BACKGROUND
crocodilians  are a group of reptiles that originated roughly  <dig> million years ago  <cit> .
they are apex predators in the marine and freshwater habitats in which they reside, and they play a major role in warm-water ecosystems throughout the world.
there are  <dig> extant species grouped into three families – crocodilidae , alligatoridae , and gavialidae   <cit> .
as evidenced by their frequent appearances in video documentaries and television programs, crocodilians are a subject of considerable human curiosity.
moreover, these reptiles have been common subjects/characters in mythology, folk tales, art , and literature suggesting that they have considerable symbolic and practical significance in the lives of humans, past and present.
crocodilians, birds, dinosaurs, and pterosaurs form a monophyletic group known as the archosauria  <cit>  of which only the crocodilian and the avian lineages are extant .
in support of this premise, molecular phylogenetic evidence from nuclear and mitochondrial dna sequencing indicates that crocodilians and birds are indeed each other's closest living relatives  <cit> .
among archosaurs, only the chicken and zebra finch have been the focus of complete genome sequencing efforts  <cit> .
genome level analyses of a crocodilian would be especially useful in leveraging information from the chicken, and a crocodilian genome would be the best possible outgroup for all genomic work within birds.
however, with the modest exception of alligator mississippiensis for which there is roughly  <dig>  mb of bac end sequence  <cit>  and  <dig> partially assembled bacs , little sequence data is available for crocodilian genomes, in part due to a relative lack of high-quality molecular tools for this important clade.
for roughly  <dig> years, bacterial artificial chromosome  libraries have been the principal molecular substrate used in physical mapping and complete eukaryote genome sequencing  <cit> .
gridding of ordered bac libraries  onto macroarrays and multiplex screening techniques have facilitated rapid gene isolation.
the utility of bac clones as substrates for end sequencing, in conjunction with advanced dna fingerprinting techniques and macroarray analysis, has permitted construction of robust physical maps and selection of minimum tiling paths  for accurate genome sequencing and assembly.
recent advances in sequencing technologies  have created powerful opportunities in which ordered bac libraries play a critical role.
a particularly promising strategy for simultaneous physical mapping and sequencing of large eukaryotic genomes involves sequencing pools of sheared, individually "bar coded" bac clones.
after sequencing, those reads sharing a bar code  are grouped together and assembled in silico, and physical maps are constructed by identifying overlapping assembled or partially assembled bac sequences  <cit> .
to expedite genome research in crocodilians, we have constructed a bac library for the australian saltwater crocodile .
the c. porosus library is only the second large-insert dna library for a crocodilian – a 10× library exists for alligator mississippiensis  <cit>  – and the only bac library for crocodilidae, the largest of the crocodilian families.
c. porosus is the largest living crocodilian and, along with a. mississippiensis, the only crocodilian species to be commercially farmed.
here we describe generation and characterization of the c. porosus bac library and demonstrate its utility as a tool for gene isolation, genome characterization, and comparative genomics.
methods
preparation of nuclei agarose plugs from crocodile blood sample
whole blood was obtained from errol, a male c. porosus from the darwin crocodile farm  <cit>  near darwin, australia.
blood was suspended in citrate buffer  containing 5% v/v dimethylsulfoxide, aliquoted into  <dig>  ml polypropylene tubes, flash frozen in liquid nitrogen, and shipped to the mississippi genome exploration laboratory  <cit> .
one of the tubes was thawed on ice and centrifuged at  <dig>  rpm in a microcentrifuge for  <dig> min.
the supernatant was decanted, the pellet was gently re-suspended in  <dig> ml of stex buffer , and the mixture was centrifuged as described above.
the blood cell pellet was re-suspended in  <dig> μl stex buffer and placed in a water bath at 45°c.
after  <dig> min, the blood suspension was mixed with an equal volume of 45°c 2% w/v cambrex  seaplaque agarose  in stex buffer.
the mixture was poured into a small petri dish so that the depth of the solution was roughly  <dig> mm.
after  <dig> min at 4°c, the resulting gel was cut into  <dig> ×  <dig> mm rectangles, and these "plugs" were transferred into a conical  <dig> ml polypropylene tube containing  <dig> ml of lysis buffer .
the capped tube was incubated at 37°c overnight with gentle agitation.
plugs were transferred into  <dig>  m edta  containing  <dig>  m phenylmethylsulfonyl fluoride and incubated at 4°c for one hour.
plugs were washed in  <dig>  medta  and then stored at 4°c in this buffer.
preparation of high-molecular-weight insert dna
a few test dna plugs were exposed to different hindiii concentrations to determine conditions providing the largest number of fragments between  <dig> to  <dig> kb .
the optimal enzyme concentration as determined in the test digests was used in a large-scale partial digest.
plugs used in the mass digestion were macerated and placed in a slot well of a 1% w/v cambrex seakem gold agarose  gel in  <dig>  × tbe buffer .
size selection of partially digested dna was performed using pulsed-field gel electrophoresis  according to chalhoub et al.
<cit> .
size-selected hindiii fragments between  <dig> and  <dig> kb were recovered from agarose by electroelution according to peterson et al.
<cit> .
bac library construction
bac library construction was performed as described in peterson et al.
<cit>  using the pindigobac- <dig> vector  and electromax dh10b t <dig> phage-resistant competent cells .
clone picking and library replication were performed using a genetix qpixii robot .
to monitor the quality of the bac library and determine mean insert size,  <dig> bac clones from every fiftieth 384-well plate were evaluated by noti digestion and pfge.
for these analyses bac dna was isolated using an autogenprep  <dig> robot .
macroarray construction
high density macroarrays were prepared using a genetix qpixii robot.
each array consisted of  <dig>  double-spotted bac clones stamped onto a  <dig>  cm <dig> hybond n+ filter .
there were enough c. porosus bac clones to produce five complete macroarrays .
stamped arrays were placed clone-side up on lb  agar containing  <dig>  mg/l chloramphenicol and incubated at 37°c overnight.
each macroarray was fixed via incubation in  <dig>  n naoh,  <dig>  m nacl for  <dig> min followed by incubation in  <dig>  m nacl,  <dig>  m tris cl for  <dig> min.
the membranes were allowed to air dry for  <dig> h, treated with  <dig>  n naoh for  <dig> min, and washed in 5× sspe  for  <dig> min.
macroarrays were air dried and stored in sealed plastic bags.
probe design and bac library screening
overgo probes  <cit>  were designed from the consensus sequences of cr1a and cr1b , two recently active cr <dig> subfamilies present in the genomes of crocodylus moreletii and osteolaemus tetraspis , and from a partial cdna sequence of the c. porosus c-mos gene .
the overgos  were labeled with 32p and hybridized to macroarrays as previously described  <cit> .
probe hybridization images were recorded and analyzed using a storm  <dig>  phosphoimager.
for the cr1-probed macroarrays, the spot densitometry tool in alphaeasefc, version  <dig> .
<dig>  was used to explore variation in spot  intensity and estimate copy number as described previously  <cit> .
positive c-mos clones were confirmed by pcr using the cmf1/cmr <dig> primer pair .
pcr was performed using the following thermocycler series: 95°c denaturation for  <dig> min,  <dig> full pcr cycles , and a final 72°c  <dig> min extension step.
subcloning and sequencing of the c. porosus c-mos gene
a bac clone containing the c. porosus c-mos gene was digested with bamhi and hindiii at 37°c for  <dig> hr followed by heating to 65°c for  <dig> min.
the cloning vector pcrii-topo  was likewise double-digested, purified by electrophoresis on a 1% w/v agarose gel, and isolated from agarose using a qiagen  qiaquick gel extraction kit.
ligation was performed at 16°c for  <dig> hrs.
the ligation mixture was used to transform chemically competent invitrogen  one shot top <dig> cells according to the manufacturer's instructions.
subclones were plated and  <dig> were screened by pcr using the c-mos primers cmf <dig> and cmr <dig> .
one positive subclone, which was shown by gel electrophoresis to contain a  <dig>  kb insert, was sent to seqwright  for cycle sequencing using the cmf <dig> and cmr <dig> primers and two additional primers .
use of a combination of primers was intended to extend the target area so that it would encompass the entire c-mos coding sequence and several hundred bases 5' and 3' of the coding region.
base-calling and assembly of sequence reads were performed using phred and phrap, respectively  <cit> .
trimming of the assembled c-mos sequence was conducted using cross_match  <cit> .
the  <dig> bp product was submitted to genbank and assigned the accession fj <dig>
RESULTS
bac library coverage
the c. porosus bac library consists of  <dig>  individual clones stored in  <dig> bar-coded 384-well microtiter plates.
noti digestion and pfge of  <dig> bac clones indicates an average clone insert size of  <dig> kb  with  <dig> % of clones possessing inserts >  <dig> kb,  <dig> % with inserts between  <dig> and  <dig> kb, and  <dig> % with inserts smaller than  <dig> kb.
internal noti sites are present in  <dig> % of pfge-examined bac clones , and  <dig> % of clones are probable false positives, i.e., they possess a vector band but no insert band.
collectively, the entire bac library represents  <dig>  gb of crocodile genomic dna .
the exact genome size of c. porosus is unknown.
however, measurements made for two other crocodylus species   <cit>  are both  <dig> mb.
assuming the c. porosus genome size is similar to those of these closely allied taxa, we estimate that the library affords  <dig> × coverage  of the c. porosus genome.
theoretically, this level of coverage affords 98% probability of finding any given genomic sequence at least once in the library  <cit> .
survey of cr <dig> elements in crocodile genome
cr <dig> elements are non-ltr retrotransposons existing in high copy numbers in bird and reptile genomes  <cit> ; there are about  <dig>  cr <dig> elements in the chicken genome  <cit> .
cr <dig> retrotransposons are considered excellent markers for molecular phylogenetic and population genetic studies  <cit> .
initial studies on the sequences from the  <dig> bac clones of alligator mississippiensis available in genbank  revealed that at least two cr <dig> subfamilies, referred to here as cr1a and cr1b, have recently been active in crocodilian genomes.
this observation is consistent with shedlock et al.
in which the authors suggested that multiple cr <dig> lineages may have been active in alligators.
in addition to a. mississippiensis, cr1a and cr1b have been identified in crocodylus moreletii, and osteolaemus tetraspis  and consensus sequences for conserved regions of these elements have been generated .
to explore cr <dig> distribution in c. porosus, macroarrays were screened with cr1b-derived overgos or a combination of cr1a and cr1b overgos.
comparison of the cr1b- and cr1a/b-probed macroarrays revealed that the cr1a and cr1b subfamilies are not distinguishable in our assay, i.e., virtually no differences in hybridization pattern and intensity were observed when comparing the cr1b and cr1a/cr1b filters .
it was clear, however, that elements similar to cr1a and b are fairly abundant in c. porosus.
examination of one-quarter of the cr1a/b-probed macroarray  indicates that  <dig> % of clones show hybridization to the cr1a/b overgos while cr1b overgo hybridization to a filter stamped with the contents of a single 384-well plate from the bac library suggests that  <dig> % of clones  are positive for the cr1b overgo .
densitometric analysis of the macroarray reveals that there is a six-fold variation in positive clone hybridization intensity.
if the lightest, but clearly positive hybridization signals represent clones containing one copy of a cr1a/b element and if the darkest clones contain six elements, there would be approximately  <dig>  copies of cr1a/b in the c. porosus genome .
however, there is the possibility that macroarray exposure conditions were such that only clones with multiple copies of cr1a/b elements appear positive; inclusion of a single-copy sequence control on the macroarray could be used to test this possibility.
moreover, the use of overgos  rather than whole cr <dig> sequences clearly limits hybridization to those elements where the regions corresponding to the overgos have been conserved.
based on analysis of  <dig>  mb of bac end sequence, shedlock  <cit>  estimated the number of cr <dig> elements in alligator mississippiensis at  <dig>  copies.
while a. mississippiensis and c. porosus are separated by  <dig> million years of evolution  <cit> , it seems reasonable that they might have similar total numbers of cr <dig> elements.
consequently, the c. porosus copy number estimate of  <dig>  should be viewed as a minimum number of cr1a/b elements, not total cr <dig> elements, in the c. porosus genome.
statistical analysis of the macroarray data indicates that cr1a/b elements are not randomly distributed throughout the c. porosus genome.
the macroarray contains  <dig>  individual clones with an average insert size of  <dig> kb.
consequently, a single macroarray represents roughly  <dig>  genome equivalents, i.e.,  ÷  <dig> mb.
if the crocodile genome contains  <dig>  copies of cr1a/b, then we would expect approximately  <dig>  copies of cr1a/b per macroarray, and if these were distributed randomly we would expect, on average,  <dig>  copies of cr1a/b per clone.
however, only  <dig> % of clones on the macroarray show hybridization to the cr1a/b probes.
to test whether such a distribution is likely by chance, we can formulate the problem as a statistical "urn model."
suppose we have  <dig>  urns, and we drop  <dig>  balls into them at random.
in such a case, classical statistical asymptotic theory  <cit>  describes the distribution of the number of occupied  urns.
in this experiment we found  <dig>  occupied and  <dig>  empty urns.
the null and alternative hypotheses are as follows:
h0: the allocation is completely random;
ha: not h <dig>
under h <dig>  the number of empty urns is  normally distributed with mean  <dig>  and standard deviation ≈  <dig>  <cit>  , so the observed number of empty urns  is more than  <dig> standard deviations above the mean, and hence is almost impossible under h <dig> .
we therefore conclude that the distribution of cr1a/b is non-random.
a project is underway that will involve sequencing some cr <dig> positive bac clones from c.porosus so that we may  compare the structure and distribution of cr <dig> elements in the alligator and crocodylus genomes.
the sequence data can then be used for evolutionary analyses among crocodilians, birds, and non-archosaur reptiles.
identification of a crocodile c-mos gene containing bac clone
a randomly selected macroarray was hybridized with the c-mos overgos , and fortuitously one of the  <dig>  double-spotted clones on the array exhibited a positive signal .
the plate and well address of the clone was determined based upon the macroarray number, the location of the positive signal on the macroarray, and the spatial relationship between the two spots .
to make sure that the correct clone was identified, a hand-held plate gridding/replicating device was used to stamp two nylon filters with the clones in the 384-well plate believed to contain the clone of interest.
blot hybridization using the c-mos overgo probes verified that the plate and well address obtained from the filter were correct .
the duplicate filter was probed with the cr1b overgos.
of note, the c-mos positive clone shows no visible hybridization with the cr1b sequence .
pcr with the cmf <dig> and cmr <dig> primers was used to independently verify the presence of the c-mos locus in the positive bac clone.
the c. porosus c-mos gene
subcloning was used to isolate a  <dig>  kb region containing the c-mos locus.
cycle sequencing using the cmf <dig>  cmf <dig>  cmr <dig>  and cmr <dig> primers  and assembly of the reads resulted in production of a continuous  <dig>  bp sequence .
this sequence contains the entire coding sequence  for c-mos as well as >  <dig> bp upstream and downstream of the cds.
like most characterized c-mos sequences  <cit> , c. porosus c-mos possesses a single orf.
the orf, which starts at base  <dig> and ends with the stop codon tga , codes for a  <dig> amino acid protein.
alignment of the c. porosus c-mos cds with those of chicken, zebra finch, and green anole orthologs  reveals considerable nucleotide and amino acid conservation .
interestingly, the c. porosus cds is six nucleotides  longer than the chicken, zebra finch, and green anole c-mos genes.
these two additional amino acids are adjacent to each other and are located at the c-terminal end of the c. porosus c-mos protein .
crocodilian sequence and comparative genomics
comparative genomics research is a burgeoning field with high potential to increase our understanding of the structure, function, and evolution behind the diversity of life.
however, the primary focus of most efforts over the past several years has been on comparisons among mammals.
for example, miller et al.
recently created a 28-way alignment of available vertebrate genomes in which only eight taxa, gallus, anolis, xenopus and five fish represent the entirety of non-mammalian vertebrates.
understanding the evolution and interrelationships among all amnotes will be severely hindered by this lack of diversity.
ongoing projects to sequence the green anole  and the painted turtle  will help correct the disparity but one significant lineage of the amniote tree remains to be addressed – crocodylia.
it is our hope that the generation of this library will facilitate genomics research in this critical lineage.
CONCLUSIONS
we have constructed a high quality  <dig> × bac library for crocodylus porosus and demonstrated the library's utility as a genomics tool.
we are currently screening the library with other genes and repeat sequences as a means of investigating the structure of the australian saltwater crocodile genome and facilitating comparative genomics research among archosaurs.
copies of the bac library, individual clones, and macroarrays can be obtained from the mississippi genome exploration laboratory  <cit> .
competing interests
the authors declare that they have no competing interests.
authors' contributions
the project was conceived through discussions between dgp and dar.
xs constructed the bac library and performed all bench research in the lab of dgp.
experiments were designed by xs with the input of dgp and dar.
dar provided sequence data for cr <dig> analyses of the library and made arrangements associated with acquisition of crocodile blood.
jab performed the statistical analysis associated with cr <dig> distribution.
all authors were involved in manuscript writing and editing.
the final version of the manuscript was read and approved by all authors.
supplementary material
additional file 1
table s <dig>  consensus sequences of highly conserved portions of crocodilian cr1a and cr1b subfamily elements.
table s <dig>  putative c-mos cds for platypus mined from the current genome assembly amended so that the two overlapping but out of frame orfs are combined into a single presumed c-mos cds.
figure s <dig>  muscle-based nucleotide alignment of the complete coding sequences of a. carolinensis, c. porosus, g. gallus, and t. guttata c-mos genes.
figure s <dig>  muscle-based amino acid alignment of the complete coding sequences of a. carolinensis, c. porosus, g. gallus, and t. guttata c-mos genes.
click here for file
acknowledgements
we thank sally isberg, lee miles, and travis glenn for helping us obtain the crocodile blood and lauren dembeck for conducting the initial cr <dig> analyses of a. mississippiensis, o. tetraspis, and c. moreletii.
this research was supported, in part, by usda award ars-58-6402-7- <dig>  and national science foundation award dbi- <dig> .
dar was supported by the eberly college of arts and sciences at wvu.
this article has been published as part of bmc genomics volume  <dig> supplement  <dig>  2009: proceedings of the avian genomics conference and gene ontology annotation workshop.
the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2164/10?issue=s2

 crocodilians are a group of reptiles that originated roughly million years ago. they are apex predators in the marine and freshwater habitats in which they reside, and they play a major role in warm-water ecosystems throughout the world. there are extant species grouped into three families – crocodilidae, alligatoridae, and gavialidae. as evidenced by their frequent appearances in video documentaries and television programs, crocodilians are a subject of considerable human curiosity. moreover, these reptiles have been common subjects/characters in mythology, folk tales, art, and literature suggesting that they have considerable symbolic and practical significance in the lives of humans, past and present. crocodilians, birds, dinosaurs, and pterosaurs form a monophyletic group known as the archosauria of which only the crocodilian and the avian lineages are extant. in support of this premise, molecular phylogenetic evidence from nuclear and mitochondrial dna sequencing indicates that crocodilians and birds are indeed each other's closest living relatives. among archosaurs, only the chicken and zebra finch have been the focus of complete genome sequencing efforts. genome level analyses of a crocodilian would be especially useful in leveraging information from the chicken, and a crocodilian genome would be the best possible outgroup for all genomic work within birds. however, with the modest exception of alligator mississippiensis for which there is roughly mb of bac end sequence and partially assembled bacs, little sequence data is available for crocodilian genomes, in part due to a relative lack of high-quality molecular tools for this important clade. for roughly years, bacterial artificial chromosome libraries have been the principal molecular substrate used in physical mapping and complete eukaryote genome sequencing. gridding of ordered bac libraries onto macroarrays and multiplex screening techniques have facilitated rapid gene isolation. the utility of bac clones as substrates for end sequencing, in conjunction with advanced dna fingerprinting techniques and macroarray analysis, has permitted construction of robust physical maps and selection of minimum tiling paths for accurate genome sequencing and assembly. recent advances in sequencing technologies have created powerful opportunities in which ordered bac libraries play a critical role. a particularly promising strategy for simultaneous physical mapping and sequencing of large eukaryotic genomes involves sequencing pools of sheared, individually "bar coded" bac clones. after sequencing, those reads sharing a bar code are grouped together and assembled in silico, and physical maps are constructed by identifying overlapping assembled or partially assembled bac sequences. to expedite genome research in crocodilians, we have constructed a bac library for the australian saltwater crocodile. the c. porosus library is only the second large-insert dna library for a crocodilian – a 10× library exists for alligator mississippiensis – and the only bac library for crocodilidae, the largest of the crocodilian families. c. porosus is the largest living crocodilian and, along with a. mississippiensis, the only crocodilian species to be commercially farmed. here we describe generation and characterization of the c. porosus bac library and demonstrate its utility as a tool for gene isolation, genome characterization, and comparative genomics. methods preparation of nuclei agarose plugs from crocodile blood sample whole blood was obtained from errol, a male c. porosus from the darwin crocodile farm near darwin, australia. blood was suspended in citrate buffer containing 5% v/v dimethylsulfoxide, aliquoted into ml polypropylene tubes, flash frozen in liquid nitrogen, and shipped to the mississippi genome exploration laboratory. one of the tubes was thawed on ice and centrifuged at rpm in a microcentrifuge for min. the supernatant was decanted, the pellet was gently re-suspended in ml of stex buffer, and the mixture was centrifuged as described above. the blood cell pellet was re-suspended in μl stex buffer and placed in a water bath at 45°c. after min, the blood suspension was mixed with an equal volume of 45°c 2% w/v cambrex seaplaque agarose in stex buffer. the mixture was poured into a small petri dish so that the depth of the solution was roughly mm. after min at 4°c, the resulting gel was cut into × mm rectangles, and these "plugs" were transferred into a conical ml polypropylene tube containing ml of lysis buffer. the capped tube was incubated at 37°c overnight with gentle agitation. plugs were transferred into m edta containing m phenylmethylsulfonyl fluoride and incubated at 4°c for one hour. plugs were washed in medta and then stored at 4°c in this buffer. preparation of high-molecular-weight insert dna a few test dna plugs were exposed to different hindiii concentrations to determine conditions providing the largest number of fragments between to kb. the optimal enzyme concentration as determined in the test digests was used in a large-scale partial digest. plugs used in the mass digestion were macerated and placed in a slot well of a 1% w/v cambrex seakem gold agarose gel in × tbe buffer. size selection of partially digested dna was performed using pulsed-field gel electrophoresis according to chalhoub et al.. size-selected hindiii fragments between and kb were recovered from agarose by electroelution according to peterson et al.. bac library construction bac library construction was performed as described in peterson et al. using the pindigobac- vector and electromax dh10b t phage-resistant competent cells. clone picking and library replication were performed using a genetix qpixii robot. to monitor the quality of the bac library and determine mean insert size, bac clones from every fiftieth 384-well plate were evaluated by noti digestion and pfge. for these analyses bac dna was isolated using an autogenprep robot. macroarray construction high density macroarrays were prepared using a genetix qpixii robot. each array consisted of double-spotted bac clones stamped onto a cm hybond n+ filter. there were enough c. porosus bac clones to produce five complete macroarrays. stamped arrays were placed clone-side up on lb agar containing mg/l chloramphenicol and incubated at 37°c overnight. each macroarray was fixed via incubation in n naoh, m nacl for min followed by incubation in m nacl, m tris cl for min. the membranes were allowed to air dry for h, treated with n naoh for min, and washed in 5× sspe for min. macroarrays were air dried and stored in sealed plastic bags. probe design and bac library screening overgo probes were designed from the consensus sequences of cr1a and cr1b, two recently active cr subfamilies present in the genomes of crocodylus moreletii and osteolaemus tetraspis, and from a partial cdna sequence of the c. porosus c-mos gene. the overgos were labeled with 32p and hybridized to macroarrays as previously described. probe hybridization images were recorded and analyzed using a storm phosphoimager. for the cr1-probed macroarrays, the spot densitometry tool in alphaeasefc, version. was used to explore variation in spot intensity and estimate copy number as described previously. positive c-mos clones were confirmed by pcr using the cmf1/cmr primer pair. pcr was performed using the following thermocycler series: 95°c denaturation for min, full pcr cycles, and a final 72°c min extension step. subcloning and sequencing of the c. porosus c-mos gene a bac clone containing the c. porosus c-mos gene was digested with bamhi and hindiii at 37°c for hr followed by heating to 65°c for min. the cloning vector pcrii-topo was likewise double-digested, purified by electrophoresis on a 1% w/v agarose gel, and isolated from agarose using a qiagen qiaquick gel extraction kit. ligation was performed at 16°c for hrs. the ligation mixture was used to transform chemically competent invitrogen one shot top cells according to the manufacturer's instructions. subclones were plated and were screened by pcr using the c-mos primers cmf and cmr. one positive subclone, which was shown by gel electrophoresis to contain a kb insert, was sent to seqwright for cycle sequencing using the cmf and cmr primers and two additional primers. use of a combination of primers was intended to extend the target area so that it would encompass the entire c-mos coding sequence and several hundred bases 5' and 3' of the coding region. base-calling and assembly of sequence reads were performed using phred and phrap, respectively. trimming of the assembled c-mos sequence was conducted using cross_match. the bp product was submitted to genbank and assigned the accession fj  bac library coverage the c. porosus bac library consists of individual clones stored in bar-coded 384-well microtiter plates. noti digestion and pfge of bac clones indicates an average clone insert size of kb with % of clones possessing inserts > kb, % with inserts between and kb, and % with inserts smaller than kb. internal noti sites are present in % of pfge-examined bac clones, and % of clones are probable false positives, i.e., they possess a vector band but no insert band. collectively, the entire bac library represents gb of crocodile genomic dna. the exact genome size of c. porosus is unknown. however, measurements made for two other crocodylus species are both mb. assuming the c. porosus genome size is similar to those of these closely allied taxa, we estimate that the library affords × coverage of the c. porosus genome. theoretically, this level of coverage affords 98% probability of finding any given genomic sequence at least once in the library. survey of cr elements in crocodile genome cr elements are non-ltr retrotransposons existing in high copy numbers in bird and reptile genomes; there are about cr elements in the chicken genome. cr retrotransposons are considered excellent markers for molecular phylogenetic and population genetic studies. initial studies on the sequences from the bac clones of alligator mississippiensis available in genbank revealed that at least two cr subfamilies, referred to here as cr1a and cr1b, have recently been active in crocodilian genomes. this observation is consistent with shedlock et al. in which the authors suggested that multiple cr lineages may have been active in alligators. in addition to a. mississippiensis, cr1a and cr1b have been identified in crocodylus moreletii, and osteolaemus tetraspis and consensus sequences for conserved regions of these elements have been generated. to explore cr distribution in c. porosus, macroarrays were screened with cr1b-derived overgos or a combination of cr1a and cr1b overgos. comparison of the cr1b- and cr1a/b-probed macroarrays revealed that the cr1a and cr1b subfamilies are not distinguishable in our assay, i.e., virtually no differences in hybridization pattern and intensity were observed when comparing the cr1b and cr1a/cr1b filters. it was clear, however, that elements similar to cr1a and b are fairly abundant in c. porosus. examination of one-quarter of the cr1a/b-probed macroarray indicates that % of clones show hybridization to the cr1a/b overgos while cr1b overgo hybridization to a filter stamped with the contents of a single 384-well plate from the bac library suggests that % of clones are positive for the cr1b overgo. densitometric analysis of the macroarray reveals that there is a six-fold variation in positive clone hybridization intensity. if the lightest, but clearly positive hybridization signals represent clones containing one copy of a cr1a/b element and if the darkest clones contain six elements, there would be approximately copies of cr1a/b in the c. porosus genome. however, there is the possibility that macroarray exposure conditions were such that only clones with multiple copies of cr1a/b elements appear positive; inclusion of a single-copy sequence control on the macroarray could be used to test this possibility. moreover, the use of overgos rather than whole cr sequences clearly limits hybridization to those elements where the regions corresponding to the overgos have been conserved. based on analysis of mb of bac end sequence, shedlock estimated the number of cr elements in alligator mississippiensis at copies. while a. mississippiensis and c. porosus are separated by million years of evolution, it seems reasonable that they might have similar total numbers of cr elements. consequently, the c. porosus copy number estimate of should be viewed as a minimum number of cr1a/b elements, not total cr elements, in the c. porosus genome. statistical analysis of the macroarray data indicates that cr1a/b elements are not randomly distributed throughout the c. porosus genome. the macroarray contains individual clones with an average insert size of kb. consequently, a single macroarray represents roughly genome equivalents, i.e., ÷ mb. if the crocodile genome contains copies of cr1a/b, then we would expect approximately copies of cr1a/b per macroarray, and if these were distributed randomly we would expect, on average, copies of cr1a/b per clone. however, only % of clones on the macroarray show hybridization to the cr1a/b probes. to test whether such a distribution is likely by chance, we can formulate the problem as a statistical "urn model." suppose we have urns, and we drop balls into them at random. in such a case, classical statistical asymptotic theory describes the distribution of the number of occupied urns. in this experiment we found occupied and empty urns. the null and alternative hypotheses are as follows: h0: the allocation is completely random; ha: not h under h the number of empty urns is normally distributed with mean and standard deviation ≈, so the observed number of empty urns is more than standard deviations above the mean, and hence is almost impossible under h. we therefore conclude that the distribution of cr1a/b is non-random. a project is underway that will involve sequencing some cr positive bac clones from c.porosus so that we may compare the structure and distribution of cr elements in the alligator and crocodylus genomes. the sequence data can then be used for evolutionary analyses among crocodilians, birds, and non-archosaur reptiles. identification of a crocodile c-mos gene containing bac clone a randomly selected macroarray was hybridized with the c-mos overgos, and fortuitously one of the double-spotted clones on the array exhibited a positive signal. the plate and well address of the clone was determined based upon the macroarray number, the location of the positive signal on the macroarray, and the spatial relationship between the two spots. to make sure that the correct clone was identified, a hand-held plate gridding/replicating device was used to stamp two nylon filters with the clones in the 384-well plate believed to contain the clone of interest. blot hybridization using the c-mos overgo probes verified that the plate and well address obtained from the filter were correct. the duplicate filter was probed with the cr1b overgos. of note, the c-mos positive clone shows no visible hybridization with the cr1b sequence. pcr with the cmf and cmr primers was used to independently verify the presence of the c-mos locus in the positive bac clone. the c. porosus c-mos gene subcloning was used to isolate a kb region containing the c-mos locus. cycle sequencing using the cmf cmf cmr and cmr primers and assembly of the reads resulted in production of a continuous bp sequence. this sequence contains the entire coding sequence for c-mos as well as > bp upstream and downstream of the cds. like most characterized c-mos sequences, c. porosus c-mos possesses a single orf. the orf, which starts at base and ends with the stop codon tga, codes for a amino acid protein. alignment of the c. porosus c-mos cds with those of chicken, zebra finch, and green anole orthologs reveals considerable nucleotide and amino acid conservation. interestingly, the c. porosus cds is six nucleotides longer than the chicken, zebra finch, and green anole c-mos genes. these two additional amino acids are adjacent to each other and are located at the c-terminal end of the c. porosus c-mos protein. crocodilian sequence and comparative genomics comparative genomics research is a burgeoning field with high potential to increase our understanding of the structure, function, and evolution behind the diversity of life. however, the primary focus of most efforts over the past several years has been on comparisons among mammals. for example, miller et al. recently created a 28-way alignment of available vertebrate genomes in which only eight taxa, gallus, anolis, xenopus and five fish represent the entirety of non-mammalian vertebrates. understanding the evolution and interrelationships among all amnotes will be severely hindered by this lack of diversity. ongoing projects to sequence the green anole and the painted turtle will help correct the disparity but one significant lineage of the amniote tree remains to be addressed – crocodylia. it is our hope that the generation of this library will facilitate genomics research in this critical lineage.  we have constructed a high quality × bac library for crocodylus porosus and demonstrated the library's utility as a genomics tool. we are currently screening the library with other genes and repeat sequences as a means of investigating the structure of the australian saltwater crocodile genome and facilitating comparative genomics research among archosaurs. copies of the bac library, individual clones, and macroarrays can be obtained from the mississippi genome exploration laboratory. competing interests the authors declare that they have no competing interests. authors' contributions the project was conceived through discussions between dgp and dar. xs constructed the bac library and performed all bench research in the lab of dgp. experiments were designed by xs with the input of dgp and dar. dar provided sequence data for cr analyses of the library and made arrangements associated with acquisition of crocodile blood. jab performed the statistical analysis associated with cr distribution. all authors were involved in manuscript writing and editing. the final version of the manuscript was read and approved by all authors. supplementary material additional file 1 table s consensus sequences of highly conserved portions of crocodilian cr1a and cr1b subfamily elements. table s putative c-mos cds for platypus mined from the current genome assembly amended so that the two overlapping but out of frame orfs are combined into a single presumed c-mos cds. figure s muscle-based nucleotide alignment of the complete coding sequences of a. carolinensis, c. porosus, g. gallus, and t. guttata c-mos genes. figure s muscle-based amino acid alignment of the complete coding sequences of a. carolinensis, c. porosus, g. gallus, and t. guttata c-mos genes. click here for file acknowledgements we thank sally isberg, lee miles, and travis glenn for helping us obtain the crocodile blood and lauren dembeck for conducting the initial cr analyses of a. mississippiensis, o. tetraspis, and c. moreletii. this research was supported, in part, by usda award ars-58-6402-7- and national science foundation award dbi-. dar was supported by the eberly college of arts and sciences at wvu. this article has been published as part of bmc genomics volume supplement 2009: proceedings of the avian genomics conference and gene ontology annotation workshop. the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2164/10?issue=s2
consequently, crocodilian genomes represent a gateway through which the molecular evolution of avian lineages can be explored. to demonstrate the utility of the library in gene isolation, we probed the c. porosus macroarrays with an overgo designed from a c-mos partial cdna. the c. porosus bac library consists of individually archived clones stored in 384-well microtiter plates. based on a genome size estimate of mb, the library affords fold coverage of the c. porosus genome. they are the only extant reptilian members of archosauria, a monophyletic group that also includes birds, dinosaurs, and pterosaurs. noti digestion of random clones indicates an average insert size of kb. a bac containing c-mos was identified and the c-mos locus was sequenced. to facilitate comparative genomics within crocodylia and between crocodilians and other archosaurs, we have constructed a bacterial artificial chromosome library for the australian saltwater crocodile, crocodylus porosus. we have demonstrated the utility of the crocodylus porosus bac library as a tool in genomics research. the results indicate that there are a minimum of cr1a/b elements in c. porosus and that their distribution throughout the genome is decidedly non-random. to investigate the utility of the library in studying sequence distribution, probes derived from cr1a and cr1b, two crocodilian cr1-like retrotransposon subfamilies, were hybridized to c. porosus macroarrays. nucleotide and amino acid sequence alignment of the c. porosus c-mos coding sequence with avian and reptilian c-mos orthologs reveals greater sequence similarity between c. porosus and birds than between c. porosus and squamates. the bac library should expedite complete genome sequencing of c. porosus and facilitate detailed analysis of genome evolution within crocodylia and between crocodilians and diverse amniote lineages including birds, mammals, and other non-avian reptiles. this is the first bac library for a crocodile and only the second bac resource for a crocodilian.
 crocodilians are an ancient vertebrate group of tremendous ecological, social, and evolutionary importance. they are the only extant reptilian members of archosauria, a monophyletic group that also includes birds, dinosaurs, and pterosaurs. consequently, crocodilian genomes represent a gateway through which the molecular evolution of avian lineages can be explored. to facilitate comparative genomics within crocodylia and between crocodilians and other archosaurs, we have constructed a bacterial artificial chromosome library for the australian saltwater crocodile, crocodylus porosus. this is the first bac library for a crocodile and only the second bac resource for a crocodilian.  the c. porosus bac library consists of individually archived clones stored in 384-well microtiter plates. noti digestion of random clones indicates an average insert size of kb. based on a genome size estimate of mb, the library affords fold coverage of the c. porosus genome. to investigate the utility of the library in studying sequence distribution, probes derived from cr1a and cr1b, two crocodilian cr1-like retrotransposon subfamilies, were hybridized to c. porosus macroarrays. the results indicate that there are a minimum of cr1a/b elements in c. porosus and that their distribution throughout the genome is decidedly non-random. to demonstrate the utility of the library in gene isolation, we probed the c. porosus macroarrays with an overgo designed from a c-mos partial cdna. a bac containing c-mos was identified and the c-mos locus was sequenced. nucleotide and amino acid sequence alignment of the c. porosus c-mos coding sequence with avian and reptilian c-mos orthologs reveals greater sequence similarity between c. porosus and birds than between c. porosus and squamates.  we have demonstrated the utility of the crocodylus porosus bac library as a tool in genomics research. the bac library should expedite complete genome sequencing of c. porosus and facilitate detailed analysis of genome evolution within crocodylia and between crocodilians and diverse amniote lineages including birds, mammals, and other non-avian reptiles. 19– may avian genomics conference and go annotation workshop starkville, ms, usa 