06Bin91 73.472A06Bin92 73.685A06Bin93 75.093A06Bin94 78.361A06Bin95 78.959A06Bin96 80.233A06Bin97 80.380A06Bin98 83.449A06Bin99 84.181

A06Bin100 85.742A06Bin101 86.206A06Bin102 87.668A06Bin103 88.558A06Bin104 88.960A06Bin105 89.786A06Bin106 91.169A06Bin107 92.634A06Bin108 93.148A06Bin109 94.975A06Bin110 96.333A06Bin111 96.773A06Bin112 98.290A06Bin113 100.426A06Bin114 102.012A06Bin115 103.726A06Bin116 104.400A06Bin117 105.537A06Bin118 107.022A06Bin119 108.046A06Bin120 109.540A06Bin121 110.847A06Bin122 115.498A06Bin123 118.339A06Bin124 118.748A06Bin125 119.054A06Bin126 119.891A06Bin127 121.366A06Bin128 122.074

A06

A07Bin01 0.000

A07Bin02 14.433A07Bin03 15.150A07Bin04 16.322A07Bin05 19.240A07Bin06 21.069A07Bin07 21.510A07Bin08 21.843A07Bin09 22.123A07Bin10 22.448A07Bin11 22.708A07Bin12 23.551A07Bin13 24.030A07Bin14 24.629A07Bin15 25.022A07Bin16 25.962A07Bin17 26.655A07Bin18 27.548A07Bin19 29.316A07Bin20 30.092A07Bin21 30.704A07Bin22 30.981A07Bin23 31.291A07Bin24 31.605A07Bin25 32.021A07Bin26 32.819A07Bin27 33.800A07Bin28 34.050A07Bin29 34.833A07Bin30 35.143A07Bin31 35.539A07Bin32 36.218A07Bin33 36.903A07Bin34 37.676A07Bin35 38.656A07Bin36 40.248A07Bin37 40.814A07Bin38 42.930A07Bin39 45.296A07Bin40 45.542A07Bin41 46.380A07Bin42 46.951A07Bin43 47.894A07Bin44 48.566A07Bin45 48.967A07Bin46 49.985A07Bin47 50.604A07Bin48 52.747A07Bin49 53.242A07Bin50 54.186A07Bin51 54.704A07Bin52 55.319A07Bin53 56.538A07Bin54 56.938A07Bin55 57.156A07Bin56 57.640A07Bin57 59.054A07Bin58 63.254A07Bin59 64.119A07Bin60 64.830A07Bin61 65.158A07Bin62 65.527A07Bin63 65.893A07Bin64 69.365A07Bin65 70.231A07Bin66 70.588A07Bin67 70.938A07Bin68 72.048A07Bin69 73.170A07Bin70 74.133A07Bin71 74.576A07Bin72 75.207A07Bin73 76.967A07Bin74 79.040A07Bin75 79.433A07Bin76 80.219A07Bin77 81.446A07Bin78 82.521A07Bin79 85.138A07Bin80 86.271A07Bin81 86.754A07Bin82 87.054A07Bin83 87.912A07Bin84 88.176A07Bin85 88.525A07Bin86 91.252A07Bin87 92.320A07Bin88 93.062A07Bin89 93.994A07Bin90 96.044A07Bin91 98.671

A07Bin92 103.960

A07A08Bin01 0.000A08Bin02 0.544A08Bin03 1.678A08Bin04 2.305A08Bin05 2.713A08Bin06 3.136A08Bin07 5.015A08Bin08 5.544A08Bin09 7.115A08Bin10 7.185A08Bin11 7.334A08Bin12 7.592A08Bin13 7.666A08Bin14 9.561A08Bin15 9.910A08Bin16 10.604A08Bin17 11.235A08Bin18 11.497A08Bin19 12.173A08Bin20 12.864A08Bin21 13.162A08Bin22 15.719A08Bin23 16.431A08Bin24 16.652A08Bin25 18.735A08Bin26 19.014A08Bin27 19.950A08Bin28 20.282A08Bin29 21.507A08Bin30 22.500A08Bin31 22.771A08Bin32 23.380A08Bin33 23.925A08Bin34 26.312A08Bin35 27.676A08Bin36 29.021A08Bin37 31.188A08Bin38 31.466A08Bin39 31.979A08Bin40 32.955A08Bin41 33.937A08Bin42 34.092A08Bin43 34.982A08Bin44 35.396A08Bin45 36.447A08Bin46 36.816A08Bin47 37.352A08Bin48 38.364A08Bin49 39.004A08Bin50 39.725A08Bin51 40.403A08Bin52 40.635A08Bin53 41.914A08Bin54 42.171A08Bin55 43.612A08Bin56 46.385A08Bin57 50.010A08Bin58 51.935A08Bin59 52.029A08Bin60 53.126A08Bin61 54.166A08Bin62 54.211A08Bin63 54.402A08Bin64 55.482A08Bin65 55.647A08Bin66 56.365A08Bin67 56.825A08Bin68 57.000A08Bin69 59.479A08Bin70 61.658A08Bin71 62.134A08Bin72 62.991


A08A09Bin01 0.000A09Bin02 0.919A09Bin03 2.553A09Bin04 2.975A09Bin05 3.917A09Bin06 5.202A09Bin07 6.100A09Bin08 6.481A09Bin09 7.420A09Bin10 10.621A09Bin11 11.000A09Bin12 11.566A09Bin13 12.010A09Bin14 12.452A09Bin15 13.463A09Bin16 14.131A09Bin17 14.420A09Bin18 14.860A09Bin19 16.018A09Bin20 18.488A09Bin21 19.051A09Bin22 19.102A09Bin23 19.413A09Bin24 19.666A09Bin25 19.991A09Bin26 20.409A09Bin27 21.147A09Bin28 21.864A09Bin29 22.149A09Bin30 22.625A09Bin31 22.820A09Bin32 23.391A09Bin33 24.996A09Bin34 25.437A09Bin35 25.527A09Bin36 25.758A09Bin37 26.029A09Bin38 26.163A09Bin39 26.706A09Bin40 26.937A09Bin41 27.037A09Bin42 27.314A09Bin43 27.443A09Bin44 27.768A09Bin45 27.868A09Bin46 28.064A09Bin47 28.561A09Bin48 28.916A09Bin49 28.965A09Bin50 29.480A09Bin51 30.339A09Bin52 30.758A09Bin53 31.049A09Bin54 31.813A09Bin55 32.581A09Bin56 32.950A09Bin57 33.006A09Bin58 33.529A09Bin59 33.671A09Bin60 33.817A09Bin61 34.376A09Bin62 35.397A09Bin63 35.410A09Bin64 36.066A09Bin65 36.611A09Bin66 37.300A09Bin67 37.459A09Bin68 37.524A09Bin69 38.441A09Bin70 38.634A09Bin71 38.913A09Bin72 39.248A09Bin73 39.903A09Bin74 40.201A09Bin75 40.589A09Bin76 40.704A09Bin77 41.035A09Bin78 41.162A09Bin79 42.140A09Bin80 43.075A09Bin81 43.836A09Bin82 44.115A09Bin83 44.510A09Bin84 44.778A09Bin85 45.166A09Bin86 45.221A09Bin87 45.397A09Bin88 45.428A09Bin89 45.728A09Bin90 45.814A09Bin91 46.121A09Bin92 46.358A09Bin93 46.423A09Bin94 46.625A09Bin95 46.865A09Bin96 47.424A09Bin97 47.581A09Bin98 47.890A09Bin99 47.927

A09Bin100 47.993A09Bin101 48.437A09Bin102 48.668A09Bin103 49.135A09Bin104 49.218A09Bin105 49.363A09Bin106 49.394A09Bin107 50.069A09Bin108 51.814A09Bin109 51.900A09Bin110 52.242A09Bin111 52.976A09Bin112 53.021A09Bin113 53.230A09Bin114 53.414A09Bin115 54.374A09Bin116 54.551A09Bin117 54.717A09Bin118 54.934A09Bin119 54.989A09Bin120 55.161A09Bin121 55.339A09Bin122 55.391A09Bin123 55.571A09Bin124 56.923A09Bin125 57.088A09Bin126 57.865A09Bin127 58.191A09Bin128 59.320A09Bin129 59.545A09Bin130 60.653A09Bin131 60.845A09Bin132 60.971A09Bin133 62.497A09Bin134 64.879A09Bin135 65.252A09Bin136 65.508A09Bin137 65.898A09Bin138 66.128A09Bin139 66.569A09Bin140 66.872A09Bin141 67.588A09Bin142 68.107A09Bin143 68.496A09Bin144 68.700A09Bin145 69.085A09Bin146 69.444A09Bin147 70.616A09Bin148 71.099A09Bin149 71.423A09Bin150 71.812A09Bin151 72.209A09Bin152 73.127A09Bin153 75.824

A09A10Bin01 0.000A10Bin02 0.100A10Bin03 1.382A10Bin04 1.392A10Bin05A10Bin06 1.900A10Bin08 2.099A10Bin07 2.199A10Bin09 4.049A10Bin10 4.059A10Bin12 5.258A10Bin11 5.268A10Bin13 6.431A10Bin14 6.441A10Bin15 8.006A10Bin16 8.016A10Bin17 9.107A10Bin18 9.117A10Bin19 11.673A10Bin20 11.683A10Bin21 12.693A10Bin22 12.703A10Bin23 12.713A10Bin24 12.723A10Bin25 13.042A10Bin26 13.052A10Bin27 13.255A10Bin28 13.848A10Bin29 14.196A10Bin30 14.322A10Bin31 14.657A10Bin32 15.167A10Bin33 15.346A10Bin34 15.607A10Bin35 16.417A10Bin36 16.764A10Bin37 17.392A10Bin38 19.291A10Bin39 19.913A10Bin40 21.426A10Bin41 23.151A10Bin42 23.614A10Bin43 23.761A10Bin44 24.507A10Bin45 26.935A10Bin46 27.653A10Bin47 31.286A10Bin48 31.667A10Bin49 32.104A10Bin50 37.676A10Bin51 38.668A10Bin52 39.099A10Bin53 40.016A10Bin54 42.501A10Bin55 43.251A10Bin56 43.975A10Bin57 44.489A10Bin58 44.499A10Bin59 46.173A10Bin60 46.656A10Bin61 48.316A10Bin62 48.326A10Bin63 49.493A10Bin64 50.949A10Bin65 50.959A10Bin66 52.300A10Bin67 53.101A10Bin68 53.606A10Bin69 53.866A10Bin70 54.515A10Bin71 55.325A10Bin72 56.103A10Bin73 58.821A10Bin74 59.560A10Bin75 61.832A10Bin76 62.273A10Bin77 62.518A10Bin78 64.363A10Bin79 64.586A10Bin80 64.703A10Bin81 64.995A10Bin82 65.610A10Bin83 66.197A10Bin84 69.039A10Bin85 69.049A10Bin86 71.228A10Bin87 72.029A10Bin88 73.687A10Bin89 74.106A10Bin90 74.685A10Bin91 75.000A10Bin92 76.273A10Bin93 76.602A10Bin94 79.516A10Bin95 79.616

A10


123

knowledge, this map is the densest genetic linkage

map constructed to date for Chinese cabbage. (De-

tailed information on the bins and SLAFs are available

in Supplementary Table 1.)

As shown in Fig. 2 and Table 1, the ten B. rapa

linkage groups varied in the number of markers, map

length, and marker density. The length of the linkage

groups ranged from 60.64 cM for A04 to 122.074 cM

for A06. The number of marker loci in the ten linkage

groups ranged from 55 to 153 for A04 and A09,

respectively. Total marker number (153) and density

(0.5 cM/marker) was highest in group A09. The

lowest marker density (1.13 cM/marker) was in link-

age group A07, and the only gap ([10 cM interval)was present on one end of this group.

Mapping QTLs for downy mildew resistance

at various plant developmental stages

Previously, the major QTL BraDM was mapped to

linkage group A08 in an AFLP-based genetic linkage

map (Yu et al. 2009). Using the same mapping

population, downymildew resistance was investigated

at four plant developmental stages using the leaf disk

assay, which demonstrated that the inheritance of

downy mildew resistance varied slightly during plant

development (Zhang et al. 2012). To further under-

stand the genetic factors involved in downy mildew

resistance, the high-density bin map developed in this

study was used to locate QTLs for resistance to this

pathogen during Chinese cabbage production.

As expected, only one major QTL for seedling

resistance, sBrDM8, was mapped between A08Bin46

and A08Bin48 on linkage group A08 with a LOD

value of 20.71 (Table 2; Fig. 3), and this was identical

to BraDM (Yu et al. 2009). To distinguish it from other

bFig. 2 Bin map for the Chinese cabbage genome based on theDH100 population. Bin names and genetic distances (in cM) are

listed on the left and right sides of the chromosomes,

respectively

Table 1 Distribution of genetic markers on the Brassica rapa genetic linkage map

Linkage

groups

No. of

SLAFs

No. of SSR

and indel

Total

markers

No.

of bin

Genetic

distance (cM)

Marker

density

(cM/bin)

GAP

([10 cM)

A01 304 15 319 139 90.72 0.65 0

A02 413 10 423 94 90.72 0.97 0

A03 457 20 477 131 68.77 0.53 0

A04 114 9 123 55 60.64 1.1 0

A05 283 15 298 100 94.5 0.95 0

A06 424 15 439 128 122.07 0.95 0

A07 288 24 312 92 103.96 1.13 1

A08 399 24 423 77 72.16 0.94 0

A09 557 52 609 153 75.82 0.5 0

A10 243 22 265 95 79.62 0.84 0

Total 3482 206 3688 1064 858.97 0.81 1

Table 2 QTLs for downy mildew resistance at four developmental stages in Chinese cabbage (Brassica rapa ssp. pekinensis)

Stages QTL Flanking marker Position (cM) CI LOD R2 (%) Additive effect

Seedling sBrDM8 A08Bin46-A01Bin48 38.352 36.816–38.364 20.71 66.9 21.77

Young plant yBrDM8 A08Bin46-A01Bin48 36.816 36.816–38.352 10.95 46.3 18.41

Rosette rBrDM8 A08Bin46-A01Bin48 36.816 36.816–38.352 23.91 64.2 21.14

rBrDM6 A06Bin27-A06Bin29 29.47 31.156–34.060 5.99 11.7 9.09

Heading hBrDM8 A08Bin45-A01Bin47 36.816 36.447–37.352 17.51 56.6 19.44

hBrDM4 A04Bin13-A04Bin14 10.471 10.044–10.471 4.6 10.2 -10.19


123

A04Bin01A04Bin02A04Bin03A04Bin04A04Bin05A04Bin06A04Bin07A04Bin08A04Bin09A04Bin10A04Bin11A04Bin12A04Bin13A04Bin14A04Bin15A04Bin16A04Bin17A04Bin18A04Bin19A04Bin20A04Bin21A04Bin22A04Bin23A04Bin24A04Bin25

A04Bin26A04Bin27A04Bin28A04Bin29A04Bin30A04Bin31A04Bin32A04Bin33A04Bin34A04Bin35A04Bin36A04Bin37A04Bin38A04Bin39A04Bin40A04Bin41

A04Bin42A04Bin43A04Bin44A04Bin45A04Bin46A04Bin47A04Bin48A04Bin49A04Bin50A04Bin51A04Bin52A04Bin53A04Bin54

A04Bin55

hBrDM4

0 2 4 6

A04

A06Bin01A06Bin02

A06Bin03A06Bin04A06Bin05A06Bin06A06Bin07A06Bin08A06Bin09A06Bin10A06Bin11A06Bin12A06Bin13

A06Bin14A06Bin15A06Bin16A06Bin17A06Bin18A06Bin19A06Bin20A06Bin21A06Bin22A06Bin23A06Bin24A06Bin25A06Bin26A06Bin27A06Bin28A06Bin29A06Bin30A06Bin31A06Bin32A06Bin33A06Bin34A06Bin35A06Bin36A06Bin37A06Bin38A06Bin39A06Bin40A06Bin41A06Bin42A06Bin43A06Bin44A06Bin45A06Bin46A06Bin47A06Bin48A06Bin49A06Bin50A06Bin51A06Bin52A06Bin53A06Bin54A06Bin55A06Bin56A06Bin57A06Bin58A06Bin59A06Bin60A06Bin61A06Bin62A06Bin63A06Bin64A06Bin65A06Bin66A06Bin67A06Bin68A06Bin69A06Bin70A06Bin71A06Bin72A06Bin73A06Bin74A06Bin75A06Bin76A06Bin77A06Bin78A06Bin79A06Bin80A06Bin81A06Bin82A06Bin83A06Bin84A06Bin85A06Bin86A06Bin87A06Bin88A06Bin89A06Bin90A06Bin91A06Bin92A06Bin93A06Bin94A06Bin95A06Bin96A06Bin97A06Bin98A06Bin99

A06Bin100A06Bin101A06Bin102A06Bin103A06Bin104A06Bin105A06Bin106A06Bin107A06Bin108A06Bin109A06Bin110A06Bin111A06Bin112A06Bin113A06Bin114A06Bin115A06Bin116A06Bin117A06Bin118A06Bin119A06Bin120A06Bin121A06Bin122A06Bin123A06Bin124A06Bin125A06Bin126A06Bin127A06Bin128

rBrDM6

0 2 4 6 8

A06A08Bin01A08Bin02A08Bin03A08Bin04A08Bin05A08Bin06A08Bin07A08Bin08A08Bin09A08Bin10A08Bin11A08Bin12A08Bin13A08Bin14A08Bin15A08Bin16A08Bin17A08Bin18A08Bin19A08Bin20A08Bin21A08Bin22A08Bin23A08Bin24A08Bin25A08Bin26A08Bin27A08Bin28A08Bin29A08Bin30A08Bin31A08Bin32A08Bin33A08Bin34A08Bin35A08Bin36A08Bin37A08Bin38A08Bin39A08Bin40A08Bin41A08Bin42A08Bin43A08Bin44A08Bin45A08Bin46A08Bin47A08Bin48A08Bin49A08Bin50A08Bin51A08Bin52A08Bin53A08Bin54A08Bin55A08Bin56A08Bin57A08Bin58A08Bin59A08Bin60A08Bin61A08Bin62A08Bin63A08Bin64A08Bin65A08Bin66A08Bin67A08Bin68A08Bin69A08Bin70A08Bin71A08Bin72

A08Bin73A08Bin74A08Bin75A08Bin76A08Bin77

hBrDM8

sBrDM8

sBrDM8

yBrDM8

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A08

hBrDM4rBrDM6hBrDM8sBrDM8yBrDM8rBrDM8

0

5

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downy mildew resistance QTL, it was given a new

name, sBrDM8. sBrDM8 showed significant

(P\ 0.001) effects on downy mildew resistance withan additive effect of 21.77 and an R2 value of 66.9 %.

For the field experiment, a total of five QTLs for

three developmental stages were located on three of

the ten Chinese cabbage chromosomes (Table 2;

Fig. 3). Major QTLs for downy mildew resistance at

the young plant, rosette, and heading stages were also

found to map very close to sBrDM8; these were

designated yBrDM8, rBrDM8, and hBrDM8, respec-

tively, and had LOD values of 10.95, 23.91, and 19.51.

This indicated that the major QTLs on A08 provide

effective downy mildew resistance at every develop-

mental stage. Two flanking markers, BIN45 and

BIN48, delimit the sBraDM8 locus within a 228 kb

region (A08.17679028-A08.17907709), that harbors

30 predicted genes. Among these genes, one serine/

threonine kinase (STK) family gene, Bra016457, was

annotated, and it is predicted to be the candidate gene

for sBraDM8. In addition, two minor QTLs, rBrDM6

and hBrDM4, were located on A06 and A04, with

LOD values of 5.99 and 4.6, respectively. These QTLs

were only effective at certain developmental stages;

rBrDM6 was effective at the rosette stage with

additive effects of 9.09 and an R2 value of 11.7 %,

while hBrDM4 showed an effect at the heading stage,

with additive effects of -10.19 and an R2 value of

10.2 %.

Development of SNP markers for sBrDM8

Using next-generation sequencing technologies, devel-

opment of SNPmarkerswill be increasingly feasible for

high-throughput molecular marker-assisted breeding

applications. The results presented above indicate that

the major QTLs on A08 are effective at every devel-

opmental stage during Chinese cabbage production. By

MQM (multiple QTL model), sBrDM8 was mapped

between A08Bin46 and A08Bin48. The interval

defined by A08Bin46 and A08Bin48 harbored SLAF

marker 12808 (A08.17679028-A08.17679278) and the

InDel marker ql933 (A08.17882485-A08. 17882821),

and defined a physical interval of*228 kb from 17.68to 17.88 M on chromosome A08. Based on SLAF

markers sequences, three putative SNP loci were

submitted to design primers for KASPTM SNP geno-

typing (Smith andMaughan 2015). ThreeSNPmarkers,

A08-709, A08-028, and A08-018, were confirmed as

beingpolymorphic in the parental lines andgave perfect

genotyping results.

A BC2 population derived from DH100-

60 9 DH100-88, which included 164 individuals,

was used to analyze marker selection power. When

the SNP marker A08-709 was used for genotypic

screening of the population, 82 plants that were

heterozygous for the resistance allele gave low disease

scores of 0–3, while in the 82 susceptible individuals

(disease scores of 5–9), 79 plants were homozygous

and three were heterozygous at the A08-709 locus

(Supplementary Table 2 and Supplementary Fig. 2).

When the SNP markers A08-028 and A08-018 were

used for genotyping, co-segregation between the

genotypes and phenotypes was observed, showing a

very high selection accuracy (Supplementary Table 2).

The three SNP markers developed in this study can be

used effectively in MAS breeding programs to select

for downy mildew resistance in B. rapa.

Discussion

In the past 25 years, more than 20molecular maps have

been developed for Chinese cabbage using a variety of

marker types. Our linkage map spans 858.97 cM, with

an average distance of 0.81 cM between adjacent

marker loci. To our knowledge, the genetic map

presented here is the densest map constructed to date

for Chinese cabbage. The average inter-locus distance

for our map is much less than the 2.13 and 2.43 cM

distances reported for the previously-published refer-

ence maps of Choi et al. (2007) andWang et al. (2011a,

b). In comparison, the previous maps of Song et al.

(1991), Choi et al. (2007), Teutonico and Osborn

(1994), Suwabe et al. (2006),Kimet al. (2006), Soengas

et al. (2007), Wang et al. (2011a, b, 2014), and Chung

et al. (2014) reported total map lengths of 1850, 1876,

1785, 1006, 1287, 663, 1182.3, 1234.2, 1086.6, and

1115.9 cM respectively, with 280, 360, 139, 262, 545,

246, 556, 507, 548, and 314 as the corresponding

number of mapped loci. The differences in map lengths

bFig. 3 QTLs for downy mildew resistance on Chinese cabbagelinkage groups A04, A06, and A08. The map distance is given

on the left in centimorgans (cM) from the top of each

chromosome. QTL likelihood maps generated by the multiple

QTL model (MQM) were plotted using Mapchart 2.2. The LOD

value threshold of 3.2 for downy mildew resistance is indicated

by the dashed line


123

between the different studies is usually attributed to

scoring errors, marker type, number of marker loci,

number of individuals in the mapping populations,

recombination frequency and LOD values, and the

software employed to calculate the map. In our study,

the recombination rates between SLAF loci were

initially calculated using HighMap software. Due to

the large number ofmarkers (3482 loci), we generated a

very long map with a total recombinational length of

3324.11 cM.

To improve the map, a bin map was constructed.

The final bin map consisted of 1064 bins, which were

manually identified (Fig. 2), and was 858.98 cM in

length. The map length was reduced by 75 %,

indicating that bin mapping is an effective method

for reducing the total genetic length. This also

indicated that the number of markers significantly

affected the map length. In addition, the Joinmap 4.0

software used in mapping maybe also one factor that

contributed to a shorter overall map length. The results

presented here accurately reflect the genetic and

polymorphic characteristics of Chinese cabbage,

adding to our understanding of the genetics of this

species and providing abundant resources for QTL

mapping, map-based gene isolation, SNP marker

development, and molecular breeding.

Different genetic systems for resistance to downy

mildew at the seedling stage versus the adult-plant stages

have been previously reported in B. oleracea. Some B.

oleracea accessions are resistant at the cotyledon stage

but are susceptible as adult plants, or vice versa (Coelho

and Monteiro 2003a, b). Monteiro et al. (2005) showed

that the response to downy mildew inoculation at the

cotyledon stage is independent of the response in adult-

stage plants. Likewise, Zhang et al. (2012) demonstrated

that the inheritance of downy mildew resistance at

different developmental stages varies slightly during

plant development in Chinese cabbage. In this study, we

confirmed that the major QTL, BraDM, identified

previously at the seedling stage (Yu et al. 2009), was

found to be effective at every developmental stage. In

addition, two minor QTLs, rBrDM6 and hBrDM4,

locatedon linkagegroupsA06andA04,were effectiveat

the rosette andheading stages. This indicates that genetic

resistance to downymildew in seedlings and adult plants

is somewhat different.

In Brassica species, several dominantly inherited R

genes have been mapped for downy mildew resis-

tance. Coelho et al. (1998) identified a single locus for

resistance to downy mildew in mature broccoli (Wang

et al. 2000), and Carlier et al. (2012) mapped the locus

Pp523 on chromosome C08. In addition, a monogeni-

cally inherited resistance gene was also mapped in B.

oleracea (Yu et al. 2013). However, relatively few

studies on downy mildew resistance have focused on

Chinese cabbage. Other than our previous work (Yu

et al. 2009, 2011), Kim et al. (2011) mapped a single

dominant gene, BrRHP1, for adult-plant resistance to

downy mildew on linkage group A01. The BrRHP1

gene originated from a downy mildew resistant inbred

line ‘RS1’. In this study, the resistance gene came

from a different line, ‘Orange Queen’, and we did not

find a QTL corresponding to BrRHP1 on A01. Our

results suggest that the locus BrDM8 (designated

sBrDM8, yBrDM8, rBrDM8, and hBrDM8 at the

seedling, young plant, rosette, and heading stages,

respectively) could be a new dominant gene. More-

over, BrDM8 was localized to a 228 kb region

between A08.17679028 and A08.17907709. The most

likely candidate gene, Bra016457, was annotated as a

serine/threonine kinase (STK) family protein gene.

Functionally, many genes in the STK family have

been previously reported to play important roles in

disease resistance; examples are Pto and Pti1, both of

which confer resistance to bacterial speck disease in

tomato (Ronald et al. 1992; Zhou et al. 1995). To

confirm the candidate gene, additional genetic analy-

ses, such as fine mapping, allelism tests, and gene

cloning are needed.

SNP markers can be used in a wide variety of

applications, including association studies (Filiault

and Maloof 2012; Dou et al. 2012), conservation

genetics (Ogden et al. 2012), and genetic diversity

analysis (Blair et al. 2013), and are fast becoming the

marker system of choice in marker-assisted plant

breeding programs (Foolad and Panthee 2012). Many

of these applications require a scalable and cost-

effective genotyping method for SNPs. KASP chem-

istry provides a versatile method of genotyping that

can be applied to both small- and large-scale projects.

Based on SLAF sequencing, two SNP markers, A08-

028 and A08-018, that are tightly linked to the downy

mildew resistance locus sBrDM were successfully

developed for KASPTM SNP genotyping, and they

showed a very high selection accuracy. The two SNP

markers developed in this study can be effectively

used in MAS-based breeding programs for resistance

against downy mildew in B. rapa.


123

Acknowledgments This work was supported by grants fromthe Program of National Natural Science Foundation (No.

31171959), the National Plan for Science and Technology

Support (2014BAD01B09), and the earmarked fund for Modern

Agroindustry Technology Research System (CARS-25-A-11).

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Construction of a sequence-based bin map and mapping of QTLs for downy mildew resistance at four developmental stages in Chinese cabbage (Brassica rapa L. ssp. pekinensis)AbstractIntroductionMaterials and methodsPlant materialsDowny mildew resistance phenotypic evaluationSLAF library construction and high-throughput DNA sequencingLinkage map constructionQTL analysisMolecular marker design and SNP assays

ResultsAnalysis of SLAF-seq data and SLAF markersConstruction of a high-density bin mapMapping QTLs for downy mildew resistance at various plant developmental stagesDevelopment of SNP markers for sBrDM8

DiscussionAcknowledgmentsReferences

construction of a sequence-based bin map and mapping of...

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