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Title
A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from
comparative mapping
Permalink
https://escholarship.org/uc/item/8gn7v5x8
Journal
BMC Genomics, 13(1)
ISSN
1471-2164
Authors
Ollitrault, Patrick
Terol, Javier
Chen, Chunxian
et al.
Publication Date
2012-11-05
DOI
http://dx.doi.org/10.1186/1471-2164-13-593
Peer reviewed
eScholarship.org
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University of California
Ollitrault et al. BMC Genomics 2012, 13:593
http://www.biomedcentral.com/1471-2164/13/593
RESEARCH ARTICLE
Open Access
A reference genetic map of C. clementina hort. ex
Tan.; citrus evolution inferences from comparative
mapping
Patrick Ollitrault1,2*, Javier Terol3, Chunxian Chen4, Claire T Federici5, Samia Lotfy1,6, Isabelle Hippolyte1,
Frédérique Ollitrault2, Aurélie Bérard7, Aurélie Chauveau7, Jose Cuenca2, Gilles Costantino8, Yildiz Kacar9, Lisa Mu5,
Andres Garcia-Lor2, Yann Froelicher1, Pablo Aleza2, Anne Boland10, Claire Billot1, Luis Navarro2, François Luro8,
Mikeal L Roose5, Frederick G Gmitter4, Manuel Talon3 and Dominique Brunel7
Abstract
Background: Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering
the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus
Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map
of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly.
Clementine is believed to be a ‘Mediterranean’ mandarin × sweet orange hybrid, and sweet orange likely arose
from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present
study were to establish a Clementine reference map using codominant markers, and to perform comparative
mapping of pummelo, sweet orange, and Clementine.
Results: Five parental genetic maps were established from three segregating populations, which were genotyped
with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers.
An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by
combining male and female Clementine segregation data. This Clementine map was compared with two pummelo
maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However,
significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed
segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed
that Clementine arose from hybridization between ‘Mediterranean’ mandarin and sweet orange. The results
identified nine recombination break points for the sweet orange gamete that contributed to the Clementine
genome.
Conclusions: A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus
reference genome sequence, was established. The high conservation of marker order observed at the interspecific
level should allow reasonable inferences of most citrus genome sequences by mapping next-generation
sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to
establish the citrus reference genome sequence appears to have been inherited primarily from the ‘Mediterranean’
mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable
extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.
Keywords: C. clementina, C. sinensis, C. maxima, SSRs, SNPs, Indels, Genetic maps
* Correspondence: patrick.ollitrault@cirad.fr
1
CIRAD, UMR AGAP, F-34398 Montpellier, France
2
IVIA, Centro Proteccion Vegetal y Biotechnologia, Ctra. Moncada-Náquera
Km 4.5, 46113 Moncada, Valencia, Spain
Full list of author information is available at the end of the article
© 2012 Ollitrault et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Ollitrault et al. BMC Genomics 2012, 13:593
http://www.biomedcentral.com/1471-2164/13/593
Background
Citrus fruits were domesticated in South East Asia several
thousand years ago and subsequently spread throughout
the world. Today, the area of citrus cultivation is primarily
found between the latitudes of 40°N and 40°S, and global
citrus production has reached 122 M tonnes [1]. The production of sweet orange, the leading varietal type,
approaches close to 69 M tonnes [1]. Small citrus fruits
(mandarin-like) are preponderant in China and very important in the Mediterranean Basin where Clementine is
the main cultivar.
Despite controversial Citrus classifications, most authors
now agree on the origin of cultivated citrus species. Scora
[2] and Barrett and Rhodes [3] were the first to suggest
that three primary Citrus species (C. medica L. – citrons,
C. reticulata Blanco – mandarins, and C. maxima L.
Osbeck – pummelos) were the ancestors of most cultivated citrus. The differentiation between these sexually
compatible taxa can be explained via the foundation effect
in three geographic zones and by an initial allopatric evolution [2,4]. Other cultivated species (referred to hereafter
as secondary species) such as C. aurantium L. (sour orange), C. sinensis (L.) Osb. (sweet orange), C. paradisi
Macf. (grapefruit), C. clementina hort. Ex Tan. (Clementine) and C. limon Osb. (lemon) originated later through
hybridization and a limited number of sexual recombination events among the basic taxa. Molecular marker
studies [5-8] generally support the role of these three taxa
as ancestors of cultivated Citrus. Furthermore, some of
these studies [8-10] highlighted the probable contribution
of a fourth taxon, C. micrantha Wester, as the ancestor of
some limes [C. aurantifolia (Christm.) Swingle].
In general, Citrus species are diploid with a basic
chromosome number x = 9 [11]. Citrus species have
small genomes. While estimating citrus genome size by
flow cytometry, Ollitrault et al. [12] found significant
genome size variation between citrus species. The largest
and smallest genomes were C. medica (average value of
398 Mb/haploid genome) and C. reticulata (average
value of 360 Mb/haploid genome), respectively. C. maxima had an intermediate genome size, with an average
value of 383 Mb/haploid genome. Interestingly, the
secondary species presented intermediate values between their putative ancestral parental taxa, C. sinensis
(370 Mb), C. aurantium (368 Mb), C. paradisi, (381 Mb)
and C. limon (380 Mb) per haploid genome.
As mentioned previously, most modern cultivars have
an interspecific origin and their genomes can be considered mosaics of large DNA fragments inherited from the
basic taxa [7]. These cultivars are generally highly heterozygous [6,7]. The C. maxima and C. reticulata gene
pools contributed to the genesis of most of the economically important species and cultivars including sweet
and sour oranges, grapefruits, tangors (mandarin × sweet
Page 2 of 20
orange hybrids), tangelos (mandarin × grapefruit hybrids)
and lemons [6,7,9]. Barkley et al. and Garcia-Lor et al.
[10,11] estimated the relative contributions of primary
species to modern cultivars. Some discrepancies have been
observed between these studies, and the detailed interspecific genome organization of cultivated secondary species
and modern cultivars is still largely unknown. As a foundational step towards deciphering the phylogenetic structures of citrus genomes and the molecular bases of
phenotypic variation, a reference whole genome sequence
of a haploid derived from Clementine was produced and
is currently being revised by the International Citrus Genome Consortium (ICGC) [13,14]. The Clementine mandarin is an interspecific hybrid that was selected one century
ago in Algeria by Father Clement as a chance offspring
among seedlings of the ‘Mediterranean’ mandarin (C. reticulata) [15]. Since that time, the Clementine has been
vegetatively propagated by grafting. In a recent large SNP
diversity survey, Ollitrault et al. [8] confirmed that the
Clementine is a ‘Mediterranean’ mandarin × sweet orange
hybrid (tangor). This conclusion is in agreement with the
hypothesis of Deng et al. and Nicolosi et al. [9,16] The
supposed parental relationships between Clementine,
sweet orange, pummelo and mandarin are summarized in
Figure 1. The Clementine genome size is estimated to be
367 Mb/haploid genome [12].
The ICGC identified the construction of a saturated
genetic map of Clementine as an essential prerequisite to
improve the sequence assembly of the haploid Clementine
reference genome. Compared with other crops, genetic
mapping in citrus is relatively less well developed. The
partial genetic maps built with codominant markers
C. reticulata
C. maxima
(Mandarins)
(Pummelos)
Interspecific
hybridizations
C.clementina
C.sinensis
(Sweet oranges)
(Clementine)
Figure 1 Assumed parentage relationships between C.
reticulata, C. maxima, C. sinensis and C. clementina. From
Ollitrault et al. [8].
Ollitrault et al. BMC Genomics 2012, 13:593
http://www.biomedcentral.com/1471-2164/13/593
(primarily SSRs) [17-19] encompass around 150 markers,
while maps based on dominant markers such as AFLPs,
[20] SRAPs, ISSRs, and RAPDs [21] include slightly more
than 200 markers. Moreover, few of the mapped markers
have been published in GenBank (or other public nucleotide databases). Within the last 15 years, the citrus community developed Simple Sequence Repeat (SSR) markers
with reference sequences that were deposited in public
databases. While a limited number of SSR markers were
obtained from genomic libraries [6,22-24], the implementation of large EST databases allowed the development of
many more SSR markers [25,26], and additional markers
have been developed from Clementine BACs end sequencing (BES; [27-29]). From the same Clementine BES database, Ollitrault et al. [30] developed 33 Indel markers to
contribute to Clementine genetic mapping. Despite these
international efforts, the number of available heterozygous
SSRs and Indels in Clementine was still insufficient to establish a saturated Clementine genetic map. SNP markers
are well adapted for high throughput methods for marker
saturation. Ollitrault et al. [8] took advantage of the Clementine BES database [27] to identify SNPs heterozygous in
Clementine, and a GoldenGate SNPs array was developed.
Interestingly, 63% of the validated SNP markers were heterozygous in the sweet orange. Therefore, these SNPs can
be used for comparative mapping between the Clementine
and sweet orange.
The primary goals of the present study were: (i) to establish a saturated reference map of Clementine using codominant markers with sequences available in public
databases; (ii) to perform comparative mapping between
sweet orange, pummelo and Clementine; and (iii) to
localize the crossover events that produced the sweet orange gamete that contributed to the Clementine genome,
and those involved in the gamete formation that gave rise
to the haploid Clementine [13] used for the citrus reference whole genome sequence [14]. The clementine reference map and the pummelo map were established from
two interspecific hybrid populations (‘Chandler’ pummelo
× ‘Nules’ Clementine – CP × NC (156 hybrids) and ‘Nules’
Clementine × ‘Pink’ pummelo – NC × PP, (140 hybrids))
with 1166 codominant markers. The sweet orange map
anchored with the Clementine map was established by
genotyping 582 segregating SNP markers from 147 progeny from crosses between sweet orange and trifoliate orange (SO × TO). This study also yielded information
regarding the magnitude and distribution of segregation
distortion within the different crosses.
Results
Polymorphism and allele calls for the SNP markers
For all SNPs, genotyping was visually confirmed, taking
advantage of the distribution of the segregating progenies relative to the parental positions. This observation
Page 3 of 20
was conducted individually for each plate of 96 genotypes. Plate/marker combinations with unclear clustering
of genotypes were removed from the analysis. No differences were found between the different sweet orange
parents or between the trifoliate orange parents of the
SO × TO progenies. Therefore, all individuals resulting
from the different crosses were considered as single family. For the selected data, the markers were assigned to
different categories based on the observed segregations,
the detection of null alleles and, finally, the type of segregation assumed according to the JoinMap nomenclature (Tables 1 and 2).
The observed segregation within a progeny permitted
identification of the null alleles in terms of homozygosity
(00) or heterozygosity (A0) in the parents (Figure 2).
These two configurations of null alleles were found for 0
and 31 markers in the Clementine, 69 and 19 in Chandler,
78 and 17 in Pink, 0 and 72 in sweet orange, and 128 and
0 in trifoliate orange, respectively (Table 2 and Additional
file 1). Markers with A0 × BB and A0 × 00 configurations
were treated as < lm × ll > and the reciprocal configurations were treated as < nn × np >. Markers with the
AB × A0 configuration were analyzed as < lm × ll > by considering (i) BA and B0 hybrids as < lm > genotypes, (ii) the
undistinguishable AA and A0 as < ll >; thus, considering
only the segregation of the AB parent. Reciprocal configurations were treated as < nn × np >.
Considering all markers (with and without null alleles),
the first category consisted of markers heterozygous in
one parent and homozygous in the other (classified as
< nn × np > or < lm × ll > in JoinMap). These markers
represented the majority of the useful markers (with 606
< nn × np > and 6 < lm × ll > in CP × NC, 8 < nn × np >
and 644 < lm × ll > in NC × PP and 1 < nn × np > and 572
< lm × ll > in SO × TO). These markers were only mapped
for the heterozygous parents. As SNP markers are diallelic,
the only other conformation encountered was < hk × hk >,
where the two parents displayed the same heterozygosity.
These markers were not frequent, and 29, 24 and 9 markers with such a configuration were observed for CP × NC,
NC × PP and SO × TO, respectively. Considering our strategy to develop independent maps for each parent, the lack
Table 1 Join map codification for the different allelic
configurations encountered for SNP markers
AA
AB
BB
A0
B0
00
AA
–
lmxll
–
lmxll
lmxll
–
AB
nnxnp
hkxhk
nnxnp
nnxnp
nnxnp
nnxnp
BB
–
lmxll
–
lmxll
lmxll
–
A0
nnxnp
lmxll
nnxnp
NO
NO
nnxnp
B0
nnxnp
lmxll
nnxnp
NO
NO
nnxnp
00
–
lmxll
–
lmxll
lmxll
–
NO: Non observed configuration.
Ollitrault et al. BMC Genomics 2012, 13:593
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Table 2 Segregation types observed for the different parents and progenies
Null allele
Nules Clementine
Chandler pummelo
Pink Pummelo
Sweet Orange
trifoliate orange
JoinMap Segregation type
Chandler x Nules
Nules x Pink
Orange x trifoliate orange
of information when assigning the parental allele for each
hybrid (only possible for the homozygous hybrid and, thus,
only half of the population) and the relatively low number
of markers with this < hk × hk > conformation, these markers were removed from the mapping analysis.
SSR and Indel genotyping
The genotyping of the CP × NC population was performed
in the framework of the ICGC. SSR analysis was performed
by six international groups (University of California at
Riverside; University of Florida; University of Cukurova–
Turkey; IVIA–Spain; INRA–France and CIRAD–France,
with the collaboration of INRAM–Morocco). The genotyping of the NC × PP was performed at CIRAD and IVIA.
Homozygous or heterozygous null alleles in the parents were assumed from the observed SSR segregations.
These two configurations of null alleles were found in 2
and 10 markers in Clementine, 9 and 4 in ‘Chandler’ and
10 and 5 in ‘Pink’, respectively (Table 2 and Additional
file 1). Loci containing null alleles were treated as previously described for SNP markers. With multiallelic
SSRs, six allelic configurations were possible. AA × AB or
SSRs
Indels
SNPs
Hom
2
0
0
Total
2
Het
10
0
31
41
Hom
9
4
69
82
Het
4
0
19
23
Hom
10
0
78
88
Het
5
0
17
22
Hom
-
-
0
0
Het
-
-
72
72
128
Hom
-
-
128
Het
-
-
0
0
nnxnp
130
20
606
756
lmxll
34
2
6
42
hkxhk
1
0
29
30
efxeg
43
3
0
46
abxcd
70
0
0
70
nnxnp
24
2
8
34
lmxll
79
15
644
738
hkxhk
3
1
24
28
efxeg
19
5
0
24
abxcd
26
0
0
26
nnxnp
-
-
1
1
lmxll
-
-
572
572
hkxhk
-
-
9
9
efxeg
-
-
0
0
abxcd
-
-
0
0
CC × AB were treated equally as < nn × np > by JoinMap,
and the two reciprocal configurations were assumed to be
< lm × ll >. Fully heterozygous configurations with four
alleles (AB × CD) or three alleles (AB×BC) were coded
< ab × cd > and < ef × eg >, respectively. Among the SSRs
successfully genotyped, the five JoinMap configurations
(nn × np, lm × ll, hk × hk, ef × eg, and ab × cd) were
encountered for 130, 34, 1, 43 and 70 markers in CP × NC
and 24, 79, 3, 19 and 26 markers in NC × PP progenies, respectively. As for SNPs, the very few markers with the
hk × hk configuration were removed from the analysis.
The nn × np and lm × ll markers were mapped for the
male or female parents, respectively. The fully heterozygous markers (< ef × eg > and < ab × cd >) were mapped
for the two parents and, therefore, allowed anchoring of
the male and female parent maps.
Only four Indel markers displayed homozygous null
alleles in ‘Chandler’ pummelo (Table 2 and Additional
file 1). No heterozygous null alleles were indicated in
‘Nules’ Clementine, ‘Chandler’ or ‘Pink’ pummelos. For
Indels, the five JoinMap configurations (nn × np, lm × ll,
hk × hk, ef × eg, and ab × cd) were encountered for 20, 2,
Ollitrault et al. BMC Genomics 2012, 13:593
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Page 5 of 20
a
NormIntensity (B)
NC x PP hybrids: B0
NC: AB
NC x PP hybrids: A0
CP and PP: 00
Norm Intensity (A)
b
NormIntensity (B)
NC x PP hybrids: BB + B0
PP: B0
NC x PP hybrids: AB
NC: AB
NC x PP hybrids: A0
Norm Intensity (A)
Figure 2 Example of segregation profiles for SNP markers with null alleles for one parent and heterozygous for the other. (a) AB × 00;
(b) AB × B0.
0, 3 and 0 markers in CP × NC and for 2, 15, 1, 5, and 0
markers in NC × PP, respectively.
Parental genetic mapping
Parental gamete genotypes were generated from the diploid data using nn × np, lm × ll, ef × eg and ab × cd scored
markers. SNP, SSR and Indel genotyping data resulted in
a matrix of 156 individuals and 872 markers for male
Clementine (CP × NC progeny), 156 individuals and 158
markers for ‘Chandler’ pummelo (CP × NC progeny), 140
individuals and 788 markers for female Clementine
(NC × PP progeny), 140 individuals and 84 markers for
‘Pink’ pummelo (NC × PP progeny), and 572 markers for
147 hybrids for sweet orange (SO × TO progeny). All of
Ollitrault et al. BMC Genomics 2012, 13:593
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these matrices were analyzed using JoinMap 4. The linkage group numbering was performed according to the
sweet orange genetic map established by the US citrus
genome working group (Mikeal Roose; personal communication). The main results of the individual mapping
analyses are given in Table 3, and detailed results are presented in Additional file 2.
‘Nules’ Clementine genetic map
The reference Clementine genetic map was obtained in
two steps. In the first step, male and female Clementine
data were analyzed separately.
Male Clementine map: Among the 872 segregating
markers, 869 (606 SNPs, 240 SSRs and 23 Indels) were
distributed into nine linkage groups (LGs) while three
markers remained ungrouped. Most of the LG conserved
their integrity until LOD=10. Only LG8 was disrupted in
three sub-groups at LOD 9.The three sub-groups corresponded to three regions of LG8 separated by relatively
wide intervals without intermediate markers. When
mapped individually they displayed conserved order and
very similar distances compared with the entire LG8.
The map spanned 1164.26 cM. The Clementine male
gametes exhibited 57% of the markers deviating from
the expected Mendelian ratio (with a 0.05 probability
threshold). Skewed markers were grouped within several
parts of the genome. The skewed markers were unequally spread throughout the linkage groups with relatively low frequencies in LG2 (3.6%) and LG8 (13.5%),
but with very high frequencies in LG4 (71.6%), LG5
(83.1%), LG7 (74.5%) and LG9 (85.6%). This distribution
of segregation distortions is detailed below in comparison with the other parents.
Female Clementine map: Among the 788 markers successfully genotyped, 783 (642 SNPs, 122 SSRs and 21
Indels) were grouped in nine LGs, while five remained
ungrouped. Most of the LG conserved their integrity until
LOD=10. Only LG8 was disrupted in two sub-groups at
LOD=8 corresponding to two regions of le LG8 separated
by a relatively wide interval without marker. When
mapped individually the sub-groups displayed conserved
order and very similar distances compared with the entire
LG8.The map size was 923.5 cM. The frequency of skewed
markers (13.0%) was much lower than that observed
among male gametes. Skewed markers were mainly concentrated in LG5 (33.3%) and LG9 (24.1%).
Despite the high frequency of skewed markers in the
male Clementine map, the colinearity between the male
and female maps was highly conserved (Additional
file 3). Therefore, the reference Clementine map was
established by joining the two data sets for each LG,
including all markers present in at least one map.
Nine hundred and sixty-one markers (677 SNPs, 258
SSRs and 26 Indels) were grouped into nine linkage
groups totaling 1084.07 cM (Figure 3 and Additional
files 2 and 4). The proportion of skewed markers
remained high (46.1% for p < 0.05). The LG size ranged from 87.5 cM (LG9) to 186.3 cM (LG3). LG7 and
LG8 possessed a relatively low density of markers
with an average of 0.45 and 0.52 markers/cM, respectively. On average, nearly one marker/cM was found
on the other LGs. Each LG exhibited a heterogeneous
density of markers (Figure 4). A few gaps larger than
10 cM were observed without mapped markers, and more
gaps between 5 cM and 10 cM were observed without markers (Figure 3). These gaps were distributed, respectively, as
Table 3 Main parameters of the six genetic maps inferred from three segregating progenies
N
LG 1
M
LG 2
D
Size
LG3
M
D
Size
M
LG 4
D
Size
M
D
LG 5
Size
M
D
Size
108.34
Clementine F
140
96
3
118.08
92
9
120.06
137
2
159.42
85
13
66.13
108
36
Clementine M
156
98
54
131.09
110
4
155.69
160
88
208.00
95
68
114.17
124
103
124.30
Clementine F+ M
296
112
42
128.46
113
15
138.92
176
86
186.32
104
58
89.49
141
71
119.93
Chandler Pummelo
156
19
0
101.79
26
9
109.39
18
2
157.23
15
0
89.93
24
3
63.29
Pink Pummelo
140
8
0
67.29
10
1
100.37
4
0
39.34
6
2
69.07
15
0
71.11
Sweet Orange
147
54
13
71.70
27
1
54.33
117
25
93.15
64
2
76.22
96
48
99.87
N
LG 6
LG 7
Size
LG 8
Size
LG 9
Size
Total
M
D
M
D
M
D
M
D
M
D
Clementine F
140
86
16
88.20
40
0
86.24
44
0
97.74
95
23
Size
79.33
783
102
Size
923.54
Clementine M
156
86
53
100.46
47
35
112.22
52
7
125.81
97
83
92.53
869
495
1164.26
Clementine F+ M
296
95
59
99.80
52
19
115.59
61
5
118.03
107
88
87.54
961
443
1084.07
Chandler Pummelo
156
19
0
64.83
8
0
53.96
16
6
115.17
6
0
73.03
151
20
828.62
Pink Pummelo
140
14
6
79.83
4
0
36.84
12
0
98.47
8
4
71.58
81
13
633.90
Sweet Orange
147
60
9
65.57
36
2
84.17
45
2
39.68
70
51
84.91
569
153
669.61
N: number of gametes; LG: linkage group; M: number of markers in the LG; D: number of markers with non-Mendelian segregation (p<0.05); Size: size of the LG in
cM; F:female; M: male.
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follows: LG1 (0, 6), LG2 (0, 7), LG3 (2, 3), LG4 (0, 0), LG5
(1, 4), LG6 (1, 2), LG7 (3, 5), LG8 (3, 4) and LG9 (0, 6). On
LG9, a special feature was observed, in which 55 markers
were mapped within a 5-cM interval.
‘Chandler’ pummelo genetic map
Among the 158 segregating markers, 151 (141 SSRs, 5
SNPs and 5 Indels) were successfully mapped in nine linkage groups (Additional files 2 and 5). One hundred and
nine of these markers were common with the Clementine
map. The level of segregation distortion was low (13.2%)
and was mainly observed on two LGs (LG2: 34.6% and
LG8: 37.5%). The total size of the map was 828.6 cM.
‘Pink’ pummelo map
Only 84 segregating markers were available for Pink
pummelo mapping. Eighty-one (67 SSRs, 7 SNPs and 7
LG1
LG2
0
*
10
30
*
*
40
*
20
50
LG3
*
110
120
130
140
150
LG5
LG6
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
LG7
*
*
90
100
The sweet orange map was only based on SNP markers.
Among the 572 segregating markers, 569 were mapped in
nine linkage groups, with a total size of 669.6 cM
(Additional files 2 and 7). Most of the LG conserved
their integrity until LOD=10. However three LG (2, 3
and 5) were disrupted in two sub-groups at LOD 9, 6
and 10 respectively. As for male and female clementine
these disruptions corresponded to relatively wide interval
without intermediate markers. When mapped individually
*
70
80
Sweet orange map
*
*
60
LG4
Indels) were mapped in nine linkage groups (Additional
files 2 and 6). Fifty-two of these markers were shared
with the Clementine map. The level of segregation distortion was similar to the Chandler pummelo map
(15.9%), but affected other LGs, mainly LG6 (42.9%) and
LG9 (50%). The map spanned 633.9 cM.
*
*
*
*
LG8
LG9
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
160
170
*
180
Figure 3 Distribution of markers in the ‘Nules’ Clementine genetic map. Red: Indels, green: SSRs, blue: SNPs, **interval between two markers
> 10 cM; *interval between two markers > 5 cM and < 10 cM.
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Number of markers in 5cM intervals
30
LG1
Page 8 of 20
LG2
LG4
LG3
20
10
60
LG5
LG6
LG7
LG8
LG9
20
10
Location in the linkage groups (5cMintervals)
Figure 4 Density of markers along the ‘Nules’ Clementine genetic map.
the sub-groups displayed conserved order and very similar
distances compared with their relative entire LGs. Four
hundred and eighteen of these markers were in common
with the reference Clementine genetic map. Segregation
distortion was relatively frequent (26.9%) and was particularly clustered in LG5 (50%) and LG9 (72.9%).
Genetic map comparisons
Analysis of colinearity between the different genetic maps
Synteny, considered as the collocation of marker in the
same chromosome, was completely conserved between
all of the parental genetic maps. The linear order of the
common markers was also highly conserved between
Figure 5 Conservation of synteny and linear order of markers in the four genetic maps. NC: ‘Nules’ Clementine, CP: ‘Chandler’ pummelo,
PP: ‘Pink’ pummelo, SO: sweet orange.
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Page 9 of 20
parents (Figure 5), with only a few cases of inverted
order in small intervals. However, the genetic distance
between markers appeared to be unequal between parents. Sweet orange in particular displayed smaller distances between shared markers than Clementine. To
avoid bias due to the different number of loci analyzed,
new genetic maps of sweet orange and Clementine
(male, female and consensus) were constructed using
only the data generated from the 418 SNP markers
that were successfully genotyped in the NC × PP,
CP × NC and SO × TO progenies. The results (Additional
file 8) confirmed that the genetic distances were generally lower (except for LG4 and LG9) in the sweet
orange map than in the Clementine reference map.
Moreover, differences were confirmed between the
male and female Clementine maps for LG3, LG4, LG7,
LG8 and LG9, with systematically lower distances in
the female map. Interestingly, markers with very strong
linkage localized in the very high marker density area
of LG9 for the Clementine and sweet orange maps
were much farther apart in ‘Chandler’ and ‘Pink’ pummelos (Figure 5).
Location of crossover events in the sweet orange gamete at
the origin of Clementine and in the Clementine gamete at
the origin of the haploid Clementine used for the reference
citrus whole genome sequence
For each linkage group, the haplotypes of sweet orange
and Clementine were inferred from SNP marker phases
given by JoinMap. The origin of Clementine from a
‘Mediterranean mandarin’ × sweet orange hybridization
was proven by Ollitrault et al. [8]. Homozygous markers in sweet oranges and Mediterranean mandarin
were used to identify the haplotype of Clementine
inherited from sweet orange. Comparison of this haplotype with the two sweet orange haplotypes allowed the
identification of nine recombination break points, one
each in LG1, LG7 and LG9, and two each in LG3, LG4
and LG5 (Figure 6a). The two Clementine haplotypes
were compared with the genotyping data of the haploid
Clementine used by the ICGC to establish the reference citrus WGS haploid sequence. This permitted the
identification of eight recombination break points, one
each in LG1, LG7 and LG8, two in LG 5 and three in
LG3 (Figure 6b). Interestingly, LG2, LG4, LG6 and
a
b
0
25
50
75
LG9
LG4
100
LG6
175
LG3
cM
LG8
LG2
150
LG7
LG1
LG5 LG1
125
Mediterranean mandarin haplotype
Sweet Orange haplotype1
Sweet Orange haplotype 2
Sweet Orange haplotype not assigned
No data
Recombination break point
Figure 6 Haplotype constitution of the sweet orange gamete at the origin of Clementine (a) and of the haploid Clementine used to
establish the reference whole citrus genome sequence (b).
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LG9 appeared to have been entirely inherited from
’Mediterranean’ mandarin without recombination.
Comparative distribution of segregation distortions
To compare the location of the genome areas affected
by segregation distortions in the different parental maps,
a rough location in the reference Clementine maps was
estimated for markers (i) mapped in sweet orange but
not in Clementine, (ii) mapped in ‘Chandler’ pummelo
but not in Clementine or sweet orange and, finally (iii)
for markers only mapped in ‘Pink’ pummelo. These location estimates were performed by applying tendency
curve equations of the location in the reference Clementine map (y axis) according to the location (x axis) for
the parent map, where additional markers were mapped.
An example of such a location is presented in Additional
file 9b. The estimated locations of all markers in the
framework of the Clementine reference map are given in
the “synthesis” column of Additional file 2. The values of
the X2 conformity test of the observed segregation
against the 1:1 Mendelian hypothesis are represented
along the linkage groups for all of the parental maps in
Additional file 9a. Skewed markers appeared to be concentrated in specific areas for the different parents.
However sporadic occurrences of a non-distorted marker within a cluster of distorted markers (CiC5563-02),
or vice versa (e.g., marker CID5573) are observed in the
Clementine reference map. Such exceptions can be
explained by the inclusion of these markers with missing
data, of probable non random origin, affecting the real
segregation ratio.
The patterns of segregation distortion are consistent
with the local selection of gametes that differ in terms of
the probability of contributing to the next generation.
Male Clementine presents the higher proportion of
skewed loci. In LG1 and at the initial part of LG5, these
distortions seem to be shared with female Clementine and
sweet orange, although at a lower intensity than in male
Clementine. Shared areas of skewed loci were also
observed for male Clementine and sweet orange at the
end of LG5 and in the middle of LG9, where high marker
density was observed. In these two regions, the magnitude
of sweet orange distortions was higher than in the male
Clementine. The very severe level of segregation distortion
observed in the middle of LG3 for male Clementine is
shared at a much lower level with sweet orange. The
skewed loci of male Pink pummelo in LG6 and LG9 were
observed in areas common with male Clementine. Distortions that were observed in Chandler in the initial part of
LG2 were not observed in the other parents.
The identification of the Clementine haplotypes inherited from ‘Mediterranean mandarin’ and sweet orange
allowed determining at each locus which allele was
inherited from both parents of Clementine. Therefore, it
Page 10 of 20
was possible to determine which parental alleles (mandarin versus sweet orange) were favored for the skewed
areas of the male and female Clementine segregations
(Figure 7). No systematic tendency was observed. For
male Clementine, the skewed segregations were globally
in favor of sweet orange alleles for LG1, LG5 and LG7,
while the skewed segregations favored mandarin alleles
in LG3, LG8 and LG9. Interestingly, in LG6 and more
markedly in LG4, a transition from positive selection for
sweet orange alleles to positive selection for mandarin
alleles was observed when moving from one end of the
LG to the other. For LG1, LG2 and LG9, similar patterns
of allele segregation were observed in female and male
gametes (but generally with a lower distortion magnitude in the female). In LG4 and LG5, the patterns between male and female Clementine were very different,
with significant distortion in opposite directions. In the
second part of LG4, the mandarin alleles were favored in
male Clementine, while sweet orange alleles were significantly favored in female Clementine. In the first part of
LG5, mandarin and sweet orange allele were favored respectively in the female and male Clementine.
Discussion
A first reference genetic map for Citrus
The reviews of citrus genetic mapping performed by
Ruiz and Asins [31], Chen et al. [19] and Roose [32]
underlined that most of the earlier citrus genetic maps
were based on intergeneric hybrids between Citrus and
Poncirus. This was due to the importance of Poncirus
trifoliata for rootstock breeding. Most of these studies
suffered from relatively low numbers of analyzed hybrids
and from the dominant nature of the markers (RAPD,
AFLP) without sequence data on the mapped fragments.
Several of the more recent maps were generated using
co-dominant markers, particularly SSRs [17-19]. However, the number of mapped markers was insufficient to
establish the nine linkage groups corresponding to the
nine chromosomes present in haploid citrus. Some recent studies also focused on the genetic mapping of Citrus varieties [17,20,21,33]. The map of Gulsen et al. [21]
was the first C. clementina map, while Bernet et al. [17]
mapped Chandler pummelo and Fortune mandarin, a C.
clementina × C. tangerina hybrid. None of these maps
encompassed enough markers with published sequences
to establish a reference citrus map useful to be combined with whole genome sequence data.
The current reference Clementine map, established
from Clementine male and female segregation, includes
961 co-dominant markers (677 SNPs, 258 SSRs and 26
Indels) spread among nine LG. The map spans 1084.1 cM,
with an average marker spacing of 1.13 cM. This is a substantially higher marker density than reported in previous
citrus maps, in which nine LG were obtained. Omura
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0.25
0.2
LG1
LG2
LG3
LG4
0.15
0.1
P=5%
0.05
0
-0.05
P=5%
-0.1
-0.15
-0.2
-0.25
0.25
0.2
LG5
LG6
LG7
LG8
LG9
0.15
0.1
P=5%
0.05
0
-0.05
-0.1
P=5%
-0.15
-0.2
-0.25
Figure 7 Distribution of the segregation distortions for female and male Clementine, along the reference Clementine genetic map. The
x axis represents the location on each linkage group (LG) and y axis represents the excess of the mandarin allele relatively to Mendelian
segregation (y = frequency of mandarin allele minus 0.5). Blue represents male Clementine segregation; red represents female Clementine
segregation. The discontinuous lines represent the threshold for significant distortion (p < 0.05).
et al. [34] established a genetic map spanning 801 cM with
120 CAPS markers. Sankar and Moore [35] published an
874 cM map including 310 markers (mostly ISSR and
RAPD). Carlos de Oliveira et al. [20]) established an
845 cM map with 227 AFLP markers and more recently using 215 markers (mostly SRAP) Gulsen et al.
[21] produced a 858 cM map.
The marker density in the current reference Clementine map varied along the genome. The density was particularly low in some regions of LG7 and LG8, with
three gaps over 10 cM between markers in each of these
LGs. The SNP markers are the most numerous markers
on the Clementine map and were randomly selected.
Therefore, these low marker density areas probably reveal highly homozygous regions of the Clementine
genome. WGS data for the diploid Clementine will be
very useful for developing targeted markers within
these "no marker" regions. At the opposite extreme,
high density areas were observed in some LGs. As
described by Lindner et al. [36] and Van Os et al. [37],
some of these high marker density regions may be
associated with centromeric locations with large physical distances, possibly corresponding to low genetic
distances. Another hypothesis is that some areas with
high marker density correspond to portions of the genome in interspecific heterozygosity. Indeed, Clementine
is considered to be a hybrid between Mediterranean
mandarin and sweet orange [8,9,16]. As sweet orange is
thought to have originated as a result of interspecific
hybridization between C. maxima and C. reticulata
gene pools [6,7,9], some parts of the Clementine genome may represent interspecific heterozygosity (C.
maxima/C. reticulata). Garcia-Lor et al. [38] showed
that the SNP/kb frequency was approximately six times
higher between C. reticulata and C. maxima that it
was within C. reticulata. Thus, randomly selected
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markers should be six times more frequent (by physical
distance unit) in those parts of the Clementine genome
involved in interspecific heterozygosity. Despite the heterogeneity of marker dispersion, the distance to the
nearest mapped marker is less than 5 cM in most locations of the Clementine genome. Moreover previous
published diversity studies done with the mapped SSRs
(5, 23–26, 28), InDels (30) and SNPs (8) gave accurate
information of their transferability and polymorphisms,
at individual locus level, within and between the principal varietal groups. Therefore, this marker framework
will be very useful for marker-trait association studies
based on linkage disequilibrium, such as QTL analysis,
bulk segregant analysis, or even genetic association
studies in the mandarin group, where strong diversity
was observed for the mapped SNP markers [8]. This
map is being used to facilitate the chromosome assembly of the reference whole genome citrus sequence
based on a haploid Clementine genotype [13,39].
Linear marker order is highly conserved between species,
but genetic distances are variable between sexes and
species
The citrus genetic maps based on dominant and mainly
cross-specific markers (such as RAPD, AFLP and ISSR)
do not permit genetic map comparisons. Multi-allelic
codominant markers, such as SSRs, are more powerful
for such applications [30]. Chen et al. [19] and Bernet
et al. [17] successfully used SSRs for citrus map comparison at the interspecific and intergeneric levels.
In the present study, the main genotyping effort concerned SNPs. Eight hundred and thirty-six SNP markers
were genotyped in the three populations. Most of these
markers were mined from Nules Clementine BAC end
sequences [8,27] and, as a result, were heterozygous for
Clementine. The development of the GoldenGate SNP
markers from the Clementine sequence without information on the interspecific variability in flanking areas
resulted in numerous homozygous null alleles in pummelo
as described by Ollitrault et al. [8] and in trifoliate orange.
Heterozygous null alleles for 72 markers were found in
sweet orange, expanding the number of markers mapped
in this species. The selected SNP markers were not efficient for pummelo or trifoliate orange mapping due to the
very low number of heterozygous loci in these species.
Moreover, the biallelic nature of SNP markers limited the
establishment of two anchored maps (male and female)
from a single cross. Therefore, comparison between Clementine and pummelo was still primarily limited to common multiallelic SSRs (109 between Clementine and
Chandler pummelo and 52 between Clementine and Pink
Pummelo). With sweet orange and Clementine maps
being developed from different populations, the 418
Page 12 of 20
common heterozygous SNPs allowed more substantial anchorage of the two maps.
The conservation of synteny was complete between
the species, with no discrepancy in marker localization
on the different linkage groups between the maps. Furthermore, the linear order of markers also appeared to
be highly conserved between C. clementina, C. sinensis
and C. maxima. This is in agreement with the conclusions of Bernet et al. [17] following their comparative
study of partial maps between three species (C. aurantium, C. maxima and P. trifoliata) and Fortune mandarin, a Clementine-derived mandarin hybrid. In the
present study, small localized inversions of marker
orders were observed between maps, particularly in
dense markers areas. Bernet et al. [17] concluded that
similar results, for local ordering changes in the integrated maps, resulted from the inclusion of markers with
missing data, and eventually different levels of distorted
segregations between populations. It is also possible that
small genotyping errors concerning the markers located
in these dense regions disturbs the mapping order
[40,41]. The fine mapping of such regions will require
larger populations than the ones genotyped in this study.
For this reason, these local inversions are not detailed in
the results of this study since artifactual origins were
quite probable. Chen et al. [19] also concluded that colinearity at the intergeneric level was highly conserved
between genetic maps of C. sinensis and P. trifoliata.
However, they also observed some inversions between
shared loci that might reveal chromosomal rearrangement events, such as translocations or inversions. Considering the data of this study and the two previous
comparative mapping studies, marker colinearity appears
highly conserved at the intrageneric level (Clementine,
mandarin, pummelo, sweet orange and sour orange), but
also between Citrus and Poncirus. This global conservation of citrus genome organization will allow reasonable
inferences of most citrus genome sequences via mapping
NGS re-sequencing data to the haploid Clementine
reference genome sequence.
Variations in LG sizes were observed between the
current male Clementine and female Clementine maps.
These variations were confirmed when the new maps
were exclusively built using the markers shared between
the three populations used for the implementation of
the Clementine and sweet orange maps. Several LGs
were longer in the male Clementine map than in the female one. This was observed in LGs with significant and
extensive segregation distortions in the male haplotype
populations compared with the female populations, and
this was also observed in LG2, where very similar patterns of low skewed loci were observed. From simulated
data, Hackett and Broadfoot [41] found that segregation
distortion (due to gametic selection) alone had very little
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effect on marker order or map length. As discussed
below, the observed distortion in Clementine probably
results from gametic rather than zygotic selection.
Therefore, it is probable that the longer LGs observed
within the male Clementine map do not result from
biased estimations due to segregation distortion, but instead reflect differential recombination rates. Such heterochiasmy between sexes is frequent in plants and
animals [42-47]. According to species, recombination
should be higher in male or in female gametes [43]. Despite the fact that heterochiasmy was documented early
in the last century [44], there is still no consensus as to
which of the several proposed hypotheses may explain
its occurrence [45]. The various models were reviewed
by Lenormand and Duteil [46]. Based on a large survey
in animals and plants, these authors concluded that sexual heterochiasmy is not influenced by the presence of
heteromorphic sex chromosomes; rather, it should result
from a male–female difference in gametic selection.
However, in this study, the citrus observations do not fit
their global model considering as Trivers [47], that
higher gametic selection in one sex reduced recombination in that sex to preserve the favorable gene combinations that confer reproductive success. Indeed, we found
(see discussion on segregation distortion below) much
more significant segregation distortion, and therefore
probable gametic selection, for Clementine male gametes
than for female gametes. The citrus data is more in
agreement with models that suggest that the sex experiencing the more intense selection, or otherwise having
the higher variance in reproductive success, should show
more recombination (as reported by Burt et al. [47]).
Important differences in LG lengths were also
observed between Clementine (male and female) and
sweet orange for LG1, LG2, LG3, LG5, LG6 and LG8.
The LGs for sweet orange were systematically shorter.
The literature on plants and animals shows that the impact of structural heterozygosity on recombination frequency is variable. Different situations have been
discussed by Parker et al. [48]. It is well established that
sequence divergence at the interspecific level has an inhibitory effect on sexual recombination [49-52]. Chetelat
et al. [52] observed a strong reduction in the recombination rate in a mapping population of an interspecific F1
tomato hybrid of Lycopersicon esculentum × Solanum
lycopersicoides. The authors concluded that the high
DNA sequence divergence between L. esculentum and S.
lycopersicoides is a better explanation of reduced recombination than structural reorganization. Previously (and
also in tomato), Liharska et al. [53] showed that the
amount of recombination in a defined genetic interval
decreased as the proportion of foreign chromatin (introgressed from close relatives of L. esculentum) increased.
The authors also mentioned that, as the donor of
Page 13 of 20
the foreign chromatin became more distantly related,
the level of observed recombination was lower. As the
Clementine is a mandarin × sweet orange hybrid, and
sweet orange arose from mandarin and pummelo gene
pools (with a higher proportion of C. reticulata; [7,9]), it
is highly probable that sweet orange contains more genome regions of interspecific heterozygosity (C. reticulata/C. maxima) than the Clementine. Therefore, it can
be hypothesized that the lower LG sizes, and the associated lower recombination rates observed in sweet orange compared with Clementine, are associated with the
relative interspecific patterns along the genome of these
two species. The area of LG9 that displays substantially
greater marker density in Clementine and sweet-orange
suggests limited recombination within a large genome
portion. Thus, two set of markers were common between
the Clementine map and the two pummelo maps
(MEST308, CIBE6092 and MEST065 for Pink pummelo
and mCrCIR07F11, JI-AAG03, MEST 308 and CIBE6092
for Chandler pummelo). Interestingly, in the pummelo
maps, these markers cover 26.5 cM and 30 cM, respectively, compared with an area concentrated within 2 cM
in the Clementine map. It appears that both Clementine
and sweet orange are strongly affected by a similar recombination limitation in LG9 for which they display
equivalent map sizes. Haplotype analysis of sweet orange
and diploid Clementine shows that the Clementine
haplotype transmitted by sweet orange was inherited primarily from one of the sweet orange haplotypes, and only
a small telomeric fragment was likely to be transmitted
from the other sweet orange haplotype. Further genome
analysis along with cytogenetic and mapping studies will
be necessary to explain the different recombination patterns observed between species.
Extensive segregation distortions are observed in specific
linkage group areas particularly when Clementine is used
as the male parent
Distortions from expected Mendelian allelic segregations
were observed for all mapped parents of the segregating
progenies. The highest rate was recorded for male Clementine with 56% skewed loci (p < 0.05). This percentage
is more than four times higher than that of female
Clementine (13%), which was equal with the estimate of
female ‘Chandler’ pummelo. Male ‘Pink’ pummelo displayed a slightly higher level of distortion than female
‘Chandler’ pummelo (16%), while sweet orange (mainly
from female data) displayed an intermediate level (27%).
Distorted loci were also observed in most of the previous citrus mapping studies [17,20,54-57]. Bernet et al.
[18] also reported a higher percentage of skewed loci in
the male parents compared to the female parents in a reciprocal cross between ‘Chandler’ pummelo and ‘Fortune’ mandarin. Since most segregation distortions affect
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the allele frequencies without disturbing the genotypic
frequency equilibrium (non significant F value–Wright
fixation index; data not shown), it is probable that gametic selection was the main factor causing skewed segregation. Bernet et al. [17] reached the same conclusion
from supporting biological data on parental fertility.
Upon cross pollination with compatible parents, the proportion of fertilized ovules is much greater than the proportion of successful male gametes. Therefore, it appears
logical that gametic selection is likely to be much more
pronounced in male gametes than in females ones. This
can result from several mechanisms such as gamete
abortion, pollen competition or, the citrus gametophytic
incompatibility system [58]. The pattern of X2 conformity test values, as well as the excess of mandarin alleles
along the linkage groups, suggests that the presence of a
small number of loci under relatively strong selection
pressure on each chromosome is more likely than selection at multiple loci. Similar patterns were observed in
tomato [52]. Identical areas of skewed loci were
observed between Clementine and sweet orange in several linkage groups (LG1, LG3, LG5 and LG9). Modern
sweet orange varieties arose from an interspecific hybrid
prototype that has undergone vegetative propagation or
propagation from seeds containing nucellar embryos
over a several thousand year period. Besides favorable
mutations and stable epigenetic variations that have
been selected by man and the environment, it is probable that without the filter of sexual reproduction, the
sweet orange genome accumulated unfavorable mutations in a heterozygous status. Some of these unfavorable mutations were likely transmitted to Clementine, as
attested by the high proportion of weak progeny
obtained from Clementine × sweet orange hybridization
(our unpublished data), which should affect both sweet
orange and Clementine segregations. Interestingly, the
gametic selections have the same orientation for male
and female Clementine in the genomic regions where
sweet orange segregations are also skewed (LG1, end of
LG5, and LG9). In other genome regions, male and female Clementine segregation distortions appeared disconnected. A very strong selection is observed in the
middle of LG3 for the male Clementine, without significant skewing in the female. The male and female distortions appeared totally opposite at the end of LG4 and in
the first part of LG5. The gametophytic incompatibility
system described in citrus [58] could be a factor for male
gametic selection. However, this may lead to a complete
exclusion of one allele for the concerned locus and
therefore, a very high distortion for the linked marker
locus. This pattern was not observed in the present
study. The gametophytic incompatibility system was also
excluded as an explanation for the segregation distortion
observed in the reciprocal crosses between ‘Fortune’
Page 14 of 20
mandarin and ‘Chandler’ pummelo [17]. Some of the
more extremely unequal allelic ratios (70/30) for the
male Clementine occurred in areas without significant
distortion (or even opposite selection) in the female.
Such differences between male and female selection may
partly explain the inconsistent results observed for trait
segregation in the reciprocal crosses. Thus, it is difficult
to infer genetic control from observed trait segregations
without concomitant marker segregation analysis. This
is particularly true if major genes controlling the studied
trait are heterozygous in the male parent. QTL analysis
may also be affected as described by Xu [59].
Haplotype structure of the diploid Clementine and the
haploid Clementine used for the implementation of the
citrus whole genome reference sequence
Clementine is thought to have been selected as a chance
seedling from a ‘Mediterranean’ mandarin by Father
Clement just over one century ago in Algeria. The mandarin female parentage was confirmed by mitochondrial
genome analysis [10]. The ‘Granito’ sour orange was initially considered to be the male parent [15]. However,
molecular studies demonstrated that the Clementine
was more likely a mandarin × sweet orange hybrid
[8,9,16]. The marker phase analysis performed from the
Clementine and sweet orange mapping data confirmed
this hypothesis, and allowed the identification of the
haplotype structures of the mandarin and sweet orange
gametes that produced the Clementine. Nine recombination break points between the two sweet orange haplotypes (one each in LG1, LG7 and LG9, and two each in
LG3, LG4 and LG5) were identified for the sweet orange
gamete that produced the Clementine.
The implementation of a reference citrus whole genome sequence has been the primary focus of the ICGC
for the last 5 years. Polymorphism in a whole genome
sequence complicates the assembly process. Assembly
contiguity and completeness is significantly lower than
would have been expected in the absence of heterozygosity [60]. Commercial citrus varieties are characterized
by high heterozygosity levels [6,7]. The comparison of
blind versus "known-haplotype" assemblies of shotgun
sequences obtained from a set of BAC clones from the
heterozygous sweet orange [61] led the ICGC to establish the reference sequence of the citrus genome from a
homozygous genotype. A haploid plant derived from the
Clementine was selected due to its immediate availability
and preexisting molecular resources [26,27,62-64]. The
selected haploid was obtained by induced gynogenesis
after in situ pollination with irradiated pollen [13]. The
haploid Clementine was genotyped using the markers
mapped in diploid Clementine and sweet orange. This
permitted the constitution of the haploid genome to be
determined according to the mandarin and sweet orange
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haplotypes constitutive of the diploid Clementine. Eight
recombination break points were identified between the
two Clementine haplotypes (one in LG1, LG7 and LG8;
two in LG 5 and three in LG3). LG2, LG4, LG6 and LG9
appear to have been entirely inherited from the ’Mediterranean’ mandarin haplotype without recombination.
Overall, a very large fraction of the genome of the haploid Clementine used for WGS was inherited from the
‘Mediterranean’ mandarin.
Conclusions
Five parental genetic maps were established from three
segregating populations that were genotyped using SNP,
SSR and Indel markers. A first medium density reference
map (961 markers for 1084.1cM) of citrus was established by joining male and female Clementine segregation data. Despite the heterogeneous dispersion of
markers, this constitutes a good framework for further
marker-trait association studies, and it has been used to
enable the chromosome assembly of the reference whole
genome citrus sequence [39]. The Clementine map was
compared with two pummelo maps (‘Chandler’ map:
151 markers for 828.6 cM; ‘Pink’ map: 81 markers for
633 cM) and a sweet orange map (569 markers for
669.6 cM). The linear order of the markers appeared
to be highly conserved at the interspecific level. This
should allow for reasonable inferences of most citrus
genome sequences via mapping NGS re-sequencing
data in the haploid Clementine reference genome sequence. Important variations between the Clementine and
sweet orange map sizes were observed, as well as variations between the male and female Clementine maps. This
suggests variations in recombination rates. The smaller
length of the sweet orange map is likely related to the
higher interspecific heterozygosity within the sweet orange
genome. Skewed segregations are numerous in the male
Clementine map, underlining the potential extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in the male parent. Genetic
mapping data confirmed that the Clementine is a hybrid
between the ‘Mediterranean’ mandarin and sweet orange.
Nine recombination break points were identified between
the two sweet orange haplotypes for the sweet orange
gamete that contributed to the Clementine genome. The
genome of the haploid Clementine used to establish the
citrus reference sequence appears to be have been primarily inherited from the ‘Mediterranean’ mandarin haplotype
of the diploid Clementine.
Page 15 of 20
genetic maps. One hundred and fifty-six hybrids of
‘Chandler’ pummelo × ‘Nules’ Clementine (CP × NC)
were produced and grown at CIRAD/INRA (Corsica),
while 140 hybrids of ‘Nules’ Clementine × ‘Pink’ pummelo (NC × PP) were obtained at IVIA. Total DNA was
extracted from fresh leaves according to Doyle and
Doyle [65]. In addition to the interspecific hybrids, total
DNA was extracted from the parental lines: diploid
‘Nules’ Clementine (IVIA-22), ‘Chandler’ pummelo
(ICVN 0100608) and ‘Pink’ Pummelo (IVIA-275). DNA
was also extracted from the haploid Clementine selected
for the whole genome sequence implementation and
‘Mediterranean’ mandarin (IVIA-154), the assumed female parent of Clementine.
Sweet orange genetic mapping
One hundred and forty seven intergeneric hybrids between sweet orange and trifoliate orange (Citrus sinensis ×
Poncirus trifoliata; SO × TO) were used for sweet orange
mapping using SNP markers shared with the Clementine
map. These hybrids were obtained at UF-CREC (Florida)
and previously used for sweet orange and trifoliate orange
mapping using SSR markers [19]. The different crosses
used were: (i) 56 hybrids of C. sinensis cv Sanford (Sa) × P.
trifoliata cv Argentina (Ar), (ii) 40 hybrids of C. sinensis
cv Fiwicke (Fi) × P. trifoliata cv Flying Dragon (FD); (iii)
15 hybrids of C. sinensis cv Ridge Pineapple (RP) × P. trifoliata cv Flying Dragon (FD), (iv) seven hybrids of C. sinensis cv Fiwicke (Fi) × P. trifoliata cv Argentina (Ar); (v) six
hybrids of C. sinensis cv Ruby (Ru) × P. trifoliata cv Flying
Dragon (FD), (vi) five hybrids of C. sinensis cv Ridge
Pineapple (RP) × P. trifoliata cv DPI0906 (Ps), (vii) five
hybrids of C. sinensis cv Ruby (Ru) × P. trifoliata Argentina cv (Ar), and (viii) 13 hybrids of P. trifoliata cv
Flying Dragon (FD) × C. sinensis Ridge cv Pineapple
(RP). Due to the nature of C. sinensis intraspecific
evolution (somatic mutations but not sexual recombination), molecular polymorphisms between sweet
orange cultivars is very rare [8,19]. Therefore, after
confirming the lack of polymorphism between parental sweet oranges at the marker loci, all of the hybrids
were considered to be derived from a single sweet orange genotype for the mapping analysis. Prior to
DNA extraction, the ploidy level of all hybrids was
estimated by flow cytometry, and only diploid hybrids
were used. Genomic DNA was isolated from tender
leaves using the CTAB method as described by
Aldrich and Cullis [66].
Materials and methods
Segregating progenies and DNA extraction
Clementine and pummelo genetic mapping
Two inter-specific segregating populations between C.
clementina and C. maxima were used to establish the
Markers
A total of 1166 markers were used to genotype the progenies. Of these markers, 837 were SNPs, 301 were SSRs
and 28 were Indels.
Ollitrault et al. BMC Genomics 2012, 13:593
http://www.biomedcentral.com/1471-2164/13/593
SNPs
CiC****-**: the 802 SNPs were mined from the Clementine BAC end sequence database [27]. These markers
are part of the 1536 total SNPs used to implement an
Illumina GoldenGate assay. These markers were
selected based on their quality and segregation in the
analyzed progenies for at least one parent. They have
been published by Ollitrault et al. [8] and the corresponding GenBank accession numbers can be found in
Additional file 1.
ACO-*-***, ADC****, Aoc****, ATGGcM155, Cax4****,
CHI-*-***, DXS-M-***, FLS-M-***; HKT1c800F141; Lap
XcF***; LCY2-*-***; LCYB-*-***, MDH-P-84; NADK2c
800F***; PKF-M-186, PSY-M-289, TRPA-M-***, TScMI
1331: These 34 SNP markers were mined by Sanger sequencing of 44 genotypes representative of Citrus and
relative diversity, and were obtained from 19 genes implicated in the primary and secondary metabolite biosynthesis pathway and salt tolerance [38]. Corresponding
GenBank accession numbers can be found in Additional
file 1. Seventeen of these SNPs have been published [8].
Details on the 17 remaining markers can be found in
Additional file 10.
Page 16 of 20
Cms** and jk-****: These seven markers were developed from genomic libraries and were published by
Ahmad et al. [71] and Kijas et al. [55], respectively.
CX****: These 70 markers were developed by Chunxian
Chen and colleagues at the CREC (Florida) from an EST
database. The corresponding GenBank accession numbers
can be found in Additional file 1. Some of the mapped
markers have been published by Chen et al. [19,25]. Data
on the remaining markers can be obtained upon request
(Chunxian Chen: cxchen@ufl.edu).
Mest****: These 73 markers were developed by Luro
and Col. at INRA/CIRAD from EST databases (France).
The corresponding GenBank accession numbers can be
found in Additional file 1. Seven of these markers were
published by Luro et al. [26]. The primer sequences of
the remaining markers can be obtained upon request
(luro@corse.inra.fr).
Indel markers
CID****: These 28 markers were developed from a Clementine BAC end sequence database [27] at IVIA/CIRAD
(Spain), and have been published by Ollitrault et al. [30].
IDCAX is an Indel marker developed by Garcia-Lor
et al. [7]. The corresponding GenBank accession numbers can be found in Additional file 1.
SSR markers
The 301 SSR markers used for mapping were developed
from genomic libraries (79), ESTs (188), and BACend
sequences (34).
CI***** and mCrCIR*****: These 57 markers were
developed by Froelicher and colleagues at CIRAD/INRA
(France) from a genomic library of ‘Cleopatra’ mandarin.
Corresponding GenBank accession numbers can be
found in Additional file 1. Most of the mapped markers
have been published [23,67-69]. Primers for the
remaining markers are given in Additional file 11.
CIBE****: These 34 markers were developed by
Ollitrault and colleagues at CIRAD/IVIA (France/Spain)
from a Clementine BAC end sequence database [27].
These markers are published in Ollitrault et al. [28]. Corresponding GenBank accession numbers can be found in
Additional file 1.
CF-*****, JI-***** and NB-****: These 59 markers were
developed by Roose and colleagues at UCR (California).
Fourteen of the markers are from genomic libraries and
45 are from ESTs. Corresponding GenBank accession
numbers can be found in Additional file 1. Only the four
NB-**** markers have been published [6]. Data on the
remaining markers can be obtained upon request
(Mikeal L. Roose <mikeal.roose@ucr.edu>).
CTV2745: This marker is closely linked to the citrus
tristeza virus immunity gene of trifoliate orange and was
developed in the Roose laboratory (UCR, California)
from a genomic sequence [70].
Genotyping methods
SSRs
SSR genotyping was performed using different methods
in different laboratories (Additional file 1).
At IVIA/CIRAD and INRA, PCR products (using
wellRED oligonucleotides, SigmaW) were separated by capillary gel electrophoresis (CEQ™ 8000 Genetic Analysis
System; Beckman Coulter Inc.) as described by Ollitrault
et al. [28]. The data collection and analysis were performed with GenomeLab™ GeXP software, version 10.0.
At CIRAD and Cukurova University, PCR products
(using tailing M13 associated with three fluorescent dyes)
were separated by electrophoresis on a Li-Cor DNA
Analyzer 4200 system (Licor Biosciences, BadHomburg,
Germany). The alleles were sized according to 50- to 350bp standards (MWG Biotech AG, Ebersberg, Germany).
SSR alleles were detected and scored using SAGA
Generation 2 software (LI-COR, USA) and controlled
visually.
At the CREC, PCR products (using tailing M13) were
separated by capillary gel electrophoresis on an ABI
3130xl Genetic Analyzer (Applied Biosystems Inc., Foster
City, CA, USA). GeneScan 3.7 NT and Genotyper 3.7
NT were used to extract the trace data and generate
the microsatellite allele tables, respectively. More details
can be found in Chen et al. [25].
At UCR, PCR products labeled by an M13-tailed primer strategy were separated using a denaturing 7% Long
Ollitrault et al. BMC Genomics 2012, 13:593
http://www.biomedcentral.com/1471-2164/13/593
Ranger (BMA, Rockland, ME, USA) polyacrylamide gel
attached to a LI-COR IR2 4200LR Global DNA sequencer dual dye system. Alleles were sized manually by
comparison with 50–350 bp size standards (LI-COR),
and then scored manually from gel image files. More
details can be found in Barkley et al. [6].
Indels
Indel markers were genotyped by Capillary Gel Electrophoresis (CEQ™ 8000 Genetic Analysis System; Beckman
Coulter Inc.) using wellRED oligonucleotides (SigmaW)
as described by Ollitrault et al. [34]. Data collection and
analysis were performed with GenomeLab™ GeXP software, version 10.0.
SNPs
All SNP markers were genotyped on a GoldenGate array
platform according to the standard Illumina GoldenGate
assay instructions (www.illumina.com). More details can
be found in Ollitrault et al. [8]. Two genotype controls
(‘Nules’ Clementine and ‘Chandler’ pummelo) were
repeated twice in each plate. The data were collected
and analyzed using the Genome Studio software (Illumina). The automatic allele calling was visually checked
for each marker/plate and corrected if necessary.
Linkage analysis and genetic mapping
The two-way pseudo-testcross mapping strategy was
used to determine the linkages in the different F1 populations from the two heterozygous parents as previously
described [72] and used in previous mapping studies in
citrus [17,19,73]. Each progeny was analyzed with JoinMap 4.0 [74]. The genotyping data were coded according
to the “CP” population option adapted for such two-way
pseudo-testcrosses with no previous knowledge of the
marker linkage phases. In the first step, JoinMap was
used to establish male and female gamete populations,
which were analyzed separately. Segregation distortion
was tested by χ2 conformity tests against the Mendelian
segregation ratio of 1:1. Linkage analysis and marker
grouping were performed using the independence LOD
and a minimum threshold LOD=4. Phases (coupling and
repulsion) of the linked marker loci were automatically
detected by the software. Map distances were established
in centiMorgans (cM) using the regression mapping algorithm and the Kosambi mapping function. Given that
missing observations have much less negative impact on
the quality of the map than errors, several authors recommend identifying suspicious data and treating them
as missing observations [75,76]. In high density genetic
mapping, a genotype error usually manifests itself as a
singleton (or a double cross-over) under a reasonably accurate ordering of the markers. A singleton is a locus
whose phase is different from both the marker phases
Page 17 of 20
immediately before and after. A reasonable strategy to
deal with genotyping errors is to remove singletons by
treating them as missing observations, and then refine
the map by running the ordering algorithm [75,76]. For
the Clementine map in which a relatively high number
of markers was genotyped, singletons were automatically
checked after a first mapping round and replaced by
missing data using an excel page routine. The Clementine maps were established from these cleaned data.
Distorted markers were not removed from the analysis
because they were very frequent for some parents.
Moreover, using JoinMap, each grouping of linked loci
was based upon a test for independence in a contingency
table. Since the test for independence is not affected by
segregation distortion like the LOD score used by other
methods of linkage analysis, a lower incidence of spurious linkage is expected [74]. The linkage maps were
drawn using the MapChart program [77]. The circle plot
diagram used to compare the marker order in four genetic maps was performed using Circos software (http://
circos.ca/). Clementine and sweet orange haplotypes
were drawn with GGT 2.0 software [78].
Additional files
Additional file 1: Origin and information for all markers. This file
contains a table showing detailed information for all markers: type of
marker (Indels, SSRs or SNPs); the type of sequence data from which the
markers were developed (genomic library, BAC end sequences, ESTs);
GenBank accession number; the laboratory in which the markers were
developed; the laboratory in which the different progenies were
genotyped, the occurrence and configuration of null allele for the
parents of analyzed progenies and the references for the papers in which
the markers were published, with an indication of the modifications (if
any) in the marker names.
Additional file 2: Detailed results of genetic mapping. This file
contains the detailed information (marker locations, X2 for Mendelian
segregation, and level of significance) on the genetic maps for male
Clementine, female Clementine, reference Clementine, sweet orange,
‘Chandler’ pummelo and ‘Pink’ pummelo. The estimated location of all
markers in the reference Clementine map is also provided (synthesis
columns).
Additional file 3: Conserved linear order between male and female
Clementine genetic maps. This file contains a figure showing the
relative positions of the markers in the female Clementine map (y axis)
and in the male Clementine map (x axis) for each linkage group.
Additional file 4: Reference Clementine genetic map. This file
contains a figure showing the nine linkage groups of the reference
Clementine genetic map and the position of each marker (blue: SNPs;
green: SSRs; red: Indels).
Additional file 5: ‘Chandler’ pummelo genetic map. This file contains
a figure showing the nine linkage groups from the ‘Chandler’ pummelo
genetic map and the position of each marker (blue: SNPs; green: SSRs;
red: Indels).
Additional file 6: ‘Pink’ pummelo genetic map. This file contains a
figure showing the nine linkage groups of the ‘Pink’ pummelo genetic
map and the position of each marker (blue: SNPs; green: SSRs; red:
Indels).
Ollitrault et al. BMC Genomics 2012, 13:593
http://www.biomedcentral.com/1471-2164/13/593
Additional file 7: Sweet orange genetic map. This file contains a
figure showing the nine linkage groups of the sweet orange genetic
map and the position of each marker (blue: SNPs).
Additional file 8: Variation of map length between male
Clementine, female Clementine, and sweet orange based only on
common SNP markers. This file contains a figure for each linkage group
showing the relative position of the markers in the female Clementine
map, the male Clementine map, and the sweet orange map in a new
mapping analysis performed using only the common markers for the
three parents. The x axis represent the location on the reference
Clementine map established from all Clementine gametes (male +
female). The relative locations in the other maps (the ratio between the
locations in the other map relative to the location in the Clementine
reference map) are shown on the y axis.
Additional file 9: Comparative distribution of the skewed markers
in the nine linkage groups for five parents. This file contains a figure
for each linkage group showing the distortion magnitude (X2 of
conformity with Mendelian segregation) for each marker and each
mapped parent. Furthermore, 9b shows an example illustrating the
method used to estimate the location in the reference Clementine map
of markers mapped in the other parents.
Additional file 10: Information on the new SNP markers included in
the GoldenGate array. This file contains information regarding the new
SNP markers included in the GoldenGate array. It includes the GenBank
accession number, the sequence surrounding the SNPs, SNP position, the
GoldenGate primers and designability rank.
Additional file 11: Characteristics and primers for the new SSR
markers developed from ‘Cleopatra’ mandarin genomic library at
CIRAD. This file contains information on the primers used for the new
SSRs developed from a Cleopatra mandarin (C. reshni) genomic library
(GenBank accession number, primer sequences, annealing temperature
and microsatellite motif).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PO managed the work, analyzed the data and wrote the manuscript. JT and
MT provided the SNP markers and contributed to data analysis. DB, ABe, ABo
and AC developed the GoldenGate array and performed the SNP
genotyping. YF developed the CP×NC population and provided DNA. PA
developed the CN×PP population and provided DNA. CC and FGG provided
the SO×TO progeny DNA and performed part of the SSR genotyping. CTF,
SL, IH, FO, GC, YK, LM, AGL, CB, LN, FL and MLR contributed to the SSR and
Indels genotyping, and JC contributed to the analysis of mapping data. All
authors have read and approved the final manuscript.
Acknowledgements
This work was principally funded by the French ANR CITRUSSEQ project. The
European Commission, under the FP6-2003- INCO-DEV-2 project CIBEWU
(n°015453), the Spanish Ministerio de Ciencia e Innovación grants, AGL200765437-C04-01/AGR and AGL2008-00596-MCI, the Spanish PSE-060000-2009-8
and IPT-010000-2010-43 projects, the Prometeo project 2008/121 Generalidad
Valenciana, the Turkish TUBITAK Project No: 108O568, the California Citrus
Research Board and UC Discovery grant itl-bio-03-10122 and the Florida
Citrus Research and Development Foundation (CRDF), grants #67 and 71 also
contributed to the work.
Author details
1
CIRAD, UMR AGAP, F-34398 Montpellier, France. 2IVIA, Centro Proteccion
Vegetal y Biotechnologia, Ctra. Moncada-Náquera Km 4.5, 46113 Moncada,
Valencia, Spain. 3IVIA, Centro de Genomica, Apartado Oficial, 46113 Moncada,
Valencia, Spain. 4Citrus Research and Education Center, University of Florida,
Lake Alfred, FL 33850 USA. 5Department of Botany and Plant Sciences,
University of California, Riverside, CA 92521 USA. 6Institut National de la
Recherche Agronomique, BP293, 14 000 Kénitra, Morocco. 7INRA, UR EPGV, 2
rue Gaston Cremieux, 91057 Evry, France. 8INRA, UR GEQA, San Giuliano,
20230 San Nicolao, France. 9Department of Horticulture, Faculty of
Page 18 of 20
Agriculture, University of Çukurova, 01330 Adana, Turkey. 10CNG, CEA/DSV/
Institut de Génomique, 2 rue Gaston Cremieux, 91057 Evry, France.
Received: 28 May 2012 Accepted: 29 October 2012
Published: 5 November 2012
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doi:10.1186/1471-2164-13-593
Cite this article as: Ollitrault et al.: A reference genetic map of C.
clementina hort. ex Tan.; citrus evolution inferences from comparative
mapping. BMC Genomics 2012 13:593.
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