全 文 ：Vol131 , No13
pp 1 275 - 282 　Mar1 , 2005作 　物 　学 　报ACTA AGRONOMICA SINICA第 31 卷 第 3 期2005 年 3 月 　275～282 页
Construction of a Molecular Marker Linkage Map and Its Use for QTL Analysis of
Erucic Acid Content in Brassica napus L1
LIU Xue2Ping , TU Jin2Xing 3 , LIU Zhi2Wen , CHEN Bao2Yuan , FU Ting2Dong
( National Key Laboratory of Crop Genetic Improvement , National Rapeseed Genetic Improvement Center , Huazhong Agricultural University , Wuhan 430070 , Hubei ,
China)
Abstract : Erucic acid is not easily absorbed and therefore is nutritionally undesirable as the long carbon2chain fatty acid in
the edible oil derived from Brassica napus1 Hence , decreasing erucic acid content is one of the important objectives in
double2low (low in erucic acid and low in glucosinolates) breeding program1 A monogene controlling erucic acid was found
in a doubled haploid (DH) population derived from a cross between a Canadian cultivar Quantum (yellow flower and low
erucic acid content) and a resynthesized B1 napus line No12127217 (white flower and high erucic acid content) 1 In order
to identify molecular markers tagging the gene controlling erucic acid , a B1 napus linkage map was constructed using the
121 DH lines of the above DH population1 A total of 207 markers were detected that comprised 102 random amplified
polymorphic DNA (RAPD) markers , 103 simple sequence repeat (SSR) markers and one morphological marker , i1e1 the
flower colour and erucic acid1 One hundred eighty2eight markers were assembled into 19 main linkage groups (LG1 -
LG19) and a minor group (A) 1 No linkage was found between the remaining 19 markers and any of the established linkage
groups1 The total map length is 1 18313 cM with an average distance of 613 cM between adjacent markers1 The marker loci
with distorted segregation ( P < 0101) accounted for 2018 % and tended to cluster in six linkage groups1 The major gene for
erucic acid was mapped on LG13 , flanked by a co2dominant SSR marker TPS039 and a dominant RAPD marker BS164a at
genetic distances of 212 cM and 1711 cM , respectively1 When QTL mapping was performed using the method of interval
mapping , only the major gene (major QTL) was detected1 The major QTL explained about 82 % of the total phenotypic
variation1 Compared with a B1 napus linkage map constructed based on a cross between N2029 and SYN1 in Brassica DB
(http :/ / www1ukcrop1net) using some common SSR markers , it was revealed that chromosomal rearrangements might have
occurred on LG131
Key words : Brassica napus ; Erucic acid content ; Molecular markers ; QTL
甘蓝型油菜遗传图谱的构建及芥酸含量的 QTL 分析
刘雪平　涂金星 Ξ 　刘志文　陈宝元　傅廷栋
(华中农业大学作物遗传改良国家重点实验室 , 国家油菜改良武汉分中心 ,湖北武汉 430070)
摘 　要 : 一个由甘蓝型油菜品种 Quantum(黄花、低芥酸)和人工合成的甘蓝型油菜品系 No12127217 (白花、高芥酸)为亲本
材料建立的 DH群体中芥酸呈现单基因的遗传模式。为了发展与芥酸紧密连锁的分子标记对其实行有效的控制 ,随机
选择 121 个结实正常的 DH系为作图群体 ,利用 SSR 和 RAPD 标记构建了一张甘蓝型油菜的遗传连锁图谱。在亲本间检
测到 207 个有多态性的遗传标记 ,其中 SSR 标记 103 个 ,RAPD 标记有 102 个及 2 个形态标记 (花色和芥酸) 。这些标记中
有 188 个标记分配到 19 个主连锁群和 1 个小的连锁群 A。这些连锁的标记跨越了 1 18313 cM ,两标记间的平均间距为
613 cM ,偏分离比例达到 2018 %( P < 0101)且主要集中在 6 个连锁群。芥酸主基因定位在LG13 上 , 一个共显性的 SSR 标
记 TPS039 和一个显性的 RAPD 标记 BS164a 在它的两侧 ,遗传距离分别为 212 cM 和 1711 cM。用区间作图对芥酸进行
QTL 扫描 ,仅扫描到该主基因 (主效QTL) ,这个QTL 解释了约 82 %的芥酸表型变异。利用共同的 SSR 标记与一张 BrassicaΞFoundation item :Supported by 973 program—the molecular biological bases of crop heterosis theory and application (973 2001CB1088) 1
Biography :LIU Xue2Ping(1975 - ) , Ph1D1 in rapeseed molecular biology field1 3　Correspondent author : TU Jin2Xing1 Tel : 027287281819 , E2mail : tujx
@mail1hzau1edu1cn
Received(收稿日期) :2004203208 ,Accepted(接受日期) :20042092131

DB (http :/ / www1ukcrop1net ,作图群体的亲本为 N2029 and SYN1) 的连锁图进行比较 ,结果表明在 LG13 上可能发生过染色
体重排。
关键词 : 甘蓝型油菜 ;芥酸含量 ;分子标记 ;QTL
中图分类号 : S565
　　Brassica napus L1 is the most important oilseed crop
species in the genus Brassica1 The oil quality is mainly
determined by the fatty acid composition :oleic (C18∶1) ,
linoleic (C18∶2) , linolenic (C18∶3) and erucic (C22∶
1) acids and some saturate fatty acids1 Erucic acid
accounted for more than 40 % of the total fatty acids in
the old B1 napus cultivars[1 ]1 Although it is controversial
about the erucic acid toxicity for human consumption , it
is certainly not easily absorbed and nutritionally
undesirable as the long carbon2chain fatty acid1 As a
result , the erucic acid content has been reduced almost to
zero in the so2called double2low (low in erucic acid and
low in glucosinolates) B1 napus cultivars for edible oil1
On the other hand , it is an important breeding objective
to achieve an erucic acid content as high as possible in
the high2erucic2acid2rapeseed (HEAR) program when the
oil is used for industrial purposes such as the production
of carburant , lubricants and surface2active agents[2 ]1
The inheritance of erucic acid content in B1 napus
has been reported by several authors[326 ] 1 Two additive
genes controlled the erucic acid content , each of them
contributed from 9 % to 14 % in different genetic
backgrounds1 As expected from an amphidiploid , the two
genes indeed resided in the A2 and C2genomes of B1
napus , respectively1 The molecular markers and genetic
linkage maps are powerful tools for improving important
agronomical traits1 Several linkage maps were constructed
using different molecular markers in B1 napus [7212 ] 1
Foisset et al1 constructed a linkage map of B1 napus
using isozyme , RAPD and RFLP markers1 The map
covered 1 765 cM with 254 markers including three PCR2
specific markers and a morphological marker in 19 linkage
groups[13 ] 1 QTL mapping was also reported for many
important agronomical traits in B1 napus [14217 ] 1
Decreasing erucic acid content is one of important
objectives in double2low rape breeding program1
Therefore , any markers linked to the erucic acid content
genes or QTLs are helpful to improve the rapeseed
quality.
In this paper , we report on the construction of a
genetic linkage map with SSR and RAPD markers in B1
napus , and the QTL analysis of erucic acid content using
the linkage map1 A co2dominant SSR marker tightly
linked with the erucic acid gene was identified1
1 　Materials and Methods
111 　Materials
　　Resynthesized B1 napus lines were produced from
the interspecific hybridizations between B1 alboglabra
Bailey and B1 rapa ( syn1 campestris ) L1 with the
objective to develop yellow2seeded B1 napus
germplasm[18 ] 1 The white2flowering and high2erucic
(about 29 %) line No12127217 is a doubled haploid
(DH) derived from the resynthesized B1 napus line
No17076 ( Chen et al1 , unpublished) 1 The yellow2
flowering and low2erucic ( < 1 %) parent used in the
cross of this study is the spring canola B1 napus cultivar
Quantum registered in Canada1 The cross was made
between Quantum ( ♀) and No12127217 ( ♂) in 19981
Doubled haploid (DH) lines were produced from the F1
plants following the microspore culture protocols as
described previously[19 ] 1 Plantlets were transplanted into
field according to the previous methods[20 ]1 Each DH line
was sown in one row in the following season and was
selfed by bagging for multiplication of the seeds1 The
flower colour of each DH line was visually recorded as
white or yellow during flowering1 All materials were
planted in the field nursery on the research farm of
Huazhong Agricultural University , Wuhan1 As the
mapping population of this study , 121 DH lines were
randomly selected from the available 600 DH lines derived
from the cross1
112 　Methods
11211 　Assay of erucic acid content 　 　The seed
samples of randomly selected from 10 plants were used to
represent each of the two parents ( No12127217 and
672 　　　　作 　　物 　　学 　　报 第 31 卷 　

Quantum) and the F1 , respectively1 The erucic acid
contents of the parents , F1 and DH lines were assayed
according to the methods as previously described[5 ]1
11212 　DNA extraction and PCR amplification 　　DNA
was extracted from young green leaves of the two parents
and the 121 DH lines following the protocol[21 ] 1 PCR
amplifications were performed in the MJ Research PTC2
2500 Peltier Thermal Cycler1 In the RAPD analysis ,
twenty micro liter reaction volumes included 60 ng DNA ,
1 ×buffer , 115 mmol/ L MgCl2 , 200 μmol/ L dNTPs
(Sangon in Shanghai) , 0145μmol/ L primer (Sangon in
Shanghai) and 1 U of Taq DNA polymerase ( MBI
afforded by Jingmei Company in Wuhan) 1 The DNA
amplification protocol was 94 ℃ for 3 min ( 1 cycle) ,
94 ℃for 30 s , 40 ℃for 30 s , 72 ℃for 45 s (35 cycles)
and 72 ℃for 10 min (1 cycle) 1 All PCR products were
separated in 112 % ( W/ V) agarose gels (with ethidium
bromide) in 1 ×TAE by electrophoresis at 90 V for 215
h1 Band sizes were estimated using Lambda DNA/ Hind
Ⅲ+ EcoR Ⅰmarker1 Gels were photographed on a UVP
system1
In the SSR analysis , ten micro liter reaction volumes
included 40 ng DNA , 1 ×buffer , 2 mmol/ L MgCl2 , 200
μmol/L dNTPs ( Sangon in Shanghai ) , 0145 μmol/ L
primer and 015 U of Taq DNA polymerase (MBI afforded
by Jingmei Company in Wuhan) 1 The DNA amplification
protocol was 94 ℃for 2 min (1 cycle) , 94 ℃for 1 min ,
60 ℃for 30 s , 72 ℃ for 45 s ( 10 cycles , - 015 ℃/
cycle) ; 94 ℃: 1 min , 55 ℃: 30 s , 72 ℃: 45 s ( 35
cycles) 1 All PCR products were separated in 6 % PAGE
gel in 015 × TBE by electrophoresis at 1 800 V for 40
min and detected by silver nitrate staining according to Lu
et al1[22 ]
11213 　Primer selection 　　The DNA concentration of
each sample was measured with the ultraviolet RNA/ DNA
measure instrument (Pharmacia Biotech , GeneQuant Ⅱ)
and then adjusted to equal concentration1 A total of 757
random 102bp (base pair) primers (Sangon in Shanghai)
were used to detect polymorphisms between the two
parental DNA samples1 The sequences of 66 SSR primers
(TPS number) were offered by Dr1 Tu in Huazhong
Agricultural University , and the other 274 primer
sequences were according to the web site http :/ /
www1ukcrop1net1 The SSR oligonucleotide primers were
synthesized by Sangon Company (Shanghai) 1
11214 　Construction of linkage groups and QTL analysis
　　Every segregation locus was tested by Chi2square
analysis (α= 0101) 1 Markers with too great distortion
values were discarded1 Linkage analysis was performed
with MAPMAKER/ EXP ,Version 310[23 ]1 Linkage groups
were established with a minimum LOD score of 410 and a
maximum recombination frequency of 0131 Two2point
command was used to establish the preliminary linkage
groups and multipoint analysis were further used to
designate the most2likely locus orders1 Multiple
segregating loci identified by the same primer were coded
with the letters a , b , etc1 , at the end of the locus name1
QTL analysis was performed by using MAPMARKER/
QTL111 (Lincoln et al1 , 1992b) [24 ] 1 A LOD score of
310 was used for detecting the existence of a putative
QTL1
2 　Results
211 　RAPD analysis
　　A total of 757 RAPD primers were randomly chosen
to screen polymorphisms between the two parents1 Seventy
of the 757 primers were selected to amplify polymorphisms
with reproducible and unambiguous band patterns1 As
some of these primers disclosed more than one
polymorphic locus , a total of 102 marker loci were scored
segregating in the mapping population1 In detail , among
the 70 primers , 47 primers detected merely a single
locus , 16 primers detected two loci , 5 primers detected
three loci , and the remaining 2 primers disclosed four
loci1
212 　SSR analysis
A total of 340 SSR primer pairs were synthesized1
Eighty2six of the 340 pairs could generate reproducible
and unambiguous band patterns1 As some of these primers
disclosed more than one polymorphic locus , a total of 103
marker loci were observed segregating in the mapping
population1 In detail , 73 primer pairs detected only a
single locus , 11 disclosed two loci , and 2 disclosed four
loci1 Twenty2nine of the 103 marker loci were dominant
and the others had co2dominant alleles1
772　第 3 期 LIU Xue2Ping et al . :Construction of a Molecular Marker Linkage Map and Its Use for QTL Analysis of ⋯⋯ 　　　

213 　Morphological marker
The F1 plants had white flowers , indicating its
dominance over yellow1 The flower colour was recorded on
the 121 DH lines1 A segregation of 58 (yellow) versus 63
(white) fit with an expected ratio of 1∶1 (χ2 = 0113 , P
= 0178) 1 The white flower was thus controlled by a
single gene1
214 　Marker segregation analysis in the mapping
population
　　In the case of no selection , the two parental alleles
were expected to fit with a ratio of 1 ∶1 at every
segregating locus in the mapping population composed of
DH lines1 Out of the total 207 markers assayed , 43
marker loci ( 2018 %) exhibited distorted segregation
(α= 0101) 1 Of the 43 distorted markers , 30 were RAPD
markers while 13 were SSR markers1 The biased
segregation was in favor of the Quantum alleles for 7414 % of the loci whereas in favor of the No12127217 alleles for2516 % of the loci1215 　Construction of linkage groupsOut of the total 207 markers , 188 markers wereassembled into 19 main linkage groups (LG1 - LG19)and a small linkage group ( A ) ( Fig11 ) 1 Themorphological marker , flower colour , was assigned on toLG131 The remaining 19 markers could not be assigned toany of the established linkage groups1 The linked locialtogether covered a genetic distance of 1 18313 cM1 Thelinkage groups varied in size from being 412 to 19215 cMlong1 The average spacing between two markers was 613cM with a range of 0 to 3714 cM1 The number of markersin one linkage group varied from 2 to 251 Markers withbiased segregation ratios tended to cluster on 6 linkagegroups1 The characters of linkage groups were summarizedin Table 11
Table 1 The genetic distances and distribution of markers in linkage groups ( L G)
LG cM Marker Deviation Max interval (cM) Min interval (cM) Average interval (cM)
LG1 19215 25 13 3714 014 717
LG2 10914 19 6 2213 014 518
LG3 4712 5 0 2712 117 914
LG4 6917 6 1 3012 712 1116
LG5 3513 5 3 1711 312 711
LG6 3013 8 1 1211 113 318
LG7 2719 7 1 1314 014 410
LG8 8 3 0 716 014 217
LG9 1713 5 1 813 214 315
LG10 1216 2 0 1216 1216 613
LG11 519 2 1 519 519 310
LG12 65 14 5 11 018 416
LG13 14913 24 4 3016 018 612
LG14 6418 7 0 1719 317 913
LG15 3015 8 0 1314 014 318
LG16 19512 22 5 2613 014 819
LG17 1212 7 0 412 014 117
LG18 2015 3 1 1016 919 618
LG19 8515 14 1 2019 018 611
A 412 2 0 412 412 211
Total 1 18313 188 43 3714 014 613
216 　Mapping the gene associated with erucic acid
21611 　Mapping erucic acid as a Mendelian trait 　　
The average erucic acid contents of Quantum , No121272
17 and the immediate F1 seeds were 0158 % , 29122 %
and 14190 % , respectively1 This indicated the embryonic
control and additive inheritance mode of the trait1 The
distribution of the erucic acid contents in the 121 DH
lines was shown in Fig121 The segregation of 57 ( low2
erucic) ∶64 ( high2erucic ) DH lines fit well with the
expected 1∶1 ratio (χ2 = 0130 , P = 0150 - 0175) , thereby supporting a monogenic difference for erucic acidbetween the two parents of this study1The first approach was to map erucic acid as aMendelian trait since the DH lines were categorized ashigh erucic acid or low erucic acid with a clear cut2offline ( Fig12) 1 The location of the gene locus on thelinkage groups was determined with MAPMAKER/ EXPversion 3101 The erucic acid locus was mapped betweenthe co2dominant SSR marker TPS039 (212 cM) and theRAPD marker BS164a ( 1711 cM) on LG131 One2way
872 　　　　作 　　物 　　学 　　报 第 31 卷 　

ANOVA was carried out at the significant threshold of
P ≤0100011 The only terminal marker on LG13 showed a significant association with erucic acid1
Fig11 Genetic linkage groups of Brassica napus using RAPD ,SSR markers and a morphological marker
Map distances in cM are indicated on the left side of linkage groups and locus names are on the right1 Multiple segregating loci identified by the
same primer were coded with the letters a ,b ,etc1 ,at the end of the locus name1NS ,OS ,RS and BS represen Na ,OL ,Ra and S ,respectively1
“3 ”　　represented the deviated markers1
972　第 3 期 LIU Xue2Ping et al . :Construction of a Molecular Marker Linkage Map and Its Use for QTL Analysis of ⋯⋯ 　　　

Fig12 The distribution of erucic acid content in DH population
derived from a cross between Quantum and No12127217
　　Of all the markers on LG13 , the co2dominant marker
TPS039 was most tightly linked with the erucic acid gene1
Of the 57 low erucic acid DH lines , one displayed the
parental No12127217 allele for TPS039 and the remaining
56 DH lines disclosed the parental Quantum allele1
Among the 64 high erucic DH lines , 60 had the
No12127217 allele whereas 4 displayed the Quantum
allele1 The genetic distance of the marker TPS039 was
212 cM from the erucic acid gene (Fig11 , on LG13) 1
21612 　Mapping the erucic acid as a quantitative trait 　
　The interval mapping method was used to localize the
putative QTL for erucic acid with MAPMARKER/
QTL1111 Scanning all of the linkage groups , one putative
major QTL was revealed in the intervals between marker
loci TPS039 and BS164a on LG13 (peak LOD 6213) 1
The QTL interval spanned 1913 cM and was responsible
for 8119 % of the total phenotypic variation1 Rescanning
of the map did not detect any other regions significantly
associated with the trait (LOD 310) when the major QTL
was fixed at its most likely position on LG131
3 　Discussions
Distorted segregation pattern of molecular markers
appears to be fairly common in many crop species1 In this
study , 43 of the 207 marker loci (2018 %) deviated from
the expected segregation ratio of 1∶1 (α= 0101) 1 Hacket
et al1 (2003) reported that the presence of segregation
distortion had little effect on marker order or length in the
construction of genetic linkage map for DH population[25 ]1
Erucic acid , as a Mendelian factor or a quantitative trait ,
was mapped at the same location on LG13 in this study ,
which might be a strong evidence that the deviated
markers had little effect on the QTL mapping1
The linkage groups covered 1 18313 cM with 188
markers in this study1 The map distances are similar with
the sum of the map distances in the diploid progenitors :
1 016 cM with 132 markers reported by Ferreira et al1
(1994 ) [8 ]　, and much less than the map distances
reported by Xu et al1 (2001) in F2 population : 1 83219
cM with 120 markers[16 ] 1 Ferreira et al1 reported that
recombination frequency of one marker was significantly
lower in DH population than that in F2 population after
compared with ten pairs of adjacent marker loci in both
populations from the same F1 plant1 He postulated that the
recombination frequency was influenced by the genetic
diversity of the parent and environmental effects on
meiosis[8 ]1 However , the reduced recombination frequency
might be derived from alterations in the chromosome
structure of resynthesized B1 napus1 On the other hand ,
DH lines only reflected the recombination frequency of
male gametes1
B1 napus linkage groups (the linkage groups codes :
N1 - N19) derived from a cross between N2029 and SYN1
in Brassica DB ( http :/ / www1ukcrop1net ) were aligned
with the linkage groups (LG1 - LG19) in this study using
the 18 common SSR markers and a morphological marker ,
i1e1 the flower colour1 They were mapped on 9 linkage
groups1 There were 5 common markers on LG13 , 4 on
LG1 , 3 on LG19 , and 2 on LG17 , respectively1 N12 ,
N14 , N15 , N16 and N18 have 1 common marker on
corresponding linkage groups ( LG12 , LG14 , LG15 ,
LG16 and LG18 ) 1 The aligned map in this study
indicated that some small rearrangements had occurred in
the genome of No1 2127217 because the same linkage
group LG13 was revealed to carry 3 SSR markers and a
morphological marker i1e1 the flower colour from two
different linkage groups (1 on N3 and 2 on N13 from the
Brassica DB ) ( Fig13 ) 1 More common markers are
needed for a comprehensive comparison1 Chen et al1
( 1988b ) previously reported about the independent
segregation of the flower colour and erucic acid content in
the early phase of the resynthesized line No12127217[26 ]1
However , Liu et al1 revealed a tight linkage relationship
between the flower colour and erucic acid content in the
C2genome of B1 napus with a recombination frequency of
082 　　　　作 　　物 　　学 　　报 第 31 卷 　

518 % in a DH population derived from a cross between
Quantum and No12127217[27 ] , which was consistent with
that reported by Woods et al1[28 ] Such a discrepancy is further evidence for possible chromosomal rearrangementsthat had occurred on LG13 of No121272171
Fig13 Chromosome rearrangement occurring in L G13 through comparative mapping between the linkage groups N3 and N13 in
Brassica DB and L G13 in this study using common SSR markers and a morphological marker i1e1the flower colour
NS ,OS ,RS and BS in this study present Na ,OL ,Ra and S ,respectively1Multiple segregating loci identified by the same primer were coded
with the letters a ,b ,etc1 ,at the end of the locus name1Map distances in cM are indicated on the left side of linkage groups and locus names
are on the right1
　　Previous cytogenetic and molecular results were
revealed that the Brassica genomes had resulted from a
common ancestor[29 ,30 ] 1 Furthermore , chromosomal
rearrangements played an important role during the
evolution of the Brassica species[31 ] 1 The A and C2
genomes of B1 napus share homoeologous regions , which
provide the opportunity for allosyndetic chromosome
pairing1 No12127217 was an artificially resynthesized B1
napus line1 Allosyndetic pairing could occur in this line
and result in chromosomal rearrangements1 Intergenomic
recombinations were observed in B1 rapa2alboglabra
addition lines ( AA + 1 chromosome from the
C2genome) [32 ]1
The two flanking markers of the erucic acid gene ,
the dominant RAPD marker BS164a and co2dominant SSR
marker TPS039 are useful to select the high2erucic plants for the industrial oil or the low2erucic plants for the edibleoil1 Further research will be to identify molecular markersfor the other gene for erucic acid in the A2genome of B1napus1References[1 ] Liu H2L (刘后利) 1 Genetic and Breeding in Brassica Rapeseed (油菜遗传育种) 1 Beijing : China Agriculture Press , 20011 202 - 203 (inChinese)[2 ] Princen L H ,Rothfus J A1 Development of new crops for industrial rawmaterials1 J Am Oil Chem Soc ,1984 ,61 :281 - 289[3 ] Harvey B L , Downey R K1 The inheritance of erucic acid content inrapeseed ( Brassica napus L1) 1 Can J Plant Sci ,1964 ,44 :104 - 111[ 4 ] Guan C2Y (官春云) ,Wang G2H (王国槐) 1 Studies on the breeding forquality characteristics in rapeseed1 Ⅱ1 Studies on the genes controllingerucic acid content in rapeseed cultivar Xiangyou No15 ( B1 napus) 1Journal of Hunan Agricultural College (湖南农学院学报) ,1986 ,1 :21 - 25 (in Chinese with English abstract)
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282 　　　　作 　　物 　　学 　　报 第 31 卷