BACKGROUND
pearl millet  is a highly cross-pollinated monocot belonging to the poaceae.
it is one of the most widely cultivated drought- and high-temperature tolerant c <dig> cereals, being grown for forage, grain and stover under dryland, rainfed and irrigated conditions in drought-prone regions of the arid and semi-arid tropics and sub-tropics, and as a mulch in conservation tillage production systems in the humid and sub-humid tropics.
it is especially important as a staple food grain, and source of feed and fodder for livestock, in hot, dry marginal agricultural production environments of africa and south asia that are home to hundreds of millions of the world’s poorest farmers  <cit> .
besides soil infertility, drought is the most important abiotic constraint to production of this crop, and is a major contributor to the instability of its grain and fodder yields.
the first molecular marker-based genetic linkage map of pearl millet, comprised largely of rflp loci supplemented by a few isozyme loci, was reported by liu et al.
<cit> .
in subsequent years, the linkage map was expanded with ssr markers  <cit> , and more recently dart markers  <cit> , and its complex relationships with the foxtail millet and rice genomes were established  <cit> .
quantitative trait loci  for disease resistance  <cit> , drought tolerance  <cit> , components of drought adaptation  <cit> , flowering time, grain and stover yield  <cit> , and ruminant nutritional quality of straw  <cit>  have been mapped, and effective marker-assisted selection for several of these traits has been demonstrated  <cit> .
these tools have already been applied for marker-assisted backcross improvement of downy mildew resistance of elite hybrid parental lines, which culminated in the  <dig> release in india of pearl millet hybrid “hhb  <dig> improved”, which was the first public-bred product of dna-marker-assisted selection to be released for cultivation in that country  <cit> .
ssr markers are one of the best options available for foreground selection in marker-assisted backcrossing programs because they are hyper-variable, multi-allelic, often co-dominant, highly reproducible, and readily multiplexed.
they are also ideal for anchoring molecular linkage maps  <cit>  that can be more highly saturated with dart  <cit> , snp  <cit> , or genotyping-by-sequencing  <cit>  markers.
est-ssrs are of particular interest for linkage map alignments, as they are readily transferable to other pedigrees  <cit>  and may functionally determine observed trait variation.
to date approximately  <dig> functional ssr primer pairs have been published for use in pearl millet  <cit> , in addition to sscp-snp  <cit> , dart  <cit> , cisp and snp  <cit>  markers.
however, much larger numbers of markers are required for their more effective application in plant breeding.
further, almost all existing pearl millet molecular markers cluster in regions proximal to the centromeres of the seven linkage groups, with very few loci mapping to distal regions of the chromosomes  <cit> .
there is an urgent need to identify larger numbers of co-dominant polymorphic markers mapping to these distal regions of the pearl millet chromosomes, which are expected to contain the vast majority of genes and gene-associated regulatory sequences.
the limited amount of sequence information in pearl millet has limited progress in gene discovery and characterization, global transcript profiling, probe design for development of gene arrays, and generation of molecular markers and their application in crop improvement programs.
interestingly, next generation sequencing  technologies are proving useful for rapidly and efficiently developing genomic resources of minor crop species.
in case of under-resourced crop species, where appropriate or adequate sequence data were not yet available, one strategy has been to sequence cdnas with ngs technologies and then align these sequences with transcript data of that species, if available, with transcript data of any related major/model crop species  <cit> , or with the aligned genome sequences of such model species  <cit> .
combining gene-based markers together with previously available marker systems will greatly assist in filling the gaps in the existing pearl millet linkage maps  <cit> , reducing linkage drag associated with marker-assisted selection, and increasing the speed and efficiency of subsequent qtl introgression programs.
while traditionally a genetic linkage map has been generated from a single population, recent efforts to create maps from multiple populations, referred to as consensus maps, have gained much interest in the scientific and breeding community.
integration of mapping data from individual maps into one consensus map has been reported in forage  <cit>  and cereal species  <cit> , including pearl millet  <cit> , and aims to determine the relative positions of transferable markers in order to compare candidate gene and qtl locations across a broad range of genetic backgrounds.
during the process of developing est resources from drought-stressed leaf and root tissues of selfed progenies from single-plant selections of two elite inbred genotypes differing in terminal drought tolerance , that are also parents of a mapping population  <cit> , a contiguous segmental substitution line set  <cit> , and several different qtl introgression line sets, we took the opportunity to develop new est-based ssr markers.
these est-ssrs, along with other pcr-compatible markers, were then mapped using four pearl millet ril mapping populations.
subsequently, a consensus map that integrates data from these four linkage maps was constructed.
RESULTS
sequence data assembly
four cdna samples synthesized from four total rna samples  derived from drought-stressed leaf and root tissues of icmb 841-p <dig> and 863b-p <dig> were sent to the j. craig venter institute  in november  <dig> for sequencing and assembly using flx/ <dig> sequencing technology.
a single full-plate run on the flx/ <dig> sequencing machine generated approximately 400 k reads with an average read length of 250–400 bp with the technology available in march  <dig>  <cit> .
the four half-plate runs of the normalized pearl millet cdna libraries on a flx/ <dig> sequencer generated an average of 184 k reads per half-plate-run while the average read length was 205 bp.
the raw ests were cleaned of rrna, vector, ligator and poor quality sequences, which resulted in a reduction in the average number of reads to 99 k per half-plate run, but an increase in the average read length to 224 bp.
cleaned ests from the four samples were assembled together using the plantta pipeline at jcvi .
this resulted in a total of  <dig>  contigs and  <dig>  singletons, i.e.
a total of  <dig>  tentative unique sequences  with an average read length of  <dig> bp.
further,  <dig>  putative snps  were identified in  <dig>  contigs that were formed from reads derived from these two inbreds.
the remaining  <dig>  contigs were either formed from sequence reads from a single genotype, or were formed from sequence reads from the two genotypes but were monomorphic with regard to putative snps when checked in silico.
development of est-ssr markers
all tuss  from the plantta pipeline were searched for class i ssrs  <cit>  using the microsatellite  program  and  <dig> class i ssrs were identified in  <dig> tuss.
nineteen tuss matching to previously published pearl millet ssr markers, based on blast search, were removed.
the remaining  <dig> tuss containing class i ssrs were analyzed using the cap <dig> program.
this yielded  <dig> non-redundant sequences, which were used for primer design with the primer <dig> program  <cit> .
these est-ssr primer pairs were given the prefix name ipes , but only  <dig>  primer pairs could be designed.
an additional  <dig> primer pairs  were designed separately from the tuss resulting from further cap <dig> assemblies of cleaned flx/ <dig> ests prepared at icrisat-patancheru.
in all,  <dig> non-redundant est-ssr primer pairs were designed, of which  <dig> est-ssr primer pairs were expected to detect class i ssr loci  and the remaining  <dig> were expected to detect class ii ssr loci .
the forward and reverse primer sequences of these newly developed ipes-series est-ssrs are given in additional file  <dig>
linkage mapping and component maps of the four recombinant inbred populations
among the  <dig> ipes primer pairs tested,  <dig> produced amplification products, out of which  <dig> were polymorphic among parents of at least one of the four rips.
apart from the newly developed est-ssr primer pairs of the ipes series, previously published est-ssr primer pairs  developed by senthilvel et al.
<cit> , genomic ssr primer pairs  developed by qi et al.
<cit>  and allouis et al.
<cit> , genomic ssr primer pairs  developed by budak et al.
<cit> , and several sts primer pairs previously developed at john innes centre, uk  were also assessed for polymorphism detection between the parents of these four rips.
among them,  <dig> primer pairs amplified.
the polymorphic markers for each of the rips were surveyed on the respective recombinant inbred line  progeny sets and then mapped using gmendel  <dig>   <cit> , mapmaker  <dig>   <cit>  and record  <cit> .
map construction was performed for each rip separately, but as the mapping was being done simultaneously for all four rips, we could identify a few of the unlinked groups for one rip as being sub-groups of larger linkage groups detected on one or more of the other three rips.
a total of  <dig> primer pairs, including  <dig> ipes,  <dig> icmp,  <dig> pmsp,  <dig> ctm and  <dig> psmp primer pairs, detected polymorphic loci mapped on one or more of the four f <dig> rips.
the details of the individual maps  for each of the four rips are:
rip a
a total of  <dig> xipes,  <dig> xicmp,  <dig> xpsmp,  <dig> xctm and  <dig> xpsmp marker loci were mapped on  <dig> linkage groups, having a total map length of 615 cm, an average length of 88 cm per linkage group, and an average inter-marker distance of 6 cm.
the linkage maps of rip a are given in additional file  <dig>
rip b
a total of  <dig> xipes,  <dig> xicmp,  <dig> xpsmp and  <dig> xctm marker loci were mapped on  <dig> linkage groups, having a total map length of 690 cm, an average length of 99 cm per linkage group, and an average inter-marker distance of 9 cm.
the linkage maps of rip b are given in additional file  <dig>
rip c
a total of  <dig> xipes,  <dig> xicmp,  <dig> xpsmp and  <dig> xctm marker loci were mapped on  <dig> linkage groups, having a total map length of 428 cm, an average length of 61 cm per linkage group, and an average inter-marker distance of 7 cm.
linkage group  <dig>  was obtained as  <dig> sub-groups containing  <dig>   <dig>  and  <dig> markers, with map lengths of  <dig>   <dig>  and 3 cm, respectively.
the linkage maps of rip c are given in additional file  <dig>
rip d
a total of  <dig> xipes,  <dig> xicmp and  <dig> xpsmp marker loci were mapped on the expected  <dig> linkage groups  and one unlinked group  with a total length of only 276 cm.
the average length for the  <dig> expected linkage groups was 29 cm, and their average inter-marker distance was 5 cm.
lg <dig> was obtained as  <dig> sub-groups, lg1a and lg1b, with  <dig> and  <dig> markers, and map lengths of  <dig> and  <dig>  cm, respectively.
lg <dig> was obtained as  <dig> sub-groups, lg6a, lg6b and lg6c, with  <dig>   <dig> and  <dig> markers, and map lengths of  <dig>   <dig> and 2 cm, respectively.
the linkage maps of rip d are given in additional file  <dig>
segregation distortion  of mapped markers ranged from 31%  to 38% .
the newly developed xipes-series markers showed distortion in the range of 25%  to 36% .
this marker distortion favored alleles of female or male parents, depending on the rip: “female parent icmb 841-p2” , “male parent prlt 2/89-33” , “male parent icmp 451-p8”  and “female parent pt 723b-p2” , for rips a, b, c and d, respectively.
a comparative map was developed using mapchart  <dig>   <cit>  with the maps of the four rips .
there were five primer pairs that detected at least two polymorphic loci:
•ipes <dig>  which detected xipes <dig>  on lg <dig> in rip b and xipes <dig>  on lg <dig> in rip c and rip d;
•ipes <dig>  which detected xipes <dig>  on lg <dig> in rip b and xipes <dig>  on lg <dig> in rip a, rip c and rip d;
•psmp <dig>  which detected xpsmp <dig>  on lg <dig> in rip c and xpsmp <dig>  on lg <dig> in rip a ;
•ipes <dig>  which detected xipes <dig>  on lg <dig> in rip b and xipes <dig>  on lg <dig> in rip c; and,
•psmp <dig>  which detected xpsmp <dig>  on lg <dig> in rip a and rip d, and xpsmp <dig>  on lg <dig> in rip b.
the numbers of common markers across the four rips were identified and displayed in a venn diagram .
in all,  <dig> marker loci were mapped among the four rips.
among these,  <dig> marker loci were shared between sets of any two or three or four rips.
there was only one polymorphic locus  shared across all four rips.
one additional primer pair  detected polymorphic loci across all four rips .
similarly,  <dig> marker loci were shared between sets of  <dig> rips,  <dig> marker loci were shared between pairs of rips, and  <dig> marker loci were unique to one or the other of the four rips.
the details of shared polymorphic loci are provided in additional file  <dig>
primer pairs previously reported to detect more than one polymorphic locus, which only detected a single mapped locus in this study, included psmp <dig>  <cit> , psmp <dig>  <cit>  and psmp <dig>  <cit> .
on average,  <dig> shared marker loci were present on each linkage group.
this substantial number of shared marker loci facilitated the production of a consensus map.
consensus map
the four maps were integrated using mergemap  <cit>  to form a consensus map comprised of the expected  <dig> linkage groups, containing  <dig> marker loci from  <dig> markers with a total map length of 899 cm .
another two markers, namely xipes <dig> and xipes <dig> belonging to lga of rip d, were not integrated in the consensus map as they were not associated with any of the seven expected linkage groups.
the map lengths of linkage groups in the consensus map were  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 130 cm for lg <dig>  lg <dig>  lg <dig>  lg <dig>  lg <dig>  lg <dig> and lg <dig> with  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> marker loci, respectively .
syntenic relationships of pearl millet with sequenced grasses
blastn results for mapped pearl millet gene-based ssr markers , for each chromosome of each of the five available sequenced cereal genomes, were combined with marker locus orders from the pearl millet linkage analysis to identify likely macro-level syntenic relationships.
possible segmentally syntenic relationships of the pearl millet linkage groups  were identified  for each of the following grasses: chromosomes of foxtail millet , chromosomes of rice , chromosomes of sorghum , chromosomes of maize  and chromosomes of brachypodium .
the blastn top hits  used for this were for the full-length sequences from which the pearl millet est-ssr primer pairs  were designed and whose corresponding loci were mapped.
pearl millet linkage groups pg_ <dig>  pg_ <dig>  pg_ <dig>  pg_ <dig>  pg_ <dig>  pg_ <dig>  pg_ <dig> and pg_a have 21/ <dig> , 17/ <dig>  6/ <dig>  9/ <dig>  9/ <dig>  16/ <dig>  10/ <dig> and 1/ <dig> marker loci, respectively, that show likely syntenic relationships with these cereals.
the orders of the loci of the gene-based markers  on the pearl millet linkage groups and the corresponding regions of chromosomal segments of the five cereal genomes are reasonably well conserved for much of the length of the pearl millet linkage groups, despite the greater degree of genomic restructuring that appears to have occurred in this species compared to better-studied grasses and which is thought responsible for the relatively large number of apparent segmental translocation, inversion and insertion events that would be required to construct the pearl millet chromosome architecture from that of the putative ancestoral grass  <cit> .
for example the order of five marker loci  on pearl millet linkage group  <dig> and its syntenic regions is highly conserved in foxtail millet, its closest relative, and less so in more distantly related grasses .
out of the  <dig> est-ssr marker loci  mapped across one or more of the four pearl millet rips, good blast hits  were detected with at least one of these five cereal genomes for  <dig> loci.
among these  <dig> loci,  <dig> % , 74% ,  <dig> % ,  <dig> %  and  <dig> %  had detectable relationships with the portions of the rice, foxtail millet, sorghum, maize and brachypodium genomes, respectively.
this suggests that pearl millet is most closely related to foxtail millet, followed by sorghum, maize, rice and brachypodium in decreasing order, which is in agreement with current understanding of grass evolution  <cit> .
linkage groups of pearl millet represented as pg_ <dig> = lg <dig>  pg_ <dig> = lg <dig>  pg_ <dig> = lg <dig>  pg_ <dig> = lg <dig>  pg_ <dig> = lg <dig>  pg_ <dig> = lg <dig>  pg_ <dig> = lg <dig> and pg_a = lga, chromosomes of foxtail millet named as si_ <dig> to si_ <dig>  chromosomes of rice as os_ <dig> to os_ <dig>  chromosomes of sorghum as sbi- <dig> to sbi- <dig>  chromosomes of maize as zm_ <dig> to zm_ <dig> and chromosomes of brachypodium as bd_ <dig> to bd_ <dig>  the numbers in the brackets indicate the number of pearl millet tuss containing mapped est-ssr markers that had significant blast hits  on the chromosome sequences of other grasses when blast search was done separately for each grass genome.
discussion
in this study we have identified high quality polymorphic est-ssrs and these have enriched the marker resources of generally marker-poor pearl millet.
the newly developed est-ssrs will be useful in genetic diversity assessment, genome mapping, qtl mapping, association mapping and marker-assisted breeding experiments.
initially,  <dig> est-ssr primer pairs were designed from the flx/ <dig> sequence data, and have been tested for amplification and ability to detect polymorphism using template dna from parental inbreds of four pearl millet ril mapping populations.
the main criteria used to select the primer pairs for genetic mapping were reproducibility, ability to produce single and/or well-defined scorable peaks with an automated florescence-based genotyping system , large repeat length , amenable for automation, product size in the range of  <dig> to 500 bp, and detecting scorable polymorphism for one of the four parental pairs tested.
these stringent criteria reduced the number of primer pairs in the working set to  <dig>  trinucleotide repeat markers were more highly polymorphic  than the dinucleotide , tetranucleotide  and pentanucleotide  repeat-based markers, as observed previously in pearl millet  <cit> .
rip a had the highest number of polymorphic marker loci , while rip d had the lowest number of polymorphic loci .
rip b had the greatest total map length; however, this total map distance was inflated by markers loosely mapping to the distal ends of several linkage groups.
it was also noted that the distribution of markers in a particular lg were not uniform across rips.
for example,  <dig> markers mapped to lg <dig> of rip a, whereas just four markers mapped to lg <dig> of rip d.
segregation distortion occurred uniformly across genomic regions, with the specific regions involved varying from rip to rip.
segregation distortion is a common phenomenon in pearl millet and has been reported in essentially all earlier mapping studies of this cross-pollinated species  <cit> .
generally, segregating populations have differential levels of segregation distortion, but rils exhibit stronger distortion of marker segregation than do earlier-generation mapping populations.
it has been suggested that involuntary selection against a few genomic regions during generation of the rils, or incompatibility between genomic regions contributed by the different parents  <cit> , contribute to the higher levels of segregation distortion observed in rips.
the bootstrap histogram  of individual lgs of the four rips revealed that the order of the markers were well conserved and all of the single-copy markers in all lgs showed unique positions except those that are very closely linked .
even these sets of closely-linked markers shared their position with markers in nearby regions.
the unique positions of these markers, in spite of the observed segregation distortion, is indicative of the stability of the pearl millet lgs, provided that there are no differences in chromosome structure such as those reported in the first rflp-based pearl millet linkage map  <cit> .
a total of  <dig> markers mapped to  <dig> loci  on the expected  <dig> linkage groups and an unlinked group  of the four rips, and these markers were relatively uniformly distributed .
the newly developed xipes-series est-ssrs have been positioned relative to previously published ssr markers and genetic linkage maps of pearl millet.
the map order of marker loci in the four rips were generally consistent with previously published ssr-based maps of pearl millet  <cit> .
rip d had an average inter-marker distance of  <dig>  cm followed by rip a with  <dig>  cm, rip c with  <dig>  cm, and rip b with  <dig>  cm.
this optimal inter-marker distance, and the uniform coverage across the nuclear genome will provide greater opportunities to locate qtls that have not been identified so far and will be particularly useful for the identification of recombination events adjacent to regions targeted for introgression in marker-assisted backcrossing programs, which are required to minimize negative linkage drag that could result from introgression of large donor segments flanking each introgression target  <cit> .
the presence of gaps in the distal regions of a few linkage groups was due to the forceful assignment of markers to the distal ends of these groups using mapmaker  <dig> .
however care was taken while assigning these markers to individual linkage groups by looking at their map positions in other rips.
xipes <dig> was assigned to the distal region of lg <dig> in rip c after considering its position in this region of lg <dig> for rip a. in the same way, a sub-group of markers linked to xipes <dig> and another sub-group of markers linked to xipes <dig> were assigned to lg <dig> for rip c and rip d, based on their linkage relationships in rip a. the presence of gaps in the sub-telomeric regions of these linkage groups is probably due to very high recombination rates in these regions , the presence of marker- or gene-poor regions immediately adjacent to the telomeres of each chromosome arm , or the absence of markers that can effectively link sub-telomeric and centromeric regions.
similar gaps were reported previously by devos et al.
<cit> , using rflp probes to establish the syntenic relationships between genetic maps of rice, foxtail millet and pearl millet.
however, other marker systems that cover the entire genome  need to be mapped in these rips to confirm this phenomenon.
an attempt has been made in this direction by supriya et al.
<cit> , using  <dig> dart and  <dig> ssr markers to cover the nuclear genome of pearl millet rip b. that study greatly extended the marker coverage in sub-telomeric regions of all seven pearl millet linkage groups.
in the current study, we have constructed a consensus map or integrated linkage map for pearl millet using mergemap, which outperforms joinmap both in terms of accuracy and running time  <cit> .
this consensus map is simply one of many possible non-conflicting linear representations of the consensus directed acyclic graphs .
however, the order of mapped loci was generally well conserved between the integrated map and the rip-specific maps, which indicates that the positions of the loci on the present integrated map can be regarded as a good “consensus map”.
unlike the integrated map published by qi et al.
<cit> , this consensus map, which is mostly based on est-ssrs  and genomic ssrs , has a more or less uniform distribution of markers across all seven expected pearl millet linkage groups.
the marker positions in the consensus map obtained using mergemap were verified using the dag files generated by the mergemap tool and by using the comparative maps  generated using mapchart.
locus order was well conserved across all of the linkage groups, except for lg <dig>  which exhibited conflicting marker positions for  <dig> loci.
there were no conflicts in marker positions for lg <dig> and lg <dig>  lg <dig> had a conflict for the marker positions of xipes <dig> and xipes0139; lg <dig> had such conflicts for xipes <dig>  xipes <dig>  xipes <dig> and xipes <dig>  on lg <dig>  the markers xpsmp <dig> and xipes <dig> mapped adjacent to each other, but their order was inverted when maps of rip a and rip c were compared.
lg <dig> had conflicts for two markers, xipes <dig> and xipes <dig>  which mapped adjacent to each other without any marker between them.
their positions were inverted when maps of rip c and rip d were compared.
for lg <dig>  the marker loci xpsmp <dig> and xipes <dig> were inverted when maps of rip a and rip c were compared with that of rip b. finally, lg <dig> had a conflict for the positions of adjacent markers xipes <dig> and xipes <dig>  which have inverted positions when maps of rip a and rip d are compared.
five ssr primer pairs detected more than one polymorphic locus, which could be due to translocation events in the genomic regions involving loci detected by primer pairs, or more likely, could be due to the presence of conserved ssrs occurring in multi-gene families or duplicated genomic regions.
the availability of published or draft genome sequences of rice, foxtail millet, sorghum, maize and brachypodium made it possible to perform blast searches of the pearl millet est sequences  against these genomes to identify possible syntenic relationships between the pearl millet linkage map and the aligned genomes of these sequenced grasses .
this allowed us to determine that previously unassigned group lga of rip d is likely to be a part of lg <dig>  a more dense or higher resolution map with additional est-based markers would give a much better picture of the possible syntenic relationships between the genome of pearl millet and those of other sequenced species.
CONCLUSIONS
linkage maps form a framework for trait mapping and qtl analysis.
the newly developed est-ssr markers , along with primer pairs for previously mapped est-ssrs and ssrs  and sts  markers were used to construct separate linkage maps of four f <dig> recombinant inbred populations.
these linkage maps were used to construct a consensus map for pearl millet with  <dig> primer pairs detecting  <dig> marker loci.
the locus order of the present consensus map is highly consistent, and is sufficiently reliable for use as a reference genetic map for pearl millet.
overall, the consensus map developed in the present study contains the largest set of mapped ssrs reported to date in pearl millet, and represents a major consolidation to existing pearl millet genetic mapping information.
the suggested syntenic relationships of the pearl millet linkage groups with chromosomes of rice, foxtail millet, sorghum, maize and brachypodium demonstrate that these grasses are closely related.
the consensus map, the four ril populations upon which it is based, and the syntenic relationships between these grasses identified based on the new pearl millet est-ssrs, will prove to be useful assets in the development of both molecular breeding for pearl millet and for comparative genetics and genomics within the grass family.

 pearl millet is a highly cross-pollinated monocot belonging to the poaceae. it is one of the most widely cultivated drought- and high-temperature tolerant c cereals, being grown for forage, grain and stover under dryland, rainfed and irrigated conditions in drought-prone regions of the arid and semi-arid tropics and sub-tropics, and as a mulch in conservation tillage production systems in the humid and sub-humid tropics. it is especially important as a staple food grain, and source of feed and fodder for livestock, in hot, dry marginal agricultural production environments of africa and south asia that are home to hundreds of millions of the world’s poorest farmers. besides soil infertility, drought is the most important abiotic constraint to production of this crop, and is a major contributor to the instability of its grain and fodder yields. the first molecular marker-based genetic linkage map of pearl millet, comprised largely of rflp loci supplemented by a few isozyme loci, was reported by liu et al.. in subsequent years, the linkage map was expanded with ssr markers, and more recently dart markers, and its complex relationships with the foxtail millet and rice genomes were established. quantitative trait loci for disease resistance, drought tolerance, components of drought adaptation, flowering time, grain and stover yield, and ruminant nutritional quality of straw have been mapped, and effective marker-assisted selection for several of these traits has been demonstrated. these tools have already been applied for marker-assisted backcross improvement of downy mildew resistance of elite hybrid parental lines, which culminated in the release in india of pearl millet hybrid “hhb improved”, which was the first public-bred product of dna-marker-assisted selection to be released for cultivation in that country. ssr markers are one of the best options available for foreground selection in marker-assisted backcrossing programs because they are hyper-variable, multi-allelic, often co-dominant, highly reproducible, and readily multiplexed. they are also ideal for anchoring molecular linkage maps that can be more highly saturated with dart, snp, or genotyping-by-sequencing markers. est-ssrs are of particular interest for linkage map alignments, as they are readily transferable to other pedigrees and may functionally determine observed trait variation. to date approximately functional ssr primer pairs have been published for use in pearl millet, in addition to sscp-snp, dart, cisp and snp markers. however, much larger numbers of markers are required for their more effective application in plant breeding. further, almost all existing pearl millet molecular markers cluster in regions proximal to the centromeres of the seven linkage groups, with very few loci mapping to distal regions of the chromosomes. there is an urgent need to identify larger numbers of co-dominant polymorphic markers mapping to these distal regions of the pearl millet chromosomes, which are expected to contain the vast majority of genes and gene-associated regulatory sequences. the limited amount of sequence information in pearl millet has limited progress in gene discovery and characterization, global transcript profiling, probe design for development of gene arrays, and generation of molecular markers and their application in crop improvement programs. interestingly, next generation sequencing technologies are proving useful for rapidly and efficiently developing genomic resources of minor crop species. in case of under-resourced crop species, where appropriate or adequate sequence data were not yet available, one strategy has been to sequence cdnas with ngs technologies and then align these sequences with transcript data of that species, if available, with transcript data of any related major/model crop species, or with the aligned genome sequences of such model species. combining gene-based markers together with previously available marker systems will greatly assist in filling the gaps in the existing pearl millet linkage maps, reducing linkage drag associated with marker-assisted selection, and increasing the speed and efficiency of subsequent qtl introgression programs. while traditionally a genetic linkage map has been generated from a single population, recent efforts to create maps from multiple populations, referred to as consensus maps, have gained much interest in the scientific and breeding community. integration of mapping data from individual maps into one consensus map has been reported in forage and cereal species, including pearl millet, and aims to determine the relative positions of transferable markers in order to compare candidate gene and qtl locations across a broad range of genetic backgrounds. during the process of developing est resources from drought-stressed leaf and root tissues of selfed progenies from single-plant selections of two elite inbred genotypes differing in terminal drought tolerance, that are also parents of a mapping population, a contiguous segmental substitution line set, and several different qtl introgression line sets, we took the opportunity to develop new est-based ssr markers. these est-ssrs, along with other pcr-compatible markers, were then mapped using four pearl millet ril mapping populations. subsequently, a consensus map that integrates data from these four linkage maps was constructed.  sequence data assembly four cdna samples synthesized from four total rna samples derived from drought-stressed leaf and root tissues of icmb 841-p and 863b-p were sent to the j. craig venter institute in november for sequencing and assembly using flx/ sequencing technology. a single full-plate run on the flx/ sequencing machine generated approximately 400 k reads with an average read length of 250–400 bp with the technology available in march. the four half-plate runs of the normalized pearl millet cdna libraries on a flx/ sequencer generated an average of 184 k reads per half-plate-run while the average read length was 205 bp. the raw ests were cleaned of rrna, vector, ligator and poor quality sequences, which resulted in a reduction in the average number of reads to 99 k per half-plate run, but an increase in the average read length to 224 bp. cleaned ests from the four samples were assembled together using the plantta pipeline at jcvi. this resulted in a total of contigs and singletons, i.e. a total of tentative unique sequences with an average read length of bp. further, putative snps were identified in contigs that were formed from reads derived from these two inbreds. the remaining contigs were either formed from sequence reads from a single genotype, or were formed from sequence reads from the two genotypes but were monomorphic with regard to putative snps when checked in silico. development of est-ssr markers all tuss from the plantta pipeline were searched for class i ssrs using the microsatellite program and class i ssrs were identified in tuss. nineteen tuss matching to previously published pearl millet ssr markers, based on blast search, were removed. the remaining tuss containing class i ssrs were analyzed using the cap program. this yielded non-redundant sequences, which were used for primer design with the primer program. these est-ssr primer pairs were given the prefix name ipes, but only primer pairs could be designed. an additional primer pairs were designed separately from the tuss resulting from further cap assemblies of cleaned flx/ ests prepared at icrisat-patancheru. in all, non-redundant est-ssr primer pairs were designed, of which est-ssr primer pairs were expected to detect class i ssr loci and the remaining were expected to detect class ii ssr loci. the forward and reverse primer sequences of these newly developed ipes-series est-ssrs are given in additional file linkage mapping and component maps of the four recombinant inbred populations among the ipes primer pairs tested, produced amplification products, out of which were polymorphic among parents of at least one of the four rips. apart from the newly developed est-ssr primer pairs of the ipes series, previously published est-ssr primer pairs developed by senthilvel et al., genomic ssr primer pairs developed by qi et al. and allouis et al., genomic ssr primer pairs developed by budak et al., and several sts primer pairs previously developed at john innes centre, uk were also assessed for polymorphism detection between the parents of these four rips. among them, primer pairs amplified. the polymorphic markers for each of the rips were surveyed on the respective recombinant inbred line progeny sets and then mapped using gmendel, mapmaker and record. map construction was performed for each rip separately, but as the mapping was being done simultaneously for all four rips, we could identify a few of the unlinked groups for one rip as being sub-groups of larger linkage groups detected on one or more of the other three rips. a total of primer pairs, including ipes, icmp, pmsp, ctm and psmp primer pairs, detected polymorphic loci mapped on one or more of the four f rips. the details of the individual maps for each of the four rips are: rip a a total of xipes, xicmp, xpsmp, xctm and xpsmp marker loci were mapped on linkage groups, having a total map length of 615 cm, an average length of 88 cm per linkage group, and an average inter-marker distance of 6 cm. the linkage maps of rip a are given in additional file rip b a total of xipes, xicmp, xpsmp and xctm marker loci were mapped on linkage groups, having a total map length of 690 cm, an average length of 99 cm per linkage group, and an average inter-marker distance of 9 cm. the linkage maps of rip b are given in additional file rip c a total of xipes, xicmp, xpsmp and xctm marker loci were mapped on linkage groups, having a total map length of 428 cm, an average length of 61 cm per linkage group, and an average inter-marker distance of 7 cm. linkage group was obtained as sub-groups containing and markers, with map lengths of and 3 cm, respectively. the linkage maps of rip c are given in additional file rip d a total of xipes, xicmp and xpsmp marker loci were mapped on the expected linkage groups and one unlinked group with a total length of only 276 cm. the average length for the expected linkage groups was 29 cm, and their average inter-marker distance was 5 cm. lg was obtained as sub-groups, lg1a and lg1b, with and markers, and map lengths of and  cm, respectively. lg was obtained as sub-groups, lg6a, lg6b and lg6c, with and markers, and map lengths of and 2 cm, respectively. the linkage maps of rip d are given in additional file segregation distortion of mapped markers ranged from 31% to 38%. the newly developed xipes-series markers showed distortion in the range of 25% to 36%. this marker distortion favored alleles of female or male parents, depending on the rip: “female parent icmb 841-p2”, “male parent prlt 2/89-33”, “male parent icmp 451-p8” and “female parent pt 723b-p2”, for rips a, b, c and d, respectively. a comparative map was developed using mapchart with the maps of the four rips. there were five primer pairs that detected at least two polymorphic loci: •ipes which detected xipes on lg in rip b and xipes on lg in rip c and rip d; •ipes which detected xipes on lg in rip b and xipes on lg in rip a, rip c and rip d; •psmp which detected xpsmp on lg in rip c and xpsmp on lg in rip a; •ipes which detected xipes on lg in rip b and xipes on lg in rip c; and, •psmp which detected xpsmp on lg in rip a and rip d, and xpsmp on lg in rip b. the numbers of common markers across the four rips were identified and displayed in a venn diagram. in all, marker loci were mapped among the four rips. among these, marker loci were shared between sets of any two or three or four rips. there was only one polymorphic locus shared across all four rips. one additional primer pair detected polymorphic loci across all four rips. similarly, marker loci were shared between sets of rips, marker loci were shared between pairs of rips, and marker loci were unique to one or the other of the four rips. the details of shared polymorphic loci are provided in additional file primer pairs previously reported to detect more than one polymorphic locus, which only detected a single mapped locus in this study, included psmp, psmp and psmp. on average, shared marker loci were present on each linkage group. this substantial number of shared marker loci facilitated the production of a consensus map. consensus map the four maps were integrated using mergemap to form a consensus map comprised of the expected linkage groups, containing marker loci from markers with a total map length of 899 cm. another two markers, namely xipes and xipes belonging to lga of rip d, were not integrated in the consensus map as they were not associated with any of the seven expected linkage groups. the map lengths of linkage groups in the consensus map were and 130 cm for lg lg lg lg lg lg and lg with and marker loci, respectively. syntenic relationships of pearl millet with sequenced grasses blastn results for mapped pearl millet gene-based ssr markers, for each chromosome of each of the five available sequenced cereal genomes, were combined with marker locus orders from the pearl millet linkage analysis to identify likely macro-level syntenic relationships. possible segmentally syntenic relationships of the pearl millet linkage groups were identified for each of the following grasses: chromosomes of foxtail millet, chromosomes of rice, chromosomes of sorghum, chromosomes of maize and chromosomes of brachypodium. the blastn top hits used for this were for the full-length sequences from which the pearl millet est-ssr primer pairs were designed and whose corresponding loci were mapped. pearl millet linkage groups pg_ pg_ pg_ pg_ pg_ pg_ pg_ and pg_a have 21/, 17/ 6/ 9/ 9/ 16/ 10/ and 1/ marker loci, respectively, that show likely syntenic relationships with these cereals. the orders of the loci of the gene-based markers on the pearl millet linkage groups and the corresponding regions of chromosomal segments of the five cereal genomes are reasonably well conserved for much of the length of the pearl millet linkage groups, despite the greater degree of genomic restructuring that appears to have occurred in this species compared to better-studied grasses and which is thought responsible for the relatively large number of apparent segmental translocation, inversion and insertion events that would be required to construct the pearl millet chromosome architecture from that of the putative ancestoral grass. for example the order of five marker loci on pearl millet linkage group and its syntenic regions is highly conserved in foxtail millet, its closest relative, and less so in more distantly related grasses. out of the est-ssr marker loci mapped across one or more of the four pearl millet rips, good blast hits were detected with at least one of these five cereal genomes for loci. among these loci, %, 74%, %, % and % had detectable relationships with the portions of the rice, foxtail millet, sorghum, maize and brachypodium genomes, respectively. this suggests that pearl millet is most closely related to foxtail millet, followed by sorghum, maize, rice and brachypodium in decreasing order, which is in agreement with current understanding of grass evolution. linkage groups of pearl millet represented as pg_ = lg pg_ = lg pg_ = lg pg_ = lg pg_ = lg pg_ = lg pg_ = lg and pg_a = lga, chromosomes of foxtail millet named as si_ to si_ chromosomes of rice as os_ to os_ chromosomes of sorghum as sbi- to sbi- chromosomes of maize as zm_ to zm_ and chromosomes of brachypodium as bd_ to bd_ the numbers in the brackets indicate the number of pearl millet tuss containing mapped est-ssr markers that had significant blast hits on the chromosome sequences of other grasses when blast search was done separately for each grass genome. discussion in this study we have identified high quality polymorphic est-ssrs and these have enriched the marker resources of generally marker-poor pearl millet. the newly developed est-ssrs will be useful in genetic diversity assessment, genome mapping, qtl mapping, association mapping and marker-assisted breeding experiments. initially, est-ssr primer pairs were designed from the flx/ sequence data, and have been tested for amplification and ability to detect polymorphism using template dna from parental inbreds of four pearl millet ril mapping populations. the main criteria used to select the primer pairs for genetic mapping were reproducibility, ability to produce single and/or well-defined scorable peaks with an automated florescence-based genotyping system, large repeat length, amenable for automation, product size in the range of to 500 bp, and detecting scorable polymorphism for one of the four parental pairs tested. these stringent criteria reduced the number of primer pairs in the working set to trinucleotide repeat markers were more highly polymorphic than the dinucleotide, tetranucleotide and pentanucleotide repeat-based markers, as observed previously in pearl millet. rip a had the highest number of polymorphic marker loci, while rip d had the lowest number of polymorphic loci. rip b had the greatest total map length; however, this total map distance was inflated by markers loosely mapping to the distal ends of several linkage groups. it was also noted that the distribution of markers in a particular lg were not uniform across rips. for example, markers mapped to lg of rip a, whereas just four markers mapped to lg of rip d. segregation distortion occurred uniformly across genomic regions, with the specific regions involved varying from rip to rip. segregation distortion is a common phenomenon in pearl millet and has been reported in essentially all earlier mapping studies of this cross-pollinated species. generally, segregating populations have differential levels of segregation distortion, but rils exhibit stronger distortion of marker segregation than do earlier-generation mapping populations. it has been suggested that involuntary selection against a few genomic regions during generation of the rils, or incompatibility between genomic regions contributed by the different parents, contribute to the higher levels of segregation distortion observed in rips. the bootstrap histogram of individual lgs of the four rips revealed that the order of the markers were well conserved and all of the single-copy markers in all lgs showed unique positions except those that are very closely linked. even these sets of closely-linked markers shared their position with markers in nearby regions. the unique positions of these markers, in spite of the observed segregation distortion, is indicative of the stability of the pearl millet lgs, provided that there are no differences in chromosome structure such as those reported in the first rflp-based pearl millet linkage map. a total of markers mapped to loci on the expected linkage groups and an unlinked group of the four rips, and these markers were relatively uniformly distributed. the newly developed xipes-series est-ssrs have been positioned relative to previously published ssr markers and genetic linkage maps of pearl millet. the map order of marker loci in the four rips were generally consistent with previously published ssr-based maps of pearl millet. rip d had an average inter-marker distance of  cm followed by rip a with  cm, rip c with  cm, and rip b with  cm. this optimal inter-marker distance, and the uniform coverage across the nuclear genome will provide greater opportunities to locate qtls that have not been identified so far and will be particularly useful for the identification of recombination events adjacent to regions targeted for introgression in marker-assisted backcrossing programs, which are required to minimize negative linkage drag that could result from introgression of large donor segments flanking each introgression target. the presence of gaps in the distal regions of a few linkage groups was due to the forceful assignment of markers to the distal ends of these groups using mapmaker. however care was taken while assigning these markers to individual linkage groups by looking at their map positions in other rips. xipes was assigned to the distal region of lg in rip c after considering its position in this region of lg for rip a. in the same way, a sub-group of markers linked to xipes and another sub-group of markers linked to xipes were assigned to lg for rip c and rip d, based on their linkage relationships in rip a. the presence of gaps in the sub-telomeric regions of these linkage groups is probably due to very high recombination rates in these regions, the presence of marker- or gene-poor regions immediately adjacent to the telomeres of each chromosome arm, or the absence of markers that can effectively link sub-telomeric and centromeric regions. similar gaps were reported previously by devos et al., using rflp probes to establish the syntenic relationships between genetic maps of rice, foxtail millet and pearl millet. however, other marker systems that cover the entire genome need to be mapped in these rips to confirm this phenomenon. an attempt has been made in this direction by supriya et al., using dart and ssr markers to cover the nuclear genome of pearl millet rip b. that study greatly extended the marker coverage in sub-telomeric regions of all seven pearl millet linkage groups. in the current study, we have constructed a consensus map or integrated linkage map for pearl millet using mergemap, which outperforms joinmap both in terms of accuracy and running time. this consensus map is simply one of many possible non-conflicting linear representations of the consensus directed acyclic graphs. however, the order of mapped loci was generally well conserved between the integrated map and the rip-specific maps, which indicates that the positions of the loci on the present integrated map can be regarded as a good “consensus map”. unlike the integrated map published by qi et al., this consensus map, which is mostly based on est-ssrs and genomic ssrs, has a more or less uniform distribution of markers across all seven expected pearl millet linkage groups. the marker positions in the consensus map obtained using mergemap were verified using the dag files generated by the mergemap tool and by using the comparative maps generated using mapchart. locus order was well conserved across all of the linkage groups, except for lg which exhibited conflicting marker positions for loci. there were no conflicts in marker positions for lg and lg lg had a conflict for the marker positions of xipes and xipes0139; lg had such conflicts for xipes xipes xipes and xipes on lg the markers xpsmp and xipes mapped adjacent to each other, but their order was inverted when maps of rip a and rip c were compared. lg had conflicts for two markers, xipes and xipes which mapped adjacent to each other without any marker between them. their positions were inverted when maps of rip c and rip d were compared. for lg the marker loci xpsmp and xipes were inverted when maps of rip a and rip c were compared with that of rip b. finally, lg had a conflict for the positions of adjacent markers xipes and xipes which have inverted positions when maps of rip a and rip d are compared. five ssr primer pairs detected more than one polymorphic locus, which could be due to translocation events in the genomic regions involving loci detected by primer pairs, or more likely, could be due to the presence of conserved ssrs occurring in multi-gene families or duplicated genomic regions. the availability of published or draft genome sequences of rice, foxtail millet, sorghum, maize and brachypodium made it possible to perform blast searches of the pearl millet est sequences against these genomes to identify possible syntenic relationships between the pearl millet linkage map and the aligned genomes of these sequenced grasses. this allowed us to determine that previously unassigned group lga of rip d is likely to be a part of lg a more dense or higher resolution map with additional est-based markers would give a much better picture of the possible syntenic relationships between the genome of pearl millet and those of other sequenced species.  linkage maps form a framework for trait mapping and qtl analysis. the newly developed est-ssr markers, along with primer pairs for previously mapped est-ssrs and ssrs and sts markers were used to construct separate linkage maps of four f recombinant inbred populations. these linkage maps were used to construct a consensus map for pearl millet with primer pairs detecting marker loci. the locus order of the present consensus map is highly consistent, and is sufficiently reliable for use as a reference genetic map for pearl millet. overall, the consensus map developed in the present study contains the largest set of mapped ssrs reported to date in pearl millet, and represents a major consolidation to existing pearl millet genetic mapping information. the suggested syntenic relationships of the pearl millet linkage groups with chromosomes of rice, foxtail millet, sorghum, maize and brachypodium demonstrate that these grasses are closely related. the consensus map, the four ril populations upon which it is based, and the syntenic relationships between these grasses identified based on the new pearl millet est-ssrs, will prove to be useful assets in the development of both molecular breeding for pearl millet and for comparative genetics and genomics within the grass family. 
pearl millet is a widely cultivated drought- and high-temperature tolerant c cereal grown under dryland, rainfed and irrigated conditions in drought-prone regions of the tropics and sub-tropics of africa, south asia and the americas. eighty-nine est-ssr marker loci from this consensus map had significant blast hits on the genome sequences of rice, foxtail millet, sorghum, maize and brachypodium with and loci, respectively. the consensus map developed in the present study contains the largest set of mapped ssrs reported to date for pearl millet, and represents a major consolidation of existing pearl millet genetic mapping information. this study increased numbers of mapped pearl millet ssr markers by >50%, filling important gaps in previously published ssr-based linkage maps for this species and will greatly facilitate ssr-based qtl mapping and applied marker-assisted selection programs. est-ssr markersestlinkage mapconsensus mapdrought stresspearl milletsynteny newly developed est-ssr markers, along with previously mapped est-ssr, genomic ssr and sts markers, were used to construct linkage maps of four f recombinant inbred populations based on crosses icmb 841-p × 863b-p, h 77/833- × prlt 2/89-, 81b-p × icmp 451-p and pt 732b-p × p1449-2-p. a consensus map of loci detected by primer pairs was constructed using mergemap to integrate the individual linkage maps. locus order in the consensus map was well conserved for nearly all linkage groups. mapped loci numbers were greatest for rip a, followed by rip b, rip c and rip d.
 pearl millet is a widely cultivated drought- and high-temperature tolerant c cereal grown under dryland, rainfed and irrigated conditions in drought-prone regions of the tropics and sub-tropics of africa, south asia and the americas. it is considered an orphan crop with relatively few genomic and genetic resources. this study was undertaken to increase the est-based microsatellite marker and genetic resources for this crop to facilitate marker-assisted breeding.  newly developed est-ssr markers, along with previously mapped est-ssr, genomic ssr and sts markers, were used to construct linkage maps of four f recombinant inbred populations based on crosses icmb 841-p × 863b-p, h 77/833- × prlt 2/89-, 81b-p × icmp 451-p and pt 732b-p × p1449-2-p. mapped loci numbers were greatest for rip a, followed by rip b, rip c and rip d. total map lengths were 615 cm, 690 cm, 428 cm and 276 cm, respectively. a total of loci detected by primer pairs were mapped among the four crosses. a consensus map of loci detected by primer pairs was constructed using mergemap to integrate the individual linkage maps. locus order in the consensus map was well conserved for nearly all linkage groups. eighty-nine est-ssr marker loci from this consensus map had significant blast hits on the genome sequences of rice, foxtail millet, sorghum, maize and brachypodium with and loci, respectively.  the consensus map developed in the present study contains the largest set of mapped ssrs reported to date for pearl millet, and represents a major consolidation of existing pearl millet genetic mapping information. this study increased numbers of mapped pearl millet ssr markers by >50%, filling important gaps in previously published ssr-based linkage maps for this species and will greatly facilitate ssr-based qtl mapping and applied marker-assisted selection programs. est-ssr markersestlinkage mapconsensus mapdrought stresspearl milletsynteny 