cotton , one of the most important economical crops in the world , provides most natural textile fiber and some of oil resources for people . cotton cultivars are diploids and tetraploids derived from natural hybridization of an african / asian a - genome and an american d - genome species . as one of the ancestors of tetraploids , the asiatic cotton ( g. arboreum l. ) has been domesticated and cultivated for almost 2000 years in china since it was first introduced from india . it is still used as germplasm resources in the present cotton breeding program due to their early maturity , strong tolerance to stress , resistance to disease and insects , high fiber strength , and excellent plasticity . so decoding the a - genome will help for the diging of potential gene in asian cotton , understanding the origin and evolution of allpolyploid , studying the genome structure of important agronomic trait and the relationship of gene structure and function , and elucidating the influence to the trait by the interaction between genes , which is important in crop improvement . the bacterial artificial chromosome ( bac ) cloning system has become an invaluable tool in many research areas of genome research because of its ability to stably maintain large dna fragments and its ease of manipulation [ 24 ] . bac libraries have been developed for a number of major crops including rice [ 57 ] , soybean [ 8 , 9 ] , wheat [ 1013 ] , maize [ 14 , 15 ] , sorghum , and tomato . for cotton , at least eight bac / bibac libraries these libraries were all made from different genotypes of ad - genome allotetraploid gossypium species . seven cotton libraries were made from upland cotton species , including tamcot hq95 , auburn 623 ( http://hbz7.tamu.edu/homelinks/bac_est/bac.htm ) , tm-1 [ 18 , 19 ] , maxxa , suyun7235 , zhongmiansuo12   and 0 - 613 - 2r , and one from g. barbadense l. cv pima90 - 53 . we report the development of a deep - coverage bac library for the diploid a - genome cotton species , which served as the maternal parent during polyploidization to generate the precursor of the commercially important allotetraploid species . g. arboreum l. cv . it is a diploid species of a - genome , widely used in breeding programs because of its high level of resistance to fusarium wilt disease and its superior fiber strength . these bac and bibac libraries will provide resources essential for advanced genomics and genetics research of cotton . etiolated cotyledons were used as the source of high - molecular - weight ( hmw ) dna preparation . nuclei were isolated , lysed and megabase dna was purified as described by yin et al . . then , megabase nuclear dna was partially digested with hindiii to identify an appropriate partial   digestion condition . six plugs were prerun and used for large - scale digestion with optimal amount of hindiii . the dna separation was performed in two stages . first , partially digested dna was put into the center wells of a 1% agarose gel and separated by pfge ( 6  v / cm , 50  s switch time , 18 hours run time , 12.5c ) . the region of the compression zone containing dna fragments in the size range from 150 to 450  kb was excised from the unstained gel and divided into three equal sections . for the second step , the three excised gel slices were embedded into a second gel and compressed by pfge ( 6  v / cm , 35  s switch time , 16 hours run time , 12.5c ) . the compressed dna band was excised and recovered from the agarose gel slices using an electroeluter model 422 ( biorad , ca , usa ) . eluted dna was ligated to vector pindigobac-5 ( hindiii - cloning ready , epicentre technologies , madison , wi , usa ) and incubated under temperature - cycle conditions . 2  l of ligation mixture was used to transform 18  l of electromax dh10b competent cells ( invitrogen , ca , usa ) by electroporation at 17  kv / cm , 100   , and 25  f . the bac clones were picked from the library and inoculated into 3  ml 2  ty medium containing chloramphenicol ( 12.5  g / ml ) and incubated at 37c for 24 hours . bac - dna was miniprepared by the method of sambrook   with some modifications . to estimate , insert size and determine distribution of clone size , a total of 103 bac clones the bac - dna was digested with 5u of noti enzyme ( 3 hours at 37c ) . the digestion products were separated by pfge ( 6  v / cm , 515  s switch time , 14 hours run time , 12.5c ) . the insert size was estimated by comparison with the midrange pfge marker ii ( new england biolabs , ma , usa ) . southern blots of size - separated bac inserts were performed by standard protocols after uv nicking the dna ( gene linker , uvp , ca , usa ) . total genomic cotton dna digested by hindiii , and haeiii used as probe and labeled with dig by standard random priming techniques ( high prime dna labeling and detection starter kit i , roche , mannheim , germany ) . six bacs selected from the library at random for stability testing ( with a volume of 5  l ) were cultured in 3  ml 2  ty medium with antibiotic at 37c for 24 hours . 5  l of this culture was used to inoculate a subsequent 3  ml 2  ty . dna samples isolated from the 1st and 5th day cultures ( 0-generation cells and 100-generation cells ) were digested with hindiii and ran on 0.8% agarose gel for 16 hours with 2.0  v / cm . the gel was stained with ethidim bromide for 30 minutes , destained in water for 30 minutes , and then photographed . under the pooling strategy by yin et al . , the bac library was arranged in two levels of pools ( column pools ; super pools ) allowing screening with various pcr - based markers ( figure 3 ) . firstly , for every 384-well plate , the clones ( 5  l cultures of each clone ) in individual row a to p in the same column were combined into a pool containing 16 individual clones by the 12-channel transferpettor . each column pool composed of 16 sequential 384-well plates in this way . in the second step , so the entire bac library of 123,648 clones was organized into 322 super pools , each consisting of 384 unique clones . as cotton leaves are particularly rich in polyphenols and polysaccharides , we modified the general library construction method to meet the needs of cotton bac library construction . modifications included using etiolated cotyledon as the dna preparation source , addition of pvp40 , and increasing the -mercaptoethanol concentration in the extraction - washing buffer . the prepared dna , consisting of about 1  mb nearly free of protein and organelle dna , was suitable for bac library construction . hmw - dna embedded in lmp agarose plugs was partially digested with the enzyme hindiii . the optimal concentration range of hindiii found to produce the maximum number of 150450  kb dna fragments was 812  u / plug . the dna fragments from the second size selection were electroeluted from the gel , and dna concentration was at least 3  ng/l . total twelve separate ligation reactions gave rise to the bac library of 123,648 individual clones stored in 322 384-well plates . to analyze the distribution of insert size and estimate the average insert size in the bac library , dna samples were isolated from 103 clones . bac - dna was digested by noti to release the insert and fractionated by pfge ( figure 1(a ) ) . statistical analysis indicated the insert size of clones ranged from 30 to 190  kb , with an average size of 100.2 kb ( figure 1(c ) ) . no clone was found without an insert . based on an a - genome size of 1697 mb , the coverage of the library is approximately 7.2 haploid genome equivalents . this accounts for an over 99% probability of hitting a specific bac clone containing any sequence in the genome . noti is a gc-8 base cutter , while cotton genome is relatively at - rich , so digestion with noti should generate one or two insert bands plus a vector band ( 7.5  kb ) , which is consistent with our data . a southern blot of the gel shown in figure 1 probed with total cotton genomic dna ( figure 1(b ) ) indicated that the source of cloned dna originated from cotton .   to test the stability of bac clones in e. coli , we analyzed the hindiii restriction patterns of six bac clones in the 0 and 100 generations . no visible changes in fingerprints were seen between 0 and 100 generations ( figure 2 ) , indicating the stability of the bacs . cotton has a relatively large genome with about 30%60% of the genome consisting of highly repetitive sequences . moreover , many homologous regions exist between the at and dt genomes of the tetraploids . therefore , construction of a bac library provides an important alternative tool for genomic research . here , we described the development and characterization of a high - quality bac library from the a - genome diploid species g. arboreum l. cv . g. arboreum l. is an old cultivated cotton species that was planted extensively in china . to date , this species is still used in the tetraploid cotton - breeding program as an elite germplasm line , due to unique properties that include early maturity , environmental resistance , biotic resistance , high fiber strength , and excellent plasticity . additionally , g. arboreum l. has historically been chosen as a model system to study fiber development . the a - genome produces spinnable fiber , whereas the d - genome alone is worthless in terms of fiber production . .   found among seven detected qtls for six fiber - related traits , five were distributed among a - subgenome chromosomes , suggesting that the a - subgenome of the allotetraploid cotton contributes to the superior fiber production . therefore , a bac library established from a - genome species is useful in cotton genome studies . screening can be performed either by hybridization on high - density filters or by pcr . both methods are feasible , but the main advantage of hybridization is the ability to combine probes for screening the entire bac library and identifying clones in a single experiment . pcr screening , however , is much more reliable , faster , and efficient with higher specificity owing to effective avoidance of false positive clones identified by repeat sequences in probes by hybridization . here , we described a three - step pcr screening procedure based on the bac library pool system . the bac pool strategy was sensitive enough to identify single positive clones among superpools containing 384 bac clones . bac clones cultured overnight served as pcr template directly , rather than using prepared bac - dna . this modification considerably simplifies the procedure and shortens the time required for library screening . with this change , the bac library can be screened using ssrs , rapd , and other pcr - based molecular tagging and will facilitate the development of whole - genome integrated physical / genetic maps of cotton . in additon , the g. arboreum l. cv . jianglinzhongmian bac library will provide a valuable tool for a diverse range of other studies including comparative genomics , physical mapping , map - based cloning of gene(s ) of qtl(s ) , and marker development based on bac - end sequencing and genoming sequencing . recently , we developed a bac library from tm-1 , which is a tetraploid genetic standard line for upland cotton . in the future , the availability of these two cotton bac libraries will allow us to perform further comparative studies between a - genome and ad - genome species and these comparisons will further reveal the evolution of cotton genome .
a bacterial artificial chromosome ( bac ) library for the a - genome of cotton has been constructed from the leaves of g. arboreum l cv . 
 jianglinzhongmian . 
 it is used as elite a - genome germplasm resources in the present cotton breeding program and has been used to build a genetic reference map of cotton . 
 the bac library consists of 123,648 clones stored in 322 384-well plates 
 . statistical analysis of a set of 103 randomly selected bac clones indicated that each clone has an average insert length of 100.2  kb per plasmid , with a range of 30 to 190  kb . 
 theoretically , this represents 7.2 haploid genome equivalents based on an a - genome size of 1697  mb . 
 the bac library has been arranged in column pools and superpools allowing screening with various pcr - based markers . in the future 
 , the a - genome cotton bac library will serve as both a giant gene resource and a valuable tool for map - based gene isolation , physical mapping and comparative genome analysis .