# Progress in Understanding and Sequencing the Genome ofBrassica rapa

Authors: Chang Pyo Hong; Soo-Jin Kwon; Jung Sun Kim; Tae-Jin Yang; Beom-Seok Park; Yong Pyo Lim
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

Brassica rapa, which is closely related to
Arabidopsis thaliana, is an important crop and a
model plant for studying genome evolution via
polyploidization. We report the current understanding of the
genome structure of B. rapa and efforts for the
whole-genome sequencing of the species. The tribe
Brassicaceae, which comprises ca. 240 species,
descended from a common hexaploid ancestor with a basic genome
similar to that of Arabidopsis. Chromosome
rearrangements, including fusions and/or fissions, resulted in
the present-day “diploid” Brassica
species with variation in chromosome number and phenotype.
Triplicated genomic segments of B. rapa are
collinear to those of A. thaliana with InDels.
The genome triplication has led to an approximately 1.7-fold
increase in the B. rapa gene number compared to
that of A. thaliana. Repetitive DNA of B.
rapa has also been extensively amplified and has
diverged from that of A. thaliana. For its
whole-genome sequencing, the Brassica rapa Genome
Sequencing Project (BrGSP) consortium has developed suitable
genomic resources and constructed genetic and physical maps.
Ten chromosomes of B. rapa are being allocated to
BrGSP consortium participants, and each chromosome will be
sequenced by a BAC-by-BAC approach. Genome sequencing of
B. rapa will offer a new perspective for plant
biology and evolution in the context of polyploidization.


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## Body

## 1. Importance ofBrassica Genomics

The genusBrassica is
one of the core genera in the tribe Brassicaceae and includes a number of crops with wide adaptation under a
variety of agroclimatic conditions. Economically, Brassica species are important sources of vegetable oil, fresh, preserved
vegetables, and condiments. B. napus, B. rapa, B. juncea, and B. carinata provide about 12% of the worldwide edible vegetable oil supply
[1]. The B. rapa and B. oleracea subspecies represent many of
the vegetables in our daily diet. In particular, B. rapa ssp. pekinensis (Chinese cabbage), on which this article focuses, is one of the most widely
used vegetable crops in northeast Asia. Moreover, Brassica species are important sources of dietary fiber, vitamin C,
and anticancer compounds [2].The genetic relationships among the different diploid and
amphidiploidBrassica species are described
by the U’s triangle [3]. Of the six widely cultivated species of Brassica, B. rapa (AA, 2n = 20), B. nigra (BB, 2n = 16), and B. oleracea (CC,
2n = 18) are monogenomic diploids. The remaining three species, B. juncea (AABB, 2n = 36), B. napus (AACC, 2n = 38), and B. carinata (BBCC, 2n = 34) exhibit stable
diploid genetics, but are allotetraploids, which have evolved via hybridization
between differing monogenomic diploids [3]. The diploid Brassica genomes range from 1.1 pg/2C (529 Mbp/1C) for B. rapa to 1.4 pg/2C (696 Mbp/1C) for B. oleracea (see Figure 1) [4]. The genomes
of the allotetraploids range from 2.2 pg/2C (1,068 Mbp/1C) for B. juncea to 2.6 pg/2C (1,284 Mbp/1C)
for B. carinata (see Figure 1).Figure 1
Genetic relationship of the different diploid and amphidiploidBrassica species. 1C, 1C nuclear DNA content (pg); GS, genome size
(Mbp) [3, 4].The genusBrassica is
characterized by morphological diversity with regard to inflorescences, leaves,
stems, roots, and terminal or apical buds [5]. For example, such morphological
diversity can be easily observed in subspecies of B. oleracea: the enlarged inflorescences of cauliflower (B.
oleracea ssp. botrytis) and broccoli (B. oleracea ssp. italica);
the enlarged stems of kohlrabi (B. oleracea ssp. gongylodes) and
marrowstem kale (B. oleracea ssp. medullosa); and the many
axillary buds of Brussels sprout (B. oleracea ssp. gemmifera) [5].
The morphological diversity in Brassica species may be linked to genomic changes associated with polyploidization [6].
The polyploidization in Brassica species
has brought about triplication of genomic segments and subsequent
rearrangements such as inversions, insertions, deletions, and substitutions [7–16], and these genetic variations may cause novel
phenotypic variations for traits among these species [5, 6]. Thus, Brassica genomics will provide us with an
understanding of the rapid phenotypic evolution of polyploid plants.
Additionally, it will help us to understand genomic changes and how they shape the
allotetrapolyploid Brassica species. For
example, a study has been done looking at rapid genomic changes and the effect of
nuclear-cytoplasm interaction in synthetic allotetrapolyploid species [17].Because of the high economic value ofBrassica species throughout the world and their potential to be models
for the study of polyploidization, genome sequencing projects for Brassica species, especially B. rapa and B. oleracea, have recently been initiated (http://www.brassica.info)
[18–20]. In particular, B. rapa ssp. pekinensis inbred line Chiffu-401-42,
discussed in this article, has been selected for Brassica-A genome sequencing in the Brassicarapa Genome
Sequencing Project (BrGSP)
(http://www.brassica.info), a component of the consortium of the Multinational Brassica Genome Project, with the goal of
completely sequencing this genome through a BAC-by-BAC approach. The BrGSP consortium has developed genomic
resources for this purpose and is proceeding with whole-genome sequencing.


## 2. Current Understanding of the Genome  Structure ofB. rapa

### 2.1. Karyotype ofB. rapa

Karyotyping is the starting point for understanding the genome
structure of a species. Moreover, it provides insight into genome evolution.
Most of the karyotypic analyses inB.
rapa have been performed on mitotic metaphase chromosomes [21–24].
However, the analyses are limited in what they can reveal about the cytological
structure of the genome because of the low resolution of the technique. For example,
different measurements of chromosome lengths and rDNA loci are obtained by this
method. Recently, the high-resolution karyotype for the B. rapa ssp. pekinensis inbred line Chiffu was determined on pachytene chromosomes by using
4′-6-diamino-2-phenylindole dihydrochloride (DAPI) staining and fluorescence in situ hybridization (FISH) of rDNAs
and pericentromeric satellite repeats [25]. By DAPI analysis, the mean lengths
of ten pachytene chromosomes ranged from 23.7 μm to 51.3 μm, with a total of
385.3 μm, a total length which
is 11.9- ∼17.5-fold longer than that of the mitotic metaphase chromosomes
reported by Lim et al. [24] and
Koo et al. [25]. In comparison,
pachytene chromosome length of A.
thaliana, Medicago truncatula,
and tomato was estimated to be about 7.4%, 15%, and 24% of the total pachytene
chromosome length, respectively (reviewed in Koo et al. [25]). In B. rapa,
the pachytene karyotype consists of two metacentric (chromosomes 1 and 6), five
submetacentric (chromosomes 3, 4, 5, 9, and 10), two subtelomeric (chromosomes
7 and 8), and one acrocentric chromosome (chromosome 2), with the corresponding centromeric index ranges
of 38.8–41.0%, 29.5–36.7%, 17.4–20.2%, and 9.38%, respectively [25]. In the chromosomal
structure at pachytene, the total length of pericentromeric heterochromatin
regions was estimated to be 38.2 μm, which is
approximately 10% of the total chromosome length [25]. In conjunction with
chromosomal structure and characteristics, 5S rDNA loci were located on
pericentromeric regions of the short arms of chromosomes 2 and 7 as well as the long arm
of chromosome 10, while
45S rDNA loci were located on the short arms of chromosomes 1, 2, 4, and 5 as well as the long arm
of chromosome 7 [24, 25].


### 2.2. Collinearity between Genomic Segments ofArabidopsis and Brassica

Brassica species are closely
related to A. thaliana, having
diverged 17-18 million years ago (MYA) from their common ancestor [16]. A. thaliana, which has been completely
sequenced, has a rather small genome (about 146 Mb) with relatively little repetitive
DNA and a high gene density [26, 27]. Protein-coding regions of the genomes of Brassica species show high sequence
conservation with those of A. thaliana,
with nucleotide sequence similarity in exons between B. oleracea and A. thaliana ranging from 75% to 90%, compared to <70% for intronic
regions [28].
This similarity allows the identification of sets of candidate genes in Brassica species and the studying of
their genome structures through comparative genomics [29]. Comparative studies
between Arabidopsis and Brassica have revealed the presence of
collinear chromosome segments (see Figure 2). Comparative genetic mapping
between diploid Brassica species and A. thaliana to identify homologous loci have
revealed many conserved blocks in their genomes [7, 8, 14, 30]. Comparative
physical mapping between Arabidopsis and Brassica further corroborated the
findings. A set of six bacterial artificial chromosomes (BACs), representing a
431-kb contiguous region of Arabidopsis chromosome 2, was mapped on chromosomes and DNA fibers of B. rapa [31]. Moreover, studies on a 222-kb gene-rich region of A. thaliana chromosome 4 and its
homologous counterparts in B. rapa or B. oleracea revealed the collinearity
of genes in homologous segments [9, 11, 13]. This finding was supported by
sequence analysis of specific homologous genomic segments [15, 16]. However,
many structural rearrangements differentiate the Brassica and Arabidopsis chromosomes (see Figure 2). Comparative genetic mapping between B. nigra and A. thaliana species revealed that the average length of conserved
segments between the two species was estimated at about 8 cM, which corresponds
to ∼90 rearrangements since the divergence of the two species [7]. In addition,
it was found that gene contents in their homologous genomic segments were also
variable with interstitial gene losses and insertions [9, 11, 13, 15, 16].An example of a comparative map ofArabidopsis and Brassica. (a) Collinearity between
genomic segments of the two species and genome triplication of Brassica revealed by comparative genetic
mapping. (b) Synteny of genes in a triplicated genomic region of Brassica.
(a)(b)


### 2.3. Genome Triplication of DiploidBrassica Species

Most of the comparative studies mentioned above demonstrated thatBrassica species contain extensively
triplicated counterparts of the corresponding homologous segments of the A. thaliana genome (see Figure 2),
thereby suggesting that diploid Brassica species may have been derived from a hexaploid ancestor: the genome which was similar
to Arabidopsis. Consistent with the
nature of genome triplication, Yang et al. [16] reported that paralogous
subgenomes of diploid Brassica species triplicated 13∼17 MYA, very soon after the Arabidopsis and Brassica divergence that occurred at 17∼18 MYA. In addition, it was reported that
after the Brassica genomes had
triplicated, their subgenomes were rearranged by inversions, translocations [7, 12, 32],  extensive interspersed gene
loss, as well as gene insertions occurred relative to the inferred structure of
the ancestral genome (see Figure 2). Additionally, such genome triplication was
extensively found across the tribe Brassicaceae [12]. In comparison with the genome of A.
thaliana, the genome triplication in Brassica species has clearly led to an increase in the genome size, resulting in a 3- to
5-fold inflation.Genome triplication events inBrassica species may also have an effect on gene expression of multicopy genes, leading
to such phenomena as pseudogenization, subfunctionalization, or neofunctionalization
in species [33–38]. For example, the MADS-box transcription
factor family, whose members control key aspects of plant vegetative and
reproductive development, shapes genetic systems by subfunctionalization [37].
It appears that after polyploid formation, considerable and sometimes very
rapid changes in genome structure and gene expression have occurred. Researchers
have hypothesized that genomic triplication in Brassica species permits mutations in loci that are normally under
tight selective constraints in Arabidopsis, and may thus result in the observed greater phenotypic
plasticity in Brassica [5]. Studies
on expression of duplicated genes in Brassica species will provide insight into the role of polyploidization in the phenotypic
divergence of the plant genus.


### 2.4. Survey of theB. rapa Genome Revealed by BAC-End Sequence Analysis

TheB. rapa genome was
surveyed via the analysis of its 12,017 HindIII
BAC-end sequences (Table 1) [39]. Analyses of BAC-end sequence or genome survey
sequences assist in understanding whole genome
structure [39–41]. It was estimated that the B.
rapa genome might contain about 43000 genes (covering 16.8% of the genome),
1.6 times more than the
A. thaliana genome. Recently, Yang et al. [16]
also estimated the gene content of B.
rapa to range from 49,000 to 63,000, based on predictions from microsynteny
studies. It has been suggested that chromosomal triplication events in Brassica have led to an increase in gene
number with subsequent gene loss [15, 16, 39, 42].Table 1
Comparison of gene, TE, and SSR abundances inB. rapa and A. thaliana.
ContentsB. rapaA. thalianaReferencesGenome size (Mb)529146[4, 27]Gene number4300∼6300026,207[16, 39, 43]TE abundance (%)(1)13.86∼7[39, 44]SSR number≈110,000 (one SSR/4.8 kb)≈36,756 (one SSR/3.2 kb)[39, 45](1)Coverage of TEs
in the genome.Transposable elements (TEs) with a predominance of
retrotransposons were estimated to occupy approximately 14% of the genome (covering
approximately 74 Mb), 8.2 times greater than that
observed previously inA. thaliana [44].
Zhang and Wessler [44] reported that TEs in B.
oleracea constituted 20% of the genome, slightly more than what was
predicted for the B. rapa genome. Of
the predicted TEs, LTR retrotransposon families were the most abundant (69.9%),
followed by non-LTR retrotransposons (13.4%), DNA transposons (11.4%), and
other retrotransposons (5.3%). In particular, Ty1/copia-like and Ty3/gypsy-like retrotransposons occupied
39.5% and 30.2% of LTR retrotransposon families, respectively. The amplification
of TEs in B. rapa, especially
retrotransposons, may have played a crucial role in both evolution and genomic
expansion.Simple sequence repeats (SSRs) have been estimated to occur with a
frequency of approximately one per 4.8 kb within theB. rapa genome, as compared to approximately one per 3.2 kb within
the A. thaliana genome [39]. Of SSRs
identified, trinucleotides were the most abundant repeat type, constituting about
37% of all SSRs, a percentage similar to those reported in other plant genomes [39, 45]. Comparison of SSR densities in different
genomic regions demonstrated that SSR density was greatest immediately in 5′-flanking
regions of predicted genes [45]. SSRs were also preferentially associated with
gene-rich regions, with pericentromeric heterochromatin SSRs mostly associated
with retrotransposons [45], suggesting that the distribution of SSRs in the
genome is nonrandom [39, 45].


### 2.5. Structure of (peri)centromeres ofB. rapa

The centromere is a dynamic and rapidly evolving structure and consists
largely of highly repetitive DNA sequences, especially tandem satellite repeats
and retrotransposons [46, 47]. Centromeric repeats characterized in plant
genomes are composed of 155∼180-bp tandem repeat motifs, including the 180-bp
pAL1 satellite in A. thaliana [48–50], the 155∼165-bp CentO satellite in rice [51, 52], the 156-bp CentC
satellite in maize [53] and the 169-bp satellite in Medicago truncatula [54, 55]. Centromeric satellite repeats of Brassica species, except for those of B. nigra, are represented by the 176-bp
CentBr [24, 25, 56–59]. The CentBr repeats in the B. rapa genome belong to two classes which have 82% sequence
similarity. The two classes are chromosome-specific, with CentBr1 found on eight
chromosomes (chromosomes 1, 3, and 5–10) and CentBr2 on two chromosomes
(chromosomes 2 and 4) [24, 25, 39]. Such distribution of the CentBr family may
reflect the predominance of CentBr1 in the Brassica genome [39]. The CentBr repeats have also undergone rapid evolution within the B. rapa genome and have diverged among
the related species of Brassicaceae [39].
Recently, Lim et al. [59]
identified and characterized the major repeats in centromeric and pericentromeric
heterochromatin of B. rapa. The
region contains CentBr arrays, 238-bp degenerate tandem repeat (TR238) arrays,
rDNAs, centromere-specific retrotransposons of Brassica (CRB), and pericentromere-specific retrotransposons
(PCRBr). In particular, CRB was a major component of all centromeres in three
diploid Brassica species and their
allotetraploid relatives, and PCRBr and TR238 were A-genome-specific [59].


## 2.1. Karyotype ofB. rapa

Karyotyping is the starting point for understanding the genome
structure of a species. Moreover, it provides insight into genome evolution.
Most of the karyotypic analyses inB.
rapa have been performed on mitotic metaphase chromosomes [21–24].
However, the analyses are limited in what they can reveal about the cytological
structure of the genome because of the low resolution of the technique. For example,
different measurements of chromosome lengths and rDNA loci are obtained by this
method. Recently, the high-resolution karyotype for the B. rapa ssp. pekinensis inbred line Chiffu was determined on pachytene chromosomes by using
4′-6-diamino-2-phenylindole dihydrochloride (DAPI) staining and fluorescence in situ hybridization (FISH) of rDNAs
and pericentromeric satellite repeats [25]. By DAPI analysis, the mean lengths
of ten pachytene chromosomes ranged from 23.7 μm to 51.3 μm, with a total of
385.3 μm, a total length which
is 11.9- ∼17.5-fold longer than that of the mitotic metaphase chromosomes
reported by Lim et al. [24] and
Koo et al. [25]. In comparison,
pachytene chromosome length of A.
thaliana, Medicago truncatula,
and tomato was estimated to be about 7.4%, 15%, and 24% of the total pachytene
chromosome length, respectively (reviewed in Koo et al. [25]). In B. rapa,
the pachytene karyotype consists of two metacentric (chromosomes 1 and 6), five
submetacentric (chromosomes 3, 4, 5, 9, and 10), two subtelomeric (chromosomes
7 and 8), and one acrocentric chromosome (chromosome 2), with the corresponding centromeric index ranges
of 38.8–41.0%, 29.5–36.7%, 17.4–20.2%, and 9.38%, respectively [25]. In the chromosomal
structure at pachytene, the total length of pericentromeric heterochromatin
regions was estimated to be 38.2 μm, which is
approximately 10% of the total chromosome length [25]. In conjunction with
chromosomal structure and characteristics, 5S rDNA loci were located on
pericentromeric regions of the short arms of chromosomes 2 and 7 as well as the long arm
of chromosome 10, while
45S rDNA loci were located on the short arms of chromosomes 1, 2, 4, and 5 as well as the long arm
of chromosome 7 [24, 25].


## 2.2. Collinearity between Genomic Segments ofArabidopsis and Brassica

Brassica species are closely
related to A. thaliana, having
diverged 17-18 million years ago (MYA) from their common ancestor [16]. A. thaliana, which has been completely
sequenced, has a rather small genome (about 146 Mb) with relatively little repetitive
DNA and a high gene density [26, 27]. Protein-coding regions of the genomes of Brassica species show high sequence
conservation with those of A. thaliana,
with nucleotide sequence similarity in exons between B. oleracea and A. thaliana ranging from 75% to 90%, compared to <70% for intronic
regions [28].
This similarity allows the identification of sets of candidate genes in Brassica species and the studying of
their genome structures through comparative genomics [29]. Comparative studies
between Arabidopsis and Brassica have revealed the presence of
collinear chromosome segments (see Figure 2). Comparative genetic mapping
between diploid Brassica species and A. thaliana to identify homologous loci have
revealed many conserved blocks in their genomes [7, 8, 14, 30]. Comparative
physical mapping between Arabidopsis and Brassica further corroborated the
findings. A set of six bacterial artificial chromosomes (BACs), representing a
431-kb contiguous region of Arabidopsis chromosome 2, was mapped on chromosomes and DNA fibers of B. rapa [31]. Moreover, studies on a 222-kb gene-rich region of A. thaliana chromosome 4 and its
homologous counterparts in B. rapa or B. oleracea revealed the collinearity
of genes in homologous segments [9, 11, 13]. This finding was supported by
sequence analysis of specific homologous genomic segments [15, 16]. However,
many structural rearrangements differentiate the Brassica and Arabidopsis chromosomes (see Figure 2). Comparative genetic mapping between B. nigra and A. thaliana species revealed that the average length of conserved
segments between the two species was estimated at about 8 cM, which corresponds
to ∼90 rearrangements since the divergence of the two species [7]. In addition,
it was found that gene contents in their homologous genomic segments were also
variable with interstitial gene losses and insertions [9, 11, 13, 15, 16].An example of a comparative map ofArabidopsis and Brassica. (a) Collinearity between
genomic segments of the two species and genome triplication of Brassica revealed by comparative genetic
mapping. (b) Synteny of genes in a triplicated genomic region of Brassica.
(a)(b)


## 2.3. Genome Triplication of DiploidBrassica Species

Most of the comparative studies mentioned above demonstrated thatBrassica species contain extensively
triplicated counterparts of the corresponding homologous segments of the A. thaliana genome (see Figure 2),
thereby suggesting that diploid Brassica species may have been derived from a hexaploid ancestor: the genome which was similar
to Arabidopsis. Consistent with the
nature of genome triplication, Yang et al. [16] reported that paralogous
subgenomes of diploid Brassica species triplicated 13∼17 MYA, very soon after the Arabidopsis and Brassica divergence that occurred at 17∼18 MYA. In addition, it was reported that
after the Brassica genomes had
triplicated, their subgenomes were rearranged by inversions, translocations [7, 12, 32],  extensive interspersed gene
loss, as well as gene insertions occurred relative to the inferred structure of
the ancestral genome (see Figure 2). Additionally, such genome triplication was
extensively found across the tribe Brassicaceae [12]. In comparison with the genome of A.
thaliana, the genome triplication in Brassica species has clearly led to an increase in the genome size, resulting in a 3- to
5-fold inflation.Genome triplication events inBrassica species may also have an effect on gene expression of multicopy genes, leading
to such phenomena as pseudogenization, subfunctionalization, or neofunctionalization
in species [33–38]. For example, the MADS-box transcription
factor family, whose members control key aspects of plant vegetative and
reproductive development, shapes genetic systems by subfunctionalization [37].
It appears that after polyploid formation, considerable and sometimes very
rapid changes in genome structure and gene expression have occurred. Researchers
have hypothesized that genomic triplication in Brassica species permits mutations in loci that are normally under
tight selective constraints in Arabidopsis, and may thus result in the observed greater phenotypic
plasticity in Brassica [5]. Studies
on expression of duplicated genes in Brassica species will provide insight into the role of polyploidization in the phenotypic
divergence of the plant genus.


## 2.4. Survey of theB. rapa Genome Revealed by BAC-End Sequence Analysis

TheB. rapa genome was
surveyed via the analysis of its 12,017 HindIII
BAC-end sequences (Table 1) [39]. Analyses of BAC-end sequence or genome survey
sequences assist in understanding whole genome
structure [39–41]. It was estimated that the B.
rapa genome might contain about 43000 genes (covering 16.8% of the genome),
1.6 times more than the
A. thaliana genome. Recently, Yang et al. [16]
also estimated the gene content of B.
rapa to range from 49,000 to 63,000, based on predictions from microsynteny
studies. It has been suggested that chromosomal triplication events in Brassica have led to an increase in gene
number with subsequent gene loss [15, 16, 39, 42].Table 1
Comparison of gene, TE, and SSR abundances inB. rapa and A. thaliana.
ContentsB. rapaA. thalianaReferencesGenome size (Mb)529146[4, 27]Gene number4300∼6300026,207[16, 39, 43]TE abundance (%)(1)13.86∼7[39, 44]SSR number≈110,000 (one SSR/4.8 kb)≈36,756 (one SSR/3.2 kb)[39, 45](1)Coverage of TEs
in the genome.Transposable elements (TEs) with a predominance of
retrotransposons were estimated to occupy approximately 14% of the genome (covering
approximately 74 Mb), 8.2 times greater than that
observed previously inA. thaliana [44].
Zhang and Wessler [44] reported that TEs in B.
oleracea constituted 20% of the genome, slightly more than what was
predicted for the B. rapa genome. Of
the predicted TEs, LTR retrotransposon families were the most abundant (69.9%),
followed by non-LTR retrotransposons (13.4%), DNA transposons (11.4%), and
other retrotransposons (5.3%). In particular, Ty1/copia-like and Ty3/gypsy-like retrotransposons occupied
39.5% and 30.2% of LTR retrotransposon families, respectively. The amplification
of TEs in B. rapa, especially
retrotransposons, may have played a crucial role in both evolution and genomic
expansion.Simple sequence repeats (SSRs) have been estimated to occur with a
frequency of approximately one per 4.8 kb within theB. rapa genome, as compared to approximately one per 3.2 kb within
the A. thaliana genome [39]. Of SSRs
identified, trinucleotides were the most abundant repeat type, constituting about
37% of all SSRs, a percentage similar to those reported in other plant genomes [39, 45]. Comparison of SSR densities in different
genomic regions demonstrated that SSR density was greatest immediately in 5′-flanking
regions of predicted genes [45]. SSRs were also preferentially associated with
gene-rich regions, with pericentromeric heterochromatin SSRs mostly associated
with retrotransposons [45], suggesting that the distribution of SSRs in the
genome is nonrandom [39, 45].


## 2.5. Structure of (peri)centromeres ofB. rapa

The centromere is a dynamic and rapidly evolving structure and consists
largely of highly repetitive DNA sequences, especially tandem satellite repeats
and retrotransposons [46, 47]. Centromeric repeats characterized in plant
genomes are composed of 155∼180-bp tandem repeat motifs, including the 180-bp
pAL1 satellite in A. thaliana [48–50], the 155∼165-bp CentO satellite in rice [51, 52], the 156-bp CentC
satellite in maize [53] and the 169-bp satellite in Medicago truncatula [54, 55]. Centromeric satellite repeats of Brassica species, except for those of B. nigra, are represented by the 176-bp
CentBr [24, 25, 56–59]. The CentBr repeats in the B. rapa genome belong to two classes which have 82% sequence
similarity. The two classes are chromosome-specific, with CentBr1 found on eight
chromosomes (chromosomes 1, 3, and 5–10) and CentBr2 on two chromosomes
(chromosomes 2 and 4) [24, 25, 39]. Such distribution of the CentBr family may
reflect the predominance of CentBr1 in the Brassica genome [39]. The CentBr repeats have also undergone rapid evolution within the B. rapa genome and have diverged among
the related species of Brassicaceae [39].
Recently, Lim et al. [59]
identified and characterized the major repeats in centromeric and pericentromeric
heterochromatin of B. rapa. The
region contains CentBr arrays, 238-bp degenerate tandem repeat (TR238) arrays,
rDNAs, centromere-specific retrotransposons of Brassica (CRB), and pericentromere-specific retrotransposons
(PCRBr). In particular, CRB was a major component of all centromeres in three
diploid Brassica species and their
allotetraploid relatives, and PCRBr and TR238 were A-genome-specific [59].


## 3. Progress ofB. rapa Genome Sequencing

### 3.1. Genomic Resources

The development of genomic resources is a prerequisite to undertaking
genome sequencing in any crop species. Genomic resources, including reference
mapping populations, DNA libraries, and DNA sequences have been developed forB. rapa ssp. pekinensis inbred line Chiffu-401-42 (Table 2). Two reference
mapping populations were derived from two B. rapa ssp. pekinensis inbred lines, Chiifu-401-42 and Kenshin-402-43 (CK), and
comprise 78 double haploid (DH) lines (the CKDH
population) and 201 recombinant inbred (RI) lines (the CKRI population). These
mapping populations have been used for construction of reference genetic maps
for genome sequencing [20]. The bacterial artificial chromosome (BAC) system,
commonly used for developing large-insert DNA libraries, is an invaluable
resource for structural and functional genomics. Three Chiffu BAC libraries
were constructed by using restriction enzymes: HindIII, BamHI, and Sau3AI, and designated as KBrH, KBrB,
and KBrS. These libraries consist of 56592, 50688, and 55296 clones with an
average insert size of 115 kb, 124 kb, and 100 kb, respectively. These BAC
libraries cover approximately 36 genome equivalents, assuming that the genome
size of Chinese cabbage is 529 Mb. Using these BAC clones, the BrGSP community has recently generated a
total of 200017 BAC-end sequences. In combination with BAC fingerprinting data,
the BAC-end sequences will give insight into the structure of the genome, be a
resource for development of genetic markers, and aid in finding the BAC clones that
correspond to the minimal tilling paths in genome sequencing [19, 60, 61]. For
functional genomics of B. rapa, 22
cDNA libraries from different tissues, including leaves, roots, cotyledons,
stems, seedlings, ovules, siliques, and anthers of Chiffu, have been
constructed, and a total of 128582 expressed sequence tags (ESTs) have been
generated from these cDNA libraries (GenBank accession number CO749247∼CO750684 and EX015357∼EX142500). Currently, the ESTs have been used for construction
of B. rapa unigene set and gene
expression microarray (http://www.brassica-rapa.org).Table 2
Genomic
resources for whole-genome sequencing ofB.
rapa.
Genomic resourcesSource materialNumberMapping populationsDH lineChiffu-401-42× Kenshin-402-4378 lines (F2 generation)RI lineChiffu-401-42× Kenshin-402-43201 lines (F8 generation)BAC librariesHindIII (KBrH)Chiffu-401-4256592 clones (115 kb(1))BamHI (KBrB)Chiffu-401-4250688 clones (124 kb(1))Sau3AI (KBrS)Chiffu-401-4255296 clones (100 kb(1))cDNA libraries22 cDNA librariesDifferent tissues
of Chiffu-401-42 and Jangwon including leaves, roots, cotyledons, stems,
seedlings, ovules, siliques, anthers—BAC-end sequencesKBrH, KBrB, and
KBrS clones200017 sequencesESTs22 cDNA clones129928 sequencesBAC shotgun
sequencesKBrH, KBrB, and
KBrS cloneson-going(2)(1)Average insert size (kb).(2)Of BACs sequenced, 511 BACs have been deposited in GenBank.


### 3.2. Genetic and Physical Mapping

Some genetic linkage maps ofB. rapa, on which genetic markers were distributed
over ten linkage groups, have been constructed since 1990 [62–67] (summarized in Table 3). The distances of genetic linkage maps ranged from
890 cM to 1850 cM. However, the genetic linkage maps may not provide direct and
accurate genetic information for the Chiffu genome sequencing because of
genetic variation between the mapping populations. For that reason, the BrGSP community has constructed the CK
genetic linkage map. Using the 78 CKDH lines, a reference genetic linkage map
has been constructed [67]. The map consists of a total of 556 markers,
including 278 AFLPs, 235 SSRs, 25 RAPDs, and 18 ESTPs/STS/CAPS markers. Ten linkage groups were identified and designated as R1
to R10 via mapping with SSR markers derived from the reference linkage map of B. napus reported previously [68]
(Table 4). The total length of the linkage map was 1182 cM with an average
interval of 2.83 cM between adjacent loci. Recently, for high-resolution
genetic mapping, the community has set a goal of developing more than 1,000 SSR
markers derived from BAC-end sequences, ESTs, and BACs. Moreover, based on the
sequence-tagged site (STS) markers, a CKRI genetic linkage map has been
constructed to be complementary to the CKDH one. The linkage groups in these
genetic maps may not correspond to the chromosomes assigned in the cytogenetic
map. Therefore, it is important to align the linkage groups on the genetic map
with chromosomes of the cytogenetic map. All ten linkage groups of a reference
genetic map of B. rapa are being assigned
to the corresponding chromosomes through fluorescence in situ hybridization (FISH) using locus-specific BAC clones as
probes (see an example in Figure 3, unpublished data).Table 3
Genetic linkage maps ofB. rapa developed since 1990.
Mapping populationPopulation typePopulation sizeNo. of lociType of markersTotal length of map (average interval)ReferencesMichihili× Spring broccoliF295280RFLP1850 cM (6.6 cM)[62]Per (winter
turnip rape)× R500 (spring yellow sarson)F291139RFLP1785 cM (13.5 cM)[63]Per
(winter turnip rape)× R500 (spring yellow sarson)F6RI87144RFLP890 cM (6.0 cM)[64]Developed from Chinese cabbage  F1 cultivar JangwonF2134545RFLP, SSR1287 cM (2.4 cM)[65]G004
(CR(a) DH line)× A9709 (CS(b) DH line)(cultivars of
Chinese cabbage)F294262RFLP, SSR, RAPD1005 cM (3.7 cM)[66]Chiffu-401-42×
Kenshin-402-43DH78556AFLP, SSR, RADP,
ESTP, STS, CAPS1182 cM (2.83 cM)[67]Table 4
The correspondence between genetic linkage groups ofB. rapa ssp. pekinensis based on B. napus reference linkage maps.
Genetic linkage map ofB. napus [68]Choi et al. [67]Kim et al. [65]Suwabe et al. [66]A1 (N1)R1R1LG6A2 (N2)R2R2LG8A3 (N3)R3R3LG1A4 (N4)R4R4LG10A5 (N5)R5R5LG3A6 (N6)R6R6LG2A7 (N7)R7R7LG4A8 (N8)R8R8LG7A9 (N9)R9R9LG5A10 (N10)R10R10LG9Figure 3
An example of an alignment of linkage group 1 in the reference genetic map to the corresponding
chromosome 5 through FISH using locus-specific BAC clones.The fingerprinted BAC map (so-called “physical map”) makes it
possible to select clones for sequencing that would ensure comprehensive
coverage of the genome and reduce sequencing redundancy [69]. In addition, the
clone-based map also enables the identification of large segments of the genome
that are repeated, thereby simplifying the sequence assembly. To construct a
deep-coverage BAC physical map of the B.
rapa genome, all BAC clones from the three BAC libraries were fingerprinted
using restriction enzyme digestion and SNaPshot [70] methodologies, and then BAC contigs have been
assembled by FingerPrinted Contigs (FPC) software (http://www.agcol.arizona.edu/software/fpc/).
This data will be open to the Brassicarapa genome sequencing consortium.


### 3.3. Approach to Genome Sequencing

Seed BACs for genome sequencing have been selected through in silico allocation ofB. rapa BAC-end sequences onto
counterpart locations of Arabidopsis chromosomes [19]. Of 91000 BAC-end sequences, a total of 45232 showed
significant sequence similarity with unique Arabidopsis sequences, and 4317 BAC clones were allocated on Arabidopsis chromosomes by significant matching with both ends
within 30–500 kb intervals, which span 93 Mb of Arabidopsis euchromatin regions (covering 78.2% of the Arabidopsis genome). However,
approximately 9.4 Mb of euchromatin regions and 16.6 Mb pericentromeric
heterochromatin regions of the Arabidopsis genome were not covered by the B. rapa BAC span (span is considered by best hit of paired ends). Based on the physical
map of B. rapa and the in silico comparative map of its
BAC-ends onto Arabidopsis chromosomes, 629 seed BACs have been selected spanning 86 Mb of Arabidopsis euchromatin regions and
scattered throughout the B. rapa genome
(http://www.brassica-rapa.org), and the BACs have been mapped on B. rapa chromosomes by STS mapping and
FISH analysis. The seed BACs which are anchored and sequenced will be used as
stepping stones for sequencing of the ten chromosomes.Considering the large genome size and the possibility of
international cooperation, a chromosome-based approach was suggested. Of ten
chromosomes (or linkage groups), eight have been allocated to the participating
countries as follow: Korea (R3 and R9), Canada (R2 and R10), UK and China (R1
and R8), USA (R6), and Australia (R7). However, R4 and R5 have remained
unassigned. Progress
of chromosome sequencing will be reported soon by each country.


## 3.1. Genomic Resources

The development of genomic resources is a prerequisite to undertaking
genome sequencing in any crop species. Genomic resources, including reference
mapping populations, DNA libraries, and DNA sequences have been developed forB. rapa ssp. pekinensis inbred line Chiffu-401-42 (Table 2). Two reference
mapping populations were derived from two B. rapa ssp. pekinensis inbred lines, Chiifu-401-42 and Kenshin-402-43 (CK), and
comprise 78 double haploid (DH) lines (the CKDH
population) and 201 recombinant inbred (RI) lines (the CKRI population). These
mapping populations have been used for construction of reference genetic maps
for genome sequencing [20]. The bacterial artificial chromosome (BAC) system,
commonly used for developing large-insert DNA libraries, is an invaluable
resource for structural and functional genomics. Three Chiffu BAC libraries
were constructed by using restriction enzymes: HindIII, BamHI, and Sau3AI, and designated as KBrH, KBrB,
and KBrS. These libraries consist of 56592, 50688, and 55296 clones with an
average insert size of 115 kb, 124 kb, and 100 kb, respectively. These BAC
libraries cover approximately 36 genome equivalents, assuming that the genome
size of Chinese cabbage is 529 Mb. Using these BAC clones, the BrGSP community has recently generated a
total of 200017 BAC-end sequences. In combination with BAC fingerprinting data,
the BAC-end sequences will give insight into the structure of the genome, be a
resource for development of genetic markers, and aid in finding the BAC clones that
correspond to the minimal tilling paths in genome sequencing [19, 60, 61]. For
functional genomics of B. rapa, 22
cDNA libraries from different tissues, including leaves, roots, cotyledons,
stems, seedlings, ovules, siliques, and anthers of Chiffu, have been
constructed, and a total of 128582 expressed sequence tags (ESTs) have been
generated from these cDNA libraries (GenBank accession number CO749247∼CO750684 and EX015357∼EX142500). Currently, the ESTs have been used for construction
of B. rapa unigene set and gene
expression microarray (http://www.brassica-rapa.org).Table 2
Genomic
resources for whole-genome sequencing ofB.
rapa.
Genomic resourcesSource materialNumberMapping populationsDH lineChiffu-401-42× Kenshin-402-4378 lines (F2 generation)RI lineChiffu-401-42× Kenshin-402-43201 lines (F8 generation)BAC librariesHindIII (KBrH)Chiffu-401-4256592 clones (115 kb(1))BamHI (KBrB)Chiffu-401-4250688 clones (124 kb(1))Sau3AI (KBrS)Chiffu-401-4255296 clones (100 kb(1))cDNA libraries22 cDNA librariesDifferent tissues
of Chiffu-401-42 and Jangwon including leaves, roots, cotyledons, stems,
seedlings, ovules, siliques, anthers—BAC-end sequencesKBrH, KBrB, and
KBrS clones200017 sequencesESTs22 cDNA clones129928 sequencesBAC shotgun
sequencesKBrH, KBrB, and
KBrS cloneson-going(2)(1)Average insert size (kb).(2)Of BACs sequenced, 511 BACs have been deposited in GenBank.


## 3.2. Genetic and Physical Mapping

Some genetic linkage maps ofB. rapa, on which genetic markers were distributed
over ten linkage groups, have been constructed since 1990 [62–67] (summarized in Table 3). The distances of genetic linkage maps ranged from
890 cM to 1850 cM. However, the genetic linkage maps may not provide direct and
accurate genetic information for the Chiffu genome sequencing because of
genetic variation between the mapping populations. For that reason, the BrGSP community has constructed the CK
genetic linkage map. Using the 78 CKDH lines, a reference genetic linkage map
has been constructed [67]. The map consists of a total of 556 markers,
including 278 AFLPs, 235 SSRs, 25 RAPDs, and 18 ESTPs/STS/CAPS markers. Ten linkage groups were identified and designated as R1
to R10 via mapping with SSR markers derived from the reference linkage map of B. napus reported previously [68]
(Table 4). The total length of the linkage map was 1182 cM with an average
interval of 2.83 cM between adjacent loci. Recently, for high-resolution
genetic mapping, the community has set a goal of developing more than 1,000 SSR
markers derived from BAC-end sequences, ESTs, and BACs. Moreover, based on the
sequence-tagged site (STS) markers, a CKRI genetic linkage map has been
constructed to be complementary to the CKDH one. The linkage groups in these
genetic maps may not correspond to the chromosomes assigned in the cytogenetic
map. Therefore, it is important to align the linkage groups on the genetic map
with chromosomes of the cytogenetic map. All ten linkage groups of a reference
genetic map of B. rapa are being assigned
to the corresponding chromosomes through fluorescence in situ hybridization (FISH) using locus-specific BAC clones as
probes (see an example in Figure 3, unpublished data).Table 3
Genetic linkage maps ofB. rapa developed since 1990.
Mapping populationPopulation typePopulation sizeNo. of lociType of markersTotal length of map (average interval)ReferencesMichihili× Spring broccoliF295280RFLP1850 cM (6.6 cM)[62]Per (winter
turnip rape)× R500 (spring yellow sarson)F291139RFLP1785 cM (13.5 cM)[63]Per
(winter turnip rape)× R500 (spring yellow sarson)F6RI87144RFLP890 cM (6.0 cM)[64]Developed from Chinese cabbage  F1 cultivar JangwonF2134545RFLP, SSR1287 cM (2.4 cM)[65]G004
(CR(a) DH line)× A9709 (CS(b) DH line)(cultivars of
Chinese cabbage)F294262RFLP, SSR, RAPD1005 cM (3.7 cM)[66]Chiffu-401-42×
Kenshin-402-43DH78556AFLP, SSR, RADP,
ESTP, STS, CAPS1182 cM (2.83 cM)[67]Table 4
The correspondence between genetic linkage groups ofB. rapa ssp. pekinensis based on B. napus reference linkage maps.
Genetic linkage map ofB. napus [68]Choi et al. [67]Kim et al. [65]Suwabe et al. [66]A1 (N1)R1R1LG6A2 (N2)R2R2LG8A3 (N3)R3R3LG1A4 (N4)R4R4LG10A5 (N5)R5R5LG3A6 (N6)R6R6LG2A7 (N7)R7R7LG4A8 (N8)R8R8LG7A9 (N9)R9R9LG5A10 (N10)R10R10LG9Figure 3
An example of an alignment of linkage group 1 in the reference genetic map to the corresponding
chromosome 5 through FISH using locus-specific BAC clones.The fingerprinted BAC map (so-called “physical map”) makes it
possible to select clones for sequencing that would ensure comprehensive
coverage of the genome and reduce sequencing redundancy [69]. In addition, the
clone-based map also enables the identification of large segments of the genome
that are repeated, thereby simplifying the sequence assembly. To construct a
deep-coverage BAC physical map of the B.
rapa genome, all BAC clones from the three BAC libraries were fingerprinted
using restriction enzyme digestion and SNaPshot [70] methodologies, and then BAC contigs have been
assembled by FingerPrinted Contigs (FPC) software (http://www.agcol.arizona.edu/software/fpc/).
This data will be open to the Brassicarapa genome sequencing consortium.


## 3.3. Approach to Genome Sequencing

Seed BACs for genome sequencing have been selected through in silico allocation ofB. rapa BAC-end sequences onto
counterpart locations of Arabidopsis chromosomes [19]. Of 91000 BAC-end sequences, a total of 45232 showed
significant sequence similarity with unique Arabidopsis sequences, and 4317 BAC clones were allocated on Arabidopsis chromosomes by significant matching with both ends
within 30–500 kb intervals, which span 93 Mb of Arabidopsis euchromatin regions (covering 78.2% of the Arabidopsis genome). However,
approximately 9.4 Mb of euchromatin regions and 16.6 Mb pericentromeric
heterochromatin regions of the Arabidopsis genome were not covered by the B. rapa BAC span (span is considered by best hit of paired ends). Based on the physical
map of B. rapa and the in silico comparative map of its
BAC-ends onto Arabidopsis chromosomes, 629 seed BACs have been selected spanning 86 Mb of Arabidopsis euchromatin regions and
scattered throughout the B. rapa genome
(http://www.brassica-rapa.org), and the BACs have been mapped on B. rapa chromosomes by STS mapping and
FISH analysis. The seed BACs which are anchored and sequenced will be used as
stepping stones for sequencing of the ten chromosomes.Considering the large genome size and the possibility of
international cooperation, a chromosome-based approach was suggested. Of ten
chromosomes (or linkage groups), eight have been allocated to the participating
countries as follow: Korea (R3 and R9), Canada (R2 and R10), UK and China (R1
and R8), USA (R6), and Australia (R7). However, R4 and R5 have remained
unassigned. Progress
of chromosome sequencing will be reported soon by each country.


## 4. Conclusions

Brassica species are
economically important crops and serve as model plants for studying phenotypic
evolution associated with polyploidization. The Brassica genomes have extensively triplicated and undergone
subsequent genome rearrangements with sequence variations. This has significantly
affected their genome structure and may underline phenotypic diversity. Genome
sequencing of B. rapa can pave the
way for elucidation of the relationship between genome evolution and phenotypic
diversity. Moreover, it enables us to search for genes and develop molecular
markers associated with agricultural traits, thereby establishing a molecular
breeding system contributing to improvement of Brassica species economically.



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*Source: 582837-2008-01-24.xml*