BACKGROUND
in vitro regeneration of cultivated tomato  has been a constant subject of research because of the commercial value of the crop.
consequently, numerous studies on plant regeneration from a wide range of tissues and organs of wild and cultivated tomato germplasm have been published  <cit> .
these studies demonstrate that organogenesis, the common tomato regeneration pathway, is strongly influenced by genotype as well as by several physical and chemical factors.
these reports also document the existence of recalcitrance , which greatly limits biotechnological breeding.
high regeneration is crucial to the success of techniques such as haploid regeneration, genetic transformation, propagation, somatic hybridization, mutation selection and germplasm storage  <cit> .
for example, the low efficiency of tomato transformation has been associated with the low regeneration potential of the cultivars used  <cit> .
in addition, in some cultivars, buds may be induced but do not develop into shoots  <cit> .
in order to increase regeneration ability in low regenerating tomato cultivars, several introgression programs have been documented  <cit> .
the process of in vitro shoot organogenesis usually involves a hormonal response of somatic cells, the dedifferentiation of differentiated cells in order to acquire organogenic competence, cell division of the responding cell and initiation and development of new shoots from the newly dividing cell, either directly or indirectly through a callus stage  <cit> .
thus, many genes may be involved at different steps of this complex process.
for instance, the cdc <dig> gene expression, which encodes p <dig>  a key cell cycle regulator, has been proposed as an indicator of the state of competence to divide  <cit> .
genes that encode or regulate cytokinins and auxin may clearly influence regeneration.
both types of growth regulators act synergistically to promote cell division and antagonistically to promote shoot and root initiation from callus cultures  <cit> .
in arabidopsis, a histidine kinase  gene that encodes a cytokinin receptor  has been identified  <cit>  and linked, like other ahks, to cell division and regulation  <cit> .
with regard to the initiation of shoot formation, the most characterized gene reported is esr <dig>  which confers, when overexpressed, cytokinin-independent shoot formation in arabidopsis root explants  <cit> .
esr <dig> encodes a transcription factor belonging to the ethylene-responsive factor  family and is classified in subgroup viii-b.
the esr <dig> gene that encodes a protein that is very similar to esr <dig> appears to have redundant functions that regulate shoot regeneration  <cit> .
the expression patterns of other arabidopsis erf viii-b subgroup genes may also be involved in early events of shoot regeneration  <cit> .
genetic analysis of regeneration in tomato suggests that dominant alleles determine high regeneration capacity  <cit> .
however, there is no consensus about the number of genes involved.
for instance, koorneef et al.
<cit>  obtained regeneration segregation ratios in accordance with either a monogenic, digenic or trigenic model depending on the tester tomato line, despite the fact that none of the lines themselves were able to regenerate shoots from root explants.
in this study, a dominant allele of s. peruvianum l. , which determines efficient shoot regeneration in tomato root explants, was mapped near the middle of chromosome  <dig>  in addition, a putative allele of rg- <dig> from s. chilense  reiche  was reported by takashina et al.
<cit>  and satoh et al.
<cit> .
both alleles may act in combination with other alleles of either tomato or the wild relatives s. peruvianum or s. chilense  <cit> .
on the other hand, torelli et al.
<cit>  identified a cdna by mrna-differential display that corresponded to the lesk <dig> gene and whose expression is specifically and transiently enhanced by the exposure to the hormonal treatment leading to caulogenesis .
this gene encodes a putative serine-threonine kinase and has been reported as an in vitro caulogenesis marker in tomato  <cit> .
despite ongoing research into the genetic control of in vitro culture traits in tomato and other crops, there is still not enough information regarding which key genes are responsible for low or high regeneration ability, nor even the number of genes involved.
the study and characterization of the reported genes and others that might be identified could greatly improve our understanding of the molecular mechanism underlying the different phases of tomato in vitro organogenesis.
in the present study, we developed two mapping populations  from s. lycopersicum  and s. pennellii correll  and conducted a qtl-based analysis.
we hereby report the identification of six qtls on five chromosomes.
these qtls present high significant lod scores and together represent a high percentage of phenotypic variance.
we also report markers associated with qtl peaks.
in addition, we located two candidate genes, rg- <dig> and lesk <dig>  and performed a preliminary search for genes situated at qtl peaks.
our findings will complement the current knowledge of the genetics of regeneration and facilitate the development of molecular markers for use in tomato breeding and gene isolation.
RESULTS
development of populations and evaluation of the regeneration ability
two mapping populations, f <dig> and bc <dig>  were obtained from a low regenerating cultivar of tomato  and the organogenic accession of s. pennellii .
the bc <dig> population was obtained using the tomato cultivar as the recurrent parent.
in the first assay, the regeneration ability of the parents and the f <dig> plant used for obtaining the mapping populations was checked by culturing leaf explants on shoot induction medium.
regeneration occurred with little callus development and can be considered as direct.
as expected, s. pennellii and f <dig> explants manifested a higher regeneration potential versus s. lycopersicum explants .
the percentage of explants with buds  in s. pennellii was 100%, whereas only 10% was obtained in tomato cv.
anl <dig> .
data obtained in f <dig> for b do not significantly differ from those obtained for s. pennellii.
the percentage of explants with shoots  and the number of regenerated plants per explant with shoots, considered to be the productivity rate , was also higher in s. pennellii and f <dig> than in cv.
anl <dig>  however, for these traits , the f <dig> values differ significantly from those of s. pennellii .
means of the traits: percentage of explants with buds , percentage of explants with shoots  and number of shoots per explant with shoots  for the parent genotypes , f <dig>  f <dig> and bc <dig>
a b and r are the percentages of explants able to develop buds and shoots, respectively.
b pr is the number of shoots per explant with shoots.
c mean values within a column separated by different letters are significantly different  according to duncan's multiple range test.
the f <dig> and bc <dig> populations were evaluated for regeneration using explants from the parents and f <dig> plants as controls .
the phenotypes are shown in additional file  <dig>  the distribution obtained for each individual trait as well as the means for controls in this assay are presented in figure  <dig>  mean values for b, r and pr in the f <dig> population are between f <dig> and tomato , but skewed towards f <dig>  for the pr trait, some f <dig> plants were in a range higher than the s. pennellii parent .
this can be considered transgressive segregation.
bc <dig> yielded mean values for b, r and pr that were intermediate between f <dig> and cv.
anl <dig> .
b and r show a high correlation , which suggests common or linked genes controlling these traits.
the correlation between pr and both b and r was lower  indicating that different genes may influence the pr trait and/or variations between different biological samples are higher in pr.
linkage maps
genetic linkage maps were constructed from  <dig> f <dig> and  <dig> bc <dig> plants genotyped with ssr, cosi, cosii, caps and aflp markers .
of the  <dig> ssr and  <dig> other markers  assayed,  <dig> ssr and  <dig>  markers exhibited codominant polymorphisms.
these markers were obtained from the sol genomics network  webpage at http://www.sgn.cornell.edu/with the exception of  <dig> ssrs that were designed following the procedure described in materials and methods .
for the f <dig> linkage map , a total of  <dig> polymorphic loci were used, including  <dig> aflp,  <dig> ssr,  <dig> cosii,  <dig> cosi and  <dig> caps markers.
the markers were aligned in  <dig> linkage groups, with lod scores ≥  <dig> .
the average number of markers per linkage group was  <dig> and markers were well distributed over all the  <dig> linkage groups.
the f <dig> map spans  <dig>  cm with an average interval of  <dig>  cm between adjacent markers.
there were five intervals >  <dig> cm in chromosomes  <dig>   <dig>   <dig> and  <dig>  a total of  <dig> polymorphic loci were used to assemble the genetic linkage map of bc <dig> , including  <dig> aflp,  <dig> ssr,  <dig> cosii,  <dig> cosi and  <dig> caps markers.
the markers were distributed over  <dig> linkage groups with lod scores ≥  <dig> .
the average number of markers per linkage group was  <dig>  the total genetic distance covered by the markers was  <dig>  cm, with an average interval of  <dig>  cm between adjacent markers.
the markers were well distributed over all the  <dig> linkage groups with only two intervals ≥  <dig> cm in chromosomes  <dig> and  <dig>  marker distribution in both maps indicates that they will be useful for tagging the traits studied.
the order and placement of ssr markers were in agreement with the s. lycopersicum x s. pennellii reference tomato-expen  <dig> map  with the exception of tahina-6- <dig> , which was expected to be positioned on chromosome  <dig>  but is positioned on chromosome  <dig>  in our f <dig> map.
distorted segregation
<dig> % of the mapped markers deviated significantly from the expected 1:2: <dig> segregation ratio for the f <dig> generation at p <  <dig>  .
segregation distorted markers  were mainly observed on chromosomes  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig> .
sdms were generally caused by a surplus of s. pennellii homozygotes, with the exception of that observed on chromosome  <dig>
in the bc <dig> population , sdms were fewer  than in f <dig>  and were observed mainly on chromosomes  <dig> ,  <dig> ,  <dig>  and  <dig> .
the distortion on chromosome  <dig> was caused by a surplus of tomato homozygotes, whereas distortions on the other chromosomes were caused by an excess of hybrid genotypes.
qtl identification
in order to identify qtls, we first used interval mapping  analysis that resulted in the identification of one qtl in the f <dig> population and six in the bc <dig> population .
the qtl identified in f <dig>  located on chromosome  <dig>  overlapped for the three traits.
in the bc <dig> analysis, this qtl also appeared for the r and pr traits.
however, in this population, another five qtls were identified on chromosomes  <dig>   <dig>   <dig>  and  <dig>  all these qtls were confirmed by restricted multiple qtl mapping  analysis .
with the exception of the qtl on chromosome  <dig>  all qtls come from s. pennellii.
these qtls were named by their origin, sp for s. pennellii or sl for s. lycopersicum, followed by rg  and the number of the chromosome on which they were located.
ain case of the absence of a peak marker, loci flanking the likely peak of a qtl are shown.
qtls for regeneration traits in the bc <dig> population
bud percentage
im analysis identified two qtls on chromosomes  <dig> and  <dig> .
sprg- <dig> has a maximum lod score of  <dig>  and is spanned by markers ssr <dig> and me17- <dig>  this qtl explained  <dig> % of the phenotypic variation of the b trait.
sprg- <dig>  with a maximum lod score of  <dig> , including just the c2_at1g <dig> marker, explained  <dig> % of the phenotypic variation in b. rmqm analysis, using c2_at2g <dig>  and c2_at1g <dig>  markers as cofactors, confirmed those qtls detected by im and detected a new one on chromosome  <dig> .
qtl characteristics are shown in table  <dig>  collectively, these qtls explained  <dig> % and  <dig> % of phenotypic variance in im and rmqm, respectively.
regeneration percentage
im analysis identified three qtls located on chromosomes  <dig>   <dig> and  <dig> denominated sprg- <dig>  sprg-4a and sprg- <dig>  respectively.
the three qtls had maximum lod scores of  <dig> ,  <dig>  and  <dig> , and each explained around 16-17% of the phenotypic variation .
rmqm analysis, using c2_at2g <dig> , tahina-4- <dig>   and tahina-7- <dig>  markers as cofactors, confirmed all qtls detected by im and detected the slrg- <dig> qtl .
in this case, the percentage of the phenotypic variation explained by each qtl was 15% for sprg- <dig>   <dig> % for sprg-4a,  <dig> % for sprg- <dig> and  <dig> % for slrg- <dig>  collectively, these qtls explained  <dig> % and  <dig> % of the phenotypic variance in im and rmqm, respectively.
productivity rate
im detected the qtls located previously for b and r on chromosomes  <dig>   <dig> and  <dig> , as well as another qtl on chromosome  <dig>  denominated sprg-4b.
the maximum phenotypic variation for pr  is explained by sprg- <dig>  and the lowest  by a qtl on chromosome  <dig>  rmqm analysis, using ssr <dig> , me20- <dig> , ssr <dig>  and tahina-7- <dig>  markers as cofactors, confirmed the qtls detected by im .
mapping tomato candidate genes
we selected the acid invertase gene linked to the rg- <dig> regeneration gene of s. chilense  <cit>  and the lesk <dig> gene, described as a marker in tomato for in vitro regeneration competence  <cit> , as the tomato candidate genes.
the amplification products of the acid invertase gene marker  produce fragments of different sizes:  <dig> bp for s. lycopersicum cv.
anl <dig> and  <dig> bp for s. pennellii .
thus, invpenn was used for mapping the bc <dig> population .
it was located in the qtl detected on chromosome  <dig>  between the c2_at5g <dig> and ssrb <dig> markers, at positions  <dig>  cm and  <dig>  cm, respectively.
for this reason, we named this qtl rg- <dig> .
the lesk <dig> gene is located in the sgn tomato-expen  <dig> map on chromosome  <dig> between markers c2_at4g <dig> and clpt-5-e <dig> .
as a result, in our bc <dig> map, lesk <dig> must be placed between c2_at4g <dig> and tahina-3- <dig> .
thus, this candidate gene is outside the located rg- <dig> qtl.
exploring qtls
the official annotation for the tomato genome provided by the international tomato annotation group at the sgn was used to carry out a preliminary search for related regeneration genes near the identified qtl peaks.
we found a histidine kinase in sprg- <dig>  several serine/threonine kinases in all identified qtls, ethylene response factors  in all identified qtls with the exception of sprg-4b, cyclines in sprg- <dig>  rg- <dig>  sprg-4a and sprg- <dig> and mads-box in sprg- <dig>  sprg-4a and sprg- <dig>
discussion
the wild tomato species s. peruvianum, s. pimpinellifolium l. and s. chilense were used as sources of regeneration genes in order to study the genetics of the in vitro regeneration in tomato  <cit> .
in this study, we used one accession of s. pennellii  as the high regeneration parent  <cit> .
this accession, along with a previously selected low regenerating tomato cultivar , was used to develop two mapping populations .
the use of the introgression lines of s. pennellii in the m <dig> tomato background  <cit>  had been previously ruled out for this analysis because of the high regeneration ability of both parent lines .
data in figure  <dig> and table  <dig> seem to indicate complete dominance for b, partial dominance for r and additive effects for pr.
this is in agreement with other reported studies on tomato where dominance, to different degrees, depending on the regeneration trait studied, was also reported  <cit> .
b and r traits show a high correlation in both populations, suggesting that common or linked genes control these traits.
the correlation between pr and both b and r was lower.
this could imply that other genes may be influencing the pr trait and/or variations between different biological samples are higher in pr .
thus, the low sample size may be also a possible explanation for the lower correlation.
some descendants in the f <dig> population showed phenotypes for the pr trait that are more extreme that those shown by the regenerating parent line .
transgressive segregation has already been described in other reports in relation to the genetic control of plant regeneration  <cit> , and suggests poligenic inheritance  <cit> .
it also suggests the existence of alleles that promote, and others that inhibit, in vitro regeneration, with only some of the alleles with positive effects occurring in the same parent  <cit> .
in fact, in this study, the slrg- <dig> qtl that contributes to regeneration came from the low regenerating parent.
plant regeneration from cultured tissues is assumed to fall under quantitative genetics  <cit> , although evidence in tomato  <cit>  and other vegetables  <cit>  indicates that just a few genes could be responsible for regeneration.
we identified  <dig> qtls in the bc <dig> analysis, which is indicative of the participation of a large number of genes in this character.
these qtls are situated on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig> .
the percentage of variance explained by each qtl ranges from  <dig>  to 27%, which is in accordance with the most common range  reported in the genetic mapping of qtls for tissue culture response in plants  <cit> .
we used three traits  as a measurement of regeneration capability that could be useful for detecting chromosome regions that act at different times.
in the f <dig> population, only the qtl of chromosome  <dig> was identified for all analyzed traits ; the sdms observed in most chromosome areas where qtls were detected in the bc <dig> population are most likely the cause .
the sdms on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig> were also observed in similar areas in the tomato-expen  <dig> map  <cit> .
sdms affect the detection power of qtls when qtls and sdms are closely linked  <cit> , as occurred in our case.
deviation from the expected segregation ratio is a common feature of inter-specific tomato crosses  <cit> .
to wit: in a f <dig> population from s. lycopersicum x s. pennellii, de vicente and tanskley  <cit>  reported a skewness rate of up to 80%.
in the bc <dig> population, three qtls were detected for b: sprg- <dig>  sprg- <dig> and slrg- <dig>  these qtls may be associated with the first stages of regeneration, that is, hormonal induction response and bud formation.
sprg- <dig>  which explained the highest percentage of variation for b , was also identified for the r and pr traits.
given that bud formation is a necessary prerequisite for the production of shoots, it was expected that this major qtl for b would be found for r and pr, which in fact turned out to be the case .
for r and pr, a common qtl on chromosome  <dig>  was also identified.
in addition, two qtls were detected for r  and pr .
all these qtls seem to be involved in the development of buds into shoots.
as can also be observed in this study, common qtls for the different regeneration traits, as well as a higher number of qtls for traits related to plant development compared to those associated with bud induction, have been reported in different studies  <cit> .
for instance, in arabidopsis, schianterelli et al.
<cit>  found a common area of chromosome  <dig> in all analyzed parameters, a peak in chromosome  <dig> and another in chromosome  <dig> when they analyzed the total number of regenerated shoots.
in wheat, ben amer et al.
<cit>  identified three qtls, two that affect green spot initiation and shoot regeneration and a third that only influences plant formation.
a partial common genetic system controlling the regeneration frequency of diverse types of explants has been reported by molina and nuez  <cit>  in melon.
this indicates that using different explants for loci detection may lead to the identification of some common qtls, but also to the possible identification of other new qtls.
root explants were used by koornneef et al.
<cit>  and satoh et al.
<cit>  for phenotyping, at which point two alleles for regeneration ability were located on chromosome  <dig> of tomato.
in the present study, leaves were used for phenotyping and a qtl  in a similar area of chromosome  <dig> was detected in addition to other qtls that influence regeneration and were identified on chromosomes  <dig>   <dig>   <dig> and  <dig>  differences in root and leaf explants for qtl identification were also found in arabidopsis thaliana  <cit> .
koornneef et al.
<cit>  located a dominant allele from s. peruvianum  near the middle of chromosome  <dig> that determines efficient shoot regeneration in tomato root explants.
satoh et al.
<cit>  mapped a putative allele  from s. chilense on this chromosome.
the acid invertase gene, reported as a marker linked to rg- <dig>  was chosen for mapping rg- <dig> in our population derived from s. pennellii.
the polymorphisms detected in our parents allow us to map this gene in the qtl detected on chromosome  <dig> that we named rg- <dig>  we consider rg- <dig> to be a putative allele of the rg- <dig> gene.
allelism must be confirmed.
the other gene chosen as a candidate was lesk <dig>  which encodes a serine/threonine kinase, and was reported as a marker of competence for in vitro regeneration in tomato  <cit> .
this gene was positioned on chromosome  <dig>  but it is not located in the rg- <dig> qtl.
the recent release of the entire genome sequence of tomato provides a powerful tool for interrogating qtl data.
in this respect, we have taken a preliminary look at genes located at the peak areas of the detected qtls, and which could be related to organogenesis.
histidine kinases were reported as cytokinin receptors  <cit> .
in our qtl peaks, only one histidine kinase is located in the sprg- <dig> qtl.
the candidate tomato gene, lesk <dig>  which has been described as a marker for in vitro competence, encodes a serine/threonine kinase.
we looked for serine/threonine kinases and found this kind of protein in all identified qtls.
other putative candidate genes could be esr <dig> and its paralogue, esr <dig>  from arabidopsis, which are the best-characterized genes related to regeneration  <cit> .
these genes code for ethylene response factors .
we found erfs, which contain the ap <dig> domain, in all analysed qtls with the exception of sprg-4b.
cyclines related to cell division  <cit>  were found in sprg- <dig>  rg- <dig>  sprg-4a and sprg- <dig>  mads-box genes, which have been correlated to adventitious regeneration induction and regulation  <cit> , were found in the sprg- <dig>  sprg-4a and sprg- <dig> qtl peaks.
CONCLUSIONS
the results obtained in this study may very well represent a significant step toward the goal of understanding the processes underlying tomato tissue culture and regeneration responses.
we have situated six qtls on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig>  five from s. pennellii and one from s. lycopersicum.
the most important qtls are sprg- <dig>  which is most likely associated with the morphogenetic response, and sprg- <dig>  which promotes bud development.
a qtl detected on chromosome  <dig>  rg- <dig>  likely contains a putative allele of the rg- <dig> and rg- <dig> genes, as is shown by mapping the acid invertase gene linked to rg- <dig>  qtls detected on chromosomes  <dig> and  <dig> most likely contain genes influencing bud formation and development, respectively.

 in vitro regeneration of cultivated tomato has been a constant subject of research because of the commercial value of the crop. consequently, numerous studies on plant regeneration from a wide range of tissues and organs of wild and cultivated tomato germplasm have been published. these studies demonstrate that organogenesis, the common tomato regeneration pathway, is strongly influenced by genotype as well as by several physical and chemical factors. these reports also document the existence of recalcitrance, which greatly limits biotechnological breeding. high regeneration is crucial to the success of techniques such as haploid regeneration, genetic transformation, propagation, somatic hybridization, mutation selection and germplasm storage. for example, the low efficiency of tomato transformation has been associated with the low regeneration potential of the cultivars used. in addition, in some cultivars, buds may be induced but do not develop into shoots. in order to increase regeneration ability in low regenerating tomato cultivars, several introgression programs have been documented. the process of in vitro shoot organogenesis usually involves a hormonal response of somatic cells, the dedifferentiation of differentiated cells in order to acquire organogenic competence, cell division of the responding cell and initiation and development of new shoots from the newly dividing cell, either directly or indirectly through a callus stage. thus, many genes may be involved at different steps of this complex process. for instance, the cdc gene expression, which encodes p a key cell cycle regulator, has been proposed as an indicator of the state of competence to divide. genes that encode or regulate cytokinins and auxin may clearly influence regeneration. both types of growth regulators act synergistically to promote cell division and antagonistically to promote shoot and root initiation from callus cultures. in arabidopsis, a histidine kinase gene that encodes a cytokinin receptor has been identified and linked, like other ahks, to cell division and regulation. with regard to the initiation of shoot formation, the most characterized gene reported is esr which confers, when overexpressed, cytokinin-independent shoot formation in arabidopsis root explants. esr encodes a transcription factor belonging to the ethylene-responsive factor family and is classified in subgroup viii-b. the esr gene that encodes a protein that is very similar to esr appears to have redundant functions that regulate shoot regeneration. the expression patterns of other arabidopsis erf viii-b subgroup genes may also be involved in early events of shoot regeneration. genetic analysis of regeneration in tomato suggests that dominant alleles determine high regeneration capacity. however, there is no consensus about the number of genes involved. for instance, koorneef et al. obtained regeneration segregation ratios in accordance with either a monogenic, digenic or trigenic model depending on the tester tomato line, despite the fact that none of the lines themselves were able to regenerate shoots from root explants. in this study, a dominant allele of s. peruvianum l., which determines efficient shoot regeneration in tomato root explants, was mapped near the middle of chromosome in addition, a putative allele of rg- from s. chilense reiche was reported by takashina et al. and satoh et al.. both alleles may act in combination with other alleles of either tomato or the wild relatives s. peruvianum or s. chilense. on the other hand, torelli et al. identified a cdna by mrna-differential display that corresponded to the lesk gene and whose expression is specifically and transiently enhanced by the exposure to the hormonal treatment leading to caulogenesis. this gene encodes a putative serine-threonine kinase and has been reported as an in vitro caulogenesis marker in tomato. despite ongoing research into the genetic control of in vitro culture traits in tomato and other crops, there is still not enough information regarding which key genes are responsible for low or high regeneration ability, nor even the number of genes involved. the study and characterization of the reported genes and others that might be identified could greatly improve our understanding of the molecular mechanism underlying the different phases of tomato in vitro organogenesis. in the present study, we developed two mapping populations from s. lycopersicum and s. pennellii correll and conducted a qtl-based analysis. we hereby report the identification of six qtls on five chromosomes. these qtls present high significant lod scores and together represent a high percentage of phenotypic variance. we also report markers associated with qtl peaks. in addition, we located two candidate genes, rg- and lesk and performed a preliminary search for genes situated at qtl peaks. our findings will complement the current knowledge of the genetics of regeneration and facilitate the development of molecular markers for use in tomato breeding and gene isolation.  development of populations and evaluation of the regeneration ability two mapping populations, f and bc were obtained from a low regenerating cultivar of tomato and the organogenic accession of s. pennellii. the bc population was obtained using the tomato cultivar as the recurrent parent. in the first assay, the regeneration ability of the parents and the f plant used for obtaining the mapping populations was checked by culturing leaf explants on shoot induction medium. regeneration occurred with little callus development and can be considered as direct. as expected, s. pennellii and f explants manifested a higher regeneration potential versus s. lycopersicum explants. the percentage of explants with buds in s. pennellii was 100%, whereas only 10% was obtained in tomato cv. anl. data obtained in f for b do not significantly differ from those obtained for s. pennellii. the percentage of explants with shoots and the number of regenerated plants per explant with shoots, considered to be the productivity rate, was also higher in s. pennellii and f than in cv. anl however, for these traits, the f values differ significantly from those of s. pennellii. means of the traits: percentage of explants with buds, percentage of explants with shoots and number of shoots per explant with shoots for the parent genotypes, f f and bc a b and r are the percentages of explants able to develop buds and shoots, respectively. b pr is the number of shoots per explant with shoots. c mean values within a column separated by different letters are significantly different according to duncan's multiple range test. the f and bc populations were evaluated for regeneration using explants from the parents and f plants as controls. the phenotypes are shown in additional file the distribution obtained for each individual trait as well as the means for controls in this assay are presented in figure mean values for b, r and pr in the f population are between f and tomato, but skewed towards f for the pr trait, some f plants were in a range higher than the s. pennellii parent. this can be considered transgressive segregation. bc yielded mean values for b, r and pr that were intermediate between f and cv. anl. b and r show a high correlation, which suggests common or linked genes controlling these traits. the correlation between pr and both b and r was lower indicating that different genes may influence the pr trait and/or variations between different biological samples are higher in pr. linkage maps genetic linkage maps were constructed from f and bc plants genotyped with ssr, cosi, cosii, caps and aflp markers. of the ssr and other markers assayed, ssr and markers exhibited codominant polymorphisms. these markers were obtained from the sol genomics network webpage at http://www.sgn.cornell.edu/with the exception of ssrs that were designed following the procedure described in materials and methods. for the f linkage map, a total of polymorphic loci were used, including aflp, ssr, cosii, cosi and caps markers. the markers were aligned in linkage groups, with lod scores ≥. the average number of markers per linkage group was and markers were well distributed over all the linkage groups. the f map spans cm with an average interval of cm between adjacent markers. there were five intervals > cm in chromosomes and a total of polymorphic loci were used to assemble the genetic linkage map of bc, including aflp, ssr, cosii, cosi and caps markers. the markers were distributed over linkage groups with lod scores ≥. the average number of markers per linkage group was the total genetic distance covered by the markers was cm, with an average interval of cm between adjacent markers. the markers were well distributed over all the linkage groups with only two intervals ≥ cm in chromosomes and marker distribution in both maps indicates that they will be useful for tagging the traits studied. the order and placement of ssr markers were in agreement with the s. lycopersicum x s. pennellii reference tomato-expen map with the exception of tahina-6-, which was expected to be positioned on chromosome but is positioned on chromosome in our f map. distorted segregation % of the mapped markers deviated significantly from the expected 1:2: segregation ratio for the f generation at p <. segregation distorted markers were mainly observed on chromosomes,,,, and. sdms were generally caused by a surplus of s. pennellii homozygotes, with the exception of that observed on chromosome in the bc population, sdms were fewer than in f and were observed mainly on chromosomes,, and. the distortion on chromosome was caused by a surplus of tomato homozygotes, whereas distortions on the other chromosomes were caused by an excess of hybrid genotypes. qtl identification in order to identify qtls, we first used interval mapping analysis that resulted in the identification of one qtl in the f population and six in the bc population. the qtl identified in f located on chromosome overlapped for the three traits. in the bc analysis, this qtl also appeared for the r and pr traits. however, in this population, another five qtls were identified on chromosomes and all these qtls were confirmed by restricted multiple qtl mapping analysis. with the exception of the qtl on chromosome all qtls come from s. pennellii. these qtls were named by their origin, sp for s. pennellii or sl for s. lycopersicum, followed by rg and the number of the chromosome on which they were located. ain case of the absence of a peak marker, loci flanking the likely peak of a qtl are shown. qtls for regeneration traits in the bc population bud percentage im analysis identified two qtls on chromosomes and. sprg- has a maximum lod score of and is spanned by markers ssr and me17- this qtl explained % of the phenotypic variation of the b trait. sprg- with a maximum lod score of, including just the c2_at1g marker, explained % of the phenotypic variation in b. rmqm analysis, using c2_at2g and c2_at1g markers as cofactors, confirmed those qtls detected by im and detected a new one on chromosome. qtl characteristics are shown in table collectively, these qtls explained % and % of phenotypic variance in im and rmqm, respectively. regeneration percentage im analysis identified three qtls located on chromosomes and denominated sprg- sprg-4a and sprg- respectively. the three qtls had maximum lod scores of, and, and each explained around 16-17% of the phenotypic variation. rmqm analysis, using c2_at2g, tahina-4- and tahina-7- markers as cofactors, confirmed all qtls detected by im and detected the slrg- qtl. in this case, the percentage of the phenotypic variation explained by each qtl was 15% for sprg- % for sprg-4a, % for sprg- and % for slrg- collectively, these qtls explained % and % of the phenotypic variance in im and rmqm, respectively. productivity rate im detected the qtls located previously for b and r on chromosomes and, as well as another qtl on chromosome denominated sprg-4b. the maximum phenotypic variation for pr is explained by sprg- and the lowest by a qtl on chromosome rmqm analysis, using ssr, me20-, ssr and tahina-7- markers as cofactors, confirmed the qtls detected by im. mapping tomato candidate genes we selected the acid invertase gene linked to the rg- regeneration gene of s. chilense and the lesk gene, described as a marker in tomato for in vitro regeneration competence, as the tomato candidate genes. the amplification products of the acid invertase gene marker produce fragments of different sizes: bp for s. lycopersicum cv. anl and bp for s. pennellii. thus, invpenn was used for mapping the bc population. it was located in the qtl detected on chromosome between the c2_at5g and ssrb markers, at positions cm and cm, respectively. for this reason, we named this qtl rg-. the lesk gene is located in the sgn tomato-expen map on chromosome between markers c2_at4g and clpt-5-e. as a result, in our bc map, lesk must be placed between c2_at4g and tahina-3-. thus, this candidate gene is outside the located rg- qtl. exploring qtls the official annotation for the tomato genome provided by the international tomato annotation group at the sgn was used to carry out a preliminary search for related regeneration genes near the identified qtl peaks. we found a histidine kinase in sprg- several serine/threonine kinases in all identified qtls, ethylene response factors in all identified qtls with the exception of sprg-4b, cyclines in sprg- rg- sprg-4a and sprg- and mads-box in sprg- sprg-4a and sprg- discussion the wild tomato species s. peruvianum, s. pimpinellifolium l. and s. chilense were used as sources of regeneration genes in order to study the genetics of the in vitro regeneration in tomato. in this study, we used one accession of s. pennellii as the high regeneration parent. this accession, along with a previously selected low regenerating tomato cultivar, was used to develop two mapping populations. the use of the introgression lines of s. pennellii in the m tomato background had been previously ruled out for this analysis because of the high regeneration ability of both parent lines. data in figure and table seem to indicate complete dominance for b, partial dominance for r and additive effects for pr. this is in agreement with other reported studies on tomato where dominance, to different degrees, depending on the regeneration trait studied, was also reported. b and r traits show a high correlation in both populations, suggesting that common or linked genes control these traits. the correlation between pr and both b and r was lower. this could imply that other genes may be influencing the pr trait and/or variations between different biological samples are higher in pr. thus, the low sample size may be also a possible explanation for the lower correlation. some descendants in the f population showed phenotypes for the pr trait that are more extreme that those shown by the regenerating parent line. transgressive segregation has already been described in other reports in relation to the genetic control of plant regeneration, and suggests poligenic inheritance. it also suggests the existence of alleles that promote, and others that inhibit, in vitro regeneration, with only some of the alleles with positive effects occurring in the same parent. in fact, in this study, the slrg- qtl that contributes to regeneration came from the low regenerating parent. plant regeneration from cultured tissues is assumed to fall under quantitative genetics, although evidence in tomato and other vegetables indicates that just a few genes could be responsible for regeneration. we identified qtls in the bc analysis, which is indicative of the participation of a large number of genes in this character. these qtls are situated on chromosomes and. the percentage of variance explained by each qtl ranges from to 27%, which is in accordance with the most common range reported in the genetic mapping of qtls for tissue culture response in plants. we used three traits as a measurement of regeneration capability that could be useful for detecting chromosome regions that act at different times. in the f population, only the qtl of chromosome was identified for all analyzed traits; the sdms observed in most chromosome areas where qtls were detected in the bc population are most likely the cause. the sdms on chromosomes and were also observed in similar areas in the tomato-expen map. sdms affect the detection power of qtls when qtls and sdms are closely linked, as occurred in our case. deviation from the expected segregation ratio is a common feature of inter-specific tomato crosses. to wit: in a f population from s. lycopersicum x s. pennellii, de vicente and tanskley reported a skewness rate of up to 80%. in the bc population, three qtls were detected for b: sprg- sprg- and slrg- these qtls may be associated with the first stages of regeneration, that is, hormonal induction response and bud formation. sprg- which explained the highest percentage of variation for b, was also identified for the r and pr traits. given that bud formation is a necessary prerequisite for the production of shoots, it was expected that this major qtl for b would be found for r and pr, which in fact turned out to be the case. for r and pr, a common qtl on chromosome was also identified. in addition, two qtls were detected for r and pr. all these qtls seem to be involved in the development of buds into shoots. as can also be observed in this study, common qtls for the different regeneration traits, as well as a higher number of qtls for traits related to plant development compared to those associated with bud induction, have been reported in different studies. for instance, in arabidopsis, schianterelli et al. found a common area of chromosome in all analyzed parameters, a peak in chromosome and another in chromosome when they analyzed the total number of regenerated shoots. in wheat, ben amer et al. identified three qtls, two that affect green spot initiation and shoot regeneration and a third that only influences plant formation. a partial common genetic system controlling the regeneration frequency of diverse types of explants has been reported by molina and nuez in melon. this indicates that using different explants for loci detection may lead to the identification of some common qtls, but also to the possible identification of other new qtls. root explants were used by koornneef et al. and satoh et al. for phenotyping, at which point two alleles for regeneration ability were located on chromosome of tomato. in the present study, leaves were used for phenotyping and a qtl in a similar area of chromosome was detected in addition to other qtls that influence regeneration and were identified on chromosomes and differences in root and leaf explants for qtl identification were also found in arabidopsis thaliana. koornneef et al. located a dominant allele from s. peruvianum near the middle of chromosome that determines efficient shoot regeneration in tomato root explants. satoh et al. mapped a putative allele from s. chilense on this chromosome. the acid invertase gene, reported as a marker linked to rg- was chosen for mapping rg- in our population derived from s. pennellii. the polymorphisms detected in our parents allow us to map this gene in the qtl detected on chromosome that we named rg- we consider rg- to be a putative allele of the rg- gene. allelism must be confirmed. the other gene chosen as a candidate was lesk which encodes a serine/threonine kinase, and was reported as a marker of competence for in vitro regeneration in tomato. this gene was positioned on chromosome but it is not located in the rg- qtl. the recent release of the entire genome sequence of tomato provides a powerful tool for interrogating qtl data. in this respect, we have taken a preliminary look at genes located at the peak areas of the detected qtls, and which could be related to organogenesis. histidine kinases were reported as cytokinin receptors. in our qtl peaks, only one histidine kinase is located in the sprg- qtl. the candidate tomato gene, lesk which has been described as a marker for in vitro competence, encodes a serine/threonine kinase. we looked for serine/threonine kinases and found this kind of protein in all identified qtls. other putative candidate genes could be esr and its paralogue, esr from arabidopsis, which are the best-characterized genes related to regeneration. these genes code for ethylene response factors. we found erfs, which contain the ap domain, in all analysed qtls with the exception of sprg-4b. cyclines related to cell division were found in sprg- rg- sprg-4a and sprg- mads-box genes, which have been correlated to adventitious regeneration induction and regulation, were found in the sprg- sprg-4a and sprg- qtl peaks.  the results obtained in this study may very well represent a significant step toward the goal of understanding the processes underlying tomato tissue culture and regeneration responses. we have situated six qtls on chromosomes and five from s. pennellii and one from s. lycopersicum. the most important qtls are sprg- which is most likely associated with the morphogenetic response, and sprg- which promotes bud development. a qtl detected on chromosome rg- likely contains a putative allele of the rg- and rg- genes, as is shown by mapping the acid invertase gene linked to rg- qtls detected on chromosomes and most likely contain genes influencing bud formation and development, respectively. 
furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: rg- and lesk which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. two linkage maps were developed and six qtls were identified on five chromosomes in the bc population by means of the interval mapping and restricted multiple qtl mapping methods. we have also located two candidate genes, discovering a putative allele of the high regeneration gene rg- in the qtl on chromosome the identified qtls could represent a significant step toward the understanding of this process and the identification of other related candidate genes. it will also most likely facilitate the development of molecular markers for use in gene isolation. as a result, we located a putative allele of rg- in the qtl detected on chromosome that we named rg- lesk which is also situated on chromosome is outside rg- in a preliminary exploration of the detected qtl peaks, we found several genes that may be related to regeneration. despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. the phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. these qtls came from s. pennellii, with the exception of the minor qtl located on chromosome which was provided by cv. we developed two mapping populations derived from a previously selected tomato cultivar with low regeneration ability and a high regeneration accession of the wild species solanum pennellii. in this study we have identified new qtls related to the complex process of regeneration from tissue culture.
 low regeneration ability limits biotechnological breeding approaches. the influence of genotype in the regeneration response is high in both tomato and other important crops. despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. the aim of this study was to localize the genetic factors affecting regeneration in tomato.  we developed two mapping populations derived from a previously selected tomato cultivar with low regeneration ability and a high regeneration accession of the wild species solanum pennellii. the phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. two linkage maps were developed and six qtls were identified on five chromosomes in the bc population by means of the interval mapping and restricted multiple qtl mapping methods. these qtls came from s. pennellii, with the exception of the minor qtl located on chromosome which was provided by cv. anl the main qtls correspond to those detected on chromosomes and in the f population, a qtl on chromosome was identified on a similar region as that detected in the bc population. marker segregation distortion was observed in this population in those areas where the qtls of bc were detected. furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: rg- and lesk which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. as a result, we located a putative allele of rg- in the qtl detected on chromosome that we named rg- lesk which is also situated on chromosome is outside rg- in a preliminary exploration of the detected qtl peaks, we found several genes that may be related to regeneration.  in this study we have identified new qtls related to the complex process of regeneration from tissue culture. we have also located two candidate genes, discovering a putative allele of the high regeneration gene rg- in the qtl on chromosome the identified qtls could represent a significant step toward the understanding of this process and the identification of other related candidate genes. it will also most likely facilitate the development of molecular markers for use in gene isolation. 