BACKGROUND
the date palm  is a critical crop tree in a large section of north africa, the middle east and south asia.
it is one of  <dig> commercially important palms  that include the monoecious oil and coconut palms.
the date palm is dioecious with separate male and female trees.
only the female bears the commercially important date fruit.
trees grown from seed require approximately 6– <dig> years to flower before gender can be determined and no sex chromosome has been identified  <cit> .
sex determination in plants is highly heterogeneous and sex chromosomes in the palms appear to have developed more than once offering an excellent model for studying this process  <cit> .
despite the clear importance of the date palm, few genetic resources exist mainly due to a long generation time of approximately 6 years.
a breeding program began in the united states in the 1940’s resulted in backcrossed varieties that exhibited depressed breeding ability and other physiological problems  <cit> .
previous research into the number of chromosomes in date palm suggests it has  <dig> chromosome pairs   <cit>  though some evidence for alternate numbers have been presented  <cit> .
technological advances allowed us to generate a draft sequence of a commercial cultivar of date palm  <cit>  that was recently improved on  <cit>  and revealed a genome size of approximately 670 mb.
the draft sequence, while critical to understanding gene content and polymorphism, does not provide structural information of the genome.
we were able to use the draft sequence to identify scaffolds containing polymorphisms segregating with date palm gender but other studies such as a search for selective sweeps and quantitative trait loci  would benefit from a genetic map.
construction of a genetic map in the absence of defined genetic resources is challenging.
specifically, outcrossed pedigrees require the identification of sufficient polymorphic markers between the male and female parents to allow map construction  <cit> .
while date palm females, which bears the fruit, are well defined by cultivar, the males are much less so as they are simply used for pollination.
the introduction of genotyping-by-sequencing  was important in overcoming these challenges and provides a method for genotyping thousands of new markers in potentially hundreds of offspring  <cit> .
accuracy in the gbs data is critical in our case as errors would result in lack of linkage or highly inflated map distances.
we have adapted the method to our requirements by using a reduced representation method of gbs.
we employed this adapted approach as each marker needed to be accurately genotyped by high depth sequencing rather than summed over a region as by andolfatto and colleagues  <cit> .
here we present a first genetic map for date palm and its comparison to the other commercially important palms.
RESULTS
genotyping
using various genotype quality requirements  such as minimum coverage and alignment scores, a total of  <dig>  single nucleotide polymorphisms  were considered as genotyped at high quality.
using this method, comparison of ‘genotypes between two khalas control libraries carried separately through the entire process revealed a genotype accuracy rate of  <dig> %.
our previous genome sequence and snp data from the khalas cultivar allowed detection of male parent specific snps where khalas was known to be homozygous.
these male parent-specific snps were used to segregate progeny by which male parent they derived from.
four major populations derived from the same female parent but different male parents were selected for use in the genetic map and contained  <dig>   <dig>   <dig>  and  <dig> individuals.
we obtained  <dig> markers that fit our quality requirements, showed similar segregation in the populations and were called in at least  <dig> of the  <dig> individuals genotyped.
markers that shared the same segregation pattern across all progeny were grouped and a single representative marker was selected from each group.
this collapsing resulted in  <dig> representative markers of which  <dig> were placed on the framework map.
markers placed on the map represented as few as one marker  and as many as  <dig> markers.
the  <dig> representative markers represent  <dig> total markers and these derive from  <dig> unique segregating parent/scaffold combinations.
that is, a scaffold could share two markers but this was only allowed if the markers segregated in different parents.
the mapped marker set derived from  <dig> scaffolds and these scaffolds span a total of  <dig> ,588 bp or ~ 20% of the 380 mb of sequence in the assembly.
genetic map features
the total genetic map length was  <dig> centimorgans  .
assuming a genome size of approximately 670 mb leads to a genetic to physical distance conversion of approximately  <dig>  cm/mb of sequence or 520 kb/cm.
inter-marker distances were evenly distributed  though some biases were observed.
the largest gap between any two markers was  <dig>  cm on linkage group   <dig>  both lg <dig> and lg <dig> had an excess of gaps greater than 3 cm with a total of  <dig> and  <dig> respectively.
distribution of marker types among the linkage groups showed pronounced marker type bias in lg <dig>   <dig>   <dig>  and  <dig>  as an example, lg <dig> shows essentially no markers heterozygous in both parents or heterozygous in the male parent for 50 cm between cm  <dig> and the end of the linkage group.
long stretches on the map without certain marker types are likely due to not enough heterozygous markers genotyped in both parents.
markers heterozygous in both parents are necessary to integrate markers homozygous in only one parent into the map.
markers heterozygous in both parents were least likely to be captured given the requirement that they had to be heterozygous in all males used to create the mapping populations.
additionally, as the cross used for the map was taken from commercial cultivar, it is possible that there are indeed large stretches of homozygosity that decrease the chance for capturing those marker types.
this both highlights the challenges of genetic mapping with outcrossed resources and the necessity of next-generation sequencing approaches to achieve high enough density to allow construction of a genetic map.
the markers in the map derived from  <dig> unique sequence scaffolds in the draft genome sequence.
if each scaffold is considered to provide a single marker, this provides a unique marker every ~210 kb of the genome sequence.
the exact number of date palm chromosomes has been uncertain with some publications reporting  <dig>   <dig>  or  <dig> pairs  <cit>  with most evidence pointing to  <dig> pairs  <cit> .
our results agree well with al salih and al-rawi’s findings of both chromosome number and predicted length  <cit> .
in their study of the ashgar cultivar they detected  <dig> chromosome pairs and measured them with microscopy.
to compare our genetic map we scaled lengths of our date palm linkage groups by adjusting the largest linkage group’s centimorgan length to their longest chromosome measured in micrometers .
this revealed very good consistency between our genetic linkage group lengths to the physical lengths of the chromosomes.
indeed, only lg <dig> and  <dig> were significantly outside the standard deviation of physical measurements for the chromosomes.
this discrepancy may be due to unplaced markers on the end of our linkage groups.
sex chromosome features
previous karyotypic analysis of the date palm genome has not identified a specific sex chromosome  <cit> .
we observed that markers from scaffolds previously segregating with gender were all localized to the lower half of lg <dig> suggesting this may be the sex chromosome.
we checked for the density of snps segregating with gender in all scaffolds and indeed the highest density was found in lg <dig> .
this region segregating with gender is approximately 26 cm in length indicating the total physical length may be as long as 13 mb or 2% of the genome.
this is larger than we originally predicted  <cit>  and may be due to the fact we utilized all markers in this region rather than collapsing to high-quality filtered representative markers as for the rest of the genome .
the use of non-quality filtered markers could lead to inclusion of genotyping errors and an inflation of the region’s size through a false increase in recombination frequency.
using markers from lg <dig> mapped to the more complete saudi date palm genome  scaffolds spanning  <dig>  mb were obtained.
if the recent draft sequence is indeed ~82% complete, this would suggest the region is approximately  <dig>  mb.
additional work will be required to determine the exact non-recombining boundaries and would benefit from large clone sequencing such as bacterial artificial chromosome libraries.
comparison of the gender segregating region to the oil palm genome suggests that it may be rearranged in date palm.
indeed, markers from lg <dig> of date palm mostly mapped to chr <dig> of oil palm though the sex determination region showed additional shorter regions of consecutive markers matching to oil palm chromosomes  <dig>   <dig> and  <dig> .
more detailed analysis at the dna sequence level should shed light on why the date palm is dioecious despite sharing such high synteny and sequence similarity with the monoecious oil palm.
comparison to the recently updated draft sequence
markers were mapped to the saudi date palm genome  <cit>  by blast  <cit>  alignment of ~1000 bp surrounding the snp.
to be considered, blast alignments were required to be unique in the genome.
<dig>  of the marker regions uniquely aligned to  <dig> scaffolds in the saudi date palm sequence.
these  <dig> scaffolds span ~304 mb  of the  <dig>  mb assembled genome.
high synteny in palm genome structure
to study palm genome structure, sequence surrounding markers on the genetic map were aligned to the recently released oil palm genome  <cit> .
2000 bp of sequence surrounding the markers were aligned to the oil palm scaffolds and oil palm chromosome locations were documented.
it is clear from this comparison  that the two genomes maintain high levels of synteny.
indeed most of the  <dig> date palm linkage group were syntenic with one of the  <dig> oil palm chromosomes .
how the two genomes diverged from  <dig> to  <dig> chromosomes is of interest.
based on telomere repeats detected in putative oil palm chromosome centromeres, it has been suggested that a robertsonian fusion caused the collapse of chromosomes into  <dig> and  <dig> in oil palm  <cit> .
our data supports this theory for oil palm chromosome  <dig>  synteny determined between the date palm genetic map and the oil palm chromosomes suggests that oil palm chromosome  <dig> constitutes a fusion of date palm chromosomes  <dig> and  <dig> whereas date palm lg <dig> shows blocks shared with oil palm chromosomes  <dig>   <dig> and  <dig>  in addition, longer stretches on oil palm chromosome  <dig> correspond to regions on date palm chromosomes  <dig> and  <dig>
the same analysis approach was used to compare available sequence markers from the coconut genetic map  <cit>  to the oil palm genome.
while only  <dig> markers from the coconut map could be unambiguously aligned to the oil palm genome, a similar pattern appears with most markers from each coconut linkage group aligning to the same oil palm genome .
despite the fact that the oil palm genome is more than double the size  of the date palm genome , long-range synteny is maintained and overall chromosome structure is very well preserved.
it will be of interest to understand if the oil palm has added nearly double the sequence to the genome while maintaining chromosome structure or the date palm has lost ancestral dna.
CONCLUSIONS
we provide a first genetic map for the commercially important date palm tree and strong evidence for identifying the sex chromosome.
using next-generation sequencing we have overcome challenges due to lack of genetic resources for this tree.
while markers on our map are not guaranteed to be polymorphic in other mapping populations, they nevertheless provide ordering of scaffolds for improved genome analysis.
additionally, by using snps from sequence scaffolds, other polymorphic markers within the same scaffolds could be selected for other populations.
the high density of the markers on the map sets a target for future date palm genome sequence projects.
future research should aim for greater than 500 kb average scaffold length to ensure that most of the genome sequences will be properly ordered on the genetic map.
to resolve more markers onto the map, it will be critical to expand these approaches with a larger pedigree  derived from a single male and female parent.
extending the methods presented here will allow for an even denser genetic map of date palm and an improved resource for quantitative trait locus mapping.
for the first time we have localized the gender segregating region in date palm to lg <dig> suggesting this region may contain the sex determination genetic alteration.
using the genetic map we estimate the size of the region to be approximately  <dig> to 13 mb and more work will be needed to ensure the true recombination boundaries.
identification of the gender segregating region will facilitate future studies on dioecy and the development of sex chromosomes.
indeed, our comparison of the region to the monoecious oil palm revealed shorter blocks of synteny in this region than the rest of the genome.
analysis of the genetic map reveals important features in the date, oil and coconut palm genomes including maintenance of long range synteny despite dramatic genome size differences.
analysis of the genetic maps combined with genomic data will assist in the comparison and improvements of these  <dig> commercially important palms.

 the date palm is a critical crop tree in a large section of north africa, the middle east and south asia. it is one of commercially important palms that include the monoecious oil and coconut palms. the date palm is dioecious with separate male and female trees. only the female bears the commercially important date fruit. trees grown from seed require approximately 6– years to flower before gender can be determined and no sex chromosome has been identified. sex determination in plants is highly heterogeneous and sex chromosomes in the palms appear to have developed more than once offering an excellent model for studying this process. despite the clear importance of the date palm, few genetic resources exist mainly due to a long generation time of approximately 6 years. a breeding program began in the united states in the 1940’s resulted in backcrossed varieties that exhibited depressed breeding ability and other physiological problems. previous research into the number of chromosomes in date palm suggests it has chromosome pairs though some evidence for alternate numbers have been presented. technological advances allowed us to generate a draft sequence of a commercial cultivar of date palm that was recently improved on and revealed a genome size of approximately 670 mb. the draft sequence, while critical to understanding gene content and polymorphism, does not provide structural information of the genome. we were able to use the draft sequence to identify scaffolds containing polymorphisms segregating with date palm gender but other studies such as a search for selective sweeps and quantitative trait loci would benefit from a genetic map. construction of a genetic map in the absence of defined genetic resources is challenging. specifically, outcrossed pedigrees require the identification of sufficient polymorphic markers between the male and female parents to allow map construction. while date palm females, which bears the fruit, are well defined by cultivar, the males are much less so as they are simply used for pollination. the introduction of genotyping-by-sequencing was important in overcoming these challenges and provides a method for genotyping thousands of new markers in potentially hundreds of offspring. accuracy in the gbs data is critical in our case as errors would result in lack of linkage or highly inflated map distances. we have adapted the method to our requirements by using a reduced representation method of gbs. we employed this adapted approach as each marker needed to be accurately genotyped by high depth sequencing rather than summed over a region as by andolfatto and colleagues. here we present a first genetic map for date palm and its comparison to the other commercially important palms.  genotyping using various genotype quality requirements such as minimum coverage and alignment scores, a total of single nucleotide polymorphisms were considered as genotyped at high quality. using this method, comparison of ‘genotypes between two khalas control libraries carried separately through the entire process revealed a genotype accuracy rate of %. our previous genome sequence and snp data from the khalas cultivar allowed detection of male parent specific snps where khalas was known to be homozygous. these male parent-specific snps were used to segregate progeny by which male parent they derived from. four major populations derived from the same female parent but different male parents were selected for use in the genetic map and contained and individuals. we obtained markers that fit our quality requirements, showed similar segregation in the populations and were called in at least of the individuals genotyped. markers that shared the same segregation pattern across all progeny were grouped and a single representative marker was selected from each group. this collapsing resulted in representative markers of which were placed on the framework map. markers placed on the map represented as few as one marker and as many as markers. the representative markers represent total markers and these derive from unique segregating parent/scaffold combinations. that is, a scaffold could share two markers but this was only allowed if the markers segregated in different parents. the mapped marker set derived from scaffolds and these scaffolds span a total of,588 bp or ~ 20% of the 380 mb of sequence in the assembly. genetic map features the total genetic map length was centimorgans. assuming a genome size of approximately 670 mb leads to a genetic to physical distance conversion of approximately  cm/mb of sequence or 520 kb/cm. inter-marker distances were evenly distributed though some biases were observed. the largest gap between any two markers was  cm on linkage group both lg and lg had an excess of gaps greater than 3 cm with a total of and respectively. distribution of marker types among the linkage groups showed pronounced marker type bias in lg and as an example, lg shows essentially no markers heterozygous in both parents or heterozygous in the male parent for 50 cm between cm and the end of the linkage group. long stretches on the map without certain marker types are likely due to not enough heterozygous markers genotyped in both parents. markers heterozygous in both parents are necessary to integrate markers homozygous in only one parent into the map. markers heterozygous in both parents were least likely to be captured given the requirement that they had to be heterozygous in all males used to create the mapping populations. additionally, as the cross used for the map was taken from commercial cultivar, it is possible that there are indeed large stretches of homozygosity that decrease the chance for capturing those marker types. this both highlights the challenges of genetic mapping with outcrossed resources and the necessity of next-generation sequencing approaches to achieve high enough density to allow construction of a genetic map. the markers in the map derived from unique sequence scaffolds in the draft genome sequence. if each scaffold is considered to provide a single marker, this provides a unique marker every ~210 kb of the genome sequence. the exact number of date palm chromosomes has been uncertain with some publications reporting or pairs with most evidence pointing to pairs. our results agree well with al salih and al-rawi’s findings of both chromosome number and predicted length. in their study of the ashgar cultivar they detected chromosome pairs and measured them with microscopy. to compare our genetic map we scaled lengths of our date palm linkage groups by adjusting the largest linkage group’s centimorgan length to their longest chromosome measured in micrometers. this revealed very good consistency between our genetic linkage group lengths to the physical lengths of the chromosomes. indeed, only lg and were significantly outside the standard deviation of physical measurements for the chromosomes. this discrepancy may be due to unplaced markers on the end of our linkage groups. sex chromosome features previous karyotypic analysis of the date palm genome has not identified a specific sex chromosome. we observed that markers from scaffolds previously segregating with gender were all localized to the lower half of lg suggesting this may be the sex chromosome. we checked for the density of snps segregating with gender in all scaffolds and indeed the highest density was found in lg. this region segregating with gender is approximately 26 cm in length indicating the total physical length may be as long as 13 mb or 2% of the genome. this is larger than we originally predicted and may be due to the fact we utilized all markers in this region rather than collapsing to high-quality filtered representative markers as for the rest of the genome. the use of non-quality filtered markers could lead to inclusion of genotyping errors and an inflation of the region’s size through a false increase in recombination frequency. using markers from lg mapped to the more complete saudi date palm genome scaffolds spanning  mb were obtained. if the recent draft sequence is indeed ~82% complete, this would suggest the region is approximately  mb. additional work will be required to determine the exact non-recombining boundaries and would benefit from large clone sequencing such as bacterial artificial chromosome libraries. comparison of the gender segregating region to the oil palm genome suggests that it may be rearranged in date palm. indeed, markers from lg of date palm mostly mapped to chr of oil palm though the sex determination region showed additional shorter regions of consecutive markers matching to oil palm chromosomes and. more detailed analysis at the dna sequence level should shed light on why the date palm is dioecious despite sharing such high synteny and sequence similarity with the monoecious oil palm. comparison to the recently updated draft sequence markers were mapped to the saudi date palm genome by blast alignment of ~1000 bp surrounding the snp. to be considered, blast alignments were required to be unique in the genome. of the marker regions uniquely aligned to scaffolds in the saudi date palm sequence. these scaffolds span ~304 mb of the  mb assembled genome. high synteny in palm genome structure to study palm genome structure, sequence surrounding markers on the genetic map were aligned to the recently released oil palm genome. 2000 bp of sequence surrounding the markers were aligned to the oil palm scaffolds and oil palm chromosome locations were documented. it is clear from this comparison that the two genomes maintain high levels of synteny. indeed most of the date palm linkage group were syntenic with one of the oil palm chromosomes. how the two genomes diverged from to chromosomes is of interest. based on telomere repeats detected in putative oil palm chromosome centromeres, it has been suggested that a robertsonian fusion caused the collapse of chromosomes into and in oil palm. our data supports this theory for oil palm chromosome synteny determined between the date palm genetic map and the oil palm chromosomes suggests that oil palm chromosome constitutes a fusion of date palm chromosomes and whereas date palm lg shows blocks shared with oil palm chromosomes and in addition, longer stretches on oil palm chromosome correspond to regions on date palm chromosomes and the same analysis approach was used to compare available sequence markers from the coconut genetic map to the oil palm genome. while only markers from the coconut map could be unambiguously aligned to the oil palm genome, a similar pattern appears with most markers from each coconut linkage group aligning to the same oil palm genome. despite the fact that the oil palm genome is more than double the size of the date palm genome, long-range synteny is maintained and overall chromosome structure is very well preserved. it will be of interest to understand if the oil palm has added nearly double the sequence to the genome while maintaining chromosome structure or the date palm has lost ancestral dna.  we provide a first genetic map for the commercially important date palm tree and strong evidence for identifying the sex chromosome. using next-generation sequencing we have overcome challenges due to lack of genetic resources for this tree. while markers on our map are not guaranteed to be polymorphic in other mapping populations, they nevertheless provide ordering of scaffolds for improved genome analysis. additionally, by using snps from sequence scaffolds, other polymorphic markers within the same scaffolds could be selected for other populations. the high density of the markers on the map sets a target for future date palm genome sequence projects. future research should aim for greater than 500 kb average scaffold length to ensure that most of the genome sequences will be properly ordered on the genetic map. to resolve more markers onto the map, it will be critical to expand these approaches with a larger pedigree derived from a single male and female parent. extending the methods presented here will allow for an even denser genetic map of date palm and an improved resource for quantitative trait locus mapping. for the first time we have localized the gender segregating region in date palm to lg suggesting this region may contain the sex determination genetic alteration. using the genetic map we estimate the size of the region to be approximately to 13 mb and more work will be needed to ensure the true recombination boundaries. identification of the gender segregating region will facilitate future studies on dioecy and the development of sex chromosomes. indeed, our comparison of the region to the monoecious oil palm revealed shorter blocks of synteny in this region than the rest of the genome. analysis of the genetic map reveals important features in the date, oil and coconut palm genomes including maintenance of long range synteny despite dramatic genome size differences. analysis of the genetic maps combined with genomic data will assist in the comparison and improvements of these commercially important palms. 
to date there has been no genetic map and no sex chromosome has been identified. here we present the first genetic map for date palm and identify the putative date palm sex chromosome. combined with the recent draft genome sequence of the same cultivar, this resource offers a critical new tool for date palm biotechnology, palm comparative genomics and a better understanding of sex chromosome development in the palms. we have integrated the genetic map, derived from the khalas cultivar, with the draft genome and placed up to 19% of the draft genome sequence scaffolds onto linkage groups for the first time. based on a modified gentoyping-by-sequencing approach we have overcome challenges due to lack of genetic resources and provide the first genetic map for date palm. comparison of the date palm linkage groups revealed significant long-range synteny to oil palm. despite its importance from antiquity, few genetic resources are available for improving the productivity and development of the dioecious date palm. the date palm is one of the oldest cultivated fruit trees. analysis of the date palm sex-determination region suggests it is telomeric on linkage group and recombination is not suppressed in the full chromosome.
 the date palm is one of the oldest cultivated fruit trees. it is critical in many ways to cultures in arid lands by providing highly nutritious fruit while surviving extreme heat and environmental conditions. despite its importance from antiquity, few genetic resources are available for improving the productivity and development of the dioecious date palm. to date there has been no genetic map and no sex chromosome has been identified.  here we present the first genetic map for date palm and identify the putative date palm sex chromosome. we placed ~ markers on the map using nearly framework markers spanning a total of 1293 cm. we have integrated the genetic map, derived from the khalas cultivar, with the draft genome and placed up to 19% of the draft genome sequence scaffolds onto linkage groups for the first time. this analysis revealed approximately ~  cm/mb on the map. comparison of the date palm linkage groups revealed significant long-range synteny to oil palm. analysis of the date palm sex-determination region suggests it is telomeric on linkage group and recombination is not suppressed in the full chromosome.  based on a modified gentoyping-by-sequencing approach we have overcome challenges due to lack of genetic resources and provide the first genetic map for date palm. combined with the recent draft genome sequence of the same cultivar, this resource offers a critical new tool for date palm biotechnology, palm comparative genomics and a better understanding of sex chromosome development in the palms. sex chromosomegenotyping by sequencingcomparative genomics 