Guo et al. BMC Genomics 2012, 13:608
http://www.biomedcentral.com/1471-2164/13/608
RESEARCH ARTICLE
Open Access
Comparative mapping in intraspecific populations
uncovers a high degree of macrosynteny
between A- and B-genome diploid species
of peanut
Yufang Guo1,2, Sameer Khanal1, Shunxue Tang1, John E Bowers1, Adam F Heesacker1, Nelly Khalilian1,
Ervin D Nagy1, Dong Zhang1, Christopher A Taylor1, H Thomas Stalker3, Peggy Ozias-Akins2* and Steven J Knapp1
Abstract
Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is an important oilseed crop with an
allotetraploid genome (AABB, 2n = 4x = 40). Both the low level of genetic variation within the cultivated gene pool
and its polyploid nature limit the utilization of molecular markers to explore genome structure and facilitate genetic
improvement. Nevertheless, a wealth of genetic diversity exists in diploid Arachis species (2n = 2x = 20), which
represent a valuable gene pool for cultivated peanut improvement. Interspecific populations have been used
widely for genetic mapping in diploid species of Arachis. However, an intraspecific mapping strategy was essential
to detect chromosomal rearrangements among species that could be obscured by mapping in interspecific
populations. To develop intraspecific reference linkage maps and gain insights into karyotypic evolution within the
genus, we comparatively mapped the A- and B-genome diploid species using intraspecific F2 populations. Exploring
genome organization among diploid peanut species by comparative mapping will enhance our understanding of
the cultivated tetraploid peanut genome. Moreover, new sources of molecular markers that are highly transferable
between species and developed from expressed genes will be required to construct saturated genetic maps for
peanut.
Results: A total of 2,138 EST-SSR (expressed sequence tag-simple sequence repeat) markers were developed by
mining a tetraploid peanut EST assembly including 101,132 unigenes (37,916 contigs and 63,216 singletons) derived
from 70,771 long-read (Sanger) and 270,957 short-read (454) sequences. A set of 97 SSR markers were also
developed by mining 9,517 genomic survey sequences of Arachis. An SSR-based intraspecific linkage map was
constructed using an F2 population derived from a cross between K 9484 (PI 298639) and GKBSPSc 30081
(PI 468327) in the B-genome species A. batizocoi. A high degree of macrosynteny was observed when comparing
the homoeologous linkage groups between A (A. duranensis) and B (A. batizocoi) genomes. Comparison of the
A- and B-genome genetic linkage maps also showed a total of five inversions and one major reciprocal
translocation between two pairs of chromosomes under our current mapping resolution.
Conclusions: Our findings will contribute to understanding tetraploid peanut genome origin and evolution and
eventually promote its genetic improvement. The newly developed EST-SSR markers will enrich current molecular
marker resources in peanut.
Keywords: Peanut (Arachis hypogaea), SSR, Genetic linkage map, Intraspecific cross, EST
* Correspondence: pozias@uga.edu
2
Department of Horticulture, The University of Georgia, Tifton, GA 31973,
USA
Full list of author information is available at the end of the article
© 2012 Guo 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.
Guo et al. BMC Genomics 2012, 13:608
http://www.biomedcentral.com/1471-2164/13/608
Background
Peanut or groundnut (Arachis hypogaea L.) is both an
important oilseed crop and a direct protein source for
human nutrition and is the only domesticated species in
the genus Arachis. It is an allotetraploid (2n = 4x = 40,
AABB) with one pair of distinctively small chromosomes
[1], and was believed to have originated from a single
hybridization event at least 3,500 years ago [2-4]. This
genus also contains additional tetraploid, diploid (2n =
2x = 20) and aneuploid (2n = 2x = 18) species. Arachis
hypogaea is a member of section Arachis, which contains species with A, B, or D genomes. The Arachis genome is characterized by the presence of a small pair of
chromosomes with a lower level of euchromatin condensation [5], whereas the B genome is distinguished by the
presence of a pair of chromosomes with a secondary
constriction proximal to the centromere [6], and has
recently been divided into subgroups [7]. Arachis duranensis and A. ipaënsis are most likely the ancestral
A- and B-genome species of cultivated peanut, respectively [2,5,8-10]. The D genome is represented by the
single species A. glandulifera [11,12].
Due to both the low level of genetic variation within
the cultivated gene pool and the polyploid nature of cultivated peanut, relatively fewer numbers of genetic linkage maps have been constructed as compared to many
other crop species [13]. Considering its relatively large
genome size (2800Mb/1C), there is still great need to
improve marker density and genome coverage [14,15].
For example, mapping of quantitative trait loci (QTL)
for late leaf spot, rust [16] and seed quality [17] traits
has only been possible at low marker density with 225
and 45 SSR loci, respectively.
A wealth of genetic diversity exists in diploid Arachis
species [18] with the potential to introgress novel alleles
into cultivated peanut [4,19]. The diploid wild species
are not commercially important in the food industry,
but they provide a valuable gene pool for sources of
resistance to many economically important pathogens and
pests [20-23]. Several linkage maps have been constructed
in both A- and B-genome diploid species [24-28], with the
first A-genome linkage map reported being composed of
11 linkage groups with 117 RFLP loci and with a genome
coverage of 1,063 cM [26]. Subsequent A-genome linkage
maps consisted of 167 RAPD and 39 RFLPs, 170 SSR, or
369 assorted markers [25,27,28]. An interspecific Bgenome linkage map has been constructed with 149 SSR
loci covering 1,294 cM [24]. Synteny between A and B
genomes was compared using diploid as well as synthetic
amphidiploid linkage maps [24,29,30].
The above-referenced diploid linkage maps were
generated from interspecific crosses. Thus, the detection
of chromosomal rearrangements within species could be
obscured, and comparative A- and B-genome linkage
Page 2 of 12
maps could be complicated due to chromosomal rearrangements associated with speciation [31-35]. Previous
comparisons were based on limited numbers of orthologous loci and markers that were primarily derived from
genomic sequences. Therefore, map coverage and resolution need to be improved using more function-related
and highly transferable markers such as EST-SSRs that
facilitate comparative and evolutionary genomics studies.
To enrich the currently available SSR marker resources
in peanut and gain clearer insights into karyotypic evolution within the genus, a new set of EST-SSR markers was
developed and mapped in an intraspecific B-genome
mapping population. Comparative mapping to intraspecific A-genome linkage maps revealed a high degree of
macrosynteny.
Results and discussion
Marker development
A total of 101,132 unigenes representing ca. 37 Mb of
the A. hypogaea genome (Additional file 1) were mined
for SSRs. We found 7,413 perfectly repeated di-, tri-,
tetra-, penta-, and hexa-nucleotide motifs (7.3% of the
unigenes contained SSRs). The SSR frequency in the
above EST resources is comparable with previous
reports in cultivated peanut [36,37], and wild Arachis
species [38]. The overall SSR density was 3,190 bp per
Mb and corresponded to approximately 1 per 5 kb of
the genic region, which is similar to a previous report of
1/5.5 kb in cultivated peanut [15]. higher than Arabidopsis [39], and barley [40] but slightly lower than that
reported for rice [41] and pepper [42], yet within the
range of most other plant species (~5%) [43]. The average SSR length was about 16 bp with almost 88% of
SSRs shorter than 22 bp. Among repeat motifs, dinucleotides were predominant (53.3%), which was inconsistent with the study of Koilkonda et al. [15], who
found trinucleotide repeats to be the most abundant
(66.8%). Discrepancies observed in various studies could
be explained by the degree of representation of dinucleotide rich UTRs in the genic sequences used or by EST
database mining software and SSR search criteria [43].
Dinucleotide repeat motifs were predominantly distributed in the UTRs while trinucleotide repeat motifs were
more frequent in exons. Considering the mode of
slippage-mediated mutations, it is unlikely that a large
proportion of the dinucleotides would be present in the
coding regions; mutations in trinucleotides or their multiples would only be tolerated if they do not disturb the
open reading frame.
In our study, the most common dinucleotide repeat
motif class was (AG)n (61.0%), while the least common
repeat class was (CG)n (1.2%). Similarly, the most abundant trinucleotide motif class was (AAG)n (35.2%), and
the least frequent was (CGA)n (0.9%). Motif classes
Guo et al. BMC Genomics 2012, 13:608
http://www.biomedcentral.com/1471-2164/13/608
Page 3 of 12
5), which was supported by the previous observations of
Moretzsohn et al. [25]. For example, SSRs with a repeat
length >26 bp showed up to 30% polymorphism between
the four tetraploid genotypes, while less than 15% polymorphism was observed for SSRs with repeat length
<20 bp. However, when all eight genotypes were combined, no trends were observed between the polymorphism and repeat length. For tetraploid genotypes, AG/CT
repeats were more polymorphic than GT/CA, while in
diploid genotypes, this effect can only be observed between interspecific genotypes in spite of the fact that the
polymorphisms among four diploid genotypes were overall high (around 70%) (Table 1). Similar observations that
AG/CT repeats were more polymorphic than GT/CA
were previously reported in peanut [25,36,48]. The polymorphisms for dinucleotide repeat motif types were generally higher than for trinucleotide repeat motif types.
However, no consistent pattern emerged for ranking of
polymorphism rate by motif type.
In addition to ESTs, a total of 9,517 genome survey
sequences (GSSs) representing ca. 5.5 Mb of the Arachis
genome were mined for SSRs. Overall, 1,168 perfectly
repeated di-, tri-, or tetra-nucleotide motifs were identified
from 960 unique sequences. By SSCP screening, 97 SSR
primer pairs can produce reliable amplification across a
panel of 12 genotypes representing different species
(Additional file 6).
(CG)n and (CGA)n are also relatively infrequent in other
plants as well as animals [44]. The motif types (AG)n
and (AAG)n have been reported as the most common
di- and tri-nucleotide repeats identified in other plant
EST databases [43,45-47], including peanut (A. hypogaea) [15,30,37,40,48,49].
We designed 2,138 EST-SSR primer pairs from the identified SSRs (Additional file 2), with 94.6% of them targeted
to amplify perfect repeats and over 70% of the primers targeted at trinucleotide motif types. By screening a panel containing eight genotypes, (Additional file 3), 15.3% of the
2,138 primer pairs didn't amplify any interpretable fragments, and 82.7% of them were fully transferable between
tetraploid and diploid species. As expected, we observed a
relatively higher transferability for EST-SSRs than for genomic SSRs; this is most likely due to greater sequence conservation within expressed regions among related species
compared to non-coding regions [25,36,43,50,51]. The frequency of polymorphism among the four tetraploid genotypes was 11.2% and less than 10.0% between paired
tetraploids. The polymorphism between two A. duranensis
accessions was 41.9% while between two A. batizocoi accessions was 21.3% (Additional file 4). The polymorphisms for
our intraspecific diploid mapping population parents were
comparable to the previous reports of interspecific diploid
mapping populations for EST-SSR markers [24,25,30]. We
also observed that the polymorphism was higher in the Agenome species than in the B-genome species [52].
Our findings also support the general theory that the
degree of polymorphism of the SSR marker increases
with the total length of the repeat [25,53,54]. A positive
correlation was observed between repeat length and
polymorphism rate, but the trend seemed more obvious
in tetraploid than in diploid genotypes (Additional file
Genetic mapping
Collectively, 2,138 newly developed EST-SSR primer
pairs (Additional file 2), 97 genomic SSR markers developed from genome survey sequences and 612 genomic
SSR primer pairs in the public domain (Additional file 7)
were screened for polymorphisms between the parents
Table 1 Effect of SSR repeat motif types on frequency of polymorphism among tetraploid and diploid genotypes
Motif
type
No. of
Between Tif-runner Between NC94022 Among four
Between 30081 Between Grif Among four Among eight
markers and GTC20 (%)
and SunOleic (%) tetraploid (%) and 9484 (%)
15036 and
diploid (%) genotypes
PI 475887 (%)
(%)
GT/CA
285
8.1
2.7
10.8
21.6
51.4
75.7
AG/CT
37
16.8
14.0
24.2
32.3
57.5
70.2
78.4
73.0
AT
69
14.5
13.0
23.2
21.7
47.8
69.6
76.8
CG
1
0
0
0
0
0
0
0
CCG/CGG
41
2.4
2.4
4.9
7.3
29.3
56.1
61.0
ACC/GGT
149
3.4
3.4
5.4
19.5
39.0
67.8
73.2
ACG/CGT
55
3.6
0
3.6
14.5
27.3
60.0
67.3
AGC/GCT
52
0
1.9
3.8
11.5
25.0
59.6
65.4
AGG/CCT
107
3.7
1.9
6.5
10.3
26.2
55.1
65.4
AAC/GTT
141
5.7
5.0
9.2
17.0
43.3
70.2
78.7
AAG/CTT
519
5.4
4.8
9.6
24.5
39.1
66.5
74.2
ACT/AGT
130
2.3
3.8
6.2
12.3
34.6
63.8
73.1
ATC/GAT
127
5.5
5.5
7.9
13.4
39.4
67.7
75.6
AAT/ATT
168
6.0
3.6
6.0
20.2
44.6
70.8
79.2
Guo et al. BMC Genomics 2012, 13:608
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Page 4 of 12
of an intraspecific A. batizocoi (BB, 2n = 2x = 20) F2
mapping population. Although A. ipaënsis is the more
likely B-genome donor than A. batizocoi to tetraploid
peanut species A. hypogaea [52,55,56], A. batizocoi
retains a high level of similarity to the B subgenome of
cultivated peanut [57]. For example, the F1 plant derived
from crossing A. hypogaea by a synthetic amphidiploid
(A. batizocoi × A. duranensis) produced bivalents, and
a few F4 plants from this cross were even able to produce two-seeded pods [58]. Furthermore, a diversity
study indicated that among all the B-genome species,
A. batizocoi showed the second closest relationship to
A. hypogaea, after A. ipaënsis [55]. Although the hypothetical B-genome donor A. ipaënsis was not used for
linkage mapping in this report because only a single
accession is available in the U.S. germplasm collection,
our A. batizocoi intraspecific map should still provide
a very close representation of the B-genome donor of
tetraploid peanut.
The screening of A. batizocoi accessions 9484 and
30081 produced 455 polymorphic EST-SSR and 171
polymorphic genomic SSR markers. After excluding
makers with numerous and/or faint bands and abnormal
segregation ratios (markers that showed extreme segregation ratios were assumed to be caused by loci with indistinguishable bands), a total of 481 markers were used
for linkage map construction. Of these, 449 loci (including 347 loci from the newly developed EST-SSR markers,
14 loci from genomic SSR markers developed from GSS
sequences, and 88 loci from the genomic SSR markers
already reported [18,25,48,49,59-62]) were mapped into
16 linkage groups (LGs), 14 of which aligned with the
10 chromosome pairs of diploid peanut numbered according to colinearity with A. duranensis (A- genome) linkage
groups from Nagy et al. [63]. The remaining two small
linkage groups had no common markers with A- genome
linkage groups, thus their chromosomal locations are
unknown. Their lengths were 1.1 cM and 9.2 cM, respectively. One LG consisted of markers GM2227 and
GM1611, and another LG was composed of markers
GM1241 and GM748.
Overall, the linkage map covered 1,278.6 cM, with
marker densities ranging from 1.1 cM/locus in LG11 to
9.2 cM/locus in LG12, giving an average density of
2.9 cM/locus for the entire map (Table 2, Figure 1). The
LGs ranged from 1.1 to 210 cM in length, and had two
(4/9B.1, 4/9B.2, and the two linkage groups described
above) to 80 (4/9B) marker loci. Gaps larger than
30.0 cM were observed only at the end of 1B (31.9 cM)
and 8B (33.6 cM) (Figure 1). The number of linkage
groups observed in this study is larger than the number
of haploid chromosomes in the diploid species (n = 10),
which may be due to insufficient markers for the
chromosome coverage. Furthermore, since the B-genome
linkage groups were named by comparing common markers to A-genome linkage groups, the chromosome location of some small linkage groups could not be
identified if they lacked anchor markers. The map
length was comparable to previously published diploid
peanut genetic maps while the density was the highest
among all B-genome linkage maps constructed to date
[24-26]. This is by far the most saturated map
Table 2 Number of loci, map length and density of each linkage group in the 9484x30081 map
Linkage group
Length (cM)
No. of marker/LG
Density (cM/locus)
Number of distorted
loci (α = 0.05)
1B
98.2
38
2.7
10
2B
138.3
35
4.1
1
3B
117.8
62
1.9
0
4/9B
209.9
80
2.7
1
4/9B.1
3.4
2
3.4
0
4/9B.2
8.6
2
8.6
0
4/9B.3
47.1
7
7.9
1
5B
114.8
52
2.3
0
5B.1
30.1
6
6.0
0
6B
164.2
44
3.8
1
7B
36.6
13
3.1
2
8B
185.1
60
3.1
0
8B.1
7.1
5
1.8
0
10B
107.1
39
2.8
2
LG11
1.1
2
1.1
0
LG12
9.2
2
9.2
0
Whole map
1278.6
449
2.9
18 (4.0%)
Guo et al. BMC Genomics 2012, 13:608
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1A
9.3
13.6
16.9
18.9
20.4
21.0
24.4
26.3
26.7
29.8
37.6
38.0
38.5
38.9
39.2
39.4
39.6
39.8
40.0
40.1
40.2
40.5
41.1
41.6
42.0
42.8
43.8
45.7
47.2
47.5
47.9
49.8
50.0
50.2
50.4
50.5
50.6
50.7
50.8
51.0
51.1
51.4
51.7
52.0
52.6
54.1
54.2
54.7
55.7
59.4
61.0
63.6
64.5
66.6
76.6
76.7
77.9
87.9
91.4
1B
GM1612
GM1750
GM2344
GM1085
GM1277
GM1620GM1275
GM38
GM1573
GM2053
GM2653
GM2061
GM1684
GM2004
GM1871
GM2132
GM2418
GM1599GM1542
GM1501
GM2350
GM1771
GM1913GM2349
GM2783
GM1879
GM2390
GM1950
GM2724
GM1444
GM933GM1339
GM1330
GM1353
GM1065
GM2840
GM1777
GM286
GM824
GM672
GM1048
GM2287GM1972
GM1233GM2527
GM1992 GM1649
GM22GM2597
GM890
GM1171
GM2015
GM917
GM170
GM2607
GM2233
GM2153
GM2173
GM1483
GM1423
GM2024
GM2603
GM1208
GM1132
GM2568
GM1504
GM2022GM1761
GM1118b
GM1331
GM825GM1030
GM24
GM1661
GM1992
0.0
1.1
GM2602GM1725d
GM2037
GM1725a
GM1401
GM1182
GM1409
GM2565a
GM475
GM24
GM2171GM1845
GM110aGM187
GM2606GM499
GM2122
GM2654
GM1610
GM443
GM1612 GM496
GM2344 GM529
GM2653GM1085
GM635
GM2132
GM1501
GM841
GM1849 GM2350
GM1290
GM1444
GM933GM1339
33.0
34.6
35.4
40.1
41.7
47.7
48.8
54.8
59.7
62.4
63.5
67.1
67.7
68.9
69.4
70.5
76.0
78.7
82.5
84.6
86.3
91.9
98.2
10A
0.0
1.9
18.6
26.8
26.9
29.7
38.4
43.2
44.5
46.0
46.6
49.0
50.1
50.6
51.1
51.5
51.6
51.7
51.8
52.0
52.3
52.6
52.9
53.9
55.1
58.4
58.9
59.9
62.1
63.0
64.9
66.0
67.0
69.5
70.6
73.0
78.5
79.1
79.4
84.7
89.0
92.2
97.3
100.2
101.1
101.3
101.8
103.9
104.7
110.2
2A
10B
GM2032
GM2166
GM857
GM1520b GM1520a
GM1886
GM1520c
GM2218
GM2148
GM2283
GM1527
GM2531
GM1453
GM1408GM2348
GM1581
GM1172 GM1460
GM2104
GM1875
GM1933
GM1526GM117
GM1303 GM1322
GM2582
GM784
GM1563
GM2207
GM2084
GM2040
GM1341
GM2383
GM2320GM1243
GM1481
GM1868
GM2722
GM2303
GM1195
GM927
GM926
GM2058
GM2029
GM2140
GM1764
GM2041
GM1624
GM1163
GM1561
GM2316
GM1107
GM810
GM2577
GM1562
GM951GM2082
GM2266
GM1373
GM950
GM2001
0.0
2.4
6.0
9.3
18.9
19.4
24.3
29.2
31.9
33.0
34.1
35.7
36.2
36.7
37.0
37.3
38.9
41.4
42.6
47.5
52.1
58.4
59.0
61.7
65.5
75.4
2B
GM1865
0.0
9.8
14.7
20.5
20.6
21.9
24.0
24.1
25.7
28.3
30.8
37.2
37.6
47.0
47.5
48.2
49.7
49.9
50.0
52.2
53.9
GM1930
GM1819
GM2016
GM733
GM2500
GM1160
GM1724
GM1852
GM2047
GM1946
GM2797 GM1714
GM2496
GM1386
GM1598
GM2491
GM1075
GM1503
GM1516 GM2455
GM2474
GM126
62.6
GM1668 GM2111
70.1
74.1
74.2
76.5
77.8
78.2
79.3
GM2257
GM2121
GM2814
GM1910
GM2098
GM1836
GM2808
87.8
88.2
92.3
96.7
96.8
97.5
99.0
103.7
GM2838
GM817
GM1655
GM1355
GM922
GM2232
GM2186
GM2845
GM1742
GM1520
GM2218
GM2217
GM1718
GM1076b
GM1527
GM450GM472
GM1016GM279
GM114GM2023
GM1563 GM2819
GM1172GM1868
GM1303 GM2411
GM238
GM1832
GM1568
GM108
GM40
GM1137
GM927
GM1074
GM1163
GM439
GM471
GM2340
100.0
100.5
GM2762
GM2763 GM2266
107.1
GM2124
0.0
3.4
GM2007
GM1840
0.0
GM1817
8.6
GM1963
Figure 1 (See legend on next page.)
3A
0.0
1.6
GM1865
GM656
10.0
GM1819
26.0
28.2
GM2196
GM87GM1860
43.0
GM509
49.1
49.6
GM1714
GM1538GM2581
54.5
GM2580GM376
60.8
GM1386
71.7
79.7
81.7
82.4
83.4
84.5
86.8
89.2
91.6
92.1
93.2
97.6
98.7
102.5
GM77a
GM1075
GM1516
GM1180
GM2655GM421
GM574
GM126
GM1598
GM430b
GM2656
GM2272 GM1668
GM534
GM1910
GM1836
GM1821
GM266
82.9
4/9B.1
4/9B.2
GM2304
GM205
GM973
GM2828
Page 5 of 12
122.8
124.4
GM431
GM1051
132.2
GM2232
138.3
GM2845
3.9
6.6
9.5
14.8
26.0
29.1
32.6
34.7
35.5
35.6
39.7
42.9
43.3
44.3
45.9
48.3
55.4
57.2
57.5
57.8
57.9
58.0
58.4
58.6
58.9
59.3
59.8
62.1
62.9
64.4
64.7
65.6
66.7
67.4
67.7
68.0
68.7
69.4
70.2
71.2
71.7
72.0
72.4
74.9
75.9
77.0
81.0
81.5
82.6
83.2
90.2
91.1
94.5
95.3
96.8
102.0
102.9
106.3
112.0
112.9
115.8
GM32
GM2599
GM2598
GM2818
GM2261
GM2372
GM1760
GM1149
GM1609
GM1191
GM1425
GM2764
GM2087
GM1266
GM2407
GM1360
GM2693GM2694
GM1733
GM2230GM2524
GM2467
GM1656
GM1487
GM828
GM2206
GM2364
GM2352
GM2184
GM2274
GM774
GM1566
GM1034
GM898
GM1618
GM1473
GM1763
GM19
GM2008
GM1583
GM1500
GM1053GM2449
GM783
GM2586
GM2402
GM1645
GM1659a
GM1681
GM1443
GM1369
GM1502 GM1717
GM2215
GM2556
GM1249
GM2772GM1726
GM2051
GM1883
GM2416
GM2027
GM743
GM2419
GM2528
GM1108GM1885
GM2069
GM866
GM2765
GM1084
GM2792a
GM714
GM1326
GM1954
GM1321
GM1652
GM2228
GM2103
GM2805
GM1213
GM1591
GM1536
GM74
GM2302
GM2301
GM1263
GM1854
GM2757
0.0
8.9
9.1
9.4
12.1
13.4
14.0
15.7
17.3
20.2
20.4
21.2
30.1
37.9
38.0
38.3
39.1
39.9
40.8
42.3
46.3
46.7
47.7
49.5
49.6
51.2
51.7
52.1
54.0
54.6
54.9
57.2
58.6
59.1
59.7
60.5
61.0
61.8
62.6
63.0
64.4
65.0
67.6
69.4
75.5
77.4
77.9
78.3
78.5
78.6
79.1
81.6
85.0
85.7
86.2
95.0
95.9
97.0
101.1
102.4
102.7
103.3
105.1
108.4
110.0
110.4
114.5
119.8
121.7
122.3
122.9
124.6
124.7
124.8
125.2
127.1
127.7
130.5
131.2
138.4
139.9
145.6
4A
3B
0.0
2.7
9.4
9.9
12.6
13.7
15.9
26.2
34.6
36.2
36.7
50.0
53.2
54.8
56.9
58.5
61.7
66.0
68.7
69.2
69.7
70.8
71.9
72.4
72.9
74.0
75.6
78.0
81.6
85.4
86.9
88.1
88.7
93.1
96.9
98.5
100.1
103.3
104.4
104.9
105.4
105.9
106.8
108.6
110.8
112.9
117.2
117.8
4/9B
4/9B.3
GM1840
GM2014
GM2091
GM1120
GM2010
GM1528
GM76
GM1647
GM1466
GM2108
GM1445
GM2448
GM1738
GM839
GM1173
GM1867
GM1565GM2246
GM1021
GM1720
GM1755
GM1296
GM2589
GM218
GM2813
GM1887
GM1959
GM2311
GM2572
GM2006
GM1465
GM1995GM1070
GM1805
GM916
GM1186GM1643
GM1167GM96
GM2142
GM1013
GM2482
GM2480
GM1554
GM162 GM1190
GM1143
GM1907
GM1919
GM1679
GM1594
GM1298
GM1729
GM1223
GM2236
GM13
GM2583
GM942
GM955
GM1062
GM1817
GM735
GM1963
GM1045
GM2755
GM2021GM2020
83.9
85.0
88.3
92.1
93.2
97.6
98.1
100.8
105.2
GM2010
GM1918
GM426
GM1670
GM76
GM682
GM1466
GM839
GM1173
GM1984
GM2246
GM2778b
GM2311
GM890
GM2542
GM2553
GM1823 GM1565
GM430a
GM952
GM2847
GM1478
GM2832
GM1462
GM2690
GM660
GM707
GM1932
GM2479
GM2113
GM1651GM1744
GM2400
GM1965
GM1861
GM425GM1922
GM2689GM1194
GM2326GM1136
GM423GM70
GM2074
GM663
GM1403
GM2488
GM110b
GM390GM1135
GM1167
GM2841
GM96
0.0
1.1
3.6
4.3
4.8
7.5
9.1
12.9
14.5
17.7
18.2
18.7
19.2
23.6
29.1
30.2
30.7
32.9
34.2
35.5
45.2
47.4
54.0
57.8
58.3
60.5
65.4
69.8
75.3
79.1
79.6
81.5
83.4
118.5
GM344
123.8
127.4
130.9
131.4
131.9
134.6
GM231
GM2778a
GM2521
GM2438
GM1338
GM2120
141.8
143.4
143.9
144.4
147.1
148.7
152.2
158.0
159.7
GM792
GM1650
GM2026
GM1893
GM2793
GM2558b
GM1906
GM66
GM2450
180.0
183.2
183.7
184.8
GM1907GM162
GM541
GM2181
GM493
192.0
195.2
GM1067
GM2106
199.8
GM942a
205.0
208.3
209.9
0.0
2.2
4.9
GM64
GM1298
GM622
GM1056
GM1045
GM2081
8.8
GM1199
20.3
GM2070
41.5
GM1459
47.1
GM1803
5A
4.0
6.0
9.5
12.4
12.7
13.7
16.8
18.0
20.6
23.8
24.2
36.3
38.3
39.5
40.8
44.2
46.6
47.0
47.6
48.7
49.8
50.7
51.0
51.5
52.9
53.1
53.2
53.4
53.5
53.7
53.8
54.4
54.8
54.9
55.0
57.1
58.7
60.4
61.3
63.8
64.7
65.9
66.2
69.1
69.4
69.5
69.7
70.5
71.1
71.3
71.5
73.2
76.7
77.4
84.4
84.8
91.0
91.1
91.7
112.0
112.1
116.4
GM32
GM2599
GM1149
GM1609
GM2372
GM1425
GM1003GM2543
GM2011
GM2355
GM2407
GM2321
GM1487
GM97GM11
GM1484
GM1566
GM1473
GM1551
GM1779bGM2088
GM415
GM1846
GM2224
GM1573
GM1361
GM469
GM49 GM1645
GM1447
GM121
GM1996
GM1717 GM1874
GM1545
GM736
GM616
GM2308
GM2688
GM2765
GM427
GM460 GM2757
GM1854
GM1642
GM2302
GM74 GM832
GM2060 GM2301
GM195
GM1321
GM1954
GM2801
GM1407
GM2730GM2729
GM655
GM866 GM211
GM877
GM2528
9A
5.3
7.4
21.7
23.7
24.7
27.0
28.9
33.3
34.4
36.6
42.0
42.3
48.3
48.9
52.3
52.6
53.2
54.5
55.1
55.5
55.6
55.7
55.9
56.6
56.7
57.3
59.9
60.2
62.4
83.0
89.2
89.3
91.1
93.0
94.1
94.2
99.4
100.5
5B
5B.1
GM2770
GM2703
GM1898
GM1728
GM1253
GM1519
GM948
GM1261
GM2793
GM1119
GM1118a
GM1317
GM1154
GM1313
GM2771
GM2663
GM2662
GM1987
GM1332
GM2092
GM2031
GM2043
GM874
GM996
GM1049
GM2820
GM1892
GM2078
GM1257
GM68
GM1577
GM2160
GM1482
GM1955
GM1967
GM16
GM28
GM1018
GM2347
GM1890 GM2429
GM1218GM2487
GM2672
GM2673GM1782
GM2137
GM2231
GM2769
GM1788
GM1227
GM1228
GM1878
GM2017
GM1843
GM1778
GM1293
GM944
GM1902
GM1912
GM1816
GM1904
GM1702
GM1162 GM1944
GM1352
0.6
2.2
2.4
4.1
6.5
7.2
7.7
8.1
8.7
8.9
9.0
9.2
9.7
10.0
10.3
10.5
11.0
11.6
11.9
12.4
13.0
13.5
14.0
14.2
14.6
15.0
15.9
16.6
17.6
18.3
19.8
19.9
20.0
20.2
20.6
21.3
21.5
21.6
21.8
22.9
23.7
24.9
25.1
26.4
27.1
28.6
28.9
29.2
30.7
31.8
32.6
34.7
34.9
38.1
41.0
41.9
42.2
42.9
47.5
48.6
49.5
50.4
53.8
68.0
68.1
68.2
77.3
GM2270c
3.9
GM2770b
14.8
20.1
23.6
28.2
35.0
35.6
37.3
44.3
51.3
54.6
59.5
62.2
67.7
70.9
72.5
73.0
74.6
75.7
76.2
76.7
78.5
81.2
83.2
83.9
85.0
86.1
87.3
88.5
89.7
92.1
95.7
97.8
101.0
101.5
102.0
102.5
105.2
114.8
8A
GM1174
GM1856GM1835
GM2438
GM1600GM324
GM2120GM1338
GM1237
GM1952GM1876
GM1559 GM1650
GM1732
GM1893
GM1796
GM66
GM2164
GM1069
GM2172
GM2839GM2359
GM1911
GM1979GM2792b
GM1834
GM2778
GM565
GM328
GM2847
GM2553 GM2292
GM1851
GM1411GM2003
GM2389GM2843
GM1291
GM1841
GM7GM1998
GM1999GM1047
GM2033
GM1803
GM2529
GM1459
GM1117
GM1329
GM1312
GM1607
GM1199
GM2081 GM1389
0.0
GM438
GM1822
GM340
GM2427
GM947b
GM947a
GM948
GM1892
GM2092
GM337
GM1989
GM1987 GM1988
GM2483
GM151
GM2771
GM1313
GM1317 GM2505
GM2298 GM1118
GM654GM504
GM1739 GM1967
GM2078 GM1423
GM68
GM1680
GM1511
GM2160
GM1577
GM2031
GM2558a
GM611
GM1908
GM1555
GM261
GM28
GM816
GM2770a
GM1018
GM1297
GM2673GM1782
GM1843
GM2769GM2137
GM1284
GM1890
6A
0.0
GM2057
GM1521
GM1495
GM1682
GM1940
GM226
GM1567
GM1548
GM1547
GM2170
GM1286
GM1385
GM1968
GM2844
GM2063
GM664GM959
GM1505
GM2701
GM1185GM92
GM2702
GM988
GM1461
GM1035GM1096
GM1962GM1896
GM83
GM2366
GM2306
GM1909
GM1617
GM1748
GM900
GM2425
GM1572
GM2817
GM255
GM847
GM1498
GM2446
GM2109
GM2110
GM2414
GM2337
GM2767
GM2066
GM1490
GM1497
GM1506
7.2
13.3
13.8
14.2
18.0
24.5
25.4
32.8
34.1
36.1
37.1
42.4
44.8
46.2
51.7
54.5
54.9
55.9
56.1
57.1
58.2
58.4
59.1
59.3
60.1
61.4
62.3
63.6
66.4
67.0
68.4
70.1
71.1
71.7
73.5
74.3
77.2
80.7
83.2
89.6
93.6
94.2
99.1
99.3
100.4
105.3
0.0
0.6
GM2557
GM175
13.3
16.8
GM1457
GM1931
32.2
GM2779
44.0
44.6
46.2
47.8
48.3
GM987
GM2062
GM2072
GM226
GM1567
57.8
GM1105
67.3
70.6
72.8
73.9
77.7
GM1187
GM1838
GM2170
GM612
GM1385
GM935GM1756
GM1968
GM45
80.4
82.4
102.9
105.1
106.2
108.4
110.0
GM452
GM1185
GM2384 GM92
GM268GM3
GM2337
GM2414
GM2110
GM2456
GM467
93.6
99.1
99.6
120.9
GM752
GM1662
127.6
130.8
131.9
132.4
134.0
135.6
GM1498
GM2817
GM1572 GM2392
GM847
GM1479
GM544
22.5
GM2381
143.4
144.5
147.2
GM1748
GM1857
GM2824
30.1
GM1546
161.6
164.2
GM1499
GM72
0.0
3.4
GM1616
GM1702
7.5
GM1944
12.2
7B
GM2099
GM1587
GM1957
GM2417
GM1238
GM1713
GM1659b
GM71
GM2713GM2712
GM1797 GM2564
GM2827
GM1509
GM1189
GM1917
GM2664GM2665
GM2176
GM1831
GM1936
GM2601
GM1839
GM2600
GM2312
GM2339 GM1934
GM1905
GM1901
GM1629
GM1240
GM1903a
GM2640
GM2644
GM1798
GM1001
GM2643
GM2832
GM1201
GM1709
GM2788 GM1613
GM2842
GM2551
GM2144
GM1281
GM2089
GM2787
GM2125
GM1092
GM2182
GM741
GM1165 GM1965
GM58
GM1136
GM2101
GM1744
GM2400
GM1844
GM883
GM1961
GM2758
GM10
GM1932
GM2055
GM1872
GM1575
GM1320
GM1628
GM1134
GM2269
GM1073
GM1239
GM2421
6B
8B
8B.1
GM2503
8.1
11.4
15.2
17.4
19.6
20.7
GM2339
GM1936
GM1477GM1937
GM1315 GM2118
GM1853
GM69
26.5
GM156
32.3
33.9
36.6
GM1347
GM2343
GM1214
0.0
2.2
GM1779a
GM2454
12.2
15.0
GM2177
GM1929
19.6
20.4
24.3
24.8
28.1
GM2791
GM2067
GM1899
GM1302
GM477
37.0
40.4
43.6
GM1140
GM1175
GM1410
61.6
64.8
67.2
69.4
69.9
71.6
75.7
76.9
78.5
79.6
81.8
82.9
83.5
87.8
91.3
92.2
92.7
93.2
95.9
97.5
99.1
99.6
100.1
101.7
103.3
104.9
107.6
108.1
109.2
114.1
117.9
119.5
120.6
122.7
123.2
123.7
125.9
132.5
135.2
136.8
147.5
GM704
GM624
GM2788
GM2785
GM1427
GM1623
GM1880
GM708
GM920
GM2605
GM1076a
GM2034
GM2551
GM437
GM1151
GM1165
GM2842GM1844
GM59 GM90
GM30
GM2089
GM2746
GM2834GM248
GM2826
GM1587
GM1957
GM871
GM2751
GM1638GM2289
GM2750
GM71
GM1509
GM1797
GM2099
GM2176
GM1917
GM2664
GM2665
GM691
GM1831
GM1469
GM1842
181.1
184.0
185.1
GM339
GM1905
GM2534
0.0
4.4
6.0
7.1
GM462
GM1239GM2269
GM945
GM1073
7A
0.9
3.1
3.4
4.0
4.6
4.9
5.0
7.3
8.8
9.5
11.5
12.3
12.6
13.6
14.4
15.4
15.5
15.6
16.7
16.8
17.8
18.6
20.7
20.9
21.2
21.9
22.0
22.6
24.7
26.7
28.1
28.7
29.4
30.9
31.1
32.0
32.1
32.2
34.6
36.9
38.4
46.5
48.9
55.6
62.3
64.3
71.3
77.2
78.4
78.5
79.6
80.6
GM2473
GM852
GM1633
GM2073
GM963
GM2146
GM2147
GM1621
GM2177
GM1929
GM1250
GM1175
GM1140
GM2454
GM1785
GM2050
GM2156
GM2199
GM1942
GM1494
GM1632
GM1111
GM1114
GM846
GM1688
GM2588
GM1023
GM1623
GM2222
GM1858
GM1076
GM1451
GM2034
GM2423GM1651
GM2326
GM1922
GM1937
GM1315
GM1829
GM1477
GM59
GM1219GM2747
GM2494
GM1701
GM802
GM1853
GM69
GM1214
GM980
GM1347
GM1348GM2526
GM840
GM953
Guo et al. BMC Genomics 2012, 13:608
http://www.biomedcentral.com/1471-2164/13/608
Page 6 of 12
(See figure on previous page.)
Figure 1 The alignment of A- and B- genome linkage maps of Arachis. Alignment of B- genome linkage groups with A- genome linkage
groups, orthologous markers are highlighted in bold. The B- genome map was based on EST-SSR markers and genomic SSR markers, and was
obtained through the analysis of 94 F2 plants from the intraspecific crossing of A. batizocoi. The A- genome map was based on EST-SSR markers
and genomic SSR markers, obtained through the analysis of 94 F2 plants from the intraspecific crossing of A. duranensis. The dotted lines indicate
the correspondences between loci.
constructed in a B-genome diploid peanut, and also
represents the first intraspecific map of a B-genome
species.
Significant segregation distortion (α ≤ 0.05) was
observed for 18 (4.0%) marker loci, distributed in
seven linkage groups representing six B-genome chromosomes (Additional file 8), which was much lower
than previously reported segregation distortion (21.5%) in
an interspecific B-genome linkage map [24]. Similarly,
high levels of skewed segregation in interspecific crosses
compared to intraspecific crosses have been reported in
both barley and cowpea [64,65]. In the A. batizocoi map, a
total of 11 markers skewed towards parental line 9484 and
two towards 30081. Linkage group 1B contained the largest number of distorted marker loci (10 marker loci comprising 55.6% of the total distorted markers). All the
marker loci on this linkage group skewed towards 9484
and clustered within a 37.1 cM genomic region, which is
37.8% of the total mapping distance for 1B and 26.3% of
the marker loci for this chromosome. Linkage groups 3B,
5B, and 8B had no markers showing segregation distortion (Additional file 8).
Synteny between A-genome and B-genome linkage maps
The B-genome linkage groups were aligned to A-genome
linkage groups by 167 putative orthologous marker loci,
10 to 25 in each chromosome (Table 3). Compared to previous reports, which identified 51 shared markers between
the A- and B-genome maps [24], or 53 SSR markers that
mapped on both A and B subgenomes in the tetraploid
map [30], our comparisons are based on a higher density,
higher information content map, and should more precisely position chromosomal rearrangement events within
defined genomic regions. Syntenic segments were supported by multiple markers in the same linear order with
some incongruities reflecting simple translocations and
inversions. A high degree of macrosynteny was observed
when comparing the nine major linkage groups identified
in the B genome to the 10 major linkage groups (1A-10A)
in the A genome (Figure 1). Linkage groups 5B and
5B.1 can both be aligned with linkage group 5A, with putative orthologous markers showing colinearity. Similarly,
LGs 8B and 8B.1 can both be aligned with linkage group
8A. Since the subgroups couldn’t be merged into one
linkage group with a minimum LOD threshold of 3.0
without a >50 cM gap between the adjacent subgroup
loci, the two subgroups were displayed separately.
Completely syntenic chromosomes
For LGs 2A and 2B, all 13 putative orthologous markers
were colinear, covering a map distance of 138.3 cM
(100%) on B2 and 103.7 cM (100%) on A2. Therefore,
2B and 2A showed a high degree of macrosynteny as
previously reported [24], although we did not detect any
split correspondence relationship with A2 and B10 as in
the previous report [24]. Ten putative orthologous markers between 10A and 10B indicated well defined macrosynteny. The common markers spanned 81.1 cM on 10B
and 75.0 cM on 10A, accounting for 75.7% and 68.0% of
the total linkage group length, respectively.
Chromosomes with inversions
The 12 putative orthologous markers between 1A and
1B were clustered into two chromosome segments. One
segment was colinear, containing 10 putative orthologous markers with 28.2 cM map coverage on 1B and
34.5 cM on 1A, accounting for 28.7% and 37.7% of the
total lengths, respectively. The other chromosome fragment had a reversed colinear order, with two putative
orthologous markers spanning 58.5 cM on 1B and 40.7
cM on 1A, accounting for 59.6% and 44.5% of the total
lengths, respectively. The reversed region defined by two
putative orthologous markers on both 1A and 1B indicated a likely inversion between these two chromosomes.
This observation was similar to previous reports [24,30].
For LGs 3B and 3A, 22 common markers were clustered into two chromosomal segments. The first of these
was extensively colinear between 3A and 3B, with 12 putative orthologous markers spanning 88.7 cM (75.3%) on
3B and 74.9 cM (51.4%) on 3A. The other fragment had
a reversed order with 10 putative orthologous markers
spanning 20.9 cM (17.7%) on 3B and 43.2 cM (29.7%)
on 3A. The chromosome segment inversion between
3A and 3B has not been previously reported.
For LGs 5A and 5B, there were 25 putative orthologous markers in total, spanning the entire 5B, 4.1 cM
(13.6%) of 5B.1, and 108.0 cM (92.8%) of 5A, with a generally colinear order, except for a chromosome segment
with nine putative orthologous marker loci spanning
31.4 cM (27.4%) in 5B, and 49.0 cM (42.1%) in the
reversed order for 5A. Since the inverted chromosome
segments accounted for nearly 30% of the total linkage
groups’ lengths and 36.0% (9 out of 25) of the total putative orthologous markers in both A and B genomes,
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there could be an inverted chromosome segment between 5A and 5B, which also has not been detected from
previous reports.
Between chromosomes 6A and 6B, there were 15 putative orthologous markers that spanned 95.6 cM in 6B
and 69.1 cM in 6A, accounting for 58.2% and 65.6% of
the total lengths on the linkage groups, respectively.
Seven putative orthologous markers were mapped in a
colinear manner at the top part of both linkage groups.
Segments containing eight putative orthologous markers
at the bottom part were involved in an inversion event.
The inverted chromosome segment on 6B was 40.5 cM
long and accounted for 24.7% of the entire 6B. The
corresponding segment on 6A was 23.2 cM, accounting
for 22.0% of the entire 6A. This inversion was reported
previously [24], but they also reported the split correspondence relationships between B6 and A10, which was
not revealed in our study.
Complex chromosome rearrangements
In addition to the observed simple inversion events, we
also found more complex chromosome rearrangements.
This intraspecific A. batizocoi genetic map has nine
major linkage groups instead of the expected 10. When
the A- and B-genome linkage groups were aligned, one
of the major B-genome linkage groups was found to correspond to both 4A and 9A and was therefore named as
4/9B. Another smaller linkage group also had putative
orthologous markers with 4A and 9A therefore it was
named 4/9B.3. The linkage group 4/9B contains 80 markers spanning 209.9 cM, while 4/9B.3 contains 7 markers
spanning 47.1 cM. In addition, there were two smaller
LGs, 4/9B.1 and 4/9B.2, containing two markers each
with genetic distances of 3.4 cM and 8.6 cM, respectively.
They were also designated as fragments of 4/9B according
to their putative orthologous markers with 4A and/or 9A.
There were several reasons that we did not further
separate 4/9B into 4B and 9B. Firstly, these linkage
groups remained inseparable even when increasing the
LOD threshold to 20. Secondly, the markers were
located densely and evenly, with no obvious gaps between two chromosome fragments. The average marker
density was 2.7 cM/locus on 4/9B and 7.9 cM on 4/9B.3,
which was comparable with the rest of the genome.
Thirdly, the effects on map order due to distorted segregation were minimal. There was only one marker that
was distorted on each of 4/9B and 4/9B.3. Double crossover events were evaluated alongside 4/9B and 4/9B.3
but no unusual segregating markers or marker blocks
were observed. Fourthly, when aligning 4/9B, 4/9B.1,
4/9B.2 and 4/9B.3 with 4A and 9A separately, by using
only putative orthologous markers, 4A with 4/9B, 4/9B.1,
4/9B.2 and 4/9B.3 showed complete colinearity, while 9A
and 4/9B, 4/9B.1, 4/9B.2 and 4/9B.3 showed a possible
Page 7 of 12
inversion. Synteny between 4A and 4B had been reported
by Moretzsohn et al. [24] and Fonceka et al. [30]. An inversion between 9A and 9B was also consistent with a
previous report [30]. Lastly, all putative orthologous
markers between 4A and 9A were interspersed alongside 4/9B, 4/9B.1, 4/9B.2 and 4/9B.3. From a previous
cytological study of the intraspecific variability of A.
batizocoi using five accessions [66], hybrids between
accessions 30081 and 9484 had reduced pollen stainability (88.6%). Moreover, cytogenetic analyses of F1s
from the same cross showed a low frequency of quadrivalents [11,66], indicating a reciprocal translocation
that would cause pairing between two non-homologous
chromosomes during meiosis. The similarity between the
two chromosomes involved in reciprocal translocation
could explain the integrated linkage group 4/9B in our
study.
A quadrivalent relationship was observed when aligning
7B and 8B with 7A and 8A. On linkage group 7B, there
were two colinearly located putative orthologous markers
with 7A, covering 3.3 cM and accounting for 9.0% of the
total length on 7B. The bottom segment had seven colinearly located putative orthologous markers with 8A,
spanning 21.4 cM, about 58.5% of the entire 7B. On 8B, a
colinear fragment with 7A was located from 2.2 cM (from
the top) to 91.2 cM (from the top), with nine putative
orthologous markers spanning a genetic distance of 89.0 cM
(48.1% of 8B), whereas the other 18 putative orthologous
markers with 8A were colinearly located from 67.2 cM
to 184.0 cM (also from the top of the linkage group),
spanning a genetic distance of 116.8 cM (63.1% of 8B).
Moreover, three colinear putative orthologous markers
with 8A also were found on 8B.1. This may indicate a
reciprocal translocation between 7B and 8B. Similar
translocations on corresponding linkage groups were
found both at the diploid level [24] and tetraploid level
(from the crosses of a tetraploid variety with a tetraploid AABB amphidiploid) [30]. However, the previous
reports did not identify the correspondence between
LGs a07 (corresponding to LG 7A in our study) and
b08 (corresponding to LG 8B in our study), which
might due to their relatively shorter chromosome coverage on LG 8B (29.8 cM in the synthetic amphidiploid
map and 86.4 cM in the diploid map versus 192.2 cM in
this report) [24,30]. Therefore, the rearrangement is most
likely a reciprocal translocation, and may have contributed to the divergence of A and B genomes, perhaps
as an ancient event that occurred before peanut
polyploidization and remained subsequent to tetraploidization of cultivated peanut [30]. Although the
chromosomes of A. hypogaea have differentiated botanical varieties and individual lines that can be
separated based on location of the secondary
constriction and symmetry of chromosomes [67],
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Page 8 of 12
Table 3 Number of common markers between the corresponding linkage groups from A- and B-genome genetic maps
Linkage group
No. of marker loci
1B
38
2B
35
3B
62
4/9B*
91
5B**
58
6B
44
Linkage group
1A
2A
3A
4A
5A
6A
7A
8A
9A
10A
Total
No. of marker loci
73
40
89
67
66
51
56
75
55
61
633
12
13
22
18
13
25
15
7B
13
7
2
8B***
65
9
21
10B
39
Total
445
10
167
*4/9B including 4/9B, 4/9B.1, 4/9B.2, and 4/9B.3.
**5B including 5B and 5B.1.
***8B including 8B and 8B.1.
introgression of disease and insect resistance traits
from wild species into the cultivated peanut has
been successful [68].
Comparative mapping summary
Comparison of the genetic linkage maps of A and B
genomes indicated that the chromosomal differences
between these two species could be explained by a
total of five inversions and one reciprocal translocation
under our current mapping resolution. Because of the
possible minor differences in ordering of tightly linked
markers, confident identification of small inversions is
more difficult than translocations. To detect the confidence of inversion, we compared our predicted inversions with previously published maps. Moretzsohn
et al. [24], used different A- and B-genome diploid
species and showed four inversions and one translocation by comparing diploid A- and B-genome maps
from interspecific mapping populations. Fonceka et al.
[30] used a synthetic allotetraploid population to compare linkage maps of the A and B subgenomes, which
revealed at least three inversions, while Burow et al.
[29] revealed four inversions. Our study identified additional translocation events by using intraspecific diploid mapping populations, and also detected a
reciprocal translocation within the B-genome species
A. batizocoi.
Chromosomal rearrangements are common within
and among A- and B-genome diploid species. For example, in an analysis to determine the intraspecific variability within the B-genome species A. batizocoi [66],
quadrivalents, hexavalents and octavalents were observed
during meiosis in F1 hybrids of different accessions, indicating one to three reciprocal translocations that
differentiate these accessions. Thus, karyotypic evolution
via translocations was considered to be an important
mechanism for species differentiation [66]. In our study,
the linkage map from A. duranensis was used as the
reference map from which the chromosome rearrangements between A and B genomes were inferred [63].
However, chromosomal rearrangements within the Agenome could exist when considering the higher genetic
diversity among the various accessions of the A-genome
species, A. duranensis. Previous research found a low frequency (0.01-0.26/PMC) of multivalents in 12 of 27
hybrids from crosses of A. duranensis accessions [69],
and quadrivalents were identified in all these 12 hybrids,
which likely represents chromosome translocations
within this species. The asymmetrical chromosomes
found in different accessions, furthermore, indicated the
presence of translocations. In addition, varied fertility of
F1s (from less than 4.7% to greater than 95%) and the
diverse morphological traits also indicated wide genetic
diversity in A. duranensis [12,69]. Univalents, laggards,
and multivalents can all be detected in intraspecific Agenome and interspecific A- by B-genome F1 hybrids,
indicating the prevalence of chromosomal rearrangements in peanut diploid species. We herein verify by
genetic mapping that a chromosomal translocation has
occurred within the B-genome species A. batizocoi.
Our findings of one reciprocal translocation between
chromosomes 7 and 8 in A-genome species A. duranensis
and B-genome species A. batizocoi is consistent with
previous reports [24,30]. The slight discrepancy of the
number of inversions between A- and B-genome chromosomes could be due to the genetic variation of the different
accessions/species used in the mapping population
development.
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Conclusions
The present study developed and characterized an extensive set of EST-SSR and genomic SSR markers. Comparative mapping of our intraspecific A- and B-genome
populations showed a high degree of macrosynteny between A- and B-genome diploid species of peanut. Consistent with previous cytological studies, it was evident
that chromosomal rearrangements were common within
and between both A- and B-genome diploid species.
Karyotypic evolution via translocations could be an important mechanism for differentiation of the species.
Our findings will facilitate an understanding of tetraploid peanut genome origin and eventually promote its
genetic improvement.
Page 9 of 12
www.gramene.org/db/markers/ssrtool) and for imperfect
motifs using FastPCR (http://primerdigital.com/fastpcr.
html). The repeat count (n) threshold for each motif
type was set for n ≥ 5. Information on repeat motif, repeat number, and SSR start and end positions within the
respective ESTs were extracted from the SSR-IT output.
The grouping of SSR motifs into respective repeat
classes was performed following the method of Jurka
and Pethiyagoda [70]. Flanking forward and reverse primers were designed using Primer3 (http://frodo.wi.mit.
edu) [71]. The parameters were set as follows: primer
length from 19 to 23 with 21 nucleotides as optimum;
amplification size of 100 to 400 base pairs; annealing
temperatures from 59°C to 63°C with a maximum difference of 3°C; and GC contents from 25% to 45%.
Methods
Plant materials
Mapping population
An F2 population consisting of 94 plants was developed
by selfing four F1 plants from the intraspecific cross of
two A. batizocoi lines PI 298639 (accession no. K 9484)
and PI 468327 (accession no. GKBSPSc 30081). The Agenome F2 mapping population also consisted of 94
plants from the intraspecific cross of A. duranensis PI
475887 and Grif 15036 [63].
Plant materials for EST-SSR characterization
Eight genotypes including four tetraploid and four
diploid accessions were used to screen all 2,138 markers. The four tetraploid genotypes in the screening
panel included Tifrunner (A. hypogaea subsp. hypogaea var. hypogaea), a runner-type peanut cultivar;
GT-C20 (A. hypogaea subsp. fastigiata var. vulgaris), a
Spanish exotic accession with reduced aflatoxin contamination; NC94022 (A. hypogaea subsp. hypogaea),
an exotic accession with higher resistance to TSWV
(tomato spotted-wilt virus) derived from var. hirsuta;
and SunOleic 97R (A. hypogaea subsp. hypogaea var.
hypogaea), a high oleic runner-type cultivar. For the
diploid genotypes, PI 475887 and Grif 15036 are Agenome germplasm accessions of A. duranensis, while
accessions 9484 and 30081 are B-genome germplasm
accessions in A. batizocoi. They are parental lines of the
respective A- and B-genome mapping populations.
SSR discovery, marker development, and length
polymorphism screening
Mining the peanut EST database for SSRs
The sequence database used for SSR marker development harbors a total of 70,771 long-read (Sanger) ESTs
and 270,957 short-read (454) ESTs assembled into
101,132 unigenes (Accession: PRJNA49471; Additional
file 1). Unigenes in the transcript assembly were
screened for perfect repeat motifs using SSR-IT (http://
Mining genome survey sequences (GSSs) for SSRs
Methylation filtered (ME) and unfiltered (U) genome
libraries were constructed from A. duranensis, A. batizocoi,
and A. hypogaea by Orion Genomics (Saint Louis,
Missouri) [72]. A total of 9,517 unique genome survey
sequences (GSS) were used for mining SSRs.
Similarly, SSR-IT was used to screen for perfect repeat
motifs (http://www.gramene.org/db/markers/ssrtool [47]),
and Primer3 (http://frodo.wi.mit.edu) [71] was used for
primer design. Primers were screened for overall amplification quality against 12 genotypes, including four diploid and eight tetraploid accessions (Additional file 6),
and with SSCP gels by silver staining according to protocols described previously [73].
SSR marker genotyping
Genomic DNA was isolated from young leaves by a
modified cetyltrimethylammonium bromide (CTAB)
method [74]. SSR markers were genotyped on an
ABI3730XL Capillary DNA Sequencer (Applied Biosystems, Foster City, CA) using forward primers labeled
with FAM, HEX, or TAMRA fluorophores. PCR was
performed in a 12 μL reaction mixture containing 1.0×
PCR buffer, 2.5 mM Mg++, 0.2 mM each of dNTPs,
5.0 pmol of each primer, 0.5 unit of Taq polymerase, and
10 ng of genomic DNA. Touchdown PCR was used to
reduce spurious amplification [75]. The SSR markers
were screened for amplification and length polymorphisms using GeneMapper 3.0 software (Applied Biosystems,
Foster City, CA).
Genetic mapping, macrosynteny analysis, and cMap
database construction
A total of 481 polymorphic markers were used to screen
94 F2 progenies for map construction. Segregation distortion at each marker locus was tested against the
expected segregation ratios (1:2:1 for codominant markers and 3:1 for dominant markers) using a chi-square
Guo et al. BMC Genomics 2012, 13:608
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goodness of fit test. Genetic maps were constructed
using Mapmaker 3.0, with error detection on [76,77].
The initial linkage groups were first determined using
the “group” application with a minimum likelihood odds
(LOD) threshold of 15 and a maximum recombination
fraction (θ) of 0.35. After aligning the draft map with the
A-genome map [63], the LOD score was relaxed to 5
with θ =0.35 for a second analysis to merge the linkage
groups that could align with the homoeologous Agenome linkage groups. Map distances (cM) were calculated using the Kosambi mapping function [78]. The
“try”, “compare”, and “ripple” commands were used to
confirm the marker order. Mapchart 2.2 was used for
the graphic visualization of the linkage groups [79].
The A. batizocoi (B-genome) linkage groups were
numbered based on colinearity to A. duranensis (Agenome) linkage groups except with the suffix “B”. The
colinear subgroups in A. batizocoi were named by identical numbers with numerical suffixes, while an unseparated linkage group was identified by using linkage
group numbers from the fused groups.
Additional files
Additional file 1: Summary of EST database for SSR discovery.
Additional file 2: List of peanut EST-SSRs.
Additional file 3: Overview of EST-SSR amplification.
Additional file 4: Summary of marker polymorphism among peanut
genotypes.
Additional file 5: Effect of SSR length on frequency of
polymorphism among tetraploid and diploid genotypes.
Additional file 6: Arachis germplasm screened for genomic SSR
markers developed from GSS sequences.
Additional file 7: Peanut genomic SSR primer pair sequences used
in this study.
Additional file 8: The distribution of distorted loci along linkage
groups for the B- genome map.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YG led the experiments and drafted the manuscript. SK, ST, JEB, AFH, NK,
EDN, DZ, CAT, and HTS participated in the experiments. POA and SJK
designed experiments, coordinated the study, POA finalized the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
This research was supported by funding from the USDA National Institute of
Food and Agriculture National Research Initiative Competitive Grants Program
(#2006-35604-17242) awarded to SJK and POA and by the National Peanut
Board, the Peanut Foundation, the Georgia Seed Development Commission,
and Georgia Research Alliance endowment funding awarded to SJK.
Author details
1
Institute of Plant Breeding, Genetics, and Genomics, 111 Riverbend Road,
The University of Georgia, Athens, GA 30602, USA. 2Department of
Horticulture, The University of Georgia, Tifton, GA 31973, USA. 3Department
of Crop Science, North Carolina State University, Raleigh, NC 27695, USA.
Page 10 of 12
Received: 11 September 2012 Accepted: 31 October 2012
Published: 10 November 2012
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doi:10.1186/1471-2164-13-608
Cite this article as: Guo et al.: Comparative mapping in intraspecific
populations uncovers a high degree of macrosynteny between A- and
B-genome diploid species of peanut. BMC Genomics 2012 13:608.
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