Wenbo Shn，Ynqin Jing，Jinlei Hn，Ki Wng，c，*

aHaixia Institute of Science and Technology（HIST），Center for Genomics and Biotechnology，F(xiàn)ujian Agriculture and Forestry University，F(xiàn)uzhou 350002，China

bInstitute of Botany，Jiangsu Province and Chinese Academy of Sciences，Nanjing 210014，China

cCollege of Crop Science，F(xiàn)ujian Agriculture and Forestry University，F(xiàn)uzhou 350002，China

?

Comprehensive cytological characterization of the Gossypium hirsutum genome based on the development of a set of chromosome cytological markers

Wenbo Shana，1，Yanqin Jiangb，1，Jinlei Hana，Kai Wanga，c，*

aHaixia Institute of Science and Technology（HIST），Center for Genomics and Biotechnology，F(xiàn)ujian Agriculture and Forestry University，F(xiàn)uzhou 350002，China

bInstitute of Botany，Jiangsu Province and Chinese Academy of Sciences，Nanjing 210014，China

cCollege of Crop Science，F(xiàn)ujian Agriculture and Forestry University，F(xiàn)uzhou 350002，China

A R T I C L E I N F O

Article history:

in revised form

14 March 2016

Accepted 29 March 2016

Available online 14 April 2016

Cotton

Karyotype

Chromosomal size variation

Cytological characterization

Fluorescence in situ hybridization

A B S T R A C T

Cotton is the world's most important natural fiber crop.It is also a model system for studying polyploidization，genomic organization，and genome-size variation.Integrating the cytological characterization of cotton with its genetic map will be essential for understanding its genome structure and evolution，as well as for performing further genetic-map based mapping and cloning.In this study，we isolated a complete set of bacterial artificial chromosome clones anchored to each of the 52 chromosome arms of the tetraploid cotton Gossypium hirsutum.Combining these with telomere and centromere markers，we constructed a standard karyotype for the G.hirsutum inbred line TM-1.We dissected the chromosome arm localizations of the 45S and 5S rDNA and suggest a centromere repositioning event in the homoeologous chromosomes AT09 and DT09.By integrating a systematic karyotype analysis with the genetic linkage map，we observed differentgenomesizesandchromosomalstructuresbetweenthesubgenomesof the tetraploid cotton and those of its diploid ancestors.Using evidence of conserved coding sequences，we suggest that the different evolutionary paths of non-coding retrotransposons account for most of the variation in size between the subgenomes of tetraploid cotton and its diploid ancestors.These results provide insights into the cotton genome and will facilitate further genome studies in G.hirsutum.

?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.

1.Introduction

Cotton（genus Gossypium）is one of the most important natural fiber and edible oil crops in the world.The genus is composed of 45 diploid and 5 tetraploid species.Among them，four species are cultivated:G.hirsutum（2n=4×=52），G.barbadense（2n=4×=52），G.arboreum （2n=2×=26），and G.herbaceum （2n=2×=26）. In contrast to the three less-utilized species（G.barbadense，G. arboreum，and G.herbaceum），G.hirsutum（or upland cotton）has been cultivated worldwide and currently accounts for the majority（＞90%）of the world's fiber production［1］.The tetraploid cotton G.hirsutum has a relatively large genome［2-4］，which emerged froman interspecific hybridization betweenG.arboreum andG.raimondii1-2millionyearsago（MYA）［5］.G.arboreumandG. raimondii（designated as A and D，and ATand DTin the tetraploid cotton，respectively）divergedfromacommonancestor5-10MYA［6］.Whole-genome sequencing has revealed that more than 60% of their genomes contain repetitive sequence［3，4，7，8］，especially in the A and ATgenomes，which is nearly twofold larger than the D and DTgenomes owing to the proliferation of retrotransposons during the past five million years［3，4，8］.In contrast，their encoding sequences are still highly conserved after long-term evolution［7，8］（or diploidization）following the formation of the tetraploid species［3，4，9-11］.

Although the relatively large genome size（885-2500 Mb）［2］and high concentration of repetitive elements have hindered studies of the cotton genome，extensive progress has been made in the study of the composition of and alterations in the cotton genome，including recent global surveys of the complexity of tetraploid cotton and its two ancestors［3，4，7，8，12］.However，conflicting values for the sizes of individual and whole genomes（Table S1）from whole-genome-sequencing（WGS）data indicate that efforts remain to be made to fill gaps and align unassembled contigs［3，4］.

Cytogenetic analysis using fluorescence in situ hybridization（FISH）is a powerful tool for the analysis and physical mapping of the genome［13，14］.Importantly，it can provide a global view of the structure of individual and whole genomes in vivo，given that the cytogenetic approach is based on the analysis of intact chromosomes.However，in contrast to the rapid progress in the molecular studies of cotton，only limited efforts have been devoted to the cytological characterization of the cotton genome. We have devoted substantial effort to the development of FISH technologies that involve mapping probes in cotton metaphase chromosome，pachytene chromosome，and extended chromatin fibers［15-17］.For the purpose of karyotype analysis and whole-genome sequencing of tetraploid cotton，we constructed a high-resolution map of the homoeologous chromosomes AT12 and DT12 based on pachytene chromosomes［18］.In the present study，to develop a global view of tetraploid cotton genome in vivo，weisolatedacompletesetofbacterialartificialchromosome（BAC）clones anchored to each of the 52 chromosomearmsof the tetraploid cotton G.hirsutum.Using telomere and centromere markers，we performed the first systematic karyotype analysis of upland cotton.We report that the subgenomes of tetraploid cotton have different chromosomal structures and sizes from those of their diploid ancestors，suggesting that the ATand DTgenomes followed different evolutionary paths after the formation of tetraploid cotton.These karyotype data and chromosome arm-specific markers provide a foundation for the cytological characterization of the cotton genome and will also improve our knowledge of the structure and evolution of the cotton genome.

2.Materials and methods

2.1.Materials

The G.hirsutum TM-1 inbred line was used for cytological studies. All BACs used for FISH mapping were identified by screening genomic BAC libraries of G.hirsutum［19］.SSR markers used for BAC screening were selected from a high-density genetic map of tetraploid cotton［20］.

The Arabidopsis telomeric sequence（TTTAGGG）was isolated by PCR as described previously［15］.BAC 97G20，containing the repetitive sequence specific to cotton centromeres［18］，was used for FISH analysis.

2.2.Chromosome preparation

Mitotic chromosome spreads were prepared as previously described［21］with several modifications.Briefly，seeds were germinated on wet filter paper in Petri dishes.Roots about 2 cm long were cut and pretreated with 25 mg mL-1cycloheximide at 30°C for 2 h to accumulate metaphase cells，and fixed in ethanol:acetic acid（3:1）fixative.Root tips were macerated in 2.0%cellulase and 0.5%pectolyase at37°C for 1 h and squashed with 45%acetic acid.After removal of cover slips，slides were dehydrated through an ethanol series（70%，90%，and 100%；5 min each）prior to use in FISH.

2.3.Fluorescence in situ hybridization

Single-ordual-colorFISHwasperformedaspreviouslydescribed［21］.For the two rounds of FISH，slides from the first-round hybridization were washed in 1×PBS to remove the cover slips. Slideswerethenwashedin1×PBSthreetimesfor15 mineachat 42°C to remove antifade solution.After cleaning，slides were dehydrated in ethanol series（70%，90%，and 100%；5 min each）and used in the second round FISH.One microgram of TM-1 Cot-1 DNA was used in FISH to block repetitive sequences that might cause nonspecific hybridization.For the ATand DTsubgenomes in tetraploid cotton，considerable conservation between homoeologous chromosomes［3，4］can cause nonspecific signals in homoeologous chromosomes under relatively low-stringency treatment［10，18］.To avoid this nonspecific hybridization，we performed high stringency hybridization（70%formamide in 2×SSC at 40°C，stringency 98.5%）［22］，so that only targets with high similarity（＞98%）to probes could hybridize stably.Biotin-and digoxigenin-labeled probes were detected using rhodamine-conjugated anti-digoxigenin（Roche Diagnostics，USA）andfluorescein-conjugatedavidin（Life Technologies，USA），respectively.Chromosomes were counterstained with DAPI（4′，6-diamidino-2-phenylindole；Sigma，USA）and antifade（Vector，USA）under a coverslip.

2.4.Image analysis

Slides were examined under an Olympus BX63 fluorescence microscope.The gray images of chromosome and FISH signal channels were captured and merged using cellSens Dimension1.9 software with an Olympus DP80 CCD camera.Final image adjustmentswereperformedusingAdobePhotoshop8.0 software.For karyotyping images，20 cells without apparent morphological distortion were analyzed.Signal position and chromosome length were measured with cellSens Dimension，andthearmratio（longarm/shortarm），totalchromosomelength（short arm+long arm），and relative chromosome length（length of the individual chromosome/total length of all chromosomes）were calculated.

3.Results and discussion

3.1.Development of chromosome arm-specific markers in G. hirsutum

Identification of individual cotton chromosomes is difficult，owing to their small size and nearly uniform appearance under microscopy（most are metacentric；see below）.In previous studies，BACs bearing molecular markers have been shown to be excellent cytological markers for the identification of individual chromosomes in cotton［21，23］.To develop reliable markers for chromosome arm identification，we isolated a set of 52 BACs hybridizing to each of the 52 chromosome arms of G.hirsutum. Simple sequence repeat（SSR）markers from the high-density genetic linkage map［20］were selected for screening the BAC library.The positive BACs were then used in FISH to test their signal strength and chromosomal localization by hybridizing them simultaneously with the chromosomally anchored BACs［23］.Finally，only the BACs that consistently produced unambiguous，brightFISHsignalswereselectedaschromosome arm-specific markers（Table 1，F(xiàn)ig.1-A，B）.

To verify that the identified BACs were indeed located on different arms of the individual chromosomes，the centromerespecific BAC 97G20［24］was hybridized with the arm-specific BACs for the individual chromosomes in a FISH experiment.As showninFig.1-A，thesetwoarm-specificBACsareclearlylocated on opposing arms of their respective chromosomes.

During interphase，chromosomes uncoil into long chromatin strings.Reducing the condensation of chromatin can markedly improve the resolving power of FISH assay［25］. However，this operation leads to a decrease in signal intensity，especially for short，single-copy probes［22，26，27］.In contrast，each BAC clone contains a large（approximately 100-kb）insert and can thus be easily detected in interphase FISH［28，29］.To verify that our chromosome arm-specific BACs could serve as reliable cytological markers in both metaphase and interphase in cotton，we performed a FISH assay on the interphase nuclei.As shown in Fig.1-B，BACs 0331 N02 and 010G17 in chromosomeDT12showedclearsignalsthatcouldbe detected in the majority of nuclei（82%，n=50）.

Table 1-Chromosome arm-specific BACs and SSR markers of G.hirsutum.

3.2.Chromosome arm-specific BACs can serve as cross-species markers in Gossypium

One important application of arm-specific BACs is their use as cross-species cytological markers for chromosome identification or comparative mapping among different species［30-35］.Incotton，upland cotton BACs have been shown to be excellent cytological markers for identifying specific chromosomes in its diploid ancestors，G.arboreum［17］and G.raimondii［36］.Diploid Gossypium species fall into eight different genome types designated as A-G and K，based on meiotic pairing behavior［37，38］.To further evaluate the use of our BACs as cytological markers in other diploids，two diploids，G.australe（G genome）and G.longicalyx（F genome），were hybridized using our TM-1 BACs.Four BACs（from chromosomes AT02，03，04 and 09）were tested in FISH using metaphase chromosomes of G. australe and G.longicalyx.Clear signals（Fig.1-C，D）were detected from all of the BACs，with the exception of 094 K09，which produced a high background in chromosomes of G. longicalyx（data not shown），suggesting the presence of high-copy-number repetitive sequences.However，relatively weak signals could still be detected when more blocking DNA （cot-1DNA，2 μg）wasadded（datanotshown）. Nonetheless，the large BAC inserts retain remnants from different wild species，making them excellent markers for the chromosome identification or comparative mapping of Gossypium species.

Fig.1-Upland cotton chromosome arm-specific BACs and their cross-hybridization in diploid cotton.（A）Twenty-six TM-1 chromosomes were FISHed with 52 chromosome arm-specific BACs.The chromosomes are positioned with the shorter arm at the top；thus，the red and green signals are derived from BACs in the short and long arms，respectively（Table 1）.The centromere-specific BAC 97G20（yellow）was used to locate the centromeres.（B）Interphase nuclei and metaphase chromosomes from root tip cells of TM-1 hybridized to BACs 0331 N02（green，arrowhead）and 010G17（red，arrow）on chromosome DT12.Two copies of the FISH signals were detected in both interphase nuclei and metaphase chromosomes.（C）and（D）Cross-hybridization using the upland cotton BACs 027E16（red，arrow）.Clear signals were detected on metaphase chromosomes of the two diploid species G.australe（G genome）（C）and G.longicalyx（F genome）（D）.The scale bars represent 10 μm.

3.3.A standard karyotype of G.hirsutum

Chromosome arm-specific BACs provide us with a powerful tool to unambiguously identify each cotton chromosome. However，in the majority of the previous karyotype analyses，the centromere and telomere were only roughly identified based on the microscopic appearance of the chromosomes（the primary constriction and the end of the chromosome，respectively）.To develop a standardized cotton karyotype，the centromere-specific BAC 97G20 and the Arabidopsis telomere repeat TTTAGGG ［39］were used to precisely locate the centromeres and ends of chromosomes in our karyotype analysis.The highly inbred G.hirsutum line TM-1 has been widelyusedincottongeneticmappingandhasbeen sequenced［1，3，4，40］.Accordingly，we used it for karyotype construction.In addition，to preserve the morphology of the chromosomes and unambiguously identify each chromosome arm，we performed a two-round sequential FISH analysis. First，the centromere and telomere probes were simultaneously used in the first-round FISH experiment（Fig.2-A）. Then the chromosome arm-specific BACs were hybridizedonto the same slide to identify the corresponding chromosome arms（Fig.2-B）.The karyotype measurements were all performed on the chromosomes derived from the first-round FISH experiment.

Fig.2-Sequential FISH for the karyotyping and rDNA loci analysis of G.hirsutum.（A and B）An example of sequential FISH for the karyotyping analysis of G.hirsutum.The telomere（green）and centromere（red）probes were first hybridized to the metaphase chromosome（A）.Then，the chromosome arm-specific BACs 045 L24（white）and 009 N05（yellow）for chromosome AT07 were hybridized to identify the individual chromosome and its arms（B）.（C-F）Sequential FISH to identify the distributions of 45S and 5S rDNA loci in upland cotton.Centromere and 45S and 5S rDNA probes were first hybridized to the metaphase chromosome（C and E）.Then，the chromosome arm-specific BACs 075 M03（AT09）and 016C21（DT09）were hybridized with the centromere BAC to identify the corresponding chromosome arms（D and F）.（G）FISHing using the probes for centromere，45S rDNA and BAC 088 L11 of chromosome DT07 to show that 45S rDNA is located on the same（long）arm as BAC 088 L11 on chromosome DT07.The scale bar represents 1 μm.

As expected，our karyotyping results showed that the lengths of all the ATchromosomes，with the exception of chromosome AT04，are greater than those of the DTchromosomes at mitotic metaphase（Table 2）.Chromosome AT04（2.52±0.39 μm）is slightly shorter than the largest DTchromosome，DT07（2.55±0.25 μm），and a t test confirmed that they are not significantly different in length（P=0.819）.The longest chromosome is AT10，which is 3.51 μm（131.0 Mb）long and has a relative length of 5.40%.The shortest chromosome is DT04，which is 1.59 μm （59.4 Mb）long and has a relative length of 2.45%.Overall，none of the chromosomes were exceptionally long or short.The majority of the chromosomes are metacentric（1.01＜arm ratio＜1.70）［41］，meaning that their arms are relatively equal in length.Three chromosomes，AT12，AT13 and DT12，with arm ratios of 1.77，1.90 and 1.74，respectively，were classified as submetacentric（1.71＜arm ratio＜3.00）［41］.Still，there are no chromosomes with an extremely long or short arm（arm ratio＞3）.

Recently，WGS data for TM-1 have been published by two groups［3，4］.However comparison reveals conflicting valuesfor the sizes of individual chromosome in these two WGS datasets，indicating that it is difficult to correctly assemble the tetraploid cotton genome（Table S1），which contains approximately 70%repetitive DNA.Still，we find a correlation between our data and those of Zhang et al.［3］.For example，all ATchromosomes are longer than DTchromosomes，and chromosome AT04 is shorter than the other ATchromosomes（Table S1）.

Table 2-Lengths and arm ratios of the mitotic chromosomes in TM-1.

The 45S and 5S rDNA are composed of long strings of satellite sequences that always reside in specific regions of the genome and are commonly used as karyotyping landmarks.Upland cotton has three major 45S rDNA loci［42］，which are located on the short arms of chromosomes AT09（Fig.2-C，D）and DT09（Fig.2-E，F(xiàn)）and the long arm of chromosome DT07（Fig.2-G）.Two major 5S rDNA loci colocalize with the two 45S rDNA-bearing chromosomes（AT09 and DT09）（Fig.2-C，E）.As in other plants［43-45］，these 5S rDNA loci are located very close to the centromere（Fig.2-C，E）.However，it is unclear whether the 5S rDNAlocalizestothesamearmasthe45SrDNAinuplandcotton.

To address this question，we performed a sequential FISH analysis in which the 45S，5S and centromere BAC probes were hybridized simultaneously in the first round of FISH，followed by a second round of FISH using the chromosome arm-specific BACs to identify chromosomes AT09 and DT09（Fig.2-C-F）. Interestingly，the45Sand5SrDNAsiteswerefoundonthesame arm of chromosome DT09（Fig.2-E，F(xiàn)）but on different arms of chromosome AT09（Fig.2-C，D）.Because AT09 and DT09 are homoeologouschromosomes，wespeculatedthat chromosome rearrangement or centromere repositioning accounts for this phenomenon.In view of the unavailability of whole-genome sequence data，we analyzed the most saturated genetic map of tetraploid cotton［46］and found no obvious regional rearrangement between chromosomes AT09 and DT09，suggesting that a centromere repositioning event may have occurred in chromosomes AT09 or DT09.

3.4.Homoeologous chromosome size variation in upland cotton

The A and D genomes of diploid cotton have undergone a dramatic genome change and acquired a twofold difference in theirgenomesizeaftertheirdivergencefromacommonancestor［7，8，12］.However，the fate of the chromosomes after they merged into one cell remains elusive.The karyotyping data from the homoeologous chromosomes analyzed in the present study provide a global view of the changes in the ATand DTchromosomal size.As showninFig.3，all of the ATchromosomes are elongated relative to the homoeologous DTchromosomes.In this respect，with the exception of chromosomes AT02，AT07，AT09，and AT11，all of the remaining chromosomes are at least 1.5-fold longer than the DThomoeologous chromosomes.Intriguingly，none of the chromosomes showed a two-fold or greater increase in size compared to their DThomoeologous chromosomes，suggesting that the ATgenome is less than twice the size of the DTgenome in tetraploid cotton.This suggestion can be confirmed with the published WGS data，in which the ATgenomes are 1.78-and 1.55-fold larger than the DTgenomes（Table S1）.

Fig.3-The size variation between ATand DThomoeologous chromosomes in upland cotton.The size ratios of the ATand DThomoeologous chromosome sets are shown above the chromosomes.

Fig.4-A schematic illustration of chromosomal size variation in tetraploid cotton compared to its diploid ancestor.The chromosomes are depicted according to the individual FISHed chromosomes using the chromosome arm-specific BACs（green and red spots）for chromosomes AT06 and A06（A），and AT08 and A08（B）.The arm lengths（μm）are shown to facilitate the comparison of the ATarms with their homologous arms in the diploid cotton G.arboreum.

There are two possible explanations for the difference in AT/DT（ratio＜2）and A/D（ratio=2）size ratios.One is that the ATor DTgenomes evolved at different rates than their diploid progenitors.The other is that they underwent different levels of chromatin condensation，leading to the variations in size measurements.Given that heterochromatin is more tightly condensed than euchromatin during metaphase，the relative physical size（in μm）of the chromosome may not accurately reflect its DNA content.As an example，an ATchromosome with twice the DNA content（base pairs）but with more heterochromatin may not show a two-fold greater physical size at metaphase than its DThomolog.However，in comparison，we found that the total length（38.91 μm）of the ATchromosomes is less than that（42.84 μm）of its diploid ancestor G.arboretum［17］.Both WGS datasets also showed that the ATgenome（Table S1）is shorter than that of G. arboreum［17］（1694 Mb）.These findings suggest that the ATand A genomes have evolved at different rates，leading to pronounced differences in DNA contents.

To further investigate the difference in chromosomal evolution between ATand A，we compared the two chromosomes AT06 and AT08 with their A chromosomes［17］because they showed the highest ratio of chromosomal size variation from their respective homoeologous chromosomes，DT06 and DT08（Fig.3）.Similarly，AT06 and AT08 also showed higher size variation than their homoeologous chromosomes in the current WGS of Zhang et al.［3］（Table S1）.The duplicated molecular loci［20］and our BAC FISH results indicate that the short and long arms of AT06 and A06 have been reversed（Fig.4-A）.Consequently，their corresponding arms have markedly different sizes（Fig.4-A）.Although we cannot exclude the possibility of centromere repositioning or other chromosomal rearrangements causing the arm-size inversion，the chromosomal size variation between chromosomes AT06 and A06 indicates that the two arms of chromosome AT06，in contrast to chromosome A06，have undergone different evolutionarypaths（expansionordeletion）.Both armsofAT08 arelongerthan their homologous arms on chromosome A08，indicating that coordinate expansion or deletion occurred along the entire lengths of chromosomes At08 or A08.

The homoeologous chromosomes AT07 and DT07 showed no appreciable change in chromosomal size（Table 2）（P=0.140）.By contrast，wefounda45SrDNAlocusattheendofthelongarmof chromosomeDT07butnotthatofchromosomeAT07.BothAand D diploids have a 45S rDNA locus on chromosome 07［17，36］. Thus，it is likely that chromosome AT07 experienced a deletion aroundtherDNAregionthatdidnotoccurinitsdiploidancestor.

Polyploidyiscommoninmanyplantsandsomeanimals［47］. After polyploidization，the genomes may undergo genetic and epigenetic changes，leading to gene expression and phenotypic variation［48］.In cotton，duplicate genes originating from the progenitors evolve independently at the same rate as those of their diploid progenitors［49］，thus maintaining high conservation in coding regions between the A and D or ATand DTgenomes［8，10，50，51］.However，it is not known whether the non-coding regions in the ATand DTgenomes followed the same evolutionary path or at the same rate as their diploid ancestors，the A and D genomes，after the formation of tetraploid cotton.Whole-genome sequencing and global mapping have revealed that the Gorge-like retrotransposon varies greatly in copy number among the A（or AT）and D（or DT）genomes and accounts for the greatest proportion of the differences in their genome sizes［8，10］.Our results reveal pronounced differences between ATand A genomes in the size of specific chromosomes and in the total genome size.This result suggests that in comparison with the diploid ancestors，Gorge-like retrotransposons in the AT（or DT）genome followed a different evolutionary path after the formation of tetraploid cotton.

Fig.5-Sizecomparisonofthegeneticandphysicallengthsofthe DTchromosomes.（A）Diagrammatic illustration of the comparison of the relative physical distance（RPD，arm length in μm/chromosome length in μm×100）and its relative genetic distance（RGD，arm length in cM/chromosome length in cM×100）for chromosome DT07.BACs and its corresponding genetic markers are indicated on the chromosome（Chr.DT07）and its linkage group（LGDT07），respectively.（B）The plotshows comparisons of the RPD and its RGD for each arm of the 13 DTchromosomes.Shortand longarms ofeachchromosome（Table 2）are plotted as upward and downward bars，respectively.

3.5.Integration of the genetic map with the cytogenetic map in tetraploid cotton

In cotton，high-density linkage maps have recently been developed［20，46］，providingafundamentalresourcefor map-based mapping，gene cloning and further whole-genome sequencing.However，it is not known whether there is an even distribution of the loci along the chromosomes.FISH using BACs anchored to genetically mapped markers has proven to be a powerful approach for evaluating the coverage of genetic markersonthelinkagemap［18，52］.ByintegratingtheBACswith thepositioninthelinkagemap，wewereabletoevaluatetheloci distribution in the current linkage map.For example，for chromosome DT07，the BACs from the long and short arms mapped to 6.6 and 52.5 cM in the linkage map［20］，respectively（Fig.5-A）.Alongwiththedeterminationofthecentromericlociin the linkage map（50.9 cM）［24］，we were able to calculate the relative physical and genetic distances for short arm of chromosome DT07.As results，the short arm of chromosome DT07 account for 48.6%and 63.7%relative physical and genetic distances，respectively（Fig.5-A，B）.Thisinconsistentresultonits physical and genetic distances indicats a uneven distribution or relatively low coverage of genetic loci on the long arm of chromosome DT07［20］.We performed the same assay for the other DTchromosomes，because the genetic positions of centromeres for only DTchromosomes were identified［24］. Overall，only two chromosomes，DT07 and DT09，showed appreciably biased distributions of their genetic loci on the current genetic map（Fig.5）.In contrast，the other chromosomesshowed nearly no bias or only a slightly biased distribution of genetic markers（0.1-10%），suggesting that the current linkage map gives relatively even coverage over the DTgenome.

Currently，we cannot evaluate the ATchromosomes because the genetic positions of the centromeres are unknown.The high levels of repeats［7］that do not undergo recombination，along withthenearlyequalnumbersofgeneticlocibetweentheATand DTgenomes［20，46］，suggest that the ATlinkage maps will have relatively low coverage.Nonetheless，our comprehensive physical characterization of the G.hirsutum genome will be of critical importance for understanding its genomic structure and evolution and will be useful for further deep genome sequencing.

Acknowledgements

WethankDr.XinlianShenforsupplyingseedsofdiploidcottonG. australe.ThisworkwassupportedbytheNationalNaturalScience Foundation of China（No.31471170），State Key Laboratory of Cotton Biology Open Fund（No.CB2015A05），the New Century Excellent Talents in University（No.NCET-10-0496），and the Open Project Program of Jiangsu Key Laboratory of Plant Functional Genomics，Yangzhou University（No.K13001）.

Supplementary data

Supplementary data for this article can be found online at http://dx.doi.org/10.1016/j.cj.2016.04.001.

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11 January 2016

at:Haixia Institute of Science and Technology（HIST），Center for Genomics and Biotechnology，F(xiàn)ujian Agriculture and Forestry University，F(xiàn)uzhou 350002，China.

E-mail address:kwang@fafu.edu.cn（K.Wang）.

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science，CAAS.1These authors equally contributed to this work.

http://dx.doi.org/10.1016/j.cj.2016.04.001

2214-5141/?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.