BACKGROUND
useful agronomic traits can be found in wild populations of crop species.
nevertheless, a large fraction of species with hermaphroditic flowers have developed genetic mechanisms that allow the pistil to recognize and reject pollen from genetically related individuals , and this may affect the efficient incorporation of such traits into crop varieties.
self-incompatibility is, in general, evolutionarily advantageous, because it promotes cross-fertilization, and thus inbreeding depression avoidance.
fabaceae is an economically important plant family with a large number of self-incompatible species , that have been reported often as showing self-incompatibility of the gametophytic type .
in gsi, if the specificity of the haploid pollen grain matches either one of the diploid pistil, an incompatible reaction occurs, leading to the degradation of the pollen tube within the pistil  <cit> .
it should be noted, however, that in all fabaceae species where pollen tube growth was assessed in controlled crosses, only in species of the genus trifolium the gsi reaction seems to be complete and takes place in the stlyle  <cit>  as observed in rosaceae , solanaceae  and plantaginaceae  si species.
in other species such as vicia faba  <cit> , lotus corniculatus  <cit> , cytisus striatus  <cit> , coronilla emerus and colutea arborescens  <cit>  there is, however a significant difference on the percentage of pollen growth in self and cross-pollinations.
in c. striatus, one of the species here studied, the percentage of ovules that are penetrated by pollen tubes is 72% in hand self-pollinated flowers compared with the  <dig> % when hand cross-pollinations are performed  <cit> .
these authors have shown that an important fraction of self pollen grains collapse along the style, as observed in rosaceae, solanaceae and plantaginaceae si species.
although the molecular characterization of the fabaceae s-locus has never been performed, some authors have suggested that in fabaceae gsi is rnase based  <cit> .
nevertheless, there are other gsi systems, such as that present in papaveraceae .
moreover, late-acting si , so called because rejection of self-pollen takes place either in the ovary prior to fertilization, or in the first divisions of the zygote  <cit> , has been described in fabaceae  <cit> .
it should be noted that, lsi can also be of the gametophytic type  <cit> .
in fabaceae, however, the genetic basis of the different mechanisms that control lsi are mostly unknown, and thus, in this work we only address the possibility that fabaceae gsi is determined by a s-rnase gene that clusters with those of the well characterized rosaceae  <cit> , solanaceae  <cit>  and plantaginaceae  <cit>  species.
the most common ancestor of fabaceae  and rosaceae species lived about 89– <dig> million years ago .
since, according to phylogenetic analyses of the t2-rnases, rnase based gsi has evolved only once, before the split of the asteridae and rosidae, about  <dig> mya  <cit> , at least some fabaceae si species are expected to have this system.
therefore, in principle, a homology based approach could be used to identify the putative pistil s-gene in fabaceae species.
three amino acid patterns , allow the distinction of s-rnase lineage genes from other t <dig> -rnase genes  <cit> .
these patterns can be used to easily identify putative s-lineage genes using blast searches.
the results can be further refined by selecting only those genes that encode basic proteins  since s-rnases have an isoelectric point between  <dig> and  <dig>  <cit> .
furthermore, the number of introns can also be used to select s-lineage genes since s-rnases have one or two introns only .
phylogenetic analyses where a set of reference genes are used, can then be performed to show that such genes belong, indeed, to the s-lineage.
nevertheless, in order to show that the identified genes are the pistil s-gene, it is necessary to show that they are highly expressed in pistils, although they can show lower expression in stigma and styles .
in malus fusca where a large number of transcriptomes  have been analysed the same pattern is observed .
moreover, it is necessary to show that they have high polymorphism levels, that there is evidence for positive selection, and that in controlled crosses they co-segregate with s-locus alleles .
the pollen component, always an f-box protein, has been identified as one gene in prunus , but multiple genes in pyrinae  and solanaceae .
f-box genes belong to a large gene family, and so far, no typical amino acid patterns have been reported for s-locus f-box protein sequences.
therefore, in non-characterized species, it is difficult to identify the pollen s-gene using sequence data alone.
in contrast to the s-rnase gene, pyrinae sfbb genes show low polymorphism and high divergence  <cit> .
pollen s-gene is , however, expected to be mainly expressed in the pollen  <cit> .
although the mechanism of self pollen tubes recognition is different when one or multiple s-pollen genes are involved  <cit> , ssk <dig>  proteins are involved in the self-incompatibility reaction in rosaceae, solanaceae and plantaginaceae species, where gsi systems are well characterized.
skp <dig> like proteins are adapters that connect diverse f-box proteins to the scf complex, and that are necessary in a wide range of cellular processes involving proteosome degradation .
ssk <dig> proteins have been described only in species having rnase based gsi  <cit> , and thus, their presence has been suggested as a marker for rnase based gsi  <cit> .
these proteins are highly conserved and have a unique c-terminus, composed of a 5– <dig> amino acid residues following the conventional “wafe” motif that is found in most plant skp <dig> proteins  <cit> .
therefore, the genes encoding such proteins can be easily retrieved using blast searches.
in solanaceae, plantaginaceae, and pyrinae, ssk <dig> proteins are expressed in pollen only  <cit> , but in prunus they are also expressed in styles  <cit> .
to identify t2-rnases that could be s-locus candidate genes in fabaceae subfamily papilionoideae, in this work, we characterized the s-lineage t2-rnase genes in five genomes of species belonging to three major subclades: trifolium pratense, medicago truncatula, and cicer arietinum from the inverted-repeat-lacking clade , glycine max from the millettioid clade, and lupinus angustifolius from the genistoid clade.
trifolium and medicago are the most closely related genera, and they share the most recent common ancestor, about  <dig> mya  <cit> .
cicer is diverging from these two genera for about  <dig> my.
glycine is diverging from species of the irlc clade for about  <dig> my, and lupinus is diverging from these for about  <dig> my  <cit> .
except for t. pratense, all these species are self-compatible.
nevertheless, the s-locus region could, in principle, be present, although the s-locus genes are expected to be non-functional  <cit> .
compatible with this view, sequences closely related to the ssk <dig> genes are here identified in t. pratense, m. truncatula, c. arietinum, and g. max genomes.
in t. pratense, m. truncatula and c. arietinum we identify s-rnase lineage genes that in phylogenetic analyses cluster with pyrinae s-rnases.
furthermore, in m. truncatula and c. arietinum genomes, where large scaffolds are available, these sequences are surrounded by f-box genes that in phylogenetic analyses cluster with s-pollen genes.
nevertheless, none of these genes show expression only in tissues related with gsi.
moreover, t. pratense genes present levels of diversity lower than those of the characterized s-rnase genes.
we also obtained a style with stigma transcriptome for cytisus striatus, a species where self-pollen grains have been reported to collapse along the style, although partially  <cit> .
once again, we found two genes that encode proteins showing the typical features of ssk <dig> genes and three t2-rnase like sequences, but none of these genes shows expression and variability levels compatible with being the s-rnase gene.
thus, we find no evidence for rnase based gsi in c. striatus.
the data here presented supports the hypothesis that fabaceae gsi is not determined by rosaceae, solanaceae, and plantaginaceae s-rnase lineage genes.
alternative hypotheses are here discussed regarding the presence of ssk <dig> genes and fabaceae gsi system.
RESULTS
ssk <dig> like genes in fabaceae
ssk <dig> genes are restricted to species having rnase based gsi  <cit> .
the presence/absence of this gene has been reported as a diagnosis marker for the presence/absence of rnase based gsi  <cit> .
the protein encoded by ssk <dig> has an unique c-terminus, composed of 5– <dig> amino acid residues, following the conventional “wafe” motif  <cit> .
in rosaceae, this amino acid tail shows the conserved sequence “gvded” .
in solanaceae and plantaginaceae this motif is not so well conserved but a d residue is always found at the last position of the motif.
it should be noted that most of the fabaceae genomes that are available are from self-compatible species, and thus, ssk <dig> genes may be non-functional, or not involved in si pathway.
therefore, when retrieving the sequences we allowed for some variability regarding these motifs .
when using these features and the ncbi flowering plant species database, we retrieved  <dig> sequences from solanaceae , plantaginaceae , rosaceae , fabaceae , malvaceae , rutaceae , euphorbiaceae  and salicaceae  species.
two other sequences, cy54873-cy <dig>  and cy41479g <dig>  were identified in the c. striatus style with stigma transcriptome.
these c. striatus sequences are incomplete at the 5′ region, since using blastx, the first  <dig> amino acids of ssk <dig> proteins are not present in these sequences.
on the other hand, these sequences are complete at the 3′ region since their putative amino acid sequence presents the rosaceae gvded motif after the wafe motif.
the phylogenetic relationship of the  <dig> ssk <dig> sequences, as well as the c-terminus sequence motif of the proteins they encode is presented in figure  <dig> .
fabaceae ssk <dig> like genes are more closely related to rosaceae ssk <dig> sequences than to those from solanaceae and plantaginaceae , according to the known relationship of the plant families.
it should be noted that only the two c. striatus deduced proteins present the rosaceae gvded motif after the wafe motif.
the t. pratense ashm <dig> , and g. max xm_ <dig> genes encode proteins that present the wafexxxxd motif, described for solanaceae and plantaginaceae ssk <dig>  the presence of ssk <dig> genes in fabaceae is, thus, consistent with the claims of rnase based gsi in fabaceae.figure  <dig> bayesian phylogenetic tree showing the relationship of ssk <dig> like genes in flowering plants presenting these genes, available at genbank .
numbers below the branches represent posterior credibility values above  <dig>  the tree was rooted using oryza sativa  and citrus maxima  genes that encode proteins not presenting the c-terminus amino acid motif following the conventional “wafe” motif.
the c-terminus amino acid motif following the conventional “wafe” of the proteins encoded by each ssk <dig> gene is also presented.
amino acids that are different from the “wafe” motif are underlined.
ssk <dig> proteins showing the rosaceae motif are also found in hevea brasiliensis  and populus trigonocarpa .
none of these species, or species of these families, has been described as having gsi.
furthermore, in citrus clementina ssk <dig> like proteins present a proline instead of a glutamic acid in the rosaceae wafegvded motif.
citrus species present gsi and cytological analysis showed that growth of pollen tubes is arrested in different regions depending on the species analysed  <cit> .
in c. clementina pollen tubes are arrested in the upper styles  <cit> .
rnase activity has been identified in stigmas and pistils of c. reticulata  <cit>  and also in ovaries of c. grandis  <cit> , but the genetic mechanism is not clear yet  <cit> .
indeed, in the comparative transcriptome analyses of stylar cells of a self-incompatible and a self-compatible cultivar of c. clementina, no t2-rnases where identified  <cit> , rising doubts if gsi is rnase based in c. clementina.
in t. cacao  a ssk <dig> like protein with the same pattern as in c. clementina has also been identified.
in this species self-pollen tubes grow to the ovary without inhibition, and self-incompatibility occurs at the embryo sac  <cit> , and not in the style.
nevertheless, other malvaceae species such as diploid species of the tarasa genera present gsi , although the genetic mechanism is unknown.table  <dig>
m. truncatula
,
c. arietinum
,
g. max
,
l. angustifolius t2-rnases
larger than 500 bp, that encode putative proteins not presenting in their amino acid sequence amino acid pattern  <dig> according to vieira,
et al.
<cit>
locus
gene code
ip
intron number
motif 1
motif 2
motif 4
location
t. pratense
tp1
tp2
tp3
tp4
tp5
tp6
tp7
tp8
tp9
tp10
tp11
tp12
tp13
tp14
tp15
m. truncatula
mt1
mt2
mt3
mt4
mt5
mt8
mt9
mt10
mt12
mt13
mt14
mt16
mt17
mettr2g <dig>
mt18
mt20
mt23
mt24
mt25
mt26
c. arietinum
ca1
ca2
ca3
ca4
ca5
ca6
ca7
ca8
ca9
ca10
ca11
ca12
ca13
ca14
ca15
ca16
ca17
ca18
ca19
ca20
ca21
g. max
gm1
gm2
gm3
gm4
gm5
gm6
gm7
l. angustifolius
la1
ip- isoelectric point.
underscored are amino acids that are not allowed in the motifs of  <cit> .
+ sequences presenting stop codons in the putative coding region.
{ sequences where gaps were introduced to avoid stop codons in the putative coding region.
> very divergent sequences that, although they present all the criteria of s-lineage s-rnase genes, were not included in phylogenetic analyses.
t. pratense, m. truncatula, c. arietinum, g. max and l. angustifóliot2-rnases-lineage genes
given the evidence for the presence of rnase based gsi in fabaceae , we attempted to identify the s-rnase gene in fabaceae species.
three main criteria were used to first identify putative s-rnase lineage genes in the t. pratense, m. truncatula, c. arietinum, g. max and l. angustifolius genomes, namely: 1) similarity at the amino acid level with s-rnases from malus and/or prunus ; 2) the gene must encode a protein where amino acid pattern  <dig> is absent, once this pattern is found in proteins encoded by non-s-rnase lineage genes only  <cit> ; and 3) the gene must encode a protein with an isoelectric point higher than  <dig> , since s-rnases are always basic proteins  <cit> .
except for t. pratense, the genomes here analyzed are from self-compatible species.
nevertheless, the s-locus region could also be present, although the s-genes could show mutations that disrupt the coding region.
for instance, in rosaceae, mutated versions of the s-rnase and/or sfb genes have been described in self-compatible species  <cit> .
table  <dig> summarizes the features of all gene sequences longer than 500 bp showing similarity at the amino acid level with s-rnases from malus and/or prunus.
although intron number was not used as a criterion for the selection of the genes, all these genes have one or two introns in the same location as those of the s-rnases  <cit> .
three t. pratense , two m. truncatula , five c. arietinum , and one g. max  genes are likely non-functional, since they present stop codons in their putative coding region.
the number of putative s-lineage genes in t. pratense, m. truncatula, and c. arietinum  is about three times larger than in g. max  or l. angustifolius .
although in c. arietinum the large number of t2-rnase lineage genes can be attributed to recent gene duplications, most of the t. pratense, and m. truncatula gene duplications are old .
three lotus corniculatus, two l. japonicus, one pisum sativum, one cajanus cajan, one lens culinaris, and one cyamopsis tetragonolobat2-rnase sequences that code for putative proteins without amino acid pattern  <dig>  and that code for basic proteins were also included in the phylogenetic analyses .figure  <dig> bayesian phylogenetic tree showing the relationship of the fabaceae s-rnase lineage genes and prunus, pyrinae, solanaceae and plantaginaceae s-rnases .
sequences were aligned using the muscle algorithm.
numbers below the branches represent posterior credibility values above  <dig>  + indicate the sequences presenting stop codons in the putative coding region.
{ indicate the sequences where gaps were introduced to avoid stop codons in the putative coding region.
the “ <dig> - 2” indicate the sequences presenting amino acid patterns  <dig> and  <dig> typical of s-rnases.
according to the phylogenetic analyses, the fabaceae sequences that show amino acid patterns  <dig> and  <dig> , that are present in rosaceae, solanaceae, plantaginaceae and rubiaceae s-rnases  <cit> , do not cluster toghether .
furthermore, fabaceae genes - tp <dig>  tp <dig>  ca <dig>  mt <dig>  mt <dig> and mt <dig>  in two of the alignment methods used , cluster with pyrinae s-rnases.
mt <dig> and mt <dig> are neighbour genes .
mt <dig> is 56164 bp apart from mt <dig> .
these genes could also represent the fabaceae s-rnase.
although, the phylogenetic relationship of m. truncatula mt <dig> gene and plantaginaceae s-rnases depends on the alignment method used, we also included this gene in the following analyses.
expression patterns of t. pratense tp <dig>  and tp <dig>  c. arietinum ca <dig> and m. truncatula mt <dig>  mt <dig>  mt <dig>  and mt <dig> genes
s-rnase expression is highest in pistils, although it can show lower expression in stigma and styles .
for t. pratense we address the expression of genes tp <dig>  and tp <dig> using cdna of styles with stigmas, ovaries, and leaves.
t <dig> gene shows expression in styles with stigmas, ovaries, and leaves .
for t <dig> gene, expression is observed in the styles with stigmas, and in leaves .
since t. pratense is a si species, these genes are thus, likely not s-rnases.
accordingly, levels of silent site  diversity for tp <dig> and tp <dig> genes are  <dig>  and  <dig> , respectively .
s-rnases show levels of silent variability higher than  <dig>   <cit> .figure  <dig> expression pattern for the t. pratense tp3
, and tp6
s-rnase lineage genes in pistils, ovaries, and leaves.
the elongation factor 1-α  gene, the positive control for cdna synthesis, is presented for these tissues .
genes similar to the s-rnase but that are not involved in gsi may, in principle, show expression in other tissues.
indeed, s-rnase lineage  <dig> genes in malus  are expressed in embryo and seeds .
this is in contrast to the s-rnase gene expression that is restricted to the stigma, styles and pistils of flowers at anthesis  <cit> .
therefore, genes showing expression in tissues other than the stigma, styles and pistils of flowers at anthesis are unlikely to be s-rnases.
for c. arietinum ca <dig> gene, blast searches against ncbi est database shows that this gene is expressed in etiolated seedlings ).
thus, this gene is likely a gene not involved in gsi.
according to m. truncatula gene expression atlas  mt <dig>  also shows expression in leaf and root tissues, among other tissues analysed.
since mt <dig>  mt <dig> and mt <dig> genes are not represented in the affymetrix genechip, used in m. truncatula gene expression atlas , we addressed their expression using blastn and the sra experiment sets for m. truncatula ).
we find evidence for expression of the three genes in this large rna-seq data set .
therefore, according to gene expression, none of these genes seems to be determining pistil gsi specificity.
f-box genes in the vicinity of the c. arietinum ca <dig> and m. truncatula mt <dig>  mt <dig>  mt <dig>  and mt <dig> genes
at the s-locus region, the s-rnase gene is always surrounded by the s-pollen gene, that can be one gene as in prunus .
it should be noted that in prunus, other f-box genes called slfls, not involved in gsi specificity determination  <cit>  are also found surrounding the s-rnase gene  <cit> .
therefore, as an attempt to identify the s-locus in fabaceae species, we identified all sfbbs/ slfs, slfls, and sfb like genes in the vicinity  of the c. arietinum ca <dig>  and m. truncatula mt <dig>  mt <dig>  mt <dig>  and mt <dig> genes .
for those gene sequences larger than 500 bp, phylogenetic inferences using reference genes  show that c. arietinum ca1_ <dig> and m. truncatula mt2_ <dig>  mt2_ <dig>  and mt7_ <dig> are f-box genes that belong to the malus, solanaceae, and plantaginaceae s-pollen and prunuss- like pollen genes clade .figure  <dig> representation of f-box sfb -sfbb- and slfl- like genes located in the  <dig> kb region surrounding the c. arietinum ca <dig> gene , and m. truncatula mt <dig>  mt <dig>  mt18
, and mt <dig> s-rnase like genes , marked in grey.
sequences assigned with # are very divergent sequences that were not included in phylogenetic analyses.
expression pattern of the c. arietinum ca1_ <dig> and m. truncatula mt2_ <dig>  mt2_ <dig>  and mt7_ <dig> genes
prunus sfb, petunia and antirrhinum slfs, and malussfbb  genes have expression restricted to pollen and anthers  <cit> .
genes showing similarity to slfs but that are not involved in gsi specificity determination  have also been described, but they have a broader pattern of expresion.
for instance, in prunus, slfl genes are expressed in pollen and anthers but also in the style  <cit> .
furthermore, in malus, slfl genes are expressed in pollen, and anthers, but also in pistils, leaves, and seeds .
therefore, we addressed the expression pattern of c. arietinum ca1_ <dig> and m. truncatula mt2_ <dig>  mt2_ <dig>  and mt7_ <dig> genes.
c. arietinum ca1_ <dig> gene is expressed in etiolated seedlings , as the s-rnase like sequence located in its vicinity.
although we do not know if this gene is also expressed in pollen and anthers, because of its expression in seeds it is likely not involved in gsi.
m. truncatulamt7_ <dig>  and mt2_ <dig> genes, according to gene expression atlas , are expressed in leafs, petiole, stems, flowers, and roots, among other tissues analyzed .
for mt2_ <dig> gene an est  also supports expression of this gene in immature seeds  <dig> to 19 days after pollination.
mt2_ <dig> gene is not represented in the affymetrix genechip, and there is no est data for this gene.
therefore, we addressed their expression using blastn and the sra srp <dig> experiment data sets for m. truncatula .
we find evidence for expression of this gene in this large rna-seq data set .
therefore according to gene expression, none of these genes seems to be determining s-pollen gsi specificity.
t2-rnases from the c. striatus style with stigma transcriptome
since we found no evidence in the available fabaceae genomes for s-rnase like genes that could be involved in gsi specificity, we performed a transcriptome analysis of c. striatus styles with stigmas.
this species has been described as having partial gsi  <cit> .
five c. striatus sequences obtained from the style with stigma transcriptome show similarity with s-rnases .
csrnase <dig>  and csrnase <dig> genes encode proteins with amino acid pattern  <dig>  that is absent from all known s-rnases  <cit> .
these genes encode putative acidic proteins , in contrast with s-rnases that are always basic proteins  <cit> .
furthermore, they share at least 85% amino acid similarity with other fabaceae proteins that are expressed in tissues other than pistils .
moreover, these genes have three introns, and known s-rnases have only one or two introns  <cit> .
therefore csrnase <dig>  and csrnase <dig> genes are not s-rnases.table  <dig>
c. striatus t2- rnases present in the style with stigma transcriptome
gene
transcriptome annotation
size
amino acid patterns
1
2
4
csrnase 1
csrnase 2
csrnase 3
csrnase4
csrnase5
underscored are the amino acids that are not allowed in the motifs of  <cit> .
na- the available sequence does not cover this region.
csrnase <dig>  and csrnase <dig> genes code for proteins that do not present amino acid pattern  <dig>  like the s-rnase gene .
because the csrnase <dig> coding sequence is incomplete, it is not possible to ascertain whether the protein encoded by this gene shows the amino acid pattern  <dig>  phylogenetic analyses of csrnase <dig>  and csrnase <dig> genes, together with the sequences of other fabaceae s-lineage genes, rosaceae, solanaceae, and plantaginaceae s-rnases show that none of these genes belong to the known s-rnase gene lineages .
csrnase <dig> gene, however, clusters with pyrinae s-rnases, and thus could represent a putative s-rnase gene .
for csrnase <dig> gene, in the 266 bp region available, there are no introns.
accordingly, in the corresponding region there are no introns at the s-rnase gene.
nevertheless, unlike the s-rnases, csrnase <dig> gene is expressed in ovaries, petals, leaves and fruits .
moreover, levels of silent site diversity for this gene are moderate , but lower than that of the s-rnase gene .
thus, csrnase <dig> gene does not present the expected features of a s-rnase gene.figure  <dig> bayesian phylogenetic trees showing the relationship of:
c. striatus csrnase1and csrnase <dig> genes and fabaceae s-rnase lineage genes, and prunus, pyrinae, solanaceae and plantaginaceae s-rnases.
sequences were aligned using the muscle algorithm; and
csrnase <dig> gene and prunus, pyrinae, solanaceae and plantaginaceae s-rnases.
the reference sequences are shaded.
numbers below the branches represent posterior credibility values above  <dig>
, csrnase1
, and csrnase2
in pollen, ovaries, leaves, fruits, petals and pistils.
the elongation factor 1-α  gene, the positive control for cdna synthesis, is presented for these tissues .
since we could not find any s-rnase candidate belonging to the rosaceae, solanaceae and plantaginaceae s-rnase lineage genes, we characterized the csrnase <dig> and csrnase <dig> genes, that do not belong to any of the known s-rnases lineages.
csrnase <dig> gene is one of the most expressed genes  at position  <dig> in additional file 7), but their genomic sequence revealed three introns .
moreover, csrnase <dig> gene is expressed in ovaries, petals, pistils, leaves and fruits , in contrast with the s-rnases that are expressed mainly in pistils  <cit> .
furthermore, levels of silent site  variability for this gene are low  which is in sharp contrast with the expectation of high levels of variability at the s-rnase gene  <cit> .
therefore, the overall evidence is that the csrnase <dig> gene is not a s-pistil gene.
for csrnase <dig> gene the genomic sequence revealed five introns , it shows expression in ovaries, petals, pistils, leaves and fruits , and low levels of silent site  variability .
therefore, csrnase <dig> gene is also not a s-pistil gene.
discussion
phylogenetic analysis of t2-rnase genes from five fabaceae genomes and one pistil transcriptome revealed more than six s-lineage genes.
the two t. pratense genes that are phylogenetically related with pyrinae s-rnases show, however, expression and polymorphism levels incompatible with being involved in gsi.
although the breeding system of the t. pratense individuals used in the polymorphism analyses was not characterized, in the literature all individuals analysed are si  <cit> .
furthermore, red clover is described as being difficult to self, because of low seed set after selfing  <cit> .
furthermore, the sequences obtained for the portuguese population for the two t. pratense genes phylogenetically related with pyrinae s-rnases, are very similar to those of the individual used for the t. pratense genome.
furthermore, none of the fabaceae t2-rnase genes phylogenetically related with known s-rnases, revealed expression patterns compatible with a candidate fabaceae s-pistil gene.
it could be argued that only t. pratense is a self-incompatible species  <cit> , and that the s-locus region may not be present in the other available genomes.
nevertheless, the presence of the same gene lineages in the t. pratense, m. truncatula and/or c. arietinum suggests that this is not the case.
in rosaceae, sc species still present the s-locus region, but s-rnase and sfb genes are non-functional  <cit> .
nevertheless, mutations at loci involved in gsi but that are unlinked to the s-locus are also observed  <cit> .
a similar pattern is also described in other si systems such as that present in brassicaceae family.
for instance, the s-locus is present in the genome of the sc arabidopsis thaliana, but the genes determining s-specificity are non-functional  <cit> .
it should be noted, however, that the si loss in m. truncatula is at least twice as old as that of a. thaliana.
therefore, genomes of sc species can also help in the identification of the putative s-locus genes.
the presence of fabaceae sequences that cluster with pyrinae s-rnases and s-pollen genes supports the hypothesis that we have identified the orthologous pyrinae s-locus region in fabaceae.
these genes in fabaceae seem to be performing functions other than gsi.
nevertheless, to exclude these genes as being the ones determining gsi, segregation analyses from controlled crosses are needed to show that these genes do not segregate as s-locus genes.
the fact that in fabaceae, the rosaceae, solanaceae, and plantaginaceae s-rnase gene lineages seem not to be involved in gsi, raises the hypothesis that in trifolium gsi could be not rnase based.
this hypothesis has been suggested before, based on the observation that on m. truncatula chromosome  <dig>  that is largely syntenic to linkage group hg <dig> of t. pratense, where the s-locus has been mapped, there are no t2-rnases exhibiting significant similarity to solanaceae, rosaceae and plantaginaceae s-rnases.
the same observation has been reported for the numerous t2-rnase like sequences in the m. truncatula genome, even for those located near f-box genes, like the s-rnases  <cit> .
nevertheless, under the current hypothesis, rnase based gsi evolved only once  <cit> .
it is, however, conceivable that the ancestral s-locus has been duplicated during evolution.
the presence of fabaceae sequences presenting motifs  <dig> and  <dig> along the phylogeny support this hypothesis.
in c. striatus, however, none of the t2-rnase genes expressed in pistils is determining gsi.
thus, there is no evidence to suggest that other t2-rnase lineage genes could be determining fabaceae gsi.
if this is the case, fabaceae gsi has evolved the novo from t2-rnase unrelated genes, and thus, the information on solanaceae, rosaceae, plantaginaceae and rubiaceae s-rnases is not useful for the identification of the fabaceae s-locus.
it is expected that the s-pistil gene is highly expressed in the tissue where gsi occurs, and transcriptome analyses of this tissue can produce a list of genes showing high expression levels, such as that we present for c. striatus .
nevertheless, expression analyses, levels of diversity, and segregation analyses in controlled crosses will be needed to identify which gene is involved in s-pistil specificity.
it should be noted that in several fabaceae species, we find ssk <dig> like genes with the typical features of those found in s-rnase based si species from other plant families.
it is conceivable that ssk <dig> like genes will be present in species where t2-rnase genes belonging to the s-lineage are present, even though such genes may not be involved in rnase based gsi.
this must be the case for c. striatus.
moreover, the presence of a ssk <dig> like gene in c. clementina where no t2-rnases were identified from the transcriptome analyses of stylar cells of a self-incompatible and a self-compatible cultivar  <cit>  offers support to this hypothesis.
the possibility that the frequency of self-incompatible species is overestimated in fabaceae should not be also, ruled out.
indeed, the presence of binucleate pollen , as well as fruit and seed production, are frequently used to assess the breeding system of a species.
nevertheless, other processes known to occur in fabaceae species can affect fruit and seed production.
for instance, papilionoideae species have a membrane at the stigmatic surface that needs to be disrupted for pollen grain germination.
in species of this subfamily the flower’s own pollen can cover the stigma at the bud stage  <cit> , but it does not germinate while the stigmatic surface is intact  <cit> .
with flowering maturation this stigmatic membrane in si species must be scratched by a pollinator that visits the flower  <cit> .
moreover, late-acting self-incompatibility  has been described in many fabaceae species such as medicago sativa  <cit> , vicia faba  <cit> , pisum sativum  <cit> , and colutea arborescens  <cit>  from the irlc clade; lotus corniculatus  <cit>  and coronilla emerus  <cit> , both from the robinoid clade; phaseolus vulgaris  <cit>  from the millettioid clade; dalbergia miscolobium  <cit>  and d.retusa  <cit>  from the dalbergioid clade; as well as in genista hirsuta, adenocarpus complicatus, retama sphaerocarpa,cytisus striatus, c. grandiflorus  <cit> , and c. multiflorus  <cit>  from the genistoid clade.
in fabaceae, lsi is due to multiple causes such as disharmony in endosperm/embryo development  <cit> , differential growth rate of the pollen tubes within the ovaries  <cit> , embryonic abortion  <cit>  and inbreeding depression  <cit> .
although the genetics and physiology of lsi is still poorly understood, it is clear that it can be genetically determined  <cit> , and that lsi and gsi can co-occur, as it happens in c. striatus  <cit> .
indeed, lsi implies similar growth of pollen tubes in the style following self- and cross-pollination , and in this species there is a significant difference in the percentage of pollen growth in self and cross-pollinations.
therefore, besides lsi, an additional partial gsi system has been inferred in c. striatus  <cit> .
similar inferences have been made for v. faba  <cit> , l. corniculatus  <cit> , c. emerus and colutea arborescens  <cit> .
CONCLUSIONS
there is no evidence for rosaceae, solanaceae, and plantaginaceae s-rnase lineage genes determining gsi in fabaceae species.
lsi is frequent in this family and may co-occur with gsi.
nevertheless, so far, in fabaceae, only trifolium species have been described as presenting gsi only.
thus, lsi or lsi in combination with gsi, will be likely the major hurdle when attempting to efficiently incorporating traits of agronomical interest from wild populations into crop varieties.

 useful agronomic traits can be found in wild populations of crop species. nevertheless, a large fraction of species with hermaphroditic flowers have developed genetic mechanisms that allow the pistil to recognize and reject pollen from genetically related individuals, and this may affect the efficient incorporation of such traits into crop varieties. self-incompatibility is, in general, evolutionarily advantageous, because it promotes cross-fertilization, and thus inbreeding depression avoidance. fabaceae is an economically important plant family with a large number of self-incompatible species, that have been reported often as showing self-incompatibility of the gametophytic type. in gsi, if the specificity of the haploid pollen grain matches either one of the diploid pistil, an incompatible reaction occurs, leading to the degradation of the pollen tube within the pistil. it should be noted, however, that in all fabaceae species where pollen tube growth was assessed in controlled crosses, only in species of the genus trifolium the gsi reaction seems to be complete and takes place in the stlyle as observed in rosaceae, solanaceae and plantaginaceae si species. in other species such as vicia faba, lotus corniculatus, cytisus striatus, coronilla emerus and colutea arborescens there is, however a significant difference on the percentage of pollen growth in self and cross-pollinations. in c. striatus, one of the species here studied, the percentage of ovules that are penetrated by pollen tubes is 72% in hand self-pollinated flowers compared with the % when hand cross-pollinations are performed. these authors have shown that an important fraction of self pollen grains collapse along the style, as observed in rosaceae, solanaceae and plantaginaceae si species. although the molecular characterization of the fabaceae s-locus has never been performed, some authors have suggested that in fabaceae gsi is rnase based. nevertheless, there are other gsi systems, such as that present in papaveraceae. moreover, late-acting si, so called because rejection of self-pollen takes place either in the ovary prior to fertilization, or in the first divisions of the zygote, has been described in fabaceae. it should be noted that, lsi can also be of the gametophytic type. in fabaceae, however, the genetic basis of the different mechanisms that control lsi are mostly unknown, and thus, in this work we only address the possibility that fabaceae gsi is determined by a s-rnase gene that clusters with those of the well characterized rosaceae, solanaceae and plantaginaceae species. the most common ancestor of fabaceae and rosaceae species lived about 89– million years ago. since, according to phylogenetic analyses of the t2-rnases, rnase based gsi has evolved only once, before the split of the asteridae and rosidae, about mya, at least some fabaceae si species are expected to have this system. therefore, in principle, a homology based approach could be used to identify the putative pistil s-gene in fabaceae species. three amino acid patterns, allow the distinction of s-rnase lineage genes from other t -rnase genes. these patterns can be used to easily identify putative s-lineage genes using blast searches. the results can be further refined by selecting only those genes that encode basic proteins since s-rnases have an isoelectric point between and. furthermore, the number of introns can also be used to select s-lineage genes since s-rnases have one or two introns only. phylogenetic analyses where a set of reference genes are used, can then be performed to show that such genes belong, indeed, to the s-lineage. nevertheless, in order to show that the identified genes are the pistil s-gene, it is necessary to show that they are highly expressed in pistils, although they can show lower expression in stigma and styles. in malus fusca where a large number of transcriptomes have been analysed the same pattern is observed. moreover, it is necessary to show that they have high polymorphism levels, that there is evidence for positive selection, and that in controlled crosses they co-segregate with s-locus alleles. the pollen component, always an f-box protein, has been identified as one gene in prunus, but multiple genes in pyrinae and solanaceae. f-box genes belong to a large gene family, and so far, no typical amino acid patterns have been reported for s-locus f-box protein sequences. therefore, in non-characterized species, it is difficult to identify the pollen s-gene using sequence data alone. in contrast to the s-rnase gene, pyrinae sfbb genes show low polymorphism and high divergence. pollen s-gene is, however, expected to be mainly expressed in the pollen. although the mechanism of self pollen tubes recognition is different when one or multiple s-pollen genes are involved, ssk proteins are involved in the self-incompatibility reaction in rosaceae, solanaceae and plantaginaceae species, where gsi systems are well characterized. skp like proteins are adapters that connect diverse f-box proteins to the scf complex, and that are necessary in a wide range of cellular processes involving proteosome degradation. ssk proteins have been described only in species having rnase based gsi, and thus, their presence has been suggested as a marker for rnase based gsi. these proteins are highly conserved and have a unique c-terminus, composed of a 5– amino acid residues following the conventional “wafe” motif that is found in most plant skp proteins. therefore, the genes encoding such proteins can be easily retrieved using blast searches. in solanaceae, plantaginaceae, and pyrinae, ssk proteins are expressed in pollen only, but in prunus they are also expressed in styles. to identify t2-rnases that could be s-locus candidate genes in fabaceae subfamily papilionoideae, in this work, we characterized the s-lineage t2-rnase genes in five genomes of species belonging to three major subclades: trifolium pratense, medicago truncatula, and cicer arietinum from the inverted-repeat-lacking clade, glycine max from the millettioid clade, and lupinus angustifolius from the genistoid clade. trifolium and medicago are the most closely related genera, and they share the most recent common ancestor, about mya. cicer is diverging from these two genera for about my. glycine is diverging from species of the irlc clade for about my, and lupinus is diverging from these for about my. except for t. pratense, all these species are self-compatible. nevertheless, the s-locus region could, in principle, be present, although the s-locus genes are expected to be non-functional. compatible with this view, sequences closely related to the ssk genes are here identified in t. pratense, m. truncatula, c. arietinum, and g. max genomes. in t. pratense, m. truncatula and c. arietinum we identify s-rnase lineage genes that in phylogenetic analyses cluster with pyrinae s-rnases. furthermore, in m. truncatula and c. arietinum genomes, where large scaffolds are available, these sequences are surrounded by f-box genes that in phylogenetic analyses cluster with s-pollen genes. nevertheless, none of these genes show expression only in tissues related with gsi. moreover, t. pratense genes present levels of diversity lower than those of the characterized s-rnase genes. we also obtained a style with stigma transcriptome for cytisus striatus, a species where self-pollen grains have been reported to collapse along the style, although partially. once again, we found two genes that encode proteins showing the typical features of ssk genes and three t2-rnase like sequences, but none of these genes shows expression and variability levels compatible with being the s-rnase gene. thus, we find no evidence for rnase based gsi in c. striatus. the data here presented supports the hypothesis that fabaceae gsi is not determined by rosaceae, solanaceae, and plantaginaceae s-rnase lineage genes. alternative hypotheses are here discussed regarding the presence of ssk genes and fabaceae gsi system.  ssk like genes in fabaceae ssk genes are restricted to species having rnase based gsi. the presence/absence of this gene has been reported as a diagnosis marker for the presence/absence of rnase based gsi. the protein encoded by ssk has an unique c-terminus, composed of 5– amino acid residues, following the conventional “wafe” motif. in rosaceae, this amino acid tail shows the conserved sequence “gvded”. in solanaceae and plantaginaceae this motif is not so well conserved but a d residue is always found at the last position of the motif. it should be noted that most of the fabaceae genomes that are available are from self-compatible species, and thus, ssk genes may be non-functional, or not involved in si pathway. therefore, when retrieving the sequences we allowed for some variability regarding these motifs. when using these features and the ncbi flowering plant species database, we retrieved sequences from solanaceae, plantaginaceae, rosaceae, fabaceae, malvaceae, rutaceae, euphorbiaceae and salicaceae species. two other sequences, cy54873-cy and cy41479g were identified in the c. striatus style with stigma transcriptome. these c. striatus sequences are incomplete at the 5′ region, since using blastx, the first amino acids of ssk proteins are not present in these sequences. on the other hand, these sequences are complete at the 3′ region since their putative amino acid sequence presents the rosaceae gvded motif after the wafe motif. the phylogenetic relationship of the ssk sequences, as well as the c-terminus sequence motif of the proteins they encode is presented in figure . fabaceae ssk like genes are more closely related to rosaceae ssk sequences than to those from solanaceae and plantaginaceae, according to the known relationship of the plant families. it should be noted that only the two c. striatus deduced proteins present the rosaceae gvded motif after the wafe motif. the t. pratense ashm, and g. max xm_ genes encode proteins that present the wafexxxxd motif, described for solanaceae and plantaginaceae ssk the presence of ssk genes in fabaceae is, thus, consistent with the claims of rnase based gsi in fabaceae.figure bayesian phylogenetic tree showing the relationship of ssk like genes in flowering plants presenting these genes, available at genbank. numbers below the branches represent posterior credibility values above the tree was rooted using oryza sativa and citrus maxima genes that encode proteins not presenting the c-terminus amino acid motif following the conventional “wafe” motif. the c-terminus amino acid motif following the conventional “wafe” of the proteins encoded by each ssk gene is also presented. amino acids that are different from the “wafe” motif are underlined. ssk proteins showing the rosaceae motif are also found in hevea brasiliensis and populus trigonocarpa. none of these species, or species of these families, has been described as having gsi. furthermore, in citrus clementina ssk like proteins present a proline instead of a glutamic acid in the rosaceae wafegvded motif. citrus species present gsi and cytological analysis showed that growth of pollen tubes is arrested in different regions depending on the species analysed. in c. clementina pollen tubes are arrested in the upper styles. rnase activity has been identified in stigmas and pistils of c. reticulata and also in ovaries of c. grandis, but the genetic mechanism is not clear yet. indeed, in the comparative transcriptome analyses of stylar cells of a self-incompatible and a self-compatible cultivar of c. clementina, no t2-rnases where identified, rising doubts if gsi is rnase based in c. clementina. in t. cacao a ssk like protein with the same pattern as in c. clementina has also been identified. in this species self-pollen tubes grow to the ovary without inhibition, and self-incompatibility occurs at the embryo sac, and not in the style. nevertheless, other malvaceae species such as diploid species of the tarasa genera present gsi, although the genetic mechanism is unknown.table m. truncatula, c. arietinum, g. max, l. angustifolius t2-rnases larger than 500 bp, that encode putative proteins not presenting in their amino acid sequence amino acid pattern according to vieira, et al. locus gene code ip intron number motif 1 motif 2 motif 4 location t. pratense tp1 tp2 tp3 tp4 tp5 tp6 tp7 tp8 tp9 tp10 tp11 tp12 tp13 tp14 tp15 m. truncatula mt1 mt2 mt3 mt4 mt5 mt8 mt9 mt10 mt12 mt13 mt14 mt16 mt17 mettr2g mt18 mt20 mt23 mt24 mt25 mt26 c. arietinum ca1 ca2 ca3 ca4 ca5 ca6 ca7 ca8 ca9 ca10 ca11 ca12 ca13 ca14 ca15 ca16 ca17 ca18 ca19 ca20 ca21 g. max gm1 gm2 gm3 gm4 gm5 gm6 gm7 l. angustifolius la1 ip- isoelectric point. underscored are amino acids that are not allowed in the motifs of. + sequences presenting stop codons in the putative coding region. { sequences where gaps were introduced to avoid stop codons in the putative coding region. > very divergent sequences that, although they present all the criteria of s-lineage s-rnase genes, were not included in phylogenetic analyses. t. pratense, m. truncatula, c. arietinum, g. max and l. angustifóliot2-rnases-lineage genes given the evidence for the presence of rnase based gsi in fabaceae, we attempted to identify the s-rnase gene in fabaceae species. three main criteria were used to first identify putative s-rnase lineage genes in the t. pratense, m. truncatula, c. arietinum, g. max and l. angustifolius genomes, namely: 1) similarity at the amino acid level with s-rnases from malus and/or prunus; 2) the gene must encode a protein where amino acid pattern is absent, once this pattern is found in proteins encoded by non-s-rnase lineage genes only; and 3) the gene must encode a protein with an isoelectric point higher than, since s-rnases are always basic proteins. except for t. pratense, the genomes here analyzed are from self-compatible species. nevertheless, the s-locus region could also be present, although the s-genes could show mutations that disrupt the coding region. for instance, in rosaceae, mutated versions of the s-rnase and/or sfb genes have been described in self-compatible species. table  summarizes the features of all gene sequences longer than 500 bp showing similarity at the amino acid level with s-rnases from malus and/or prunus. although intron number was not used as a criterion for the selection of the genes, all these genes have one or two introns in the same location as those of the s-rnases. three t. pratense, two m. truncatula, five c. arietinum, and one g. max genes are likely non-functional, since they present stop codons in their putative coding region. the number of putative s-lineage genes in t. pratense, m. truncatula, and c. arietinum is about three times larger than in g. max or l. angustifolius. although in c. arietinum the large number of t2-rnase lineage genes can be attributed to recent gene duplications, most of the t. pratense, and m. truncatula gene duplications are old. three lotus corniculatus, two l. japonicus, one pisum sativum, one cajanus cajan, one lens culinaris, and one cyamopsis tetragonolobat2-rnase sequences that code for putative proteins without amino acid pattern and that code for basic proteins were also included in the phylogenetic analyses.figure bayesian phylogenetic tree showing the relationship of the fabaceae s-rnase lineage genes and prunus, pyrinae, solanaceae and plantaginaceae s-rnases. sequences were aligned using the muscle algorithm. numbers below the branches represent posterior credibility values above + indicate the sequences presenting stop codons in the putative coding region. { indicate the sequences where gaps were introduced to avoid stop codons in the putative coding region. the “ - 2” indicate the sequences presenting amino acid patterns and typical of s-rnases. according to the phylogenetic analyses, the fabaceae sequences that show amino acid patterns and, that are present in rosaceae, solanaceae, plantaginaceae and rubiaceae s-rnases, do not cluster toghether. furthermore, fabaceae genes - tp tp ca mt mt and mt in two of the alignment methods used, cluster with pyrinae s-rnases. mt and mt are neighbour genes. mt is 56164 bp apart from mt. these genes could also represent the fabaceae s-rnase. although, the phylogenetic relationship of m. truncatula mt gene and plantaginaceae s-rnases depends on the alignment method used, we also included this gene in the following analyses. expression patterns of t. pratense tp and tp c. arietinum ca and m. truncatula mt mt mt and mt genes s-rnase expression is highest in pistils, although it can show lower expression in stigma and styles. for t. pratense we address the expression of genes tp and tp using cdna of styles with stigmas, ovaries, and leaves. t gene shows expression in styles with stigmas, ovaries, and leaves. for t gene, expression is observed in the styles with stigmas, and in leaves. since t. pratense is a si species, these genes are thus, likely not s-rnases. accordingly, levels of silent site diversity for tp and tp genes are and, respectively. s-rnases show levels of silent variability higher than.figure expression pattern for the t. pratense tp3, and tp6 s-rnase lineage genes in pistils, ovaries, and leaves. the elongation factor 1-α gene, the positive control for cdna synthesis, is presented for these tissues. genes similar to the s-rnase but that are not involved in gsi may, in principle, show expression in other tissues. indeed, s-rnase lineage genes in malus are expressed in embryo and seeds. this is in contrast to the s-rnase gene expression that is restricted to the stigma, styles and pistils of flowers at anthesis. therefore, genes showing expression in tissues other than the stigma, styles and pistils of flowers at anthesis are unlikely to be s-rnases. for c. arietinum ca gene, blast searches against ncbi est database shows that this gene is expressed in etiolated seedlings ). thus, this gene is likely a gene not involved in gsi. according to m. truncatula gene expression atlas mt also shows expression in leaf and root tissues, among other tissues analysed. since mt mt and mt genes are not represented in the affymetrix genechip, used in m. truncatula gene expression atlas, we addressed their expression using blastn and the sra experiment sets for m. truncatula ). we find evidence for expression of the three genes in this large rna-seq data set. therefore, according to gene expression, none of these genes seems to be determining pistil gsi specificity. f-box genes in the vicinity of the c. arietinum ca and m. truncatula mt mt mt and mt genes at the s-locus region, the s-rnase gene is always surrounded by the s-pollen gene, that can be one gene as in prunus. it should be noted that in prunus, other f-box genes called slfls, not involved in gsi specificity determination are also found surrounding the s-rnase gene. therefore, as an attempt to identify the s-locus in fabaceae species, we identified all sfbbs/ slfs, slfls, and sfb like genes in the vicinity of the c. arietinum ca and m. truncatula mt mt mt and mt genes. for those gene sequences larger than 500 bp, phylogenetic inferences using reference genes show that c. arietinum ca1_ and m. truncatula mt2_ mt2_ and mt7_ are f-box genes that belong to the malus, solanaceae, and plantaginaceae s-pollen and prunuss- like pollen genes clade.figure representation of f-box sfb -sfbb- and slfl- like genes located in the kb region surrounding the c. arietinum ca gene, and m. truncatula mt mt mt18, and mt s-rnase like genes, marked in grey. sequences assigned with # are very divergent sequences that were not included in phylogenetic analyses. expression pattern of the c. arietinum ca1_ and m. truncatula mt2_ mt2_ and mt7_ genes prunus sfb, petunia and antirrhinum slfs, and malussfbb genes have expression restricted to pollen and anthers. genes showing similarity to slfs but that are not involved in gsi specificity determination have also been described, but they have a broader pattern of expresion. for instance, in prunus, slfl genes are expressed in pollen and anthers but also in the style. furthermore, in malus, slfl genes are expressed in pollen, and anthers, but also in pistils, leaves, and seeds. therefore, we addressed the expression pattern of c. arietinum ca1_ and m. truncatula mt2_ mt2_ and mt7_ genes. c. arietinum ca1_ gene is expressed in etiolated seedlings, as the s-rnase like sequence located in its vicinity. although we do not know if this gene is also expressed in pollen and anthers, because of its expression in seeds it is likely not involved in gsi. m. truncatulamt7_ and mt2_ genes, according to gene expression atlas, are expressed in leafs, petiole, stems, flowers, and roots, among other tissues analyzed. for mt2_ gene an est also supports expression of this gene in immature seeds to 19 days after pollination. mt2_ gene is not represented in the affymetrix genechip, and there is no est data for this gene. therefore, we addressed their expression using blastn and the sra srp experiment data sets for m. truncatula. we find evidence for expression of this gene in this large rna-seq data set. therefore according to gene expression, none of these genes seems to be determining s-pollen gsi specificity. t2-rnases from the c. striatus style with stigma transcriptome since we found no evidence in the available fabaceae genomes for s-rnase like genes that could be involved in gsi specificity, we performed a transcriptome analysis of c. striatus styles with stigmas. this species has been described as having partial gsi. five c. striatus sequences obtained from the style with stigma transcriptome show similarity with s-rnases. csrnase and csrnase genes encode proteins with amino acid pattern that is absent from all known s-rnases. these genes encode putative acidic proteins, in contrast with s-rnases that are always basic proteins. furthermore, they share at least 85% amino acid similarity with other fabaceae proteins that are expressed in tissues other than pistils. moreover, these genes have three introns, and known s-rnases have only one or two introns. therefore csrnase and csrnase genes are not s-rnases.table c. striatus t2- rnases present in the style with stigma transcriptome gene transcriptome annotation size amino acid patterns 1 2 4 csrnase 1 csrnase 2 csrnase 3 csrnase4 csrnase5 underscored are the amino acids that are not allowed in the motifs of. na- the available sequence does not cover this region. csrnase and csrnase genes code for proteins that do not present amino acid pattern like the s-rnase gene. because the csrnase coding sequence is incomplete, it is not possible to ascertain whether the protein encoded by this gene shows the amino acid pattern phylogenetic analyses of csrnase and csrnase genes, together with the sequences of other fabaceae s-lineage genes, rosaceae, solanaceae, and plantaginaceae s-rnases show that none of these genes belong to the known s-rnase gene lineages. csrnase gene, however, clusters with pyrinae s-rnases, and thus could represent a putative s-rnase gene. for csrnase gene, in the 266 bp region available, there are no introns. accordingly, in the corresponding region there are no introns at the s-rnase gene. nevertheless, unlike the s-rnases, csrnase gene is expressed in ovaries, petals, leaves and fruits. moreover, levels of silent site diversity for this gene are moderate, but lower than that of the s-rnase gene. thus, csrnase gene does not present the expected features of a s-rnase gene.figure bayesian phylogenetic trees showing the relationship of: c. striatus csrnase1and csrnase genes and fabaceae s-rnase lineage genes, and prunus, pyrinae, solanaceae and plantaginaceae s-rnases. sequences were aligned using the muscle algorithm; and csrnase gene and prunus, pyrinae, solanaceae and plantaginaceae s-rnases. the reference sequences are shaded. numbers below the branches represent posterior credibility values above, csrnase1, and csrnase2 in pollen, ovaries, leaves, fruits, petals and pistils. the elongation factor 1-α gene, the positive control for cdna synthesis, is presented for these tissues. since we could not find any s-rnase candidate belonging to the rosaceae, solanaceae and plantaginaceae s-rnase lineage genes, we characterized the csrnase and csrnase genes, that do not belong to any of the known s-rnases lineages. csrnase gene is one of the most expressed genes at position in additional file 7), but their genomic sequence revealed three introns. moreover, csrnase gene is expressed in ovaries, petals, pistils, leaves and fruits, in contrast with the s-rnases that are expressed mainly in pistils. furthermore, levels of silent site variability for this gene are low which is in sharp contrast with the expectation of high levels of variability at the s-rnase gene. therefore, the overall evidence is that the csrnase gene is not a s-pistil gene. for csrnase gene the genomic sequence revealed five introns, it shows expression in ovaries, petals, pistils, leaves and fruits, and low levels of silent site variability. therefore, csrnase gene is also not a s-pistil gene. discussion phylogenetic analysis of t2-rnase genes from five fabaceae genomes and one pistil transcriptome revealed more than six s-lineage genes. the two t. pratense genes that are phylogenetically related with pyrinae s-rnases show, however, expression and polymorphism levels incompatible with being involved in gsi. although the breeding system of the t. pratense individuals used in the polymorphism analyses was not characterized, in the literature all individuals analysed are si. furthermore, red clover is described as being difficult to self, because of low seed set after selfing. furthermore, the sequences obtained for the portuguese population for the two t. pratense genes phylogenetically related with pyrinae s-rnases, are very similar to those of the individual used for the t. pratense genome. furthermore, none of the fabaceae t2-rnase genes phylogenetically related with known s-rnases, revealed expression patterns compatible with a candidate fabaceae s-pistil gene. it could be argued that only t. pratense is a self-incompatible species, and that the s-locus region may not be present in the other available genomes. nevertheless, the presence of the same gene lineages in the t. pratense, m. truncatula and/or c. arietinum suggests that this is not the case. in rosaceae, sc species still present the s-locus region, but s-rnase and sfb genes are non-functional. nevertheless, mutations at loci involved in gsi but that are unlinked to the s-locus are also observed. a similar pattern is also described in other si systems such as that present in brassicaceae family. for instance, the s-locus is present in the genome of the sc arabidopsis thaliana, but the genes determining s-specificity are non-functional. it should be noted, however, that the si loss in m. truncatula is at least twice as old as that of a. thaliana. therefore, genomes of sc species can also help in the identification of the putative s-locus genes. the presence of fabaceae sequences that cluster with pyrinae s-rnases and s-pollen genes supports the hypothesis that we have identified the orthologous pyrinae s-locus region in fabaceae. these genes in fabaceae seem to be performing functions other than gsi. nevertheless, to exclude these genes as being the ones determining gsi, segregation analyses from controlled crosses are needed to show that these genes do not segregate as s-locus genes. the fact that in fabaceae, the rosaceae, solanaceae, and plantaginaceae s-rnase gene lineages seem not to be involved in gsi, raises the hypothesis that in trifolium gsi could be not rnase based. this hypothesis has been suggested before, based on the observation that on m. truncatula chromosome that is largely syntenic to linkage group hg of t. pratense, where the s-locus has been mapped, there are no t2-rnases exhibiting significant similarity to solanaceae, rosaceae and plantaginaceae s-rnases. the same observation has been reported for the numerous t2-rnase like sequences in the m. truncatula genome, even for those located near f-box genes, like the s-rnases. nevertheless, under the current hypothesis, rnase based gsi evolved only once. it is, however, conceivable that the ancestral s-locus has been duplicated during evolution. the presence of fabaceae sequences presenting motifs and along the phylogeny support this hypothesis. in c. striatus, however, none of the t2-rnase genes expressed in pistils is determining gsi. thus, there is no evidence to suggest that other t2-rnase lineage genes could be determining fabaceae gsi. if this is the case, fabaceae gsi has evolved the novo from t2-rnase unrelated genes, and thus, the information on solanaceae, rosaceae, plantaginaceae and rubiaceae s-rnases is not useful for the identification of the fabaceae s-locus. it is expected that the s-pistil gene is highly expressed in the tissue where gsi occurs, and transcriptome analyses of this tissue can produce a list of genes showing high expression levels, such as that we present for c. striatus. nevertheless, expression analyses, levels of diversity, and segregation analyses in controlled crosses will be needed to identify which gene is involved in s-pistil specificity. it should be noted that in several fabaceae species, we find ssk like genes with the typical features of those found in s-rnase based si species from other plant families. it is conceivable that ssk like genes will be present in species where t2-rnase genes belonging to the s-lineage are present, even though such genes may not be involved in rnase based gsi. this must be the case for c. striatus. moreover, the presence of a ssk like gene in c. clementina where no t2-rnases were identified from the transcriptome analyses of stylar cells of a self-incompatible and a self-compatible cultivar offers support to this hypothesis. the possibility that the frequency of self-incompatible species is overestimated in fabaceae should not be also, ruled out. indeed, the presence of binucleate pollen, as well as fruit and seed production, are frequently used to assess the breeding system of a species. nevertheless, other processes known to occur in fabaceae species can affect fruit and seed production. for instance, papilionoideae species have a membrane at the stigmatic surface that needs to be disrupted for pollen grain germination. in species of this subfamily the flower’s own pollen can cover the stigma at the bud stage, but it does not germinate while the stigmatic surface is intact. with flowering maturation this stigmatic membrane in si species must be scratched by a pollinator that visits the flower. moreover, late-acting self-incompatibility has been described in many fabaceae species such as medicago sativa, vicia faba, pisum sativum, and colutea arborescens from the irlc clade; lotus corniculatus and coronilla emerus, both from the robinoid clade; phaseolus vulgaris from the millettioid clade; dalbergia miscolobium and d.retusa from the dalbergioid clade; as well as in genista hirsuta, adenocarpus complicatus, retama sphaerocarpa,cytisus striatus, c. grandiflorus, and c. multiflorus from the genistoid clade. in fabaceae, lsi is due to multiple causes such as disharmony in endosperm/embryo development, differential growth rate of the pollen tubes within the ovaries, embryonic abortion and inbreeding depression. although the genetics and physiology of lsi is still poorly understood, it is clear that it can be genetically determined, and that lsi and gsi can co-occur, as it happens in c. striatus. indeed, lsi implies similar growth of pollen tubes in the style following self- and cross-pollination, and in this species there is a significant difference in the percentage of pollen growth in self and cross-pollinations. therefore, besides lsi, an additional partial gsi system has been inferred in c. striatus. similar inferences have been made for v. faba, l. corniculatus, c. emerus and colutea arborescens.  there is no evidence for rosaceae, solanaceae, and plantaginaceae s-rnase lineage genes determining gsi in fabaceae species. lsi is frequent in this family and may co-occur with gsi. nevertheless, so far, in fabaceae, only trifolium species have been described as presenting gsi only. thus, lsi or lsi in combination with gsi, will be likely the major hurdle when attempting to efficiently incorporating traits of agronomical interest from wild populations into crop varieties. 
in t. pratense, m. truncatula, and c. arietinum we identify s-rnase lineage genes that in phylogenetic analyses cluster with pyrinae s-rnases. to address if other t2-rnases could be determining fabaceae gsi, here we obtained a style with stigma transcriptome of cytisus striatus, a species that shows significant difference on the percentage of pollen growth in self and cross-pollinations. in m. truncatula and c. arietinum genomes, where large scaffolds are available, these sequences are surrounded by f-box genes that in phylogenetic analyses also cluster with s-pollen genes. therefore, to characterize the fabaceae s-pistil gene, expression analyses, levels of diversity, and segregation analyses in controlled crosses are needed for those genes showing high expression levels in the tissues where gsi occurs. the m. truncatula and c. arietinum s-rnase and s-pollen like genes phylogenetically related to pyrinae s-genes, are also expressed in tissues other than those involved in gsi. moreover, levels of diversity are lower than those observed for other s-rnase genes. we first identify ssk like genes, in the trifolium pratense, medicago truncatula, cicer arietinum, glycine max, and lupinus angustifolius genomes. gametophytic self-incompatibilitymolecular evolutions-rnase like genestrifolium pratensemedicago truncatulacicer arietinumcytisus striatusissue-copyright-statement© the author 2015 we find no evidence for fabaceae gsi being determined by rosaceae, solanaceae, and plantaginaceae s-rnase lineage genes. expression and polymorphism analyses of the c. striatus s-rnase like genes revealed that none of these genes, is the s-pistil gene. then, we characterize the s-lineage t2-rnase genes in these genomes. fabaceae species are important in agronomy and livestock nourishment. there is no evidence that t2-rnase lineage genes could be determining gsi in c. striatus. in t. pratense the s-rnase lineage genes show, however, expression in tissues not involved in gsi. we address the hypothesis of fabaceae gametophytic si being rnase based, by recruiting the same s-rnase lineage gene of rosaceae, solanaceae or plantaginaceae si species.
 fabaceae species are important in agronomy and livestock nourishment. they have a long breeding history, and most cultivars have lost self-incompatibility, a genetic barrier to self-fertilization. nevertheless, to improve legume crop breeding, crosses with wild si relatives of the cultivated varieties are often performed. therefore, it is fundamental to characterize fabaceae si system. we address the hypothesis of fabaceae gametophytic si being rnase based, by recruiting the same s-rnase lineage gene of rosaceae, solanaceae or plantaginaceae si species.  we first identify ssk like genes, in the trifolium pratense, medicago truncatula, cicer arietinum, glycine max, and lupinus angustifolius genomes. then, we characterize the s-lineage t2-rnase genes in these genomes. in t. pratense, m. truncatula, and c. arietinum we identify s-rnase lineage genes that in phylogenetic analyses cluster with pyrinae s-rnases. in m. truncatula and c. arietinum genomes, where large scaffolds are available, these sequences are surrounded by f-box genes that in phylogenetic analyses also cluster with s-pollen genes. in t. pratense the s-rnase lineage genes show, however, expression in tissues not involved in gsi. moreover, levels of diversity are lower than those observed for other s-rnase genes. the m. truncatula and c. arietinum s-rnase and s-pollen like genes phylogenetically related to pyrinae s-genes, are also expressed in tissues other than those involved in gsi. to address if other t2-rnases could be determining fabaceae gsi, here we obtained a style with stigma transcriptome of cytisus striatus, a species that shows significant difference on the percentage of pollen growth in self and cross-pollinations. expression and polymorphism analyses of the c. striatus s-rnase like genes revealed that none of these genes, is the s-pistil gene.  we find no evidence for fabaceae gsi being determined by rosaceae, solanaceae, and plantaginaceae s-rnase lineage genes. there is no evidence that t2-rnase lineage genes could be determining gsi in c. striatus. therefore, to characterize the fabaceae s-pistil gene, expression analyses, levels of diversity, and segregation analyses in controlled crosses are needed for those genes showing high expression levels in the tissues where gsi occurs. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords gametophytic self-incompatibilitymolecular evolutions-rnase like genestrifolium pratensemedicago truncatulacicer arietinumcytisus striatusissue-copyright-statement© the author 2015 