RESEARCH ARTICLE Open Access
Leaf morphology in Cowpea [Vigna unguiculata
(L.) Walp]: QTL analysis, physical mapping and
identifying a candidate gene using synteny with
control of leaf shape in legumes.
Pottorff et al. BMC Genomics 2012, 13:234
http://www.biomedcentral.com/1471-2164/13/1/234USA
Full list of author information is available at the end of the articleKeywords: QTL analysis, Leaf morphology, Genomics, Genetics, Physical map, Synteny, Candidate genes, Cowpea,
Legumes, EZA1/SWINGER
* Correspondence: philip.roberts@ucr.edu; timothy.close@ucr.edu
1Department of Botany & Plant Sciences, University of California Riverside,
Riverside, CA, USA
4Department of Nematology, University of California Riverside, Riverside, CA,model legume species
Marti Pottorff1, Jeffrey D Ehlers1,2, Christian Fatokun3, Philip A Roberts4* and Timothy J Close1*
Abstract
Background: Cowpea [Vigna unguiculata (L.) Walp] exhibits a considerable variation in leaf shape. Although
cowpea is mostly utilized as a dry grain and animal fodder crop, cowpea leaves are also used as a high-protein pot
herb in many countries of Africa.
Results: Leaf morphology was studied in the cowpea RIL population, Sanzi (sub-globose leaf shape) x Vita 7
(hastate leaf shape). A QTL for leaf shape, Hls (hastate leaf shape), was identified on the Sanzi x Vita 7 genetic map
spanning from 56.54 cM to 67.54 cM distance on linkage group 15. SNP marker 1_0910 was the most significant
over the two experiments, accounting for 74.7% phenotypic variance (LOD 33.82) in a greenhouse experiment and
71.5% phenotypic variance (LOD 30.89) in a field experiment. The corresponding Hls locus was positioned on the
cowpea consensus genetic map on linkage group 4, spanning from 25.57 to 35.96 cM. A marker-trait association of
the Hls region identified SNP marker 1_0349 alleles co-segregating with either the hastate or sub-globose leaf
phenotype. High co-linearity was observed for the syntenic Hls region in Medicago truncatula and Glycine max. One
syntenic locus for Hls was identified on Medicago chromosome 7 while syntenic regions for Hls were identified on
two soybean chromosomes, 3 and 19. In all three syntenic loci, an ortholog for the EZA1/SWINGER (AT4G02020.1)
gene was observed and is the candidate gene for the Hls locus. The Hls locus was identified on the cowpea
physical map via SNP markers 1_0910, 1_1013 and 1_0992 which were identified in three BAC contigs; contig926,
contig821 and contig25.
Conclusions: This study has demonstrated how integrated genomic resources can be utilized for a candidate gene
approach. Identification of genes which control leaf morphology may be utilized to improve the quality of cowpea
leaves for vegetable and or forage markets as well as contribute to more fundamental research understanding the© 2012 Pottorff 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.
Pottorff et al. BMC Genomics 2012, 13:234 Page 2 of 12
http://www.biomedcentral.com/1471-2164/13/1/234Background
Cowpea [Vigna unguiculata (L.) Walp] exhibits a consid-
erable variation in leaf shape. Cowpea leaves are com-
pound, having two asymmetrical side leaflets and one
central terminal leaflet which is symmetrical. Typically,
the central leaflet of the trifoliate is used in classifying
the leaf shape due to variability of the side leaflets. In
cowpea, the leaf shape is important for taxonomic classi-
fication and also for distinguishing cowpea varieties.
However, there isn’t a central naming convention for
cowpea leaves nor detailed descriptions of the leaf
shapes, thus, many researchers name the leaf shapes dif-
ferently. The two largest cowpea germplasm agencies are
the International Institute of Tropical Agriculture (IITA)
and the United States Department of Agriculture
(USDA). IITA, which houses 14,500 cowpea accessions
from 65 different countries, classifies cowpea leaf shapes
into four categories, sub-globose, sub-hastate, globose
and hastate/lanceolate (http://genebank.iita.org). The
USDA, which houses 6,8411 cowpea accessions from 50
countries, classifies cowpea leaf shapes into five categor-
ies; globose, hastate, sub-globose, sub-hastate, strip and
ovate-lanceolate (http://www.ars-grin.gov/cgi-bin/npgs/
html/desclist.pl?188).
Multipurpose cowpea
Cowpea is a multipurpose crop; the entire plant can be
used for either human or livestock consumption. In
2009, cowpea dry grain production was estimated at
5,249,571 tons worldwide (http://faostat.fao.org). Al-
though cowpea is not one of the highest production
crops worldwide, nearly 90% of cowpea is produced in
West Africa, which is estimated at 4,447,358 tons
(http://faostat.fao.org). Cowpea is mainly grown in semi-
arid regions by subsistence farmers, who sell the fresh or
dried seeds, fresh pods and leaves as vegetables and the
green or dried leftover parts of the plant, leaves and
stems (haulms), can be used as fodder for livestock [1].
Young cowpea leaves are eaten as a pot herb and
enjoyed in many parts of Africa. The freshly harvested
leaves are sold in local markets in many parts of Ghana,
Mali, Benin, Cameroon, Ethiopia, Uganda, Kenya, Tanza-
nia and Malawi [2]. Cowpea shoots and leaves are rich
sources of calcium, phosphorous and Vitamin B [3]. The
young leaves are especially important in drought-prone
regions of Sub-Saharan Africa to tide local populations
over during the “hungry period” which occurs after plant-
ing but before the main harvest of fresh pods and dry
grains. In Mozambique, dried cowpea seeds are mainly
consumed by the poorer classes of people, whereas all so-
cial strata consume cowpea leaves eaten as a vegetable
(personal communication, Rogerio Chiulele). Importantly,
farmers can harvest and sell the young tender cowpea
leaves while waiting for the cowpea grain crop to mature,which helps provide income to buy staple foods. Cowpea
seedlings and tender young leaves are also a local delicacy
and inherent to Zimbabwean cultures (personal commu-
nication, Wellington Muchero).
Dual purpose cowpea varieties which are bred for
quality seeds, vegetables and fodder may add to a farm-
er’s revenue. For example, in Nigeria, farmers who sold
dried cowpea fodder during the peak of the drought sea-
son saw a 25% increase to their annual income [4].
Although there is no emphasis in breeding cowpeas
for the shape of their leaves, leaf shape is important for
classifying and distinguishing cowpea varieties. The
shape of the leaves may also be potentially useful as a
morphological or physical marker used during the selec-
tion process if it is closely linked with an agronomic trait
of interest. Interestingly, many wild cowpea relatives
have the narrow or hastate leaf shape whereas most cul-
tivated varieties of cowpea have the more common ovate
or sub-globose leaf shape. However, any possible adap-
tive advantage for narrow leaves in wild cowpea has not
been investigated. The hastate leaf shape was reported to
be dominant to the ovate leaf shape in several studies
[5–10]. This may indicate that the hastate shape is an-
cestral to the ovate leaf shape and the preponderance of
the latter in most cultivated cowpea is due to direct or
indirect selection by humans over time.
Molecular genetic tools and genomic resources have
been developed for cowpea with an objective of enhan-
cing breeding programs for the improvement of cowpea
varieties for the United States, India, Brazil, and numer-
ous countries in Africa and Asia. These integrated gen-
omic resources include a 1536 SNP genotyping platform,
an EST-derived SNP consensus genetic map, known syn-
tenic relationships between cowpea, Medicago trunca-
tula, Glycine max and Arabidopsis thaliana, and a
cowpea EST sequence collection housed in HarvEST:
Cowpea database (http://harvest.ucr.edu) [11,12]. A cow-
pea physical map has been partially anchored to the
cowpea consensus genetic map using the same SNP
markers (UCR cowpea group, unpublished) and is avail-
able publically (http://phymap.ucdavis.edu/cowpea). In
addition, about 500 diverse cowpea accessions have been
SNP-genotyped (UCR cowpea group, unpublished data)
and a first draft of the cowpea genome, vs.0.02, has been
assembled (www.harvest-blast.org). These resources will
enable dissection of underlying genetic components of
target agronomic traits using Quantitative Trait Locus
(QTL) analysis and Association Mapping. The identified
and confirmed QTLs will facilitate cultivar improvement
using marker-assisted breeding.
In this study, we analyzed the genetics of leaf morph-
ology in a segregating cowpea RIL population, Sanzi (sub-
globose) x Vita7 (hastate). A QTL was identified for the
“hastate leaf shape” locus, Hls, which was positioned on
the cowpea consensus genetic map and cowpea physical
map. A candidate gene was identified using syntenic rela-
tionships between cowpea, soybean and Medicago. In
addition, a SNP marker was found which co-segregated
Pottorff et al. BMC Genomics 2012, 13:234 Page 3 of 12
http://www.biomedcentral.com/1471-2164/13/1/234with the leaf morphology genotypes and phenotype, which
could be used as a molecular marker for breeding pur-
poses. Future perspectives for this study are to fine map
the Hls locus and identify cowpea candidate genes which
would be utilized for more basic studies on leaf morph-
ology in cowpea.
Results and discussion
Inheritance of leaf morphology
The inheritance of leaf morphology was studied using
phenotypic data from one greenhouse experiment and
one field experiment on the cowpea RIL population,
Sanzi (sub-globose) x Vita 7 (hastate). The hastate and
sub-globose leaf shape segregated 58:60 in the green-
house experiment and 59:57 in the field experiment (x2
1:1 = 0.03, p-value = 0.85) which fit the proposed model
that the leaf shape is a qualitative trait (Table 1).
Several other researchers have studied the inheritance
of the leaf shape in cowpea (hastate x ovate leaf shape)
and reported that it was a qualitative trait [7,8,10,13]. Al-
though the F1 generation was not assessed in the current
study, the majority of researchers studying cowpea leaf
shape have concluded that the hastate leaf shape is dom-
inant to the more common ovate or sub-globose leaf
shape [5–10]. However, Saunders et al. (1960b) reported
that the hastate leaf shape was incompletely dominant to
the ovate leaf shape.
QTL analysis
QTL analysis of the two phenotypic datasets identified
one major QTL with a large effect for leaf shape morph-
ology. The leaf morphology QTL spanned 11 cM dis-
tance on the Sanzi x Vita 7 individual genetic map from
56.54 cM to 67.54 cM on linkage group 15 (Figure 1,
Tables 2, 3). SNP marker 1_0910 was the most signifi-
cant marker in both of the datasets, accounting for
74.7% of the phenotypic variance (LOD 33.82) in the
greenhouse experiment and 71.5% phenotypic variance
(LOD 30.89) in the field experiment (Table 3). We
propose the designation Hls (hastate leaf shape) for the
QTL identified.
Other researchers studying the inheritance of the hast-
ate leaf shape in cowpea have reported a single
Table 1 Inheritance of leaf shape in Sanzi x Vita 7
population
Experiment Hastate Sub-globose Ratio x2 p-valueGreenhouse 58 60 1:1 0.03 0.85
Field 59 57 1:1 0.03 0.85dominant gene controlling the hastate leaf shape over
the ovate or sub-globose leaf shape. Several gene sym-
bols have been proposed, the first being L, which is a
dominant gene controlling lanceolate leaf shape [14].
Ojomo et al. (1977) proposed the gene symbol Ha for
the hastate leaf shape and Kolhe et al. (1970) proposed
Nlf for narrow leaf shape. Fery (1980) proposed the gene
symbol, La, for the narrow leaf shape. However, all of
the studies investigating the narrow leaf shape used dif-
ferent cowpea accessions to make their populations.
Whether these many studies are describing the same leaf
shape locus or whether they are describing multiple in-
dependent loci remains inconclusive. Interestingly,
Ogundiwin et al. (2005) identified one major QTL for
the hastate leaf shape, designated La, in Vigna unguicu-
lata ssp. textilus. Subspecies textilus is closely related to
cultivated cowpea (V. unguiculata ssp. unguiculata);
however, it does not easily hybridize. La could possibly
be the syntenic locus of Hls in V. textilus.
The corresponding location of Hls was identified on
the cowpea consensus genetic map. SNP markers which
identified the Hls locus in the Sanzi x Vita 7 genetic map
were aligned with the cowpea consensus genetic map
(Table 3). The Hls locus spans from 25.57 cM to
35.96 cM on the cowpea consensus genetic map linkage
group 4 (Table 3). The length of Hls on the individual
genetic map, 11 cM, is nearly the same as on the cowpea
consensus genetic map, 10.39 cM which may reflect ac-
curacy of marker order (Table 3). The Hls locus on the
cowpea consensus genetic map has several SNP markers
which were not present in the Sanzi x Vita 7 population
because of lack of polymorphism in the individual popu-
lation (Table 3). In addition, there was a slight difference
in the order of the SNP markers in the Sanzi x Vita7
population versus the cowpea consensus genetic map
due to the merging of twelve individual genetic maps.
Marker-trait association analysis
Seventeen diverse cowpea genotypes which have either
the hastate or sub-globose leaf shape were used in a
marker-trait association study to identify a SNP marker in
the Hls region linked with the leaf shape phenotype. The
hastate genotypes used for the analysis were selected from
the USDA GRIN cowpea accession database and under
their naming convention were classified as “strip” leaved.
Vita 7, PI 632869, PI 632870, PI 632871, PI 632900, PI
632876, PI 632901, PI 632899 and PI 598341 were chosen
for the hastate leaf shape phenotype (Additional file 1). PI
632882, CB27, Bambey 21, PI 418979, PI 448337 and PI
448682 were chosen from the USDA GRIN database and
under their naming convention were classified as “sub-
globose” leaf shape (Additional file 1). Accessions desig-
nated “TVNu” are wild cowpeas, many of which have the
hastate leaf shape.
Pottorff et al. BMC Genomics 2012, 13:234 Page 4 of 12
http://www.biomedcentral.com/1471-2164/13/1/234The alleles of SNP marker 1_0349 (35.9 cM position)
co-segregated perfectly with the hastate or sub-globose
leaf phenotype (boxed in green in Figure 2). The allele
for the hastate genotype at this locus was the thymine
nucleotide (color coded blue in Figure 2). The allele for
the sub-globose genotype was the cytosine nucleotide
(color coded red in Figure 2). The thymine/cytosine SNP
for 1_0349 is at position 2122 in the cowpea P12 assem-
bly unigene 8605 and can be viewed in HarvEST:Cowpea
Figure 1 Hls locus on the Sanzi x Vita 7 genetic map. Using Interval Ma
shown), Hls mapped to linkage group 15 on the Sanzi x Vita 7 genetic map
data are plotted in blue and the field experiment data in green. SNP marke
The LOD significance threshold of 2.0 is indicated by a horizontal dotted lin
Table 2 QTL analysis of the Hls locus in the Sanzi x Vita 7 pop
Experiment Analysis 1_0106 1_1316
Greenhouse IM LOD 27.32 28.8
IM R2 66.2 69.1
KW test statistic 76.12 78.68
KW p-value 0.0001 0.0001
Field IM LOD 27.29 28.77
IM R2 66.2 69.1
KW test statistic 76.08 78.62
KW p-value 0.0001 0.0001
IM = Interval Mapping analysis, KW = Kruskal-Wallis analysis.(http://harvest.ucr.edu) (Additional file 2). The marker-
trait association narrowed the Hls QTL to a 0.3 cM re-
gion and was defined by flanking SNP markers 1_0083
and 1_0417 (Figure 2).
Candidate gene analysis using synteny with M. truncatula
and G. max
The Hls locus was compared with the soybean, Medicago
and Arabidopsis genomes to determine if a syntenic
pping and Kruskal-Wallis analysis (only Interval Mapping analysis
, spanning from 56.54 cM to 67.54 cM. The greenhouse experiment
rs 1_0992 and 1_0910 are highlighted in red on the linkage group.
e on the graph.
ulation
1_0417 1_0349 1_0992 1_0910
24.18 24.18 31.21 33.82
62.7 62.7 71.9 74.7
71.38 71.38 81.74 84.91
0.0001 0.0001 0.0001 0.0001
22.44 22.44 28.57 30.89
59.9 59.9 68.7 71.5
68.30 68.30 78.15 81.31
0.0001 0.0001 0.0001 0.0001
relationship exists. A high co-linearity or a conservation
of gene order utilizing the EST-derived SNP markers with
the HarvEST:Cowpea database and are publicly available
(http://harvest.ucr.edu). Due to limited resolution in the
Table 3 The Hls locus in the Sanzi x Vita 7 genetic map, cowpea consensus genetic map and cowpea physical map
Sanzi x Vita 7 genetic map Cowpea consensus genetic map Cowpea physical map
LG cM Locus LOD LG cM Locus Contig BAC clone(s)
15 56.55 1_0106 27.32 4 25.57 1_0106 383 CM056F01, CM067G06, CM007L11
N/A 4 27.60 1_0678 1014 CH021P21
N/A 4 27.90 1_1209 N/A
N/A 4 29.30 1_0117 N/A
N/A 4 29.51 1_0128 N/A
15 63.65 1_1316 28.80 4 31.88 1_1316 N/A
N/A 4 32.21 1_0157 N/A
N/A 4 33.57 1_0038 926 CM002I07, CM052G13
N/A 4 34.09 1_1013 926 CM050B03, CH004H23, CH046B08
15 67.54 1_0910 33.82 4 34.09 1_0910 821 CH050F07
15 67.20 1_0992 31.21 4 34.69 1_0992 25 CM041C03
N/A 4 35.66 1_0083 N/A
15 66.46 1_0349 24.18 4 35.87 1_0349 N/A
15 66.46 1_0417 24.18 4 35.96 1_0417 N/A
SNP markers are aligned in the order defined by the cowpea consensus genetic map.
Pottorff et al. BMC Genomics 2012, 13:234 Page 5 of 12
http://www.biomedcentral.com/1471-2164/13/1/234any of the sequenced genomes might reveal candidate
genes. Synteny was examined using EST-derived SNP
markers previously BLASTed and aligned to the soybean,
Medicago and Arabidopsis genomes which are housed inFigure 2 Marker-trait association in the Hls locus. The Hls locus on the
along with cowpea genotypes which differ in hastate or sub-globose leaf s
indicate the “BB” allele and grey colored blocks indicate that the locus has
below: “S” indicates a sub-globose leaf shape and “H” indicates the hastate
1_0349 (35.90 cM position) which is boxed in green. SNP marker 1_0349 co
corresponding leaf phenotype. The allele for the hastate leaf genotype at t
sub-globose genotype is the cytosine nucleotide, color coded red. The thy
assembly unigene 8605 and can be viewed in HarvEST:Cowpea (http://harvsoftware images, not all markers are presented in the
screenshot images output from Harvest:Cowpea. How-
ever, the cowpea consensus genetic map vs. 4 [12] has
been used in fidelity. In order to view each individualcowpea consensus genetic map linkage group 4 is depicted vertically
hape. Red colored blocks indicate the “AA” allele, blue colored blocks
no detected SNP. Leaf shapes for cowpea accessions are labeled
leaf shape. A marker-trait association was found for SNP marker
-segregated with the hastate and sub-globose leaf genotypes and the
his locus is the thymine nucleotide, color coded blue. The allele for the
mine/cytosine SNP for 1_0349 is at position 2122 in the cowpea P12
est.ucr.edu).
cin
rev
Pottorff et al. BMC Genomics 2012, 13:234 Page 6 of 12
http://www.biomedcentral.com/1471-2164/13/1/234Figure 3 Synteny of the Hls locus with Medicago truncatula and Gly
M. truncatula and cowpea and G. max using EST-derived SNP markers pmarker, the linkage group must be magnified in the Har-
vEST:Cowpea database.
The Hls locus was examined for synteny with the Arabi-
dopsis genome; however very low synteny was displayed
at the macro level between cowpea and Arabidopsis so no
further examination was pursued (Additional file 3).
A high co-linearity was observed for the Hls locus with
Medicago chromosome 7 (Figure 3, Table 4). Eight Medi-
cago genes orthologous to cowpea SNP markers were
which spans 25.57 cM to 35.96 cM on linkage group 4 of the cowpea cons
syntenic locus spanned from Medicago locus Medtr7g084010 to Medtr7g1
syntenic region of Hls, Medtr7g133020, which was annotated as an ortholo
were identified for the Hls locus in soybean chromosomes 3 and 19. The sy
locus Glyma03g34240 to Glyma03g38550. An orthologous candidate gene
Glyma03g38320, which was annotated as an ortholog of the Arabidopsis E
chromosome 19 spanned from Glyma19g36180 to Glyma19g41150 where
Glyma19g40430, was observed. The syntenic map was drawn using HarvES
value of −10 and a minimum number of 10 lines drawn per linkage group
truncatula and G. max chromosomes.
Table 4 The Hls syntenic region in Medicago truncatula chrom
Medicago locus Position (bp) Phytozome anno
Medtr7g084010 MtChr7: 18093097–18096342 Glycosyltransferas
Medtr7g127710 MtChr7: 30002559–30004421 Small nuclear ribo
Medtr7g130340 MtChr7: 30448639–30451565 Tetrahydrofolate d
Medtr7g132610 MtChr7: 30739419–30778183 Histidine kinase
Medtr7g132800 MtChr7: 30863955–30868447 Glycosyl hydrolase
Medtr7g133020 MtChr7: 30974729–30981121 SWN (SWINGER); t
Medtr7g134340 MtChr7: 31708007–31710614 Peptidyl-prolyl cis
Medtr7g134420 MtChr7: 31747440–31752793 Papain family cyst
Medtr7g134530 MtChr7: 31793943–31799643 ATP-dependent Re max. Synteny was examined for the Hls locus between cowpea and
iously BLASTed and aligned to the sequenced genomes. The Hls locusidentified in the syntenic region of Medicago chromosome
7 (Table 4). The syntenic region spanned from
Medtr7g084010 locus to Medtr7g134530 locus which cor-
responded to 29.30 cM to 35.96 cM of the Hls locus on
the cowpea consensus genetic map (Tables 3, 4). The re-
gion which spanned from Medicago genes orthologous to
cowpea SNP markers 1_1013 to 1_0349 were in the same
linear order as on the cowpea consensus genetic map
(Tables 3, 4). The region spanning between Medicago
ensus genetic map was syntenic with Medicago chromosome 7. The
34530. A candidate gene was identified in the highly significant
g of the Arabidopsis EZA1/SWINGER (SWN) gene. Two syntenic loci
ntenic region in soybean chromosome 3 spanned from the soybean
was observed in the most significant region of the syntenic Hls locus,
ZA1/SWINGER (SWN) gene. The syntenic Hls locus on soybean
another soybean ortholog of the EZA1/SWINGER (SWN) gene,
T:Cowpea database (http://harvest.ucr.edu) using a cut off e-score
. Colored lines indicate cowpea genes orthologous to genes on M.
osome 7
tation Cowpea SNP LG cM
e 1_1316 4 31.88
nucleoprotein G 1_0117 4 29.30
ehydrogenase/cyclohydrolase 1_1013 4 34.09
1_0910 4 34.09
family 3 C terminal domain 1_0992 4 34.69
ranscription factor N/A N/A N/A
-trans isomerase 1_0083 4 35.66
eine protease 1_0417 4 35.96
NA helicase 1_0349 4 35.87
genes orthologous to cowpea SNP markers 1_0910 (most
significant marker in the QTL analysis) and 1_0349 (co-
segregated with leaf genotype and phenotype) was exam-
ined for genes known to be associated with the molecular
control of leaf morphology in other plant species [15] on
the Medicago genome browser on the Phytozome web-
page (http://www.phytozome.net). The Medicago locus
Medtr7g133020 was observed between Medicago genes
orthologous to cowpea SNP markers 1_0992 and 1_0083
and was annotated as an ortholog of the Arabidopsis gene
AT4G02020.1 aka EZA1 or SWINGER (SWN) (Table 4).
Medtr7g133020 has a SET domain (protein lysine methyl-
transferase enzyme) with two copies of a cysteine rich
motif and is annotated as KOG: 1079; transcriptional re-
pressor EZA1 (http://www.phytozome.net) (accessed April
2012).
The Hls region was examined for synteny with the
soybean genome and was found to be highly co-linear
with soybean chromosomes 3 and 19 (Figure 3, Table 5).
Eight Medicago genes orthologous to cowpea SNP mar-
kers identified the region from locus Glyma03g34240 to
Glyma03g38550 as the Hls syntenic locus in soybean
chromosome 3 (Table 5). The soybean syntenic locus
corresponded to 27.60 cM to 35.96 cM region in the Hls
locus and was also in the same general marker order as
the cowpea consensus genetic map (Table 5). The region
spanning between orthologous soybean genes to cow-
pea SNP markers 1_1013 and 1_0349 was examined for
leaf morphology candidate genes on the soybean gen-
ome browser on the Phytozome webpage (http://www.
phytozome.net). Soybean locus Glyma03g38320 was
observed flanked by orthologous genes for cowpea
SNP markers 1_1013 and 1_0417 and was annotated
as an ortholog of EZA1/SWINGER (SWN) gene.
Glyma03g38320 has a SET domain (protein lysine methyl-
transferase enzyme) and two copies of a cysteine rich motif
and is annotated as KOG: 1079; transcriptional repressor
EZA1 (http://www.phytozome.net) (accessed April 2012).
The Hls syntenic region in soybean chromosome 19 was
identified by thirteen out of fourteen SNP markers, span-
ning from Glyma19g36180 to Glyma19g41150 which cor-
responded to 24.10 cM to 39.80 cM on the cowpea
consensus genetic map (Table 5). The syntenic region in
soybean between orthologous cowpea SNP markers
1_0910 and 1_0349 was examined for known leaf develop-
ment genes using the soybean genome browser on the
Table 5 The Hls syntenic region in Glycine max chromosomes 3 and 19
G. max chromosome G. max locus Location (bp) Phytozome annotation Cowpea SNP LG cM
3 Glyma03g34240 Gm03: 41726178–41732134 Protein phosphatase type 2A 1_1209 4 27.90 cM
3 Glyma03g34420 Gm03: 41865023–41866819 UDP glycosyl transferase 1_0678 4 27.60 cM
S
G
6
T
E
C
A
6
4
S
U
G
P
6
6
T
6
H
G
E
Pottorff et al. BMC Genomics 2012, 13:234 Page 7 of 12
http://www.biomedcentral.com/1471-2164/13/1/2343 Glyma03g35490 Gm03: 42670842–42672212
3 Glyma03g36050 Gm03: 43046482–43052190
3 Glyma03g36560 Gm03: 43503702–43504835
3 Glyma03g37080 Gm03: 43844395–43846689
3 Glyma03g38320 Gm03: 44664969–44672254
3 Glyma03g38520 Gm03: 44857426–44863787
3 Glyma03g38550 Gm03: 44884051–44889833
19 Glyma19g36180 Gm19: 43520883–43522581
19 Glyma19g36250 Gm19: 43594256–43596114
19 Glyma19g38130 Gm19: 45131688–45132559
19 Glyma19g38170 Gm19: 45154806–45156026
19 Glyma19g38720 Gm19: 45583969–45589659
19 Glyma19g39170 Gm19: 45946131–45951841
19 Glyma19g39240 Gm19: 45993099–45993972
19 Glyma19g39570 Gm19: 46201543–46203746
19 Glyma19g39710 Gm19: 46301684–46304736
19 Glyma19g40080 Gm19: 46544712–46546719
19 Glyma19g40090 Gm19: 46547961–46552179
19 Glyma19g40300 Gm19: 46736904–46743350
19 Glyma19g40430 Gm19: 46838345–4684472119 Glyma19g41120 Gm19: 47437575–47443343 C
19 Glyma19g41150 Gm19: 47465990–47471582 Amall nuclear ribonucleoprotein G 1_0117 4 29.30 cM
lycosyl transferase 1_1316 4 31.88 cM
0S ribosomal protein 1_0157 4 32.21 cM
etrahydrofolate dehydrogenase 1_1013 4 34.09 cM
ZA1 (SWINGER); transcription factor N/A N/A N/A
ysteine proteinase 1_0417 4 35.96 cM
TP-dependent RNA helicase 1_0349 4 35.87 cM
0S ribosomal protein 1_0106 4 25.57 cM
0S ribosomal protein S23 1_0061 2 24.10 cM
mall nuclear ribonucleoprotein G 1_0117 4 29.30 cM
biquitin extension protein 2 (UBQ2) 1_0128 4 29.51 cM
lycosyl transferase 1_1316 4 31.88 cM
rotein phosphatase 1_1349 3 39.80 cM
0S ribosomal protein L21 1_0157 4 32.21 cM
0S ribosomal protein L19 1_0038 4 33.57 cM
etrahydrofolate dehydrogenase 1_1013 4 34.09 cM
0S ribosomal protein L19 1_0038 4 33.57 cM
istidine kinase 1_0910 4 34.09 cM
lycosyl hydrolase family 1_0992 4 34.69 cM
ZA1 (SWINGER); transcription factor N/A N/A N/Aysteine proteinase 1_0417 4 35.96 cM
TP-dependent RNA helicase 1_0349 4 35.87 cM
Pottorff et al. BMC Genomics 2012, 13:234 Page 8 of 12
http://www.biomedcentral.com/1471-2164/13/1/234Phytozome webpage (http://www.phytozome.net). Gly-
ma19g40430 locus was observed flanked by soybean genes
orthologous to SNP markers 1_0992 and 1_0417 and was
annotated as an ortholog of the Arabidopsis EZA1/
SWINGER (SWN) gene (Table 5). Glyma19g40430 has a
SET domain (protein lysine methyltransferase enzyme)
and two copies of a cysteine rich motif and is annotated as
KOG: 1079; transcriptional repressor EZA1 (http://www.
phytozome.net) (accessed April 2012).
The candidate gene approach using syntenic relation-
ships between cowpea, soybean and Medicago for the
Hls locus identified orthologous candidate genes for the
Arabidopsis gene AT4G02020.1 or EZA1/SWINGER
(SWN). EZA1/SWINGER (SWN) is one of three Arabi-
dopsis E(Z) orthologs of the Drosophila melanogaster
gene ENHANCER OF ZESTE [E(Z)], which includes
CURLY LEAF (CLF) and MEDEA (MEA) [16]. EZA1/
SWINGER (SWN) is an H3K27 methyltransferase tran-
scription factor and belongs to the Polycomb group pro-
teins (Pc-G). Pc-Gs are involved in epigenetic regulation
of developmental processes and are highly conserved in
plants, animals and humans. In plants, Pc-G proteins are
essential in regulating processes such as seed develop-
ment [17], flower organ development [18–20] and leaf
development [18,21].
CLF and SWN are expressed throughout many phases
of plant development and have been shown to be involved
in regulating leaf development. CLF is expressed during
leaf and flower development [18] and EZA1/SWINGER is
expressed in regions of dividing cells and meristems dur-
ing vegetative and reproductive development [19]. CLF
has been shown to directly target and repress the floral
homeotic gene, AGAMOUS (AG), and a homeobox gene,
SHOOTMERISTEMLESS (STM) [20,21]. SWN has been
shown to have partially redundant functions with CLF and
therefore may also be involved in regulating leaf develop-
ment [19]. A clf swn double mutant produced narrow
cotyledons, hypocotyls and roots and as it matured, the
cotyledons developed finger-like growth on the margins as
well as other abnormalities such as the shoot apex not
developing leaves but a disorganized mass of undifferenti-
ated tissue [19]. The fact that EZA1/SWINGER has been
associated with leaf development in Arabidopsis makes it
a plausible candidate gene for regulating leaf morphology
in cowpea.
The combination of the marker-trait association and
the identity of candidate genes in the syntenic loci
enabled us to narrow the Hls region on the consensus
genetic map, from 10.39 cM to approximately 1.87 cM
distance. The narrowest distance between flanking mar-
kers to an orthologous candidate gene was in the Medi-
cago locus, where Medtr7g133020 was flanked by SNP
markers 1_0992 (34.69 cM position) and 1_0083
(35.66 cM position) which narrowed it to a 0.97 cMregion. In soybean chromosome 19, the EZA1/
SWINGER ortholog Glyma19g40430 was flanked by
SNP markers 1_0992 (34.69 cM position) and 1_0417
(35.96 cM position) which narrowed the region to
1.37 cM. The furthest distance between flanking markers
to orthologous candidate genes was in the syntenic locus
in soybean chromosome 3, where Glyma03g38320 was
flanked by SNP marker 1_1013 (34.09 cM position) and
1_0417 (35.96 cM position) with an approximate dis-
tance of 1.87 cM. On average, the most significant re-
gion in the Hls locus was narrowed to a 1.4 cM distance
using the position of the candidate genes to narrow the
QTL region. Assuming that the co-linearity of these
three syntenous regions is upheld when extrapolated
back to cowpea; the cowpea ortholog of EZA1/
SWINGER should be present in this narrowed region.
Differences in marker significance under different ana-
lyses may be of interest. For example, SNP marker
1_0910 was the most significant in the QTL analysis
while SNP marker 1_0349 co-segregated with the geno-
type and phenotype for leaf shape. QTL analysis often
identifies large confidence intervals depending on the
heritability of the trait and because all genes on a
chromosome will show some linkage amongst them-
selves, a QTL will be associated with several markers
[22]. This was the case for SNP markers 1_0349 and
1_0910, which are 1.08 cM distance apart on the individ-
ual genetic map and 1.78 cM on the cowpea consensus
genetic map (Table 3). We have found that small pheno-
typing differences between experiments may move the
most significant marker by 1 cM or more. The marker-
trait association in which SNP marker 1_0349 co-
segregated with the genotype and phenotype for leaf
shape utilized a simplified haplotype analysis, where un-
related individuals were examined for inheritance of
alleles within a specific region. The synteny study
revealed that Medicago and soybean orthologs to cow-
pea SNP markers 1_0083, 1_0092, 1_1013 and 1_0417
were flanking the EZA1 candidate genes (Tables 4, 5,
Additional file 4). These four markers flank the most sig-
nificant marker from the QTL analysis, 1_0910, and
1_0349 which co-segregated with the genotype and
phenotype for leaf shape (Additional file 4). By utilizing
QTL analysis, marker-trait association and candidate
gene analysis using synteny, validation was provided that
the genetic determinant is most likely located within a
1.37 cM region of closely linked markers.
Leaf morphology candidate genes BLAST to cowpea
genomic resources
The genomic sequences for Medtr7g133020, Gly-
ma03g38320, Glyma19g40430 and the Arabidopsis EZA1
gene (AT4G02020.1) were BLASTed to the cowpea gen-
ome vs. 02 (www.harvest-blast.org) and HarvEST:Cowpea
database (http://harvest.ucr.edu) to identify orthologous
cowpea sequences. The Medtr7g133020 and AT4G02020.1
genomic sequences returned a high BLAST alignment with
contig C27495629 (Table 6). The genomic sequences for
Glyma03g38320 and Glyma19g40430 returned a high
which was closely linked with the EZA1 candidate gene in
700 bp for clones in the minimum tiling path (MTP) of
BAC contigs in the cowpea physical map. However, none
of the BESs of clones in either contig25 or contig821
yielded cowpea EZA1 genes when BLASTed to the Har-
vEST:Cowpea database. Future perspectives for enhancing
R g
Pottorff et al. BMC Genomics 2012, 13:234 Page 9 of 12
http://www.biomedcentral.com/1471-2164/13/1/234two out of three of the syntenic loci, was identified in
BAC clone CM041C03 of contig25 (Table 3). Contig25
has 731 overlapping BAC clones and 1843 non-repeated
bands which estimated the length as 3,022,520 bp (http://
phymap.ucdavis.edu/cowpea) (Table 3). The combined
length of the two BAC contigs which span the most sig-
nificant region of the Hls QTL is 3,232,440 bp. Since SNP
marker 1_0992 was closely linked to the EZA1/SWINGER
candidate gene in the Hls syntenic locus in Medicago
chromosome 7 and soybean chromosome 19, the cowpea
EZA1 gene may be located on BAC contig25. Currently,
there are BAC-end sequences (BES) of approximately
Table 6 Medicago, soybean and Arabidopsis EZA1/SWINGE
EZA1(SWINGER) ortholog Cowpea genome
Medtr7g133020 C27495629
Glyma03g38320 C27664167alignment with contig C27664167 and scaffold28398
(Table 6). All genomic sequences when BLASTed to Har-
vest:Cowpea database returned the best alignment with
cowpea unigene 21752 which was annotated as an EZA1
ortholog (Table 6). Interestingly, unigene 21752 was
obtained from leaf and shoot meristems used for a ma-
ture pre-flowering developmental stage cDNA library
from cowpea varieties DanIla, Tvu11986, Tvu7778 and
12008D (http://harvest.ucr.edu). The genomic and uni-
gene sequences identified for the cowpea ortholog for
EZA1 will enable future studies to clone and confirm the
candidate gene.
Hls in the cowpea physical map
The cowpea physical map (http://phymap.ucdavis.edu/
cowpea) which has been partially anchored to the cowpea
consensus genetic map via the same SNP markers was
used to identify BAC contigs which span the Hls region.
Significant markers from the QTL study and closely
linked markers from the cowpea consensus genetic map
identified several BAC contigs which incompletely span
the Hls region (Table 3). The most significant SNP marker
from the QTL analysis, 1_0910, was identified in BAC
clone CH050F07 of contig821 (Table 3). Contig821 has
four overlapping BAC clones and 128 non-repeating
bands which estimated the contig size at 209,920 bp
(http://phymap.ucdavis.edu/cowpea). SNP marker 1_0992Glyma19g40430 scaffold28398
AT4G02020.1 C27495629the cowpea physical map may include sequencing BAC
clones within the MTP of each BAC contig which would
enable the direct identification of genes of interest.
To test the candidacy of the cowpea EZA1 gene for
the Hls locus, a molecular marker could be developed
and mapped to ensure it co-locates in the Hls locus in
the Sanzi x Vita 7 population. Additionally, the cowpea
EZA1 gene would need to be cloned and sequenced
from both parents to determine the allelic variation for
phenotype followed by complementation tests to validate
gene function.
Conclusion
This study has identified one major QTL, Hls, which is
associated with the hastate and sub-globose leaf shape in
the cowpea RIL population Sanzi x Vita 7. Our candidate
gene approach utilized mapping the locus and a marker-
trait association to narrow the QTL locus of 11 cM to one
marker which co-segregated with the trait. The conserved
gene order amongst closely related species, cowpea and
soybean, and members within the same legume family,
cowpea, Medicago and soybean, enabled the identification
of a candidate gene for the Hls locus. Future goals will be
to utilize the molecular marker which co-segregated with
leaf shape in MAS breeding efforts. A more fundamental
study could also be undertaken to determine if the candi-
date gene EZA1/SWINGER is the genetic determinant
governing leaf morphology in cowpea.
Methods
Plant population
Leaf morphology was studied in a cowpea RIL popula-
tion which was developed from an intraspecific cross of
Sanzi x Vita 7. The population consisted of 122 RILs
which were advanced by single seed descent to the F10
generation. Sanzi is a local landrace from Ghana which
has a prostrate sprawling architecture, grayish-purple
seeds, and a sub-globose leaf shape. Vita 7 (PI 580806/
TVu-8461) is an IITA advanced breeding line from Ni-
geria with an upright bush type architecture, beige seeds
enes BLAST to cowpea genomic resources
e-score Cowpea unigene e-score
1.00E-15 21752 4.00E-11
7.00E-30 21752 1.00E-176.00E-36 21752 6.00E-10
3.00E-22 21752 9.00E-21
and hastate leaf shape (IITA germplasm database online;
http://genebank.iita.org). The Sanzi x Vita 7 population
was received from Christian Fatokun, IITA, Ibadan, Ni-
geria. All cowpea accessions were available from the
University of California Riverside cowpea germplasm
collection.
Phenotyping
The terminal central leaflet was observed and classified
as “hastate” or “sub-globose” (Figure 4) five weeks after
markers. The map consists of nineteen linkage groups
and spans approximately 753 cM total distance.
Cowpea consensus genetic map
The cowpea consensus genetic map vs. 4, which is an
updated version of the Muchero et al. 2009 map, was
used for this study [12]. The consensus version 4 map
consists of ten RIL populations and two F4 breeding
populations, which has increased the marker density and
improved the marker order. The map is 680 cM in
Pottorff et al. BMC Genomics 2012, 13:234 Page 10 of 12
http://www.biomedcentral.com/1471-2164/13/1/234germination for each of the RILs. Two sets of pheno-
typic data were obtained; one dataset during a green-
house experiment and the second dataset during a field
experiment. The greenhouse study, which phenotyped
118 out of 122 RILs, was conducted from February to
April 2010 in Riverside, California. Seedlings were trans-
planted into 3785 cm3 pots and watered daily, with day
and night temperatures set to 28°C and 16°C, respect-
ively. The field experiment, which phenotyped 116 out
of 122 RILs, was conducted at the Citrus Research
Center-Agricultural Experiment Station (CRC-AES) in
Riverside CA, from July to September 2010. Twenty-five
seeds per replicate were planted for each RIL in a rando-
mized complete block design using four replicates. Seeds
were machine-planted in single rows on pre-irrigated
raised beds spaced 76 cm apart with 10 cm spacing be-
tween seeds.
SNP genotyping
The Sanzi x Vita 7 population was genotyped at the F8
generation using bi-allelic SNP markers from the 1536
Illumina GoldenGate Assay as previously described [11].
All genotypes used for the marker-trait association study
were SNP genotyped at the F8 generation or above as
previously described [11].
Genetic map
A SNP genetic map was developed previously for the
Sanzi x Vita 7 RIL population and is included in the
cowpea consensus genetic map vs.4 [12]. The individual
map was generated using 122 RILs and 416 SNPFigure 4 Hastate and sub-globose leaf shapes segregating in the Sanlength and contains 1107 markers with an average of
0.65 cM between markers. The current SNP-based cow-
pea linkage map is included in a publicly available
browser called HarvEST:Cowpea, which can be down-
loaded from http://harvest.ucr.edu or viewed online at
www.harvest-web.org.
Statistical analysis
The Kruskal-Wallis and Interval Mapping analysis
packages of MapQTL 5.0 software were used to conduct
the QTL analysis [23]. A QTL was considered significant
if the same QTL was identified using both phenotypic
datasets and if the statistical tests for the markers met
significance thresholds for both Kruskal-Wallis and
Interval Mapping analyses. A significance threshold was
set to 0.05 for Kruskal-Wallis analysis and LOD thresh-
olds for the Interval Mapping analysis were calculated
using 1000 permutations at the 0.05 significance level. A
95% confidence interval was used to estimate the left
and right margins of the QTL using 1-LOD and 2-LOD
of the most likely position. QTLs were visualized using
MapChart 2.2 software [24].
Synteny
Synteny was examined for cowpea with G. max, M.
truncatula and A. thaliana using EST-derived SNP mar-
kers previously BLASTed and aligned to the sequenced
genomes. Annotations for the soybean and Medicago
loci were taken directly from the Phytozome website
(www.phytozome.org). Syntenic relationships amongst
the different genomes can be examined in the HarvEST:zi x Vita7 population.
9. Fery RL: The genetics of cowpea: a review of the world literature. In
10:660–666.
Pottorff et al. BMC Genomics 2012, 13:234 Page 11 of 12
http://www.biomedcentral.com/1471-2164/13/1/234Cowpea database (http://harvest.ucr.edu). Syntenic maps
were drawn using HarvEST:Cowpea using a cut-off e-
score value of -10, with a minimum number of 10 lines
drawn per linkage group.
Marker-trait association
Genotypic data comprised of cowpea varieties and SNP
marker information in the Hls locus were visualized
using GGT 2.0 software [25]. The cowpea consensus
genetic map vs.4 [12] was loaded into the software to
visualize linkage groups.
Cowpea physical map
The physical map was developed using an advanced
African breeding line IT97K-499-35 (http://phymap.
ucdavis.edu/cowpea). It consists of two BAC clone li-
braries developed using restriction enzymes HindIII and
MboI (Amplicon Express, Pullman, WA). Contigs were
assembled using the snapshot method of DNA finger-
printing [26] and completed at University of California
Davis by Ming Cheng Luo. The final physical map is an
assembly of 43,717 BACs with an 11x genome depth of
coverage. The size of the BAC clones was estimated by
multiplying the number of unique bands generated from
the fingerprinting assay by 1640 bp (personal communica-
tion, Ming Cheng Luo).
Additional files
Additional file 1: Cowpea accessions with a hastate or sub-globose
leaf phenotype.
Additional file 2: SNP marker 1_0349 sequence. cDNA sequence of
P12 assembly unigene 8605 which is housed in Harvest:Cowpea database
(http://harvest.ucr.edu). The SNP (thymine/cytosine) is located at position
2122, parenthesized, underlined and in bold.
Additional file 3: Synteny of the Hls locus with A. thaliana. Synteny
was examined for the Hls locus between cowpea and A. thaliana using
EST-derived SNP markers previously BLASTed and aligned to the
sequenced genome. The Hls locus on the cowpea consensus genetic
map, linkage group 4 (25.57 cM – 35.96 cM position), showed very low
synteny with the Arabidopsis genome. The syntenic map was drawn
using HarvEST:Cowpea database (http://harvest.ucr.edu) using a cut off
e-score value of −10 and a minimum number of 10 lines drawn per
linkage group.
Additional file 4: Summary of significant markers in the Hls locus.
Abbreviations
BAC: Bacterial artificial chromosome; BES: BAC end sequence; bp: Base pairs;
cM: Centimorgans; EST: Expressed sequence tags; EZA1: ENHANCER OF
ZESTE; LG: Linkage group; LOD: Logarithm of (base 10) of odds; MAS: Marker-
assisted selection; Mb: Megabases; MTP: Minimum tiling path; Pc-
G: Polycomb-group protein; QTL: Quantitative trait locus; RIL: Recombinant
inbred line; SNPs: Single nucleotide polymorphism; SWN: SWINGER.
Competing interests
The author(s) declare that they have no competing interests.Authors’ contributions
MP conducted the greenhouse and field experiments. MP analyzed the
genetic inheritance, QTL analysis, marker-trait association, candidate gene16. Guitton A, Berger F: Control of reproduction by Polycomb Group
complexes in animals and plants. Int J Dev Biol 2005, 49:707–716.
17. Wang D, Tyson MD, Jackson SS, Yadegari R: Partially redundant functions of
two SET-domain polycomb-group proteins in controlling initiation of seed
development in Arabidopsis. Proc Natl Acad Sci 2006, 103:13244–13249.Cowpea Research, Production and Utilization. Edited by Singh SR, Rachie KO.
Chichester: John Wiley and Sons; 1985:25–62.
10. Oluwatosin OB: Inheritance of genes for leaflet shape and leaflet shape
modifier in cowpea. Afr Crop Sci J 2002, 10:133–137.
11. Muchero W, Diop NN, Bhat PR, Fenton RD, Wanamaker S, Pottorff M, Hearne
S, Cisse N, Fatokun C, Ehlers JD: A consensus genetic map of cowpea
[Vigna unguiculata (L) Walp.] and synteny based on EST-derived SNPs.
Proc Natl Acad Sci 2009, 106:18159–18164.
12. Lucas MR, Diop NN, Wanamaker S, Ehlers JD, Roberts PA, Close TJ:
Cowpea–soybean synteny clarified through an improved genetic map.
Plant Genome J 2011, 4:218–225.
13. Saunders AR: Inheritance in the cowpea III: mutations and linkages. S Afr J
Agric Sci 1960, 3:327–348.
14. Harland SC: Inheritance of certain characters in the cowpea (Vigna
sinensis). J Genet 1919, 8:101–132.
15. Barkoulas M, Galinha C, Grigg SP, Tsiantis M: From genes to shape:
regulatory interactions in leaf development. Curr Opin Plant Biol 2007,analysis using synteny and comparison of the cowpea consensus genetic
map and physical map. CF provided the RIL population. MP, JDE, PAR and
TJC participated in the design, interpretation of data and writing of the
manuscript. All authors read and approved the final manuscript.
Funding declaration
This work was supported in part by the Generation Challenge Program
through a grant from the Bill and Melinda Gates Foundation, the U.S.
Agency for International Development Collaborative Research Support
Program Grants GDG-G-00-02-00012-00 and EDH-A-00-07-00005 and the
University of California Agricultural Experiment Station.
Author details
1Department of Botany & Plant Sciences, University of California Riverside,
Riverside, CA, USA. 2Bill & Melinda Gates Foundation, Seattle, WA, USA.
3International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria.
4Department of Nematology, University of California Riverside, Riverside, CA,
USA.
Received: 21 February 2012 Accepted: 29 May 2012
Published: 12 June 2012
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doi:10.1186/1471-2164-13-234
Cite this article as: Pottorff et al.: Leaf morphology in Cowpea [Vigna
unguiculata (L.) Walp]: QTL analysis, physical mapping and identifying a
candidate gene using synteny with model legume species. BMC
Genomics 2012 13:234.
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