BACKGROUND
pearl millet  is an annual, diploid, highly allogamous cereal with seven chromosome pairs  <cit> .
it can be grown in a vast range of environmental conditions including environments that are characterized by frequent drought events and poor soil fertility  <cit> .
this is one reason that pearl millet is one of the most important staple food crops in west and central africa   <cit> .
the other reason is that pearl millet grain has relatively high nutritional values for a cereal.
its grain has higher protein and fat content than wheat or rice and its amino acid composition is more appropriate for human nutrition than that of wheat or polished rice  <cit> .
cultivated pearl millet displays tremendous phenotypic variability for traits such as flowering time, panicle length, grain and stover characteristics, tolerance to drought, pests, and diseases, as well as nutritional value .
efficient and systematic exploitation of this diversity is the key to any crop improvement program  <cit> .
this, however, requires in a first step the assessment of genetic diversity and population structure of the species under consideration.
for pearl millet, several studies have examined these issues.
<cit>  determined the influence of farmer management on pearl millet landrace diversity in two villages in north-eastern nigeria.
<cit>  assessed the genetic diversity within and between ten indian pearl millet landraces.
the genetic diversity of  <dig> wild and  <dig> cultivated genotypes of pearl millet from niger was analyzed by  <cit>  examined the phylogeny and origin of pearl millet.
however, to our knowledge, no earlier study examined the genetic diversity and population structure of pearl millet across a wide geographic range in wca.
the distribution area of pearl millet in wca harbours a wide range of climatic and environmental conditions as well as diverse farmer preferences and pearl millet utilization habits .
this may lead to local adaptation and thereby to population structure.
however, no earlier study examined systematically the forces that may have affected or led to the observed population structure in pearl millet.
the objectives of our research were to  assess the geographical distribution of genetic diversity,  assess the population structure of pearl millet from wca, and  identify those geographical parameters and environmental factors from the location at which landraces were sampled, as well as those phenotypic traits that may have affected or led to this population structure.
RESULTS
the heritabilities of the four phenotypic traits assessed for the  <dig> pearl millet in-breds ranged from  <dig>  for sv to  <dig>  for pl and was  <dig>  for ph and  <dig>  for ft. the geographic and environmental parameters as well as the phenotypic traits available for all pearl millet inbreds showed a continous distribution .
the correlation between the geographic and environmental parameters and examined phenotypic traits ranged from - <dig>  between latitude and precipitation to  <dig>  between latitude and mean annual temperature.
for the  <dig> ssr markers examined in our study, the number of alleles ranged from  <dig> to  <dig> , with an average of  <dig> .
gene diversity d was lowest for markers psmp <dig> and psmp <dig>  and highest for marker psmp <dig> .
the average d value across all pearl millet inbreds was  <dig> .
the first two principal components, which explained  <dig>  and  <dig> % of the total genetic variation, revealed no obvious clusters .
the structure analysis resulted in five sub-groups and one admixed group.
these groups, which comprised between  <dig> and  <dig> inbreds , were located in different sectors of the pca.
the gene diversity d of the sub-groups ranged from  <dig>   to  <dig>   and the number of group-specific alleles varied between  <dig>  and  <dig> .
the overall fixation index fst was  <dig> .
the number of alleles per locus of the pearl millet subsets of size  <dig>   <dig>   <dig>  ...,  <dig> which maximize the gene diversity d ranged from  <dig>  to  <dig> , where gene diversity d varied between  <dig>  and  <dig>  .
the amova with the country of origin as hierarchy level revealed that most of the genetic variation was present between inbreds derived from landraces of the same country as well as within landraces but only a small proportion of the total genetic variation was attributable to countries .
the same trend was observed with respect to the agro-ecological zone of origin as hierarchy level .
plotting the structure results on the geographic map revealed no obvious association of sub-group membership probability and country of origin or agro-ecological zone of origin .
the pairwise pearson's correlation coefficient between mrd and the geographic or phenotypic trait-based distances was highest for sbd  and lowest for ph  .
the highest correlation with sbd was observed for pl  and the lowest for annual temperature .
the tests of association between the q matrix from structure and geographic or environmental parameters as well as phenotypic traits were significant  for ft, rrp, pl, and country of origin.
* p <  <dig> , ** p <  <dig> × 10− <dig>  *** p <  <dig> × 10−10
†p value for the association of the corresponding parameters with the q matrix from structure based on a multivariate linear regression.
discussion
genetic diversity of the examined pearl millet germplasm
irrespective of the considered hierarchy level, the amova revealed that about four times more variation was present between landraces than within landraces .
our finding is in good accordance with the results of  <cit>  who observed that about  <dig>  times more variation was present between indian pearl millet landraces than within the landraces.
consequently, we observed realistic estimates of within and between landrace variation despite the fact that we examined inbreds derived from landraces as well as that the average number of inbreds per landrace was only  <dig> .
long-term selection gain requires genetic variability  <cit> .
therefore, it was important to examine the genetic diversity of the pearl millet inbreds analyzed in our study.
since estimates of genetic diversity d are not affected by differences in sample size, direct comparisons between different studies are possible.
across the  <dig> pearl millet inbreds examined, we observed a total gene diversity d of  <dig>  .
this value is higher than the gene diversity estimates observed in the study of  <cit>   for  <dig> genotypes of  <dig> allogamous cultivated pear millet landracs from niger, based on ssr marker.
this difference might be explained by the fact that in the latter study only genotypes from niger were examined, whereas the inbreds of our study were derived from landraces from a much larger area of wca.
furthermore, we observed a higher d value than  <cit>   for a pearl millet world collection.
this finding might be due to the fact that we used a higher proportion of di-nucleotide repeat ssr markers, which tend to be more variable than ssrs with longer repeat motifs , than  <cit> .
our observation on d was supported by the results on the average number of alleles per locus.
although our population size was smaller than that of the other studies and we examined inbreds instead of heterozygous genotypes, we observed a considerably higher number of alleles per locus   than previously re-ported by  <cit>   and  <cit>  .
these findings on d and the average number of alleles per locus suggested that the pearl millet inbreds examined in this study is a valuable resource for increasing the genetic diversity in pearl millet breeding pro-grams.
spatial distribution of genetic diversity
we observed differences between the genetic diversity parameters gene diversity d, average number of alleles per locus, and the number of group specific alleles a between the pearl millet inbreds originating from different wca countries .
however, these differences are to a large extent  only due to the fact that the number of inbreds originating from each of the ten countries differed considerably.
furthermore, the amova revealed that less than one percent of the variation was found between countries .
this observation is in contrast to findings of  <cit> , who observed statistically different d values for pearl millet genotypes from different countries.
in addition, that study reported coefficients of differentiation between the genotypes from different countries  that suggested the presence of considerable variation between countries.
these contrasting findings compared to our results might be due to the fact that our sampling was extremely unbalanced with respect to the number of inbreds per country.
another explanation might be that our study was based on ssr markers, whereas  <cit>  examined isozyme markers that may not be selectively neutral and reveal only a low number of alleles per locus.
in wca, pearl millet is cultivated throughout three agro-ecological zones  <cit> .
the adaptation of pearl millet to these different environments has the potential to lead to genetic differentiation.
however, amova revealed that almost none of the ssr genotype variation observed could be attributed to differences between the agro-ecological zones .
our observation suggested that establishing core-collections based on the information of the agro-ecological zone of origin  might be sub-optimal.
this was supported by our observation that the three agro-ecological zones contributed different numbers of inbreds to the pearl millet subsets maximizing the gene diversity d .
our findings might be explained by the habit of farmers, especially in the sudano-sahelian and in the sudanian zones, to grow not only one landrace but several .
in order to fill the hungry period, an early maturing landrace is cultivated, and in addition a landrace with late maturity that has a high yield potential in years with good growing conditions but might fail in years with severe terminal drought.
also the highly allogamous behaviour of the crop combined with an overlapping flowering time of early and late but photo-sensitive landraces in certain years with late planting dates has the potential to diluting differentiation between agro-ecological zones.
finally, our observation might be explained by the fact that the environmental conditions within each agro-ecological zone are too heterogeneous  to permit detectable genetic differentiation between landraces of different agro-ecological zones.
inference of population structure
due to the fact that neither country nor agro-ecological zone of origin revealed a clear sub-grouping of the pearl millet inbreds examined in our study .
we used the software structure  <cit>  to infer the population structure.
the results of this analysis indicated that the  <dig> pearl millet inbreds of our study belong to five sub-groups .
the grouping of the pearl millet inbreds by structure was in fair accordance with the results of the pca .
the five groups showed no distinct clusters but were located in different sectors of the pca.
our finding together with an overall fst value of  <dig>  suggested that the sub-groups of pearl millet are not as differentiated as in maize .
our finding of five sub-groups for pearl millet inbreds from wca was in the range of previously reported numbers of sub-groups.
<cit>  identified based on isozyme markers two distinct sub-groups in pearl millet inbreds from west africa, whereas  <cit>  reported three sub-groups for pearl millet landraces from niger.
in contrast,  <cit>  identified based on phenotypic data of morphological and disease resistance traits ten clusters of pearl millet landraces from burkina faso.
these differences in the number of sub-groups compared to our study, are most likely due to the fact that earlier studies examined a lower number of accessions than we did.
in addition, the above mentioned studies were based either on phenotypic data or a relatively low number of isozyme markers, while we used ssr markers to examine pearl millet inbreds.
association of population structure and geographical, environmental, and phenotypic parameters
in the harshest production environments in wca, farmers rely in highly cross-pollinated pearl millet landraces with site-specific adaptation and good production stability that are rarely outyielded by on-station-bred improved cultivars  <cit> .
thereby, farmers, together with the environmental factors, shape the pearl millet populations .
identifying the variables that are correlated with population structure, thus, has the potential to help identify adaptive traits as well as the environmental conditions which are the driving factors of adaptation.
we observed that the phenotypic traits ft, ppr, and pl were significantly associated with the q matrix from structure  using a multivariate linear regression model.
for ft, our observation is in accordance with observations in other plant species such as maize  <cit>  or arabidopsis  <cit> .
this finding can be explained by the fact that ft is in most plant species under divergent selection as it is the key adaptive trait enabling plants to flower at the optimum time for pollination and seed development  <cit> .
in wca, the growing conditions of pearl millet are characterized, among other hazards, by highly variable beginnings of the rainy season  <cit> .
photoperiod-sensitive flowering, in our study measured as ppr, has the potential to enhance adaptation to such environments.
this is due to the fact that it leads to simultaneous flowering of genotypes in the target region, independent of the individual date of sowing in different fields.
this in turn, is expected to lead to divergence and thereby might explain why ppr is associated with population stucture in pearl millet .
our finding that pl was significantly associated with the q matrix from structure  might be explained by the fact that farmers preferences seem to differ largely in different regions of wca .
this has the potential to lead to population stucture.
the preference for a long panicle in certain regions of wca is mostly due to practical reasons.
in these areas, the harvested pearl millet panicles are usually tied up into bundles for transport from the field to the grain store, and this can be more easily done with long panicles, where short panicles require usually a bag for transportation.
we observed that the phenotypic traits ft, rrp, and pl were significantly associated with the q matrix from structure, but not the environmental factors  that are expected to influence these traits in natural populations such as latitude, temperature, or precipitation .
this was also true, if the phenotypic traits were used as cofactors when examining the environmental factors .
our results suggested that for pearl millet landraces natural selection is less important than human selection in shaping populations which in turn can be explained by the fact that pearl millet landraces are no natural populations.
CONCLUSIONS
our findings of high d values as well as a high average number of alleles per locus suggested that the pearl millet inbreds examined in this study is a valuable resource for increasing the genetic diversity in pearl millet breeding programs.
furthermore, the results of this study suggested that for pearl millet landraces natural selection is less important than human selection in shaping populations.

 pearl millet is an annual, diploid, highly allogamous cereal with seven chromosome pairs. it can be grown in a vast range of environmental conditions including environments that are characterized by frequent drought events and poor soil fertility. this is one reason that pearl millet is one of the most important staple food crops in west and central africa. the other reason is that pearl millet grain has relatively high nutritional values for a cereal. its grain has higher protein and fat content than wheat or rice and its amino acid composition is more appropriate for human nutrition than that of wheat or polished rice. cultivated pearl millet displays tremendous phenotypic variability for traits such as flowering time, panicle length, grain and stover characteristics, tolerance to drought, pests, and diseases, as well as nutritional value. efficient and systematic exploitation of this diversity is the key to any crop improvement program. this, however, requires in a first step the assessment of genetic diversity and population structure of the species under consideration. for pearl millet, several studies have examined these issues. determined the influence of farmer management on pearl millet landrace diversity in two villages in north-eastern nigeria. assessed the genetic diversity within and between ten indian pearl millet landraces. the genetic diversity of wild and cultivated genotypes of pearl millet from niger was analyzed by examined the phylogeny and origin of pearl millet. however, to our knowledge, no earlier study examined the genetic diversity and population structure of pearl millet across a wide geographic range in wca. the distribution area of pearl millet in wca harbours a wide range of climatic and environmental conditions as well as diverse farmer preferences and pearl millet utilization habits. this may lead to local adaptation and thereby to population structure. however, no earlier study examined systematically the forces that may have affected or led to the observed population structure in pearl millet. the objectives of our research were to assess the geographical distribution of genetic diversity, assess the population structure of pearl millet from wca, and identify those geographical parameters and environmental factors from the location at which landraces were sampled, as well as those phenotypic traits that may have affected or led to this population structure.  the heritabilities of the four phenotypic traits assessed for the pearl millet in-breds ranged from for sv to for pl and was for ph and for ft. the geographic and environmental parameters as well as the phenotypic traits available for all pearl millet inbreds showed a continous distribution. the correlation between the geographic and environmental parameters and examined phenotypic traits ranged from - between latitude and precipitation to between latitude and mean annual temperature. for the ssr markers examined in our study, the number of alleles ranged from to, with an average of. gene diversity d was lowest for markers psmp and psmp and highest for marker psmp. the average d value across all pearl millet inbreds was. the first two principal components, which explained and % of the total genetic variation, revealed no obvious clusters. the structure analysis resulted in five sub-groups and one admixed group. these groups, which comprised between and inbreds, were located in different sectors of the pca. the gene diversity d of the sub-groups ranged from to and the number of group-specific alleles varied between and. the overall fixation index fst was. the number of alleles per locus of the pearl millet subsets of size..., which maximize the gene diversity d ranged from to, where gene diversity d varied between and. the amova with the country of origin as hierarchy level revealed that most of the genetic variation was present between inbreds derived from landraces of the same country as well as within landraces but only a small proportion of the total genetic variation was attributable to countries. the same trend was observed with respect to the agro-ecological zone of origin as hierarchy level. plotting the structure results on the geographic map revealed no obvious association of sub-group membership probability and country of origin or agro-ecological zone of origin. the pairwise pearson's correlation coefficient between mrd and the geographic or phenotypic trait-based distances was highest for sbd and lowest for ph. the highest correlation with sbd was observed for pl and the lowest for annual temperature. the tests of association between the q matrix from structure and geographic or environmental parameters as well as phenotypic traits were significant for ft, rrp, pl, and country of origin. * p <, ** p < × 10− *** p < × 10−10 †p value for the association of the corresponding parameters with the q matrix from structure based on a multivariate linear regression. discussion genetic diversity of the examined pearl millet germplasm irrespective of the considered hierarchy level, the amova revealed that about four times more variation was present between landraces than within landraces. our finding is in good accordance with the results of who observed that about times more variation was present between indian pearl millet landraces than within the landraces. consequently, we observed realistic estimates of within and between landrace variation despite the fact that we examined inbreds derived from landraces as well as that the average number of inbreds per landrace was only. long-term selection gain requires genetic variability. therefore, it was important to examine the genetic diversity of the pearl millet inbreds analyzed in our study. since estimates of genetic diversity d are not affected by differences in sample size, direct comparisons between different studies are possible. across the pearl millet inbreds examined, we observed a total gene diversity d of. this value is higher than the gene diversity estimates observed in the study of for genotypes of allogamous cultivated pear millet landracs from niger, based on ssr marker. this difference might be explained by the fact that in the latter study only genotypes from niger were examined, whereas the inbreds of our study were derived from landraces from a much larger area of wca. furthermore, we observed a higher d value than for a pearl millet world collection. this finding might be due to the fact that we used a higher proportion of di-nucleotide repeat ssr markers, which tend to be more variable than ssrs with longer repeat motifs, than. our observation on d was supported by the results on the average number of alleles per locus. although our population size was smaller than that of the other studies and we examined inbreds instead of heterozygous genotypes, we observed a considerably higher number of alleles per locus than previously re-ported by and. these findings on d and the average number of alleles per locus suggested that the pearl millet inbreds examined in this study is a valuable resource for increasing the genetic diversity in pearl millet breeding pro-grams. spatial distribution of genetic diversity we observed differences between the genetic diversity parameters gene diversity d, average number of alleles per locus, and the number of group specific alleles a between the pearl millet inbreds originating from different wca countries. however, these differences are to a large extent only due to the fact that the number of inbreds originating from each of the ten countries differed considerably. furthermore, the amova revealed that less than one percent of the variation was found between countries. this observation is in contrast to findings of, who observed statistically different d values for pearl millet genotypes from different countries. in addition, that study reported coefficients of differentiation between the genotypes from different countries that suggested the presence of considerable variation between countries. these contrasting findings compared to our results might be due to the fact that our sampling was extremely unbalanced with respect to the number of inbreds per country. another explanation might be that our study was based on ssr markers, whereas examined isozyme markers that may not be selectively neutral and reveal only a low number of alleles per locus. in wca, pearl millet is cultivated throughout three agro-ecological zones. the adaptation of pearl millet to these different environments has the potential to lead to genetic differentiation. however, amova revealed that almost none of the ssr genotype variation observed could be attributed to differences between the agro-ecological zones. our observation suggested that establishing core-collections based on the information of the agro-ecological zone of origin might be sub-optimal. this was supported by our observation that the three agro-ecological zones contributed different numbers of inbreds to the pearl millet subsets maximizing the gene diversity d. our findings might be explained by the habit of farmers, especially in the sudano-sahelian and in the sudanian zones, to grow not only one landrace but several. in order to fill the hungry period, an early maturing landrace is cultivated, and in addition a landrace with late maturity that has a high yield potential in years with good growing conditions but might fail in years with severe terminal drought. also the highly allogamous behaviour of the crop combined with an overlapping flowering time of early and late but photo-sensitive landraces in certain years with late planting dates has the potential to diluting differentiation between agro-ecological zones. finally, our observation might be explained by the fact that the environmental conditions within each agro-ecological zone are too heterogeneous to permit detectable genetic differentiation between landraces of different agro-ecological zones. inference of population structure due to the fact that neither country nor agro-ecological zone of origin revealed a clear sub-grouping of the pearl millet inbreds examined in our study. we used the software structure to infer the population structure. the results of this analysis indicated that the pearl millet inbreds of our study belong to five sub-groups. the grouping of the pearl millet inbreds by structure was in fair accordance with the results of the pca. the five groups showed no distinct clusters but were located in different sectors of the pca. our finding together with an overall fst value of suggested that the sub-groups of pearl millet are not as differentiated as in maize. our finding of five sub-groups for pearl millet inbreds from wca was in the range of previously reported numbers of sub-groups. identified based on isozyme markers two distinct sub-groups in pearl millet inbreds from west africa, whereas reported three sub-groups for pearl millet landraces from niger. in contrast, identified based on phenotypic data of morphological and disease resistance traits ten clusters of pearl millet landraces from burkina faso. these differences in the number of sub-groups compared to our study, are most likely due to the fact that earlier studies examined a lower number of accessions than we did. in addition, the above mentioned studies were based either on phenotypic data or a relatively low number of isozyme markers, while we used ssr markers to examine pearl millet inbreds. association of population structure and geographical, environmental, and phenotypic parameters in the harshest production environments in wca, farmers rely in highly cross-pollinated pearl millet landraces with site-specific adaptation and good production stability that are rarely outyielded by on-station-bred improved cultivars. thereby, farmers, together with the environmental factors, shape the pearl millet populations. identifying the variables that are correlated with population structure, thus, has the potential to help identify adaptive traits as well as the environmental conditions which are the driving factors of adaptation. we observed that the phenotypic traits ft, ppr, and pl were significantly associated with the q matrix from structure using a multivariate linear regression model. for ft, our observation is in accordance with observations in other plant species such as maize or arabidopsis. this finding can be explained by the fact that ft is in most plant species under divergent selection as it is the key adaptive trait enabling plants to flower at the optimum time for pollination and seed development. in wca, the growing conditions of pearl millet are characterized, among other hazards, by highly variable beginnings of the rainy season. photoperiod-sensitive flowering, in our study measured as ppr, has the potential to enhance adaptation to such environments. this is due to the fact that it leads to simultaneous flowering of genotypes in the target region, independent of the individual date of sowing in different fields. this in turn, is expected to lead to divergence and thereby might explain why ppr is associated with population stucture in pearl millet. our finding that pl was significantly associated with the q matrix from structure might be explained by the fact that farmers preferences seem to differ largely in different regions of wca. this has the potential to lead to population stucture. the preference for a long panicle in certain regions of wca is mostly due to practical reasons. in these areas, the harvested pearl millet panicles are usually tied up into bundles for transport from the field to the grain store, and this can be more easily done with long panicles, where short panicles require usually a bag for transportation. we observed that the phenotypic traits ft, rrp, and pl were significantly associated with the q matrix from structure, but not the environmental factors that are expected to influence these traits in natural populations such as latitude, temperature, or precipitation. this was also true, if the phenotypic traits were used as cofactors when examining the environmental factors. our results suggested that for pearl millet landraces natural selection is less important than human selection in shaping populations which in turn can be explained by the fact that pearl millet landraces are no natural populations.  our findings of high d values as well as a high average number of alleles per locus suggested that the pearl millet inbreds examined in this study is a valuable resource for increasing the genetic diversity in pearl millet breeding programs. furthermore, the results of this study suggested that for pearl millet landraces natural selection is less important than human selection in shaping populations. 
the distribution area of pearl millet in west and central africa harbours a wide range of climatic and environmental conditions as well as diverse farmer preferences and pearl millet utilization habits which have the potential to lead to local adaptation and thereby to population structure. the objectives of our research were to assess the geographical distribution of genetic diversity in pearl millet inbreds derived from landraces, assess the population structure of pearl millet from wca, and identify those geographical parameters and environmental factors from the location at which landraces were sampled, as well as those phenotypic traits that may have affected or led to this population structure. our study was based on a set of inbred lines derived from different pearl millet landraces from wca. our results suggested that for pearl millet natural selection is compared to artificial selection less important in shaping populations. we observed that the phenotypic traits flowering time, relative response to photoperiod, and panicle length were significantly associated with population structure but not the environmental factors which are expected to influence these traits in natural populations such as latitude, temperature, or precipitation.
 the distribution area of pearl millet in west and central africa harbours a wide range of climatic and environmental conditions as well as diverse farmer preferences and pearl millet utilization habits which have the potential to lead to local adaptation and thereby to population structure. the objectives of our research were to assess the geographical distribution of genetic diversity in pearl millet inbreds derived from landraces, assess the population structure of pearl millet from wca, and identify those geographical parameters and environmental factors from the location at which landraces were sampled, as well as those phenotypic traits that may have affected or led to this population structure. our study was based on a set of inbred lines derived from different pearl millet landraces from wca.  five sub-groups were detected within the entire germplasm set by structure. we observed that the phenotypic traits flowering time, relative response to photoperiod, and panicle length were significantly associated with population structure but not the environmental factors which are expected to influence these traits in natural populations such as latitude, temperature, or precipitation.  our results suggested that for pearl millet natural selection is compared to artificial selection less important in shaping populations. 