BACKGROUND
cowpea  exhibits a considerable variation in leaf shape.
cowpea leaves are compound, 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 classification 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 differently.
the two largest cowpea germplasm agencies are the international institute of tropical agriculture  and the united states department of agriculture .
iita, which houses  <dig>  cowpea accessions from  <dig> different countries, classifies cowpea leaf shapes into four categories, sub-globose, sub-hastate, globose and hastate/lanceolate .
the usda, which houses  <dig>  cowpea accessions from  <dig> countries, classifies cowpea leaf shapes into five categories; globose, hastate, sub-globose, sub-hastate, strip and ovate-lanceolate .
multipurpose cowpea
cowpea is a multipurpose crop; the entire plant can be used for either human or livestock consumption.
in  <dig>  cowpea dry grain production was estimated at  <dig> , <dig> tons worldwide .
although cowpea is not one of the highest production crops worldwide, nearly 90% of cowpea is produced in west africa, which is estimated at  <dig> , <dig> tons .
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 , can be used as fodder for livestock  <cit> .
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, tanzania and malawi  <cit> .
cowpea shoots and leaves are rich sources of calcium, phosphorous and vitamin b  <cit> .
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 planting 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 social strata consume cowpea leaves eaten as a vegetable .
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 .
dual purpose cowpea varieties which are bred for quality seeds, vegetables and fodder may add to a farmer’s revenue.
for example, in nigeria, farmers who sold dried cowpea fodder during the peak of the drought season saw a 25% increase to their annual income  <cit> .
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 selection 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 cultivated varieties of cowpea have the more common ovate or sub-globose leaf shape.
however, any possible adaptive 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  <cit> .
this may indicate that the hastate shape is ancestral 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 enhancing breeding programs for the improvement of cowpea varieties for the united states, india, brazil, and numerous countries in africa and asia.
these integrated genomic resources include a  <dig> snp genotyping platform, an est-derived snp consensus genetic map, known syntenic relationships between cowpea, medicago truncatulaglycine max and arabidopsis thaliana, and a cowpea est sequence collection housed in harvest:cowpea database   <cit> .
a cowpea physical map has been partially anchored to the cowpea consensus genetic map using the same snp markers  and is available publically .
in addition, about  <dig> diverse cowpea accessions have been snp-genotyped  and a first draft of the cowpea genome, vs. <dig> , has been assembled .
these resources will enable dissection of underlying genetic components of target agronomic traits using quantitative trait locus  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 morphology in a segregating cowpea ril population, sanzi  x vita <dig> .
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 relationships between cowpea, soybean and medicago.
in addition, a snp marker was found which co-segregated with the leaf morphology genotypes and phenotype, which could be used as a molecular marker for breeding purposes.
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 morphology in cowpea.
RESULTS
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  x vita  <dig> .
the hastate and sub-globose leaf shape segregated 58: <dig> in the greenhouse experiment and 59: <dig> in the field experiment  which fit the proposed model that the leaf shape is a qualitative trait .
several other researchers have studied the inheritance of the leaf shape in cowpea  and reported that it was a qualitative trait  <cit> .
although the f <dig> generation was not assessed in the current study, the majority of researchers studying cowpea leaf shape have concluded that the hastate leaf shape is dominant to the more common ovate or sub-globose leaf shape  <cit> .
however, saunders et al.
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 morphology.
the leaf morphology qtl spanned 11 cm distance on the sanzi x vita  <dig> individual genetic map from  <dig>  cm to  <dig>  cm on linkage group  <dig> .
snp marker 1_ <dig> was the most significant marker in both of the datasets, accounting for  <dig> % of the phenotypic variance  in the greenhouse experiment and  <dig> % phenotypic variance  in the field experiment .
we propose the designation hls  for the qtl identified.
im = interval mapping analysis, kw = kruskal-wallis analysis.
snp markers are aligned in the order defined by the cowpea consensus genetic map.
other researchers studying the inheritance of the hastate leaf shape in cowpea have reported a single dominant gene controlling the hastate leaf shape over the ovate or sub-globose leaf shape.
several gene symbols have been proposed, the first being l, which is a dominant gene controlling lanceolate leaf shape  <cit> .
ojomo et al.
proposed the gene symbol ha for the hastate leaf shape and kolhe et al.
proposed nlf for narrow leaf shape.
fery  proposed the gene symbol, la, for the narrow leaf shape.
however, all of the studies investigating the narrow leaf shape used different cowpea accessions to make their populations.
whether these many studies are describing the same leaf shape locus or whether they are describing multiple independent loci remains inconclusive.
interestingly, ogundiwin et al.
identified one major qtl for the hastate leaf shape, designated la, in vigna unguiculata ssp.
textilus.
subspecies textilus is closely related to cultivated cowpea ; 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  <dig> genetic map were aligned with the cowpea consensus genetic map .
the hls locus spans from  <dig>  cm to  <dig>  cm on the cowpea consensus genetic map linkage group  <dig> .
the length of hls on the individual genetic map, 11 cm, is nearly the same as on the cowpea consensus genetic map,  <dig>  cm which may reflect accuracy of marker order .
the hls locus on the cowpea consensus genetic map has several snp markers which were not present in the sanzi x vita  <dig> population because of lack of polymorphism in the individual population .
in addition, there was a slight difference in the order of the snp markers in the sanzi x vita <dig> 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  <dig>  pi  <dig>  pi  <dig>  pi  <dig>  pi  <dig>  pi  <dig>  pi  <dig>  pi  <dig> and pi  <dig> were chosen for the hastate leaf shape phenotype .
pi  <dig>  cb <dig>  bambey  <dig>  pi  <dig>  pi  <dig> and pi  <dig> were chosen from the usda grin database and under their naming convention were classified as “sub-globose” leaf shape .
accessions designated “tvnu” are wild cowpeas, many of which have the hastate leaf shape.
the alleles of snp marker 1_ <dig>  co-segregated perfectly with the hastate or sub-globose leaf phenotype .
the allele for the hastate genotype at this locus was the thymine nucleotide .
the allele for the sub-globose genotype was the cytosine nucleotide .
the thymine/cytosine snp for 1_ <dig> is at position  <dig> in the cowpea p <dig> assembly unigene  <dig> and can be viewed in harvest:cowpea  .
the marker-trait association narrowed the hls qtl to a  <dig>  cm region and was defined by flanking snp markers 1_ <dig> and 1_ <dig> .
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 relationship exists.
a high co-linearity or a conservation of gene order utilizing the est-derived snp markers with any 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 in the harvest:cowpea database and are publicly available .
due to limited resolution in the software images, not all markers are presented in the screenshot images output from harvest:cowpea.
however, the cowpea consensus genetic map vs.  <dig>  <cit>  has been used in fidelity.
in order to view each individual marker, the linkage group must be magnified in the harvest:cowpea database.
the hls locus was examined for synteny with the arabidopsis genome; however very low synteny was displayed at the macro level between cowpea and arabidopsis so no further examination was pursued .
a high co-linearity was observed for the hls locus with medicago chromosome  <dig> .
eight medicago genes orthologous to cowpea snp markers were identified in the syntenic region of medicago chromosome  <dig> .
the syntenic region spanned from medtr7g <dig> locus to medtr7g <dig> locus which corresponded to  <dig>  cm to  <dig>  cm of the hls locus on the cowpea consensus genetic map .
the region which spanned from medicago genes orthologous to cowpea snp markers 1_ <dig> to 1_ <dig> were in the same linear order as on the cowpea consensus genetic map .
the region spanning between medicago genes orthologous to cowpea snp markers 1_ <dig>  and 1_ <dig>  was examined for genes known to be associated with the molecular control of leaf morphology in other plant species  <cit>  on the medicago genome browser on the phytozome webpage .
the medicago locus medtr7g <dig> was observed between medicago genes orthologous to cowpea snp markers 1_ <dig> and 1_ <dig> and was annotated as an ortholog of the arabidopsis gene at4g <dig>  aka eza <dig> or swinger  .
medtr7g <dig> has a set domain  with two copies of a cysteine rich motif and is annotated as kog: 1079; transcriptional repressor eza <dig>  .
the hls region was examined for synteny with the soybean genome and was found to be highly co-linear with soybean chromosomes  <dig> and  <dig> .
eight medicago genes orthologous to cowpea snp markers identified the region from locus glyma03g <dig> to glyma03g <dig> as the hls syntenic locus in soybean chromosome  <dig> .
the soybean syntenic locus corresponded to  <dig>  cm to  <dig>  cm region in the hls locus and was also in the same general marker order as the cowpea consensus genetic map .
the region spanning between orthologous soybean genes to cowpea snp markers 1_ <dig> and 1_ <dig> was examined for leaf morphology candidate genes on the soybean genome browser on the phytozome webpage .
soybean locus glyma03g <dig> was observed flanked by orthologous genes for cowpea snp markers 1_ <dig> and 1_ <dig> and was annotated as an ortholog of eza1/swinger  gene.
glyma03g <dig> has a set domain  and two copies of a cysteine rich motif and is annotated as kog: 1079; transcriptional repressor eza <dig>  .
the hls syntenic region in soybean chromosome  <dig> was identified by thirteen out of fourteen snp markers, spanning from glyma19g <dig> to glyma19g <dig> which corresponded to  <dig>  cm to  <dig>  cm on the cowpea consensus genetic map .
the syntenic region in soybean between orthologous cowpea snp markers 1_ <dig> and 1_ <dig> was examined for known leaf development genes using the soybean genome browser on the phytozome webpage .
glyma19g <dig> locus was observed flanked by soybean genes orthologous to snp markers 1_ <dig> and 1_ <dig> and was annotated as an ortholog of the arabidopsis eza1/swinger  gene .
glyma19g <dig> has a set domain  and two copies of a cysteine rich motif and is annotated as kog: 1079; transcriptional repressor eza <dig>  .
the candidate gene approach using syntenic relationships between cowpea, soybean and medicago for the hls locus identified orthologous candidate genes for the arabidopsis gene at4g <dig>  or eza1/swinger .
eza1/swinger  is one of three arabidopsis e orthologs of the drosophila melanogaster gene enhancer of zeste , which includes curly leaf  and medea   <cit> .
eza1/swinger  is an h3k <dig> methyltransferase transcription factor and belongs to the polycomb group proteins .
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 development  <cit> , flower organ development  <cit>  and leaf development  <cit> .
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  <cit>  and eza1/swinger is expressed in regions of dividing cells and meristems during vegetative and reproductive development  <cit> .
clf has been shown to directly target and repress the floral homeotic gene, agamous , and a homeobox gene, shootmeristemless   <cit> .
swn has been shown to have partially redundant functions with clf and therefore may also be involved in regulating leaf development  <cit> .
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 undifferentiated tissue  <cit> .
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  <dig>  cm to approximately  <dig>  cm distance.
the narrowest distance between flanking markers to an orthologous candidate gene was in the medicago locus, where medtr7g <dig> was flanked by snp markers 1_ <dig>  and 1_ <dig>  which narrowed it to a  <dig>  cm region.
in soybean chromosome  <dig>  the eza1/swinger ortholog glyma19g <dig> was flanked by snp markers 1_ <dig>  and 1_ <dig>  which narrowed the region to  <dig>  cm.
the furthest distance between flanking markers to orthologous candidate genes was in the syntenic locus in soybean chromosome  <dig>  where glyma03g <dig> was flanked by snp marker 1_ <dig>  and 1_ <dig>  with an approximate distance of  <dig>  cm.
on average, the most significant region in the hls locus was narrowed to a  <dig>  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 analyses may be of interest.
for example, snp marker 1_ <dig> was the most significant in the qtl analysis while snp marker 1_ <dig> co-segregated with the genotype 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 themselves, a qtl will be associated with several markers  <cit> .
this was the case for snp markers 1_ <dig> and 1_ <dig>  which are  <dig>  cm distance apart on the individual genetic map and  <dig>  cm on the cowpea consensus genetic map .
we have found that small phenotyping differences between experiments may move the most significant marker by 1 cm or more.
the marker-trait association in which snp marker 1_ <dig> co-segregated with the genotype and phenotype for leaf shape utilized a simplified haplotype analysis, where unrelated individuals were examined for inheritance of alleles within a specific region.
the synteny study revealed that medicago and soybean orthologs to cowpea snp markers 1_ <dig>  1_ <dig>  1_ <dig> and 1_ <dig> were flanking the eza <dig> candidate genes .
these four markers flank the most significant marker from the qtl analysis, 1_ <dig>  and 1_ <dig> which co-segregated with the genotype and phenotype for leaf shape .
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  <dig>  cm region of closely linked markers.
leaf morphology candidate genes blast to cowpea genomic resources
the genomic sequences for medtr7g <dig>  glyma03g <dig>  glyma19g <dig> and the arabidopsis eza <dig> gene  were blasted to the cowpea genome vs.  <dig>  and harvest:cowpea database  to identify orthologous cowpea sequences.
the medtr7g <dig> and at4g <dig>  genomic sequences returned a high blast alignment with contig c <dig> .
the genomic sequences for glyma03g <dig> and glyma19g <dig> returned a high alignment with contig c <dig> and scaffold <dig> .
all genomic sequences when blasted to harvest:cowpea database returned the best alignment with cowpea unigene  <dig> which was annotated as an eza <dig> ortholog .
interestingly, unigene  <dig> was obtained from leaf and shoot meristems used for a mature pre-flowering developmental stage cdna library from cowpea varieties danila, tvu <dig>  tvu <dig> and 12008d .
the genomic and unigene sequences identified for the cowpea ortholog for eza <dig> will enable future studies to clone and confirm the candidate gene.
hls in the cowpea physical map
the cowpea physical map  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 .
the most significant snp marker from the qtl analysis, 1_ <dig>  was identified in bac clone ch050f <dig> of contig <dig> .
contig <dig> has four overlapping bac clones and  <dig> non-repeating bands which estimated the contig size at  <dig>  bp .
snp marker 1_ <dig> which was closely linked with the eza <dig> candidate gene in two out of three of the syntenic loci, was identified in bac clone cm041c <dig> of contig <dig> .
contig <dig> has  <dig> overlapping bac clones and  <dig> non-repeated bands which estimated the length as  <dig> ,520 bp  .
the combined length of the two bac contigs which span the most significant region of the hls qtl is  <dig> ,440 bp.
since snp marker 1_ <dig> was closely linked to the eza1/swinger candidate gene in the hls syntenic locus in medicago chromosome  <dig> and soybean chromosome  <dig>  the cowpea eza <dig> gene may be located on bac contig <dig>  currently, there are bac-end sequences  of approximately 700 bp for clones in the minimum tiling path  of bac contigs in the cowpea physical map.
however, none of the bess of clones in either contig <dig> or contig <dig> yielded cowpea eza <dig> genes when blasted to the harvest:cowpea database.
future perspectives for enhancing the 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 eza <dig> 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  <dig> population.
additionally, the cowpea eza <dig> 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.
CONCLUSIONS
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  <dig>  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 candidate gene eza1/swinger is the genetic determinant governing leaf morphology in cowpea.

 cowpea exhibits a considerable variation in leaf shape. cowpea leaves are compound, 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 classification 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 differently. the two largest cowpea germplasm agencies are the international institute of tropical agriculture and the united states department of agriculture. iita, which houses cowpea accessions from different countries, classifies cowpea leaf shapes into four categories, sub-globose, sub-hastate, globose and hastate/lanceolate. the usda, which houses cowpea accessions from countries, classifies cowpea leaf shapes into five categories; globose, hastate, sub-globose, sub-hastate, strip and ovate-lanceolate. multipurpose cowpea cowpea is a multipurpose crop; the entire plant can be used for either human or livestock consumption. in cowpea dry grain production was estimated at, tons worldwide. although cowpea is not one of the highest production crops worldwide, nearly 90% of cowpea is produced in west africa, which is estimated at, tons. 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, can be used as fodder for livestock. 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, tanzania and malawi. cowpea shoots and leaves are rich sources of calcium, phosphorous and vitamin b. 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 planting 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 social strata consume cowpea leaves eaten as a vegetable. 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. dual purpose cowpea varieties which are bred for quality seeds, vegetables and fodder may add to a farmer’s revenue. for example, in nigeria, farmers who sold dried cowpea fodder during the peak of the drought season saw a 25% increase to their annual income. 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 selection 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 cultivated varieties of cowpea have the more common ovate or sub-globose leaf shape. however, any possible adaptive 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. this may indicate that the hastate shape is ancestral 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 enhancing breeding programs for the improvement of cowpea varieties for the united states, india, brazil, and numerous countries in africa and asia. these integrated genomic resources include a snp genotyping platform, an est-derived snp consensus genetic map, known syntenic relationships between cowpea, medicago truncatulaglycine max and arabidopsis thaliana, and a cowpea est sequence collection housed in harvest:cowpea database. a cowpea physical map has been partially anchored to the cowpea consensus genetic map using the same snp markers and is available publically. in addition, about diverse cowpea accessions have been snp-genotyped and a first draft of the cowpea genome, vs., has been assembled. these resources will enable dissection of underlying genetic components of target agronomic traits using quantitative trait locus 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 morphology in a segregating cowpea ril population, sanzi x vita. 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 relationships between cowpea, soybean and medicago. in addition, a snp marker was found which co-segregated with the leaf morphology genotypes and phenotype, which could be used as a molecular marker for breeding purposes. 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 morphology in cowpea.  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 x vita. the hastate and sub-globose leaf shape segregated 58: in the greenhouse experiment and 59: in the field experiment which fit the proposed model that the leaf shape is a qualitative trait. several other researchers have studied the inheritance of the leaf shape in cowpea and reported that it was a qualitative trait. although the f generation was not assessed in the current study, the majority of researchers studying cowpea leaf shape have concluded that the hastate leaf shape is dominant to the more common ovate or sub-globose leaf shape. however, saunders et al. 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 morphology. the leaf morphology qtl spanned 11 cm distance on the sanzi x vita individual genetic map from  cm to  cm on linkage group. snp marker 1_ was the most significant marker in both of the datasets, accounting for % of the phenotypic variance in the greenhouse experiment and % phenotypic variance in the field experiment. we propose the designation hls for the qtl identified. im = interval mapping analysis, kw = kruskal-wallis analysis. snp markers are aligned in the order defined by the cowpea consensus genetic map. other researchers studying the inheritance of the hastate leaf shape in cowpea have reported a single dominant gene controlling the hastate leaf shape over the ovate or sub-globose leaf shape. several gene symbols have been proposed, the first being l, which is a dominant gene controlling lanceolate leaf shape. ojomo et al. proposed the gene symbol ha for the hastate leaf shape and kolhe et al. proposed nlf for narrow leaf shape. fery proposed the gene symbol, la, for the narrow leaf shape. however, all of the studies investigating the narrow leaf shape used different cowpea accessions to make their populations. whether these many studies are describing the same leaf shape locus or whether they are describing multiple independent loci remains inconclusive. interestingly, ogundiwin et al. identified one major qtl for the hastate leaf shape, designated la, in vigna unguiculata ssp. textilus. subspecies textilus is closely related to cultivated cowpea; 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 genetic map were aligned with the cowpea consensus genetic map. the hls locus spans from  cm to  cm on the cowpea consensus genetic map linkage group. the length of hls on the individual genetic map, 11 cm, is nearly the same as on the cowpea consensus genetic map,  cm which may reflect accuracy of marker order. the hls locus on the cowpea consensus genetic map has several snp markers which were not present in the sanzi x vita population because of lack of polymorphism in the individual population. in addition, there was a slight difference in the order of the snp markers in the sanzi x vita 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 pi pi pi pi pi pi pi and pi were chosen for the hastate leaf shape phenotype. pi cb bambey pi pi and pi were chosen from the usda grin database and under their naming convention were classified as “sub-globose” leaf shape. accessions designated “tvnu” are wild cowpeas, many of which have the hastate leaf shape. the alleles of snp marker 1_ co-segregated perfectly with the hastate or sub-globose leaf phenotype. the allele for the hastate genotype at this locus was the thymine nucleotide. the allele for the sub-globose genotype was the cytosine nucleotide. the thymine/cytosine snp for 1_ is at position in the cowpea p assembly unigene and can be viewed in harvest:cowpea. the marker-trait association narrowed the hls qtl to a  cm region and was defined by flanking snp markers 1_ and 1_. 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 relationship exists. a high co-linearity or a conservation of gene order utilizing the est-derived snp markers with any 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 in the harvest:cowpea database and are publicly available. due to limited resolution in the software images, not all markers are presented in the screenshot images output from harvest:cowpea. however, the cowpea consensus genetic map vs. has been used in fidelity. in order to view each individual marker, the linkage group must be magnified in the harvest:cowpea database. the hls locus was examined for synteny with the arabidopsis genome; however very low synteny was displayed at the macro level between cowpea and arabidopsis so no further examination was pursued. a high co-linearity was observed for the hls locus with medicago chromosome. eight medicago genes orthologous to cowpea snp markers were identified in the syntenic region of medicago chromosome. the syntenic region spanned from medtr7g locus to medtr7g locus which corresponded to  cm to  cm of the hls locus on the cowpea consensus genetic map. the region which spanned from medicago genes orthologous to cowpea snp markers 1_ to 1_ were in the same linear order as on the cowpea consensus genetic map. the region spanning between medicago genes orthologous to cowpea snp markers 1_ and 1_ was examined for genes known to be associated with the molecular control of leaf morphology in other plant species on the medicago genome browser on the phytozome webpage. the medicago locus medtr7g was observed between medicago genes orthologous to cowpea snp markers 1_ and 1_ and was annotated as an ortholog of the arabidopsis gene at4g aka eza or swinger. medtr7g has a set domain with two copies of a cysteine rich motif and is annotated as kog: 1079; transcriptional repressor eza. the hls region was examined for synteny with the soybean genome and was found to be highly co-linear with soybean chromosomes and. eight medicago genes orthologous to cowpea snp markers identified the region from locus glyma03g to glyma03g as the hls syntenic locus in soybean chromosome. the soybean syntenic locus corresponded to  cm to  cm region in the hls locus and was also in the same general marker order as the cowpea consensus genetic map. the region spanning between orthologous soybean genes to cowpea snp markers 1_ and 1_ was examined for leaf morphology candidate genes on the soybean genome browser on the phytozome webpage. soybean locus glyma03g was observed flanked by orthologous genes for cowpea snp markers 1_ and 1_ and was annotated as an ortholog of eza1/swinger gene. glyma03g has a set domain and two copies of a cysteine rich motif and is annotated as kog: 1079; transcriptional repressor eza. the hls syntenic region in soybean chromosome was identified by thirteen out of fourteen snp markers, spanning from glyma19g to glyma19g which corresponded to  cm to  cm on the cowpea consensus genetic map. the syntenic region in soybean between orthologous cowpea snp markers 1_ and 1_ was examined for known leaf development genes using the soybean genome browser on the phytozome webpage. glyma19g locus was observed flanked by soybean genes orthologous to snp markers 1_ and 1_ and was annotated as an ortholog of the arabidopsis eza1/swinger gene. glyma19g has a set domain and two copies of a cysteine rich motif and is annotated as kog: 1079; transcriptional repressor eza. the candidate gene approach using syntenic relationships between cowpea, soybean and medicago for the hls locus identified orthologous candidate genes for the arabidopsis gene at4g or eza1/swinger. eza1/swinger is one of three arabidopsis e orthologs of the drosophila melanogaster gene enhancer of zeste, which includes curly leaf and medea. eza1/swinger is an h3k methyltransferase transcription factor and belongs to the polycomb group proteins. 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 development, flower organ development and leaf development. 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 and eza1/swinger is expressed in regions of dividing cells and meristems during vegetative and reproductive development. clf has been shown to directly target and repress the floral homeotic gene, agamous, and a homeobox gene, shootmeristemless. swn has been shown to have partially redundant functions with clf and therefore may also be involved in regulating leaf development. 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 undifferentiated tissue. 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  cm to approximately  cm distance. the narrowest distance between flanking markers to an orthologous candidate gene was in the medicago locus, where medtr7g was flanked by snp markers 1_ and 1_ which narrowed it to a  cm region. in soybean chromosome the eza1/swinger ortholog glyma19g was flanked by snp markers 1_ and 1_ which narrowed the region to  cm. the furthest distance between flanking markers to orthologous candidate genes was in the syntenic locus in soybean chromosome where glyma03g was flanked by snp marker 1_ and 1_ with an approximate distance of  cm. on average, the most significant region in the hls locus was narrowed to a  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 analyses may be of interest. for example, snp marker 1_ was the most significant in the qtl analysis while snp marker 1_ co-segregated with the genotype 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 themselves, a qtl will be associated with several markers. this was the case for snp markers 1_ and 1_ which are  cm distance apart on the individual genetic map and  cm on the cowpea consensus genetic map. we have found that small phenotyping differences between experiments may move the most significant marker by 1 cm or more. the marker-trait association in which snp marker 1_ co-segregated with the genotype and phenotype for leaf shape utilized a simplified haplotype analysis, where unrelated individuals were examined for inheritance of alleles within a specific region. the synteny study revealed that medicago and soybean orthologs to cowpea snp markers 1_ 1_ 1_ and 1_ were flanking the eza candidate genes. these four markers flank the most significant marker from the qtl analysis, 1_ and 1_ which co-segregated with the genotype and phenotype for leaf shape. 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  cm region of closely linked markers. leaf morphology candidate genes blast to cowpea genomic resources the genomic sequences for medtr7g glyma03g glyma19g and the arabidopsis eza gene were blasted to the cowpea genome vs. and harvest:cowpea database to identify orthologous cowpea sequences. the medtr7g and at4g genomic sequences returned a high blast alignment with contig c. the genomic sequences for glyma03g and glyma19g returned a high alignment with contig c and scaffold. all genomic sequences when blasted to harvest:cowpea database returned the best alignment with cowpea unigene which was annotated as an eza ortholog. interestingly, unigene was obtained from leaf and shoot meristems used for a mature pre-flowering developmental stage cdna library from cowpea varieties danila, tvu tvu and 12008d. the genomic and unigene sequences identified for the cowpea ortholog for eza will enable future studies to clone and confirm the candidate gene. hls in the cowpea physical map the cowpea physical map 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. the most significant snp marker from the qtl analysis, 1_ was identified in bac clone ch050f of contig. contig has four overlapping bac clones and non-repeating bands which estimated the contig size at  bp. snp marker 1_ which was closely linked with the eza candidate gene in two out of three of the syntenic loci, was identified in bac clone cm041c of contig. contig has overlapping bac clones and non-repeated bands which estimated the length as,520 bp. the combined length of the two bac contigs which span the most significant region of the hls qtl is,440 bp. since snp marker 1_ was closely linked to the eza1/swinger candidate gene in the hls syntenic locus in medicago chromosome and soybean chromosome the cowpea eza gene may be located on bac contig currently, there are bac-end sequences of approximately 700 bp for clones in the minimum tiling path of bac contigs in the cowpea physical map. however, none of the bess of clones in either contig or contig yielded cowpea eza genes when blasted to the harvest:cowpea database. future perspectives for enhancing the 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 eza 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 population. additionally, the cowpea eza 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.  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 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 candidate gene eza1/swinger is the genetic determinant governing leaf morphology in cowpea. 
one syntenic locus for hls was identified on medicago chromosome while syntenic regions for hls were identified on two soybean chromosomes, and in all three syntenic loci, an ortholog for the eza1/swinger gene was observed and is the candidate gene for the hls locus. this study has demonstrated how integrated genomic resources can be utilized for a candidate gene approach. the corresponding hls locus was positioned on the cowpea consensus genetic map on linkage group spanning from to  cm. the hls locus was identified on the cowpea physical map via snp markers 1_ 1_ and 1_ which were identified in three bac contigs; contig contig and contig a qtl for leaf shape, hls, was identified on the sanzi x vita genetic map spanning from  cm to  cm distance on linkage group snp marker 1_ was the most significant over the two experiments, accounting for % phenotypic variance in a greenhouse experiment and % phenotypic variance in a field experiment. a marker-trait association of the hls region identified snp marker 1_ 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. cowpea exhibits a considerable variation in leaf shape. leaf morphology was studied in the cowpea ril population, sanzi x vita. 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 control of leaf shape in legumes. 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.
 cowpea 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.  leaf morphology was studied in the cowpea ril population, sanzi x vita. a qtl for leaf shape, hls, was identified on the sanzi x vita genetic map spanning from  cm to  cm distance on linkage group snp marker 1_ was the most significant over the two experiments, accounting for % phenotypic variance in a greenhouse experiment and % phenotypic variance in a field experiment. the corresponding hls locus was positioned on the cowpea consensus genetic map on linkage group spanning from to  cm. a marker-trait association of the hls region identified snp marker 1_ 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 while syntenic regions for hls were identified on two soybean chromosomes, and in all three syntenic loci, an ortholog for the eza1/swinger 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_ 1_ and 1_ which were identified in three bac contigs; contig contig and contig  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 control of leaf shape in legumes. qtl analysisleaf morphologygenomicsgeneticsphysical mapsyntenycandidate genescowpealegumeseza1/swinger 