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New Biotechnology �Volume 26, Number 5 �November 2009 RESEARCH PAPER

A microsatellite (SSR) based linkage mapof Brassica rapa

Rahul Kapoor1, Surindar Singh Banga2 and Shashi Kaur Banga2

1University Seed Farm, Ladhowal, Punjab Agricultural University, Ludhiana, Punjab, India2Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, India

In the present study we describe the construction of a genetic linkage map for the Brassica rapa (AA)

genome that will act as a key resource in undertaking future structural and functional genomic studies in

B. rapa. A F2 mapping population consisting of 48 F2 individual plants developed following

hybridization of 2 inbred lines Bathari mandi and IC 331817 was used to construct the map. The map

comprises 53 SSR markers derived from 3 different public domain resources. Nine linkage groups along

with a small subgroup were identified and designated as R1–R9 through alignment and orientation using

SSR markers in common with existing B. rapa reference linkage maps. The total length of the genetic

linkage map was 354.6 cM with an average interval of 6.6 cM between adjacent loci. The length of linkage

groups ranged from 28.0 cM to 44.2 cM for R6 and R1A, respectively. The number variability of markers in

the 9 linkage groups ranged from 3 for R6 to 10 for R1. Of the 53 SSR markers assigned to the linkage

groups, only 5 (9.4%) showed deviation from the expected segregation ratio. The development of this

map is vital to the genome integration and genetic information and will enable the international

research community to share resources and data for the improvement of B. rapa and other cultivated

Brassica species.

IntroductionThe genus Brassica is one of the core genera in the tribe Brassicaceae

and includes several crops with wide adaptation under a variety of

agroclimatic conditions. Of the six cultivated species of Brassica, B.

rapa (AA, 2n = 20), B. nigra (BB, 2n = 16) and B. oleracea (CC,

2n = 18), are monogenomic diploids which have contributed, by

hybridization, to the allopolyploids (amphiploids) B. juncea

(AABB, 2n = 36), B. napus (AACC, 2n = 38) and B. carinata (BBCC,

2n = 34). Economically, Brassica species are important sources of

vegetable oil, fresh and preserved vegetables and condiments. B.

napus, Brassica rapa, B. juncea and B. carinata provide about 12% of

the worldwide edible vegetable oil supply [1]. B. rapa (AA) and B.

oleracea (CC) provide many vegetables that contribute to a healthy

human diet, being a valuable source of dietary fiber, vitamin C and

other health enhancing factors such as anticancer compounds [2].

Corresponding author: Kapoor, R. (rahul200189@yahoo.co.in)

1871-6784/$ - see front matter � 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.09.003

B. rapa comprises several morphologically diverse crops, including

Chinese cabbage, pakchoi, turnip and broccoletto, as well as

oilseeds that include yellow and brown Sarsons [3]. The B. rapa

(A genome) therefore has worldwide importance in agriculture,

with the quality and economic value of derived products such as

processed vegetable edible oil through the crops B. napus (oilseed

rape, Canola) and B. juncea (mustard oil).

Assigning molecular markers to the linkage groups and con-

structing genetic maps is an important step toward analysing the

genomes of crop species. Such maps provide a better insight into

genome organization, evolution of the crop species and synteny

with related species. From the crop improvement point of view,

the genetic maps are useful for tagging and cloning genes of

economically important traits, marker assisted breeding and gene

pyramiding. The high degree of neutral DNA polymorphisms of

most Brassica species [4] has facilitated the development of many

genetic linkage maps, where common sets of DNA markers and/or

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RESEARCH PAPER New Biotechnology � Volume 26, Number 5 �November 2009

FIGURE 1

Brassica rapa specific SSR primer ENA17 showing polymorphism in B. rapa F2population.

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parental genotypes have been used along with common nomen-

clature [5–7]. Several genetic linkage maps based on a range of

marker types, including Restriction Fragment Length Polymorph-

ism (RFLPs), Random Amplified Polymorphic DNA (RAPD), Simple

Sequence Repeats (SSRs) and Amplified Fragment Length Poly-

morphisms (AFLPs), have been produced for B. rapa [8–15]. PCR

based markers have been widely used in developing genetic link-

age maps for B. oleracea [16,7], B. nigra [17], B. juncea [18] and B.

napus [19–21]. However, there is only limited published data on

SSR-based genetic maps in B. rapa [13], especially the markers that

may provide anchors to the B. rapa genome and are quickly

transferable to other populations.

In recent years, SSRs or microsatellites have been recognized as

useful molecular markers in marker assisted selection (MAS), the

analysis of genetic diversity, population analysis and other pur-

poses in various species [22]. SSR markers are a class of co-domi-

nant markers that are readily transferable because they exhibit a

high degree of polymorphism and locus specificity. For B. rapa,

several SSRs have recently been described, of which 90% success-

fully amplified in other Brassica species, and 40% amplified in

Arabidopsis [23,24]. In the present study we have generated a

detailed linkage map of B. rapa using genome specific SSR markers.

To establish the identity of linkage groups corresponding to R1–

R10, we used SSR markers from [23,25].

Materials and methodsPlant materialMapping population consisting of 48 individual F2 plants was

developed following hybridization of 2 inbred lines Bathari mandi

and IC 331817. Bathari mandi is a land race of yellow sarson

collected from Himalayas while IC 331817 is a brown sarson type.

Both of the genotypes differ significantly for their morphophy-

siological traits.

Plant DNA extractionDNA was isolated using a standard procedure [26]. Young leaves

were collected, ground to powder using liquid nitrogen and auto-

claved in pestle mortar. Pre-warmed (658C) CTAB extraction buffer

(900 ml) was added and samples incubated at 658C for 45 min.

TABLE 1

Salient features of genetic linkage map of Brassica rapa

Linkage groupof B. rapa

Number ofmarkers assigned

Density(markers/cM)

R1 10 4.40

R1A 5 8.84

R2 5 6.46

R3 4 7.40

R4 4 8.90

R5 6 5.90

R6 3 9.33

R7 4 8.07

R8 5 6.46

R9 7 5.85

a Adjacent markers >1 cM.b Distance between adjacent markers �15 cM.

240 www.elsevier.com/locate/nbt

Eight hundred microliters chloroform was added by centrifugation

at 12,000 rpm for 20 min and supernatant was transferred to a

fresh tube. DNA precipitation and RNAse treatments were as per

standard procedures.

Assessment of quantity and quality of DNADNA quantity and quality was assessed by electrophoresis in

agarose gel (0.8%) stained with ethidium bromide 1%, using

0.5� TBE electrophoresis buffer. Quantitative estimates of sample

DNA were made by visual comparison with DNA of known con-

centration.

PCR amplification and SSR analysisA total of 130 SSR primer sequences were selected from the

information available in public domain [23,25,15]. DNA amplifi-

cation was carried out in volumes of 20 ml, containing 2.1 units of

Taq polymerase, 0.38 mM of primers, 5.0 mM of dNTPs, 0.5 mM of

MgCl2, 1� PCR buffer and 45 ng of genomic DNA as templates.

The PCR profile was: initial 4 min at 948C and 40 cycles each with

1 min DNA denaturation at 948C, 1 min at appropriate annealing

Number ofintervalsa

Number ofgapsb

Length oflinkage group (cM)

6 1 44.0

3 1 44.2

4 1 32.3

3 1 29.6

3 1 35.6

5 1 35.3

2 1 28.0

3 1 32.3

4 1 32.3

5 1 41.0

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temperature and 2 min extension at 728C with final extension of

7 min at 728C. PCR was performed in a 96-well microtiter plate in

MJ Research, PTC 200TM or Applied Biosystems thermal cycler.

Products were analyzed by agarose gel electrophoresis.

Linkage analysis and map constructionMarkers that were reproducibly polymorphic between the parent

lines were scored in the F2 population. Linkage analysis and map

FIGURE 2

A SSR map of Brassica rapa showing the location of 53 SSR markers based on 48 F2markers in cM (Kosambi function) are shown on the left side of each chromosomeLinkage group R4; (E) Linkage group R5; (F) Linkage group R6; (G) Linkage group

construction were performed using Mapmaker software [27]. Seg-

regating data were sorted according to locus order for each linkage

group using MS Excel. This facilitated detection of errors asso-

ciated with putative ‘double recombinant’ events and guided

visual checking of original autoradiographs and revision of data

points where these had been misscored or typed. All editing

operations were recorded and are traceable. The mapping function

of Kosambi [28] was used because of the independent crossover

plants of Bathari mandi� IC 331817 population. The genetic distances of SSR

. (A) Linkage group R1, R1A; (B) Linkage group R2; (C) Linkage group R3; (D)R7; (H) Linkage group R8; (I) Linkage group R9.

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events in different meiotic phases during the development from F1

to F2. Two point, three point and multipoint analysis was used to

determine the best order of marker loci within the linkage groups.

Maximum LOD score of 2.0–2.5 and recombination fraction of 40–

50 were used. The most possible order of marker in each group was

determined using ‘compare’ and ‘ripple’ commands. In case of

more than one possible arrangement of linkage groups, the one

with smallest genetic linkage distance between adjacent marker

loci of the linkage groups was chosen to construct the genetic map.

Linkage maps were visualized using MapChart Version 2.1 [29].

Results and discussionPolymorphism surveyThe polymorphism survey using microsatellite (SSR) based primers

for assaying F2 population was carefully analyzed (see supplemen-

tary data). Of the 130 SSR primers selected from information

available in public domain, 62 (47.7%) showed polymorphic

banding pattern in F2 population of a cross between B. rapa cv.

Bathari mandi � B. rapa cv. IC 331817. Both of the genotypes differ

significantly for their morphophysiological traits. Perhaps it was

the reason that a high frequency of SSR primers used in the study

was polymorphic. Of 62 polymorphic SSR markers, 53 were

assigned to 9 linkage groups of B. rapa and the remaining 68

SSR primer pairs were either unspecific as shown by smear or

superfluous bands (20 primer pairs) or did not amplify at all (48

primer pairs). Ten SSR markers (Ra2G03, Ra2C05, Ra2A06, Ra2G05,

Ra1A06, Ra1G07, BRMS 44, GOL2, KBF19 and KBHB20) detected

more than one segregating locus (Fig. 1).

Linkage group assembly and distribution of markersA total of 53 SSR loci were assigned to 9 linkage groups with LOD

score value of 2.0. The genetic map had a total map length of

354.6 cM, with an average distance of 6.6 cM between two loci

(Table 1). The whole B. rapa genome could be assembled into nine

(R1–R9) linkage groups (Fig. 2A–I). No SSR marker could be

anchored to R10. A subgroup (R1A) was created within R1 linkage

group because of unavailability of linked SSR markers that could

fill the large gap. The length of linkage groups ranged from 28.0 cM

242 www.elsevier.com/locate/nbt

for R6 to 44.2 cM for R1A. The number variability of markers in the 9

linkage groups ranged from 3 for R6 to 10 for R1. The number of

intervals as determined on the basis of mapped points in the

linkage groups varied from two for linkage group R6 to six for

R1. As far as gaps are concerned, one each for all the linkage groups

was observed, that too at the position of first loci. Skewed segrega-

tion of markers is a common feature in most of the Brassica linkage

maps. Of the 53 SSR markers assigned to the linkage groups, only 5

(9.4%) showed deviation from the expected segregation ratios of

1:2:1 or 3:1 supporting the findings by [8,11,13,14]. However,

some markers showed conspicuously high x2 values which was

in each case caused by the reduced frequency of any one of the

parental allele as shown in [30]. Besides biased selection of parental

genotypes during F2 population development, other reported

explanations for the frequent occurrence of segregation distortion

include loss of chromosomes [31], the presence of gene conversion

events [32] and homologous recombination [33]. The markers

showing distorted segregation were distributed randomly along

all the linkage groups, except R6.

ConclusionThe use of 53 SSR markers enabled us to align and orientate 9

linkage groups of B. rapa and more SSR primers are now proposed

to be used to saturate this framework linkage map to ensure greater

genome coverage. The linkage map reported here is therefore a key

resource in undertaking future structural and functional genomic

studies in B. rapa.

AcknowledgementWe thank Kuldeep Singh (Molecular Geneticist, School of

Agricultural Biotechnology, PAU, Ludhiana, Punjab, India) for his

immense support in providing data analysis with microsatellite

primers and use of MapMaker software in developing a linkage

map.

Appendix A. Supplementary dataSupplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.nbt.2009.09.003.

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