Gossypium genus belongs to the Malvaceae family, which contains 46 diploid (2n = 2× = 26,) species and 5 tetraploid (2n = 4× = 52). The Gossypium genus are believed to have originated from a common ancestor approximately 5～10 million years ago，of which eight diploid genomes, designated as A to G and K, have been found across Africa, Asia Australia and North America (Phillips,1963; Wendel ,1989; Fryxell,1992; Dejooded and Wendel, 1992), and 5 tetraploid species were named (AD)₁ (G. hirsutum), (AD)₂ (G. barbadense), (AD)₃ (G. tomentosum), (AD)₄ (G. mustelinum) and (AD)₅ (G. darwinii) respectively(Wendel ,1989). At present, (AD)₁ and (AD)₂ are major cultivated species, which are extensive cultivated in the world. Previous studies had shown that diploid cotton A and D genome species are the respective donor species of A and D sub-genome of the allotetraploid cotton species, and the A sub-genome chromosomes are longer than the D sub-genome chromosomes(Phillips,1966; Endrizzi,1985).

The Fluorescence in situ hybridization (FISH) technology was introduced to plant research in 1985 (Rayburn,1985), which has applied to various aspects such as repeat sequences positioning, chromosome identification, cell genetic map construction, the origin of polyploid genome evolution and phylogenetic relationships and so on (Snowdon et al., 1997 ; Tang et al., 2000; Jiang and Gill,2006; Wang k et al.,2008; Nemeth et al., 2015; Melo et al., 2015).

FISH technology have developed suceessfully on other crops (Mukai et al.,1993; Cheng et al., 2002 ; Xiong et al., 2004). But research on cotton far lags behind those, main because cotton cell has more density cytoplasm, more hard cell wall, more number of chromoses and smaller size compare with other crops, which lead to cotton cell processing more difficult in prepare of high quality chromosome slides. In recent years, with the breakthrough of the bottleneck technology, cotton FISH research is developing rapidly(Liu et al., 2005 ; Wang et al., 2009; Wu et al., 2008 ; Gan et al., 2013 ; Zhang et al., 2014; Cui et al., 2015).

At present, about the great theories on Gossypium such as origin of species, evolution and classification had already formed the basic agreement. However, there still exist dissensions on the specific donors of the 5 tetraploid cotton species, and the germplasm disproportionation in the process of origin, and the homologous degree between different genome species of Gossypium or within one genome species(Liu et al., 2003 ;Wang KB et al., 2008). Our laboratory has made a great progress on using FISH technology to research the origin and evolution of cotton genomes and the interspecific genetic relationship or the genonomy of Gossypium (Wang et al., 1999; Wang et al., 2001; Liu et al., 2005; Wu et al., 2008; Gan et al., 2013).

In this study, GISH(Genomic in situ hybridization, one of the FISH technology) was used to the two cultivated alltetraploid cottons somatic metaphase chromosomes of (AD)₁ and (AD)₂， and used All 3 diploid A and 13 diploid D cotton gDNA (genomic DNA) as probe blocking with ssDNA, in order to further explore the specific A and D sub-genome progenitor donors of (AD)₁ and (AD)₂, and their genetic relationships; homologous degree between each tested cotton genome species. Wished to provide a beneficial enlightenment and help for the genetic improvement and suggest the preferred Gossypium species for genome sequencing.

1 Results and Analysis

1.1 GISH of (AD)₁ and (AD)₂ with all 3 diploid A cotton gDNA as probes respctively

GISH of the somatic metaphase chromosomes of (AD)₁and (AD)₂ both with all 3 diploid A (A₁, A₂ and A_1-a) gDNA as probes respectively, blocking with ssDNA(salmon sperm DNA).The results showed that the red hybridization signals were mainly distributed on the longer 13 pairs of A sub-genome chromosomes of (AD)₁ and (AD)₂ (As shown in Figure 1), and this visually confirmed the allodidiploid origin of the tetraploid cottons (Wendel et al., 2002). Besides, three pairs of crimson signals were detected only with the A_1-a gDNA probe both in (AD) ₁ and (AD) ₂ (Figure 1-c, f ), which were named “GISH-NORs” (Liu et al., 2005) , of which one in the A sub-genome chromosomes (green arrows), and two in the D sub-genome chromosomes (white arrows), and it significantly different from the A₁ and A₂ gDNA probes, the specific distributions about GISH-NORs were shown in Table 1.

1.2 GISH of (AD)₁ and (AD)₂ with all 13 diploid D cotton gDNA as probes respectively

Except the D₆(G.gossypiodies) gDNA probe, GISH of the somatic metaphase chromosomes of (AD)₁ and (AD)₂ both with other 12 diploid D gDNA as probe respectively, blocking with ssDNA (As shown in Figure 2 and Figure 3), the red fluorescence signals were observed on the short D sub-genome chromosomes of (AD)₁ and (AD)₂. In addition, three pairs of major GISH-NORs were detected, of which one in the longer A sub-genome chromosomes (green arrows showed in each image), and other two in the D sub-genome chromosomes (white arrows in each image),and the specific distribution about GISH-NORs were shown in Table 1. However, there were some differences on the fluorescence signal strength generated by each D genome species probe. GISH of (AD)₁ and (AD)₂ with D₆ gDNA probe respectively, the red fluorescence signals were not only distribute on the D sub-genome chromosomes but also on A sub-genomes (As shown in Figure 2-h and Figure 3-h), and we could not distinguish the A and D sub-genome of (AD) ₁ and (AD) ₂, and three pairs of major GISH-NORs were also observed. This showed a great difference between the D₆ genome and the other 12 D genomes, and the result illustrated that diploid D₆ genome may contain plenty of A genome repeat sequences, it is a very special genome species in the diploid D genomes.

1.3 DA value analysis based on GISH of (AD)₁ and (AD)₂ with diploid A, D genome species probes

DA (distinguishing ability) value reflect the genetic relationship between the diploid A or D genomes and the tetraploid genomes (Markova et al., 2007). DA values (As shown in Table 2) generated by A₁, A₂ and A_1-a gDNA probes to (AD)₁ was 0.361, 0.358 and 0.369 respectively. It showed the A_1-a gDNA possess the strongest ability to recognize the A sub-genome chromosomes of (AD) ₁, while the A₁ and A₂ gDNA possess same ability to recognize the A sub-genome chromosomes of (AD) ₁, that’s to say the A_1-a genome had closer genetic relationship with the A sub-genome of (AD)₁. And DA values generated by A₁, A₂ and A_1-a gDNA probes to (AD)₂ were 0.343, 0.346 and 0.352 respectively. the results also showed the A_1-a gDNA possess the strongest ability to recognize the A sub-genome chromosomes of (AD)_2, therefore, the A_1-a genome also had closer genetic relationship with the A sub-genome of (AD)₂.

DA values of (AD)₁ generated by all 13 diploid D gDNA probes respectively were shown in Table 2. Because of the hybridization signals generated by D₆ gDNA probe were distributed on both A and D sub-genome chromosomes of (AD)₁, therefore the DA value generated by D₆ gDNA probe(DA value was 0.402) was much higher than the other 12 D gDNA probes in (AD)₁. Except the D₆ gDNA probe, the highest DA value was 0.286 generated by D_3-d gDNA probe, and then followed by D₄, D₁, D_5, D₁₁, D_2-1, D_2-2, D₈, D_3-k, D₉, D₁₀ and D₇ probes. This showed that the D_3-d gDNA probe possess the strongest ability to recognize the D sub-genome chromosomes of (AD)₁, while the other D gDNA probes show weaker ability to recognize the D sub-genome chromosomes of (AD) ₁, therefore the D_3-d genome had closer genetic relationship with the D sub-genome of (AD)₁.

DA values of (AD) ₂ generated by all 13diploid D gDNA probes also be shown in Table 2. Except the D₆ gDNA probe(DA value was 0.387), the highest DA value generated by D₅ gDNA probe was 0.263, and then followed by D_3-d, D₁, D₄, D_2-1, D_2-2, D₁₁, D₈, D_3-k, D₁₀, D₉ and D₇ probes. which was also higher than those of the other 12 D gDNA probes. The results showed that the D₅ gDNA possess the strongest ability to recognize the D sub-genome chromosomes of (AD)₂, while the other 12 D gDNA show weaker ability to recognize the D sub-genome chromosomes of (AD)₂, so the D₅ genome species had closer genetic relationship with the D sub-genome of (AD)₂.

2 Discussion

2.1 The A sub-genome progenitor of (AD)₁ and (AD)₂

Since the discovery that allotetraploid Gossypium genomes contain both A and D genomes, investigators had attempted to look for which one of the modern diploid A and D genome species can be best served as the progenitor genome donors of allopolyploid cottons.

Which one of the diploid A genome species was the really donor of the A sub-genome of allopolyploid cottons? Many previous researches suggested that G. herbaceum (A₁) was the donor or the similar ancestors of the allopolyploid A sub-genome (Beasley, 1940; Gerstel, 1953; Phillips, 1963; 1964). However, the subsequent research had shown that there existed much differences between G. herbaceum (A₁) and the A sub-genome of allopolyploid cotton, whether in the chromosome or molecular level (Wendel, 1989; Wendel and Albert, 1992ï¼›Cronn, et al., 1996). And cell cytogenetic and comparative mapping research also revealed that there existed at least two large translocations between their genomes (Gerstel, 1953ï¼›Small, et al., 1998ï¼›Liu and Wendel, 2001). Branch taxonomy analyses of the molecular sequences had showed that G. herbaceum(A₁) was not the actual progenitor of the A sub-genome of allotetraploid cottons (Endrizzi, et al., 1985; Wendel and Cronn, 2002). In the evolutionary process of allotetraploid cottons, G. herbaceum(A₁) and G. arboretum(A₂) were the phylogenetically sisters between each other and hence were genealogical equidistant to the A sub-genome of the allotetraploid cottons (Cronn et al., 1996; Liu and Wendel, 2001; Wendel, 1989; Wendel and Albert, 1992).

In our experiment, GISH of (AD) ₁ and (AD) ₂ both with all 3 A genome gDNA as probes, 13 pairs of A sub-genome chromosomes were painted with red fluorescence signals, and the DA value was very similar between A₁ and A₂ gDNA probe, there was no significant difference between A₁ and A₂ both in GISH of (AD)₁ and (AD)₂, while the DA value of A_1-a gDNA probe was higher than A₁ and A₂ gDNA probe, the A_1-a gDNA had the strongest ability to recognize the A sub-genome chromosomes of (AD) ₁ and (AD)₂, so we considered that A_1-a genome species was most likely to be the A sub-genome progenitor donor of allotetraploid cotton (AD)₁ and (AD)₂.

The A_1-a genome had been divided into the variant of A₁ genome in the taxonomy(Stewart, 1987). Many researches always used the varieties of A₁ and A₂ genome species to study the origination and evolution of the A sub-genome of allotetraploid cottons, but they seldom and nearly didn’t use the A_1-a genome as the research materials, and even some researchers took the A_1-a and A₁ genome to lump together (Stewart, 1987; Wendel,1989; Wendel and Albert, 1992; Wendel et al.,1994, 1995; Wendel and Wessler, 2000; Wendel and Cronn, 2002; Wang et al., 2004). In this experiment, when using A_1-a gDNA as probe we found that the GISH result was obviously distinguished from the A₁ gDNA probe, and Wang et al (1995) had also found the karyotype parameters exist significant differences between A₁ and A_1-a genome. The A_1-a genome made a strong independence from A₁ genome, and we suggested that A_1-a genome should be given the “species” level in the classification of gossypium, that’s means the A_1-a genome possessed the same status with A₁ and A₂ genome.

2.2 The D sub-genome progenitor of (AD)₁ and (AD)₂

Since the discovery that allopolyploid Gossypium species contain two genomes whose progenitors presently occur in different hemispheres, investigators had attempted to provide pieces to the puzzle of the polyploid origin. A diverse array of tools had been used in an effort to examine this issue, from early study methods by comparative morphology, cytology, cytogenetic, comparative phytochemistry, and protein electrophoretic methods to modern phylogenetic investigations using DNA sequencing of homologous genes. Several investigators proposed that allopolyploid cottons formed more than once, they suggested the best models of the potential D subgenome ancestral donor of allopolyploid cottons were D₁ (G. thurberii), D_3-d(G. davidsonii), D_3-k (G. klotzschianum) and D₅(G. raimondii), so they considered the allopolyploid cottons are polyphyletic (Kammacher, 1960; Sherwin, 1970; Johnson, 1975; Umbeck, 1985; Stewart, 1987; Da and Bertrand,1995). However, Most authors proposed that allopolyploid cottons formed only once, and that D₅ was more similar to the D subgenome of allotetraploid cottons than other D genome diploids, they considered the allopolyploid cottons are monophyletic (Phillips,1963, 1964, 1966; Endrizzi et al., 1985; Wendel, 1989; Cronn et al.,1996; Seelanan et al., 1997; Small and Wendel, 1999, 2000; Wendel and Albert et al., 1992 ;Wendel et al., 1995; Wendel and Cronn, 2002; Admas, 2003).

We compared the GISH signals and DA values of (AD)₁ and (AD)₂ chromosomes generated by different diploid D genome species. The DA values of D₆(G. gossypioides) were much higher than those of other D genome species both in (AD)₁ and (AD)₂, but the signals were distributed on both A and D subgenome chromosomes of (AD)₁ and (AD)₂, and the signal intensity was much weaker than that produced by other D genome species such as D_3-d (G. davidsonii) and D₄ (G. aridum) in (AD)₁, or D₁ (G. thurberi), D_3-d (G. davidsonii) and G. raimondii (D₅) in (AD)₂. Therefore, the D₆ genome species cannot be the D subgenome progenitor donor of (AD)₁ and (AD)₂.

The signal intensity in D_3-d probe was more stronger than any other D genome species probes in GISH of (AD)₁, and the DA value of D_3-d probe was much higher than other D genome species too. This indicated that D_3-d genome species has the strongest ability to recognize the D subgenome chromosomes of (AD)₁, so we suggested that D_3-d but not D₅ was the possible D subgenome progenitor donor of (AD)₁.

And on the other hand, the signal intensity in D₅ probe was more stronger than any other D genome probes in GISH of (AD)₂, and the DA value of D₅ probe was also much higher than other D genome probes.This indicated that D₅ genome species has the strongest ability to recognize the D subgenome chromosomes of (AD)₂, so we thought that the D₅ is the possible D subgenome progenitor donor of (AD)₂.

Previous GISH studies had shown that diploid D₁ and D_3-d genome species was the D sub-genome progenitor donor of (AD)₄ (G. mustelinum) and (AD)₅ (G. darwinii) respectively (Wu et al., 2010; 2013), combined with the results of this experiment, allopolyploid cottons maybe formed with different D genome species as the progenitor donor, these results supported the hypothesis that allopolyploid cottons are polyphyletic.

3 Materials and Methods

We used allotetraploid cultivated cotton species (2n = 4x = 52) (AD)₁ (G. hirsutum var. Zhongmiansuo 16) and (AD)₂ (G. barbadense var. Xinhai 7) as target chromosomes. The Probes were made from diploid cotton (2n = 2x = 26) genomic DNA (gDNA), including all 3 A genome species G. herbaceum var. hongxing (A₁), and G. arboreum var. shixiya 1 (A₂), and G. herbaceum wild var. arfrium (A_1-a); and all 13 D genome species, including G. thurberi (D₁), G. armourianum (D_2-1), G. harknessii (D_2-2), G. davidsonii (D_3-d), G. klotzschianum (D_3-k), G. aridum (D₄), G. raimondii (D₅), G. gossypiodies (D₆), G. lobatum (D₇), G. trilobum (D₈), G. laxum (D₉), G. turneri (D₁₀) and G. schwendimanii (D₁₁). All of the plant materials are being taken from National Wild Cotton Germplasm Nursery in Sanya city, Hainan, China.

Total gDNA was extracted and purificated from immature leaves using the CTAB method (Song et al., 1999). The purified total gDNA was cut off with 120℃ 10 min, and examined with 0.8% agarose gel electrophoresis for their fragment size, which was generally appropriate in 300-600bp, and then tagged with markers. The gDNA probes were labeled with DIG-High-Prime labeling system (Roche Company, Germany), according to the standard operating procedures. The preparation of mitotic metaphase chromosomes which derived from the root tip cells and the procedure of GISH referenced to the method of Wang (Wang et al., 1999).

The hybridization signals were observed using a fluorescence microscope (Ziess Axioskop 2 plus). Images were captured by ISIS (in situ imaging system) software by adjusting their brightness and contrast. And also used this software to calculate the DA (distinguishing ability) value which was described by Markova et al (2007). And used Adobe Photoshop 7.0 software makes the plate.

Acknowledgment

This work was supported by the Exploring and Ultilization of Economic Crop Germplasm Resources(2013BAD01B03), the National Agriculture Research System of Cotton (CARS-18-36) and the Subproject of the Major Projects of Genetically Modified Organisms Breeding (2011ZX08005002-009) and also the Science and Technology Plan of the Jiangxi Province (20141BBF60011).

Reference

Adams K.L., Cronn, R., Percifield R., and Wendel J.F., 2003,Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing, Proc. Nat. Acad. Sci. USA, 100(8): 4649-4654

http://dx.doi.org/10.1073/pnas.0630618100 PMid:12665616 PMCid:PMC153610

Beasley J.O. , 1940, The origin of American tetraploid Gossypium species, Am. Nat., 74(7): 285-286

http://dx.doi.org/10.1086/280895

Cheng Z.K., Dong F.G., Langdon T., Ouyang S., Buell C.R., Gu M.H., Blattner F.R.,and Jiang J.M., 2002, Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon, Plant Cell,14(8):1691-1704

http://dx.doi.org/10.1105/tpc.003079 PMid:12172016 PMCid:PMC151459

Cronn R.C., Zhao X.P., Paterson A.H., and Wendel J.F., 1996, Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons, J. Mol. Evol., 42(6): 685-705

http://dx.doi.org/10.1007/BF02338802 PMid:8662014

Cui X.L., Liu F.,Liu Y.L.,Zhou Z.L.,Zhao Y.Y.,Wang C.Y.,Wang X.X.,Cai X.Y.,Wang Y.H.,Meng F.,Peng R.H.,and Wang K.B., 2015, Construction of cytogenetic map of Gossypium herbaceum chromosome 1 and its integration with genetic maps, Mol. Cytogenetic, 8:2-10

http://dx.doi.org/10.1186/s13039-015-0106-y PMid:25628758 PMCid:PMC4307992

Da Rocha P.S.C.F., and Bertrand H., 1995, Structure and comparative analysis of the rDNA intergenic spacer of Brassica rapa:Implication for the function and evolution of the Cruciferae spacer, Eur. J. Biochem, 229(2): 550-557

http://dx.doi.org/10.1111/j.1432-1033.1995.tb20497.x PMid:7744079

Dejooded D.R., and Wendel J.F., 1992, Genetic diversity and origin of the Hawaiian Islands cotton,Gossypium tomentosum, Amer. J. Bot., 79: 1311-1319

http://dx.doi.org/10.2307/2445059

Endrizzi J.E., Turcotte E.L., and Kohel R.J., 1985, Genetics, cytogenetics, and evolution of Gossypium, Adv. Genet.,23:271-375

http://dx.doi.org/10.1016/S0065-2660(08)60515-5

Fryxell P.A., 1992, A revised taxonomic interpretation of Gossypium L.(Malvaceae), Rheedea,2(2): 108-165

Gan Y., Liu F., Chen D.,Wu Q., Qin Q.,Wang C., Li S., Zhang X.,Wang Y.,and Wang K., 2013, Chromosomal Locations of 5S and 45S rDNA in Gossypium Genus and Its Phylogenetic Implications Revealed by FISH, PloS one, 8(6): e68207

http://dx.doi.org/10.1371/journal.pone.0068207 PMid:23826377 PMCid:PMC3695094

Gerstel D.U., 1953, Chromosomal translocations in interspecific hybrids of the genus Gossypium,Evolution, 7: 234-244

http://dx.doi.org/10.2307/2405734

Jiang J.M., and Gill B.S.,2006, Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research, Genome, 49(9):1057-1068

http://dx.doi.org/10.1139/g06-076 PMid:17110986

Johnson B.L., 1975, Gossypium palmeri and a polyphyletic origin of the New World cottons, Bull Torrey Bot. Club, 102(6): 340-349

http://dx.doi.org/10.2307/2484760

Kammacher P., 1960, Observations cytologiques sur deux hybrides F₁ entre especes cultivees tetraploides de cotonniers et l’espece diploide sauvage Gossypium raimondii, UlB. Rev. Cytol. Biol. Veg., 22(5): 1-31

Liu B., and Wendel J.F., 2001, Intersimple sequences repeat (ISSR) polymorphisms as a genetic marker system in cotton,Molecular Ecology Notes, 1(3):205-208

http://dx.doi.org/10.1046/j.1471-8278.2001.00073.x

Liu S.H., Wang K.B., Song G.L., Wang C.Y., Liu F., Li S.H., Zhang X.D., and Wang Y.H., 2005, Primary investigation on GISH-NOR in cotton, Chinese Science Bulletin, 50(5): 425-429

http://dx.doi.org/10.1007/BF02897457 http://dx.doi.org/10.1360/982004-582

Markova M., Michu E., Vyskot B., Janousek B., and Zluvova J., 2007, An interspecific hybrid as a tool to study phylogenetic relationships in plants using the GISH technique, Chromosome Research, 15(8):1051-1059

http://dx.doi.org/10.1007/s10577-007-1180-8 PMid:18075777

Melo C.A., Silva G.S., and Souza M.M., 2015, Establishment of the genomic in situ hybridization (GISH) technique for analysis in interspecific hybrids of Passiflora,Genet. Mol. Res., 14(3):2176-2188

http://dx.doi.org/10.4238/2015.March.27.4 PMid:25867365

Mukai Y.,Nakahara Y.,and Yamamoto M., 1993, Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes, Genome,36(3):489-494

http://dx.doi.org/10.1139/g93-067 PMid:18470003

Nemeth C., Yang C.Y., Kasprzak P., Hubbart S., Scholefield D., Mehra S., Skipper E., and King J., 2015, Generation of amphidiploids from hybrids of wheat and related species from the genera Aegilops, Secale, Thinopyrum, and Triticum as a source of genetic variation for wheat improvement, Genome ,58 (2): 71-79

http://dx.doi.org/10.1139/gen-2015-0002 PMid:26053312

Phillips L.L., 1963, The cytogenetics of Gossypium and the origin of new world cotton, Evolution,17:460-496

http://dx.doi.org/10.2307/2407096

Phillips L.L., 1966, The cytology and phylogenetics of the diploid species of Gossypium, Amer. J. Bot., 53(4): 328-335

http://dx.doi.org/10.2307/2439872

Phillips L.L., 1964, Segregation in new alloploids of Gossypiumâ…¤. Mutivalent formation in new world×Asiatic and new world×wild American hexaploids,Ame. J. Bot., 51(3):324-329

Rayburn A.L., and Gill B.S.,1985, Use of biotin labeled probes to map specific DNA sequences on wheat chromosomes, J. Hered., 76:78-81

Seelanan T., Schnabel A., and Wendel J.F., 1997, Congruence and consensus in the cotton tribe, Syst. Bot., 22(2): 259-290

Sherwin K.H., 1970, Winds across the Atlantic-possible African origins for some Pre-Columbia New World CULTIGENS, Res. Rec. Univ. Mus. S. 111. Univ. Mso-Am. Stand, 6:1-33

Small R.L., and Wendel J.F., 1999, The mitochondrial genome of allotetraploid cotton (Gossypium L.), J. Hered., 90(1): 251-253

http://dx.doi.org/10.1093/jhered/90.1.251 PMid:9987935

Small R.L., Ryburn J.A., and Cronn R.C., Seelanan T., Wendel J. F., 1998, The tortoise and the hare: Choosing between noncoding plastome and nuclear Adh sequences for phylogeny reconstruction in a recently diverged plant group. Am. J. Bot., 85(9): 1301-1315

http://dx.doi.org/10.2307/2446640 PMid:21685016

Small R.L., and Wendel J. F., 2000, Copy number lability and evolutionary dynamics of the Adh gene family in diploid and tetraploid cotton (Gossypium), Genetics, 155(4):1913-1926

Snowdon R.J., Kohler W., Friedt W., and Kohler A.,1997, Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids, Theor. Appl. Genet., 95(8): 1320-1324

http://dx.doi.org/10.1007/s001220050699

Song G.L., Cui R.X., Wang K.B.,Li S.H.,Zhang J.F., and Guo J.H., 1999, Analysis of genetic diversity of Australian species of Gossypium using RAPD, Cotton Science, 11(2): 65-69

Stewart J.M., Craven L.A., and Fryxell P.A., 1987, Gossypium germplasm from Australia, Plant Genetic Resources Newsletter, 69: 44-47

Tang S., Li X., and Jia X., 2000, Genomic in situ hybridization (GISH) analyses of thinopyrum intermedium, its partial amphiploid zhong 5, and disease-resistant derivatives in wheat. Theor. Appl. Genet., 100(3): 344-352

http://dx.doi.org/10.1007/s001220050045

Umbeck P.F., and Stewart J.M., 1985, Substitution of cotton cytoplasms from wild diploid species for cotton germplasm improvement, Crop Sci., 25(6): 1015-1019

http://dx.doi.org/10.2135/cropsci1985.0011183X002500060028x

Wang K., Guan B., Guo W.Z.,Zhou B.L., Hu Y.,Zhu Y.C., and Zhang T.Z.,2008, Completely distinguishing individual A-genome chromosomes and their karyotyping analysis by multiple bacterial artificial chromosome–fluorescence in situ hybridization, Genetics, 178(2): 1117-1122

http://dx.doi.org/10.1534/genetics.107.083576 PMid:18287408 PMCid:PMC2248359

Wang K., Yang Z.J., Shu C.S., Hu J.,Lin Q.Y.,Zhang W.P.,Guo W.Z.,and Zhang T.Z., 2009, Higher axial-resolution and sensitivity pachytene fluorescence in situ hybridization protocol in tetraploid cotton, Chromosome Research, 17(8): 1041-1050

http://dx.doi.org/10.1007/s10577-009-9085-3 PMid:19844799

Wang C.Y., Wang K.B., Wang W.K., Li M.X., Song G.L., Cui R.X., Li S.H., Zhang, X.D., and Zhang J.M., 1999, Protocol of cotton FISH of somatic chromosomes with gDNA as probes, Cotton Science, 11(2):79-83

Wang K.B., Du X.M., and Song G.L.,2004, The present situation and development of the cotton germplasm creation, Plant genetic resources, 5(supplement): 23-28

Wang K.B., Song G.L., Wang C.Y., Liu F., Li S.H., Zhang X.D., and Wang Y.H., 2008, FISH-based karyotype of Gossypium herbaceum generated with 45s rDNA and gDNA of Gossypium raimondii as probes, Cotton Science, 20(4): 264-273 

Wang K.B., Wang W.K., Wang C.Y., Song G.L., Cui R.X., Li S.H., and Zhang X.D., 2001, Studies of FISH and karyotype of Gossypium barbadense, Acta Genetica Sinica, 28(1): 69-75

Wu Q., Cheng H., Liu F., Wang S.F., Wang C.Y., Song G.L., Li S.H., Zhang X.D., Wang Y.H., Ma Z.Y., and Wang, K.B., 2010, Screen of FISH marker of chromosomes at Gossypium D genome species, Chinese Science Bulletin, 55(21): 2099-2105

Wu Q., Liu F., Li S., Song G., Wang C., Zhang X., Wang Y., Stelly D., and Wang K., 2013,Uniqueness of the Gossypium mustelinum genome revealed by GISH and 45S rDNA FISH, J. Integr. Plant. Biol., 55(7):654-662

http://dx.doi.org/10.1111/jipb.12084 PMid:23758934 PMCid:PMC3810724