Chromosomal Locations of 5S and 45S rDNA in Gossypium Genus and Its Phylogenetic Implications Revealed by FISH

We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A1D1). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G . incanum (E4), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G . raimondii (D5) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution.


Introduction
Cotton (Gossypium) is an important economic fiber crop. The genus of Gossypium comprises about 50 species throughout tropical and subtropical regions of the world, including five tetraploid (2n=4x=52) species and about 45 diploid (2n=2x=26) species. The taxonomic and evolution study on Gossypium genus has been an important subject of investigation due to its economic significance. In the late 1800's or early 1900's, the taxonomy was mainly based on morphological characteristics and geographical distributions, however, it has been confusion due to un-consensus characteristics used by different taxonomists. With development of cytological methods, the diploid species have been classified genetically into seven genome types, i.e. A, B, C, D, E, F, G and K genomes [1,2]. The evolutionary history of Gossypium genus was reconstruct based on geography, morphology, cytogenetics and molecular data. However, due to the continuous recombination and exchange, great differences existed between existing cotton species and their ancestors in terms of physiological feature, agronomic trait and morphology. Therefore, more interpretation about the phylogenetic and interspecific evolution in Gossypium genus is quite necessarily to be clarified.
Ribosomal DNA (rDNA) has highly conserved repetitive sequences in the plant genome, and the polymorphism or conservatism of their copy number and chromosomal localization are visual and comparative [3][4][5]. By comparing the number and distribution characteristics of rDNA sites on the chromosomes among species, interspecific phylogenetic relationships and the related mechanism of speciation and chromosomal evolution could be revealed [6]. Recently, the physical FISH location of rDNA in plants have provided much information to the evolutional relationship of many close species and the origin of allopolyploid [7][8][9][10][11][12].
In genus of Gossypium, research on rDNA location in the early days was mainly focused on G. hirsutum (upland cotton) due to its economic importance and to the abundant genetic materials created. 5S rDNA and 18S-28S rDNA were located to chromosomes of G. hirsutum by FISH on chromosomes of the meiosis metaphase [13][14][15]. Later, the number and copy number of 5S and 18S-28S rDNA in tetraploid G. hirsutum, diploid species of A and D genomes, were revealed by FISH on the metaphase chromosomes of mitosis [16]. Recently, the number and copy numer of 5S and 45S rDNA, the 5S-bearing and 45S-bearing chromosomes of other tetraploid species and diploid species of A and D genomes have been revealed [17][18][19].
In order to further understand the cytogenetics and evolution of Gossypium genus, the distribution of 5S and 45S rDNA was analyzed by cocktail FISH for the four tetraploid species, as well as 13 diploid species and one variation representing diploid A, B, D, E, F and G genome. Combined with rDNA distribution in previous reports [17][18][19], the chromosome evolution of rDNA loci of Gossypium genus would be determined. Also, the phylogenetic implication based on rDNA patterns could be inferred to gain further insight into the evolutionary history of Gossypium genomes.

Plant materials and clones
The species and their genomes and accessions (cultivars) used in this study were shown in Table 1. The plant materials are maintained perennially in the National Wild Cotton Nursery in Sanya City, Hainan Island, sponsored and owned by the Institute of Cotton Research of Chinese Academy of Agricultural Sciences (ICR-CAAS), and at the same time, some of them are as well conserved in pots in greenhouse of ICR-CAAS at Anyang City, Henan Province, China.
For diploid D genome species and D t subgenome of tetraploid species, four types of probes were used, including 5S rDNA, 45S rDNA, BAC clone 150D24 and some D h chromosome-specific BAC clones. For diploid A genome and A t subgenome of tetraploid species, three types of probes were used, including 5S rDNA, 45S rDNA and some A h chromosome-specific BAC clones. For the diploid B, E, F and G genome species, only 5S rDNA and 45S rDNA were used. The 5S and 45S rDNA derived from Arabidopsis thaliana were kindly provided by Professor Yunchun Song of Wuhan University, China. The BAC clone 150D24 which contains centromere-specific repeats in D subgenome and D genome of Gossypium was screened from Pima 90-53 BAC library [20] to indicate centromere position. The A h (D h ) chromosome-specific BAC clones used to identify individual chromosome were kindly provided by Professor Tianzhen Zhang of Nanjing Agricultural University, China [21].

DNA probes preparation
The probes 5S, 45S rDNA and BAC DNA were isolated using a standard alkaline extraction [22]. 45S rDNA and BAC clone 150D24 were labeled by standard Dig-nick translation reactions, whereas 5S rDNA and some A h (D h ) subgenome chromosome-specific BAC clones [21] were labeled with Biotinnick translation reactions, according to the manufacturer's instructions (Roche Diagnostics, USA).

Chromosome preparation and FISH
Preparation of mitotic chromosomes and the FISH procedure were conducted according to [23] with some modifications. Digoxigenin-labeled and biotin-labeled probes were detected

The number of 5S and 45S rDNA in Gossypium genus
Three 45S rDNA loci and two 5S rDNA loci were detected in all three tetraploid species (Figure 1 a, b, c). Similarly, the number of 45S rDNA loci was detected three, two, four and three in D 5 , D 6 , D 9 , D 11 , respectively, while only one 5S rDNA locus was observed in the four D genome species (Figure 1d-1g). In A 1 and its variant A 1-a , three 45S loci and one 5S rDNA locus were found, respectively (Figure 1 h, 1i). In B 1 , B 3 , E 2 , E 3 , F 1 and G 3 , three 45S loci and one 5S rDNA locus were observed (Figure 1j-1m, 1p), while three 45S loci and two 5S rDNA loci in E 4 ( Figure 1n) as well as four 45S loci and one 5S rDNA locus in G 1 were observed (Figure 1q).

The location of 5S and 45S rDNA in Gossypium genus
To further identify the rDNA locations specific to individual chromosomes or even to arms in D subgenome of tetraploid and D genome cottons, the individual chromosome-specific BAC clones and a D genome centromere-specific BAC clone (150D24) as BAC-FISH probes were used in the experiments. And for D t subgenome of tetraploid, the individual chromosome BAC clones was used as BAC-FISH probes. Figure 2a-2w showed the 45S and 5S rDNA locations specific to individual chromosomes or even to arms in the three tetraploid cottons (A 1 D 1 , A 3 D 3 , A 4 D 4 and A 2 D 2 ) and the four D genome cottons (D 9 , D 11 , D 6 , D 5 ). In both A 1 D 1 (Figure 2a-2c) and A 3 D 3 ( Figure  2d-2f), 45S and 5S rDNA, syntenic with BAC clones specific to chromosomes A h 09, D h 07 and D h 09 (h indicates A 1 D 1 ), respectively, were located to the corresponding chromosomes and chromosomal arms. According to the homology within D subgenomes, chromosomes bearing with 45S locus of A 3 D 3 were named as A tt 09, D tt 07 and D tt 09 (tt indicates A 3 D 3 ), respectively. So that in these two species, three 45S loci were observed at the end of the short arm of chromosomes, whereas two 5S loci were co-localized with the 45S locus and was found interstitial on the short arm of chromosomes A t 09 and D t 09 (t indicates tetraploid species), respectively, suggesting a synteny relationship for 5S and 45S rDNA. In A 4 D 4 , three 45S loci, syntenic with BAC clones specific to chromosomes A h 07, A h 09 and A h 08 respectively were found located at the end of the short arm of chromosomes of A subgenome (Figure 2g-2i), while the two 5S loci, syntenic with BAC clones specific to chromosomes A h 09 and D h 09, respectively, were located to the end of the short arm of chromosomes (Figure 2h, 2j). Likewise, chromosomes bearing 45S and 5S rDNA loci were named as A m 07, A m 09, A m 08 and D m 09, respectively (m indicates A 4 D 4 ). And so, 5S and 45S rDNA, being positioned on the chromosome A m 09, showed a synteny relationship, and the other 5S rDNA was positioned on the on the chromosome D m 09 showing no synteny relationship with any of 45S loci. In addition, 5S and 45S rDNA on the A subgenome of A 2 D 2 was located to the same chromosome A b 09 (b indicates A 2 D 2 ) (Figure 2k). Figure 2l-2w showed the 45S and 5S rDNA locations specific to individual chromosomes or even to arms in the four D genome cottons (D 9 , D 11 , D 6 , D 5 ). In D 9 , 45S and 5S rDNA, syntenic with BAC clones specific to chromosomes D h 05, D h 07, D h 09 and D h 12, respectively, were located to the corresponding chromosomes and chromosomal arms (Figure 2l-2o). According to the homology between D genomes and D subgenome of A 1 D 1 , chromosomes bearing 45S locus of D 9 were named as D 9 05, D 9 07, D 9 09 and D 9 12, respectively. The result showed that four 45S rDNA loci were found at the end of short arm of chromosomes D 9 05, D 9 07, D 9 09 and D 9 12, with one 5S rDNA locus was found interstitial to the short arm of chromosome D 9 09. Likewise, 45S and 5S rDNA, syntenic with BAC clones specific to chromosomes D h 05, D h 07 and D h 09, were found respectively located to the corresponding chromosomes and chromosomal arms of D 11 (Figure 2p-2r). Chromosomes bearing with 45S locus of D 11 were named as D 11 05, D 11 07 and D 11 09, respectively. Three 45S rDNA loci were showed at the end of short arm of chromosomes D 11 05, D 11 07 and D 11 09, and one 5S rDNA locus was found interstitial to the short arm of chromosome D 11 09. Also, 45S and 5S rDNA, syntenic with BAC clones specific to chromosomes D h 07 and D h 09, respectively, were located to the corresponding chromosomes and chromosomal arms of D 6 (Figure 2s, 2t). Chromosomes bearing with 45S locus of D 6 were named as D 6 07 and D 6 09, respectively. Therefore, two 45S rDNA loci were seen at the end of short arm of chromosomes D 6 07 and D 6 09, and one 5S rDNA locus was found interstitial to the short arm of chromosome D 6 09. In D 5 , two 45S loci and one 5S rDNA, syntenic with BAC clones specific to chromosomes D h 09 and D h 11, respectively, were located to the corresponding chromosomes (Figure 2u, 2w). Since the chromosome bearing the third 45S could not be identified with BAC clones derived from G. hirsutum (Gan unpublished), the third 45S was therefore identified to chromosome 02 ( Figure 2v) with BAC clone screened from A 2 D 2 (Qinqin unpublished). According to the homology between D genome and D subgenome, chromosomes bearing with 45S locus of D 5 were named as D 5 09, D 5 11 and D 5 02, respectively. And, three 45S loci were shown at the end of short arm of chromosomes D 5 09, D 5 11 and D 5 02, while one 5S rDNA locus was found interstitial to the short arm of chromosome D 5 09. Figure 3a-3f showed the 45S and 5S rDNA locations specific to individual chromosomes in A 1 and A 1-a . Three 45S and one 5S rDNA, syntenic with BAC clones specific to chromosomes A h 05, A h 07 and A h 09, were located to the corresponding chromosomes. According to the homology between A genomes and A subgenome, chromosomes bearing 45S locus were named as A 1 05, A 1 07 and A 1 09 (for A 1 ) (Figure 3a-3c), A 1-a 05, A 1-a 07 and A 1-a 09 (for A 1-a ) (Figure 3d-3f), respectively.   Therefore, in both A 1 and A 1-a , three 45S rDNA loci were revealed at the end of short arm of chromosomes A 1 05 (A 1-a 05), A 1 07 (A 1-a 07) and A 1 09 (A 1-a 09), and one 5S locus was located on chromosomes A 1 09 ( Figure 3c) and A 1-a 09 (Figure 3c), respectively. There is limited genomic information available for species of B, E, F and G genomes, and the chromosome identification for BAC clones is unavailable either, and so the location of 5S and 45S rDNA has had been identified by using rDNA-FISH with only 5S and 45S rDNA probed. In B 1 and B 3 , one 5S locus and two 45S loci were observed at the end of chromosomes, while one 45S locus was found nearby centromere, displaying the satellite-intermediate type, which is extremely rare in Gossypium species (Figure 1j, 1k). In E 2 , E 3 , E 4 , 5S and 45S were both located at the end of chromosomes (Figure 1l-1n). In F 1 , two 45S rDNA loci and the third 45S rDNA locus syntenic with 5S rDNA were located to the end and near centromere of chromosomes, respectively, while the 5S rDNA locus of F 1 was positioned outside the 45S rDNA and to the end of chromosome (Figure 1o). In G 1 and G 3 , 45S and 5S rDNA were located at the end and near centromere of chromosomes, respectively (Figure 1p, 1q). And the relationship between 5S and 45S is nonsyntenic in B 1 , B 3 , E 2 and E 3 , while it showed syntenic in F 1 , G 1 and G 3 . Among two 5S loci in E 4 , one is synteny with 45S rDNA while the other one is not synteny with 45S rDNA.

Chromosome distribution of rDNA in Gossypium genus
In order to compare and analyze the evolutional relationship among cotton species, the chromosomal distribution of 5S and 45S for 17 species and one variant in the present study and 8 species presented in previous papers [17][18][19] are summarized in Table 2 and displayed schematically in Figure 4.
The 5S rDNA loci maintained high homogeneity in the number (except E 4 ), distribution and the copy number among 25 species and one variant, only varied partly in copy numbers in five tetraploid species (Table 2 and Figure 4). In all diploid species except E 4 , only one 5S rDNA locus was found. In tetraploid species and diploid species of A and D genome, all 5S rDNA loci were located to chromosomes 09 (A t 09/D t 09/A g 09/D g 09). The number of 45S rDNA is conserved in some species but is various in other species, no matter related with their genome resource. Even the same number of 45S loci was observed, they were still positioned to different chromosomes. Beside the number and chromosomes bearing 45S, the copy number (identified by signal intensity) of 45S rDNA was observed to be similar to some extend, but varied to a great extent. They varied in either different species or different rDNA Pattern in Gossypium Genus PLOS ONE | www.plosone.org chromosomes of the same species. In addition, the syntenic relationship between 5S and 45S rDNA were divided into three groups. One is syntenic for A, D, F and G genome, and the second is nonsyntenic for B and two species of E genome. Then the third one is both syntenic and nonsyntenic in E 4 which has two 5S rDNA loci.

Discussion
Genomic and evolutionary researches in Gossypium have obtained great progress with morphological characteristics, geographical distribution, cytogenetic and molecular data. And the evolutionary history has been built into phylogenetic tree [24]. However, our understanding of evolution and chromosome structure is still extremely limited. Most of Gossypium species have rather small chromosomes, which in many cases are similar in shape and size, and therefore are difficult to be distinguished. In this study, we used chromosome-specific BAC clones from G. hirsutum, 5S and 45S rDNA as multiple probes to have FISH located precisely the 5S and 45S rDNA of tetraploid species and species of their donor genome (A and D genome). And species of other genomes are FISH located with 5S and 45S rDNA as double probes. In general, 5S and 45S rDNA were revealed for both conservation and polymorphism in the number of rDNA loci, the number of rDNA repeats, chromosome-bearing rDNA and the synteny relationship between 5S and 45S rDNA. The rDNA pattern is generally in accordance with phylogeny with some disagreements. It is noted that, the chromosomes bearing 5S and 45S rDNA have been identified accurately for A and D genome as well as the tetraploid species using chromosomespecific BAC clones. Therefore, location of 5S and 45S rDNA in our study provided more detailed and comprehensive information for the evolution of Gossypium genus than the previous reports, which provided insights into molecular evolutionary changes.

Chromosomal patterns of 5S and 45S rDNA
The 5S rDNA loci among 25 cotton species (Table 2 & Figure 4) were observed to be highly conserved in this study. Only one 5S rDNA locus was found in all diploid species except E 4 and two loci in tetraploid species. The high conservation of 5S rDNA is a common phenomenon in plant genera such as Oryza genus [11], Arichis genus [8], and Quadrifaria group of Paspalum genus [25]. The conserved distribution of 5S rDNA might be associated with their location at pericentromeric regions [26], which rarely related to the chromosomal structure rearrangement [27]. Also, the recombination intercalary site of  The phylogeny is according to the reference of Wendel [24]. The words under the chromosomes indicated the chromosomes bearing the rDNA locus. 5S rDNA: green signals; 45S rDNA: red signals. * and ** indicate the differences according 45S rDNA pattern in the present study from previous report [24], respectively; *** indicates G. herbaceum var. africanum. chromosome is much lower than that of ones at the end of chromosome in upland cotton [21]. The distribution of 45S rDNA among 25 cotton species (Table 2 & Figure 4) is both conserved and polymorphic, in accordance with other reports showing similar pattern [11,16]. The number variations of rDNA among plants of the same ploidy level have been attributed to chromosomal rearrangements, transposable events and gene silence [28]. According to the data of the whole genome sequencing of G. raimondii which is the smallest Gossypium species, a high proportion of transposable elements such as the gypsy and copia-like LTRs were found [29], suggesting that the most possible mechanisms associated with 45S rDNA variation could be transposon mobility. Taking conservative karyotypes of Gossypium interspecies into account, major chromosomal structural rearrangements are not frequent among species. Therefore, in Gossypium genus, the key mechanism facilitating diversification of 45S rDNA distribution patterns should be considered as transpositions rather than chromosome rearrangements. Moreover, the copy numbers of 45S rDNA loci was discrepant in the different chromosomes of the same species or the corresponding chromosomes of different species. The copy numbers of rDNA repeats might be amplified or decreased by unequal crossing over to the extent that these new sites can be detected by FISH.
Besides the numbers and copy numbers of rDNA, some tendencies of syntenic relationship between 5S and 45S rDNA have been as well indicated. It is syntenic for A, D, F and G genome, but nonsyntenic for B and E genome. The diploid species were divided into Australian species (C, G and K genome), American species (D genome) and Africa-Asian species (A, B, E and F genome), and the last one is considered as original species [30]. Species of B and E genome could be the most original according to the chromosomal pairing analysis in interspecies hybridization [31], electrophoretic analysis of seed protein [32] and phenotypic relationship analysis [33]. According to the evolution pattern, more advanced species evolved from the distribution center to the edge of the expansion. It is considered that Africa species (A, B, E and F genome) originated from Africa which was a distribution centre of cotton [30]. Therefore, the nonsynteny relationship in species of B and E genomes might be related the original types.

Phylogenetic implications with rDNA pattern for Gossypium genus
Phylogenetic implications for tetraploid species. The variations in rDNA distribution are with phylogenetic implications, for the closeness of taxa is correlated to the similarity of their rDNA FISH patterns [6]. Theoretically, the rDNA loci of tetraploid cottons should be the sum of rDNA loci of its putative diploid ancestors (A and D genome). In the present study, the sum of 5S rDNA loci of species of A and D genome was equal to that of tetraploid cottons, while 45S rDNA loci decreased in tetraploid cottons. The nonadditive contribution of rDNA during the evolution of polyploidy species has been described in several plant genera [10,16,28,34]. Several hypotheses may explain the reason. Decreases in site number could arise to stabilize new genomes by the formation of translocations with breakpoints proximal to the rDNA sites during the formation of polyploidy [35]. And, deletions may have eliminated loci in modern tetraploid cottons. Also, new 45S rDNA loci may have been formed by transposition of sequences containing rDNA repeats in the modern A and Ddiploids.
Additionally, the copy number of 45S rDNA locus on chromosome A t 09 in tetraploid species is much higher than that of chromosome A g 09 in donor genome, but that of 5S rDNA locus of D t 09 in tetraploid species reduced obviously relative to that of D g 09 in donor genome. It is possible that the copy number of 45S rDNA locus on chromosome A g 09 in original parental species is very high, but unfortunately the species died out [35]. Or the copy number of 45S rDNA locus on chromosome A t 09 could increase after the formation of polyploidy or the subsequent evolution. Compared to parental species, 5S rDNA locus in polyploidy species according to the researches on other genus reported tended to eliminate [6,16,[34][35][36]. Some hypotheses may explain why 5S rDNA locus disappeared or their copy number reduced following polyploidization of tetraploid cottons. Firstly, the copy number of 5S rDNA locus could be very low in the original parental species which may extinct [37]. Secondly, the copy number decreased during the course of the formation of tetraploid species. Thirdly, the copy number did not decrease in the modern tetraploid species evolved from the original ones. And the related mechanisms account for the changes could be unequal crossing over, gene exchange and transposons events, and so on [3].
Phylogenetic implication for American diploid species (D genome). Thirteen species of D genome containing six subsections, have received considerable phylogenetic attentions [30,[38][39][40], but evolutionary relationships among these subsections still have not enough evidence [24]. Some evolutionary evidences could be obtained from the rDNA patterns which were revealed both conserved and changeable in ten species of six subsections studied here. The evidence is that it is conserved in the chromosomal location, copy number and synteny relationship of 5S and 45S rDNA on chromosomes D g 07 and D g 09 in ten species except D 5 and D 3-d (Table 2 and Figure 4). D 5 , divided into subsection Austroamericana Fryxell at the end of D genome [1], the 45S loci on chromosomes D 5 09, D 5 11 and D 5 02 differ greatly with other species (Table 2 and Figure 4), suggesting the greater evolutional history than that of other D-genome species. D 6 is the sole representative of subsection Selera Fryxell with distinctive morphological characteristics [41]. It has only two 45S loci on chromosomes D 6 07 and D 6 09 ( Table 2 and Figure 4), which is in agreement with that of D 6 and could be used as the base of D genome species [34]. D 1 and its sister species D 8 , the two representative of subsection Houzingenia [30,39,42], both has four 45S loci on chromosomes D 1 07(D 8 07), D 1 09(D 8 09), D 1 03(D 8 03) and D 1 11(D 8 11) (Table 2 and Figure 4 [17]). The latter two 45S loci could be more unique to the two species rather than other species, which is in accordance with their clades showing different from other three clades in cladogram [24]. The rest six species, derived from the same clade but rDNA Pattern in Gossypium Genus PLOS ONE | www.plosone.org being divided into three subsection [24], have common 45S loci on chromosomes D g 07, D g 09 and D g 05 supporting the same clade, however the variation of the last 45S locus distribution suggested two divisions among the six species as the following: On one hand, D 3-k and D 3-d of subsection Integrifolia Todaro and D 9 of subsection Erioxylum all have four 45S rDNA on the chromosomes D g 07, D g 09, D g 05 and D g 12, suggesting that they might be closer than with other three species. And copy numbers of the 45S locus on chromosome D 3-d 09 is of much less than other nine species of D genome including D 3-k ( Table 2 Figure 4 * [18]). On the other hand, the other three species, D 2-1 of subsection Caducibracteolata Mauer as well as D 4 and D 11 of subsection Erioxylum, all have three 45S loci on the chromosomes D g 07, D g 09 and D g 05 ( Table 2 Figure 4 ** [17]), suggesting that they might be closer than their relationship previously reported [24].
Phylogenetic implication for African-Asian diploid species (A, B, E and F genome). The A, B, E and F genomes belong to the same clade, which are different from other genomes, according to the phylogenesis history of Gossypium genus [24]. Species of these four genomes have same numbers of 45S and 5S loci (except E 4 ) but varied significantly in synteny relationship. A and F genomes with the same rDNA pattern could associate with their proposed sister relationship [24]. And the 5S locus in F 1 was found near the satellite outside the 45S rather than near centromere inside the 45S unlike other species. Philips [31] proposed that F 1 should be removed from E genome and classified as F genome, as F 1 could be a new cellular type according to cytology research. F 1 with the synteny relationship of 5S and 45S is different from E genome with the nonsynteny relationship, providing more visual evidence of the further establishment of F genome. Besides, the 5S and 45S rDNA at telomere ends in F 1 are close to each other, which is rare in Gossypium species though it has been reported in other plants [43][44][45], suggesting it may relate to the stabilization of centromeric fission products [45,46].
And, the similar distribution of 5S and 45S loci was observed in B 1 and B 3 . It was not extensively accepted that B 3 was classified into B genome at the early days. As far as phenotypic traits, B 3 grows likewise A 1 (with yellow crown, apetalous basis points and five-room capsule), other than any species of B genome (with ivory petal, large basis points of petals and three-room capsule). From the synteny relationship of 5S and 45S, B 3 could be confirmed to be in B genome. And the nonsynteny relationship of 5S and 45S could be considered as the classification basis of B genome. So the rDNA identification for all species of B genome has the cytogenetic evidences for the classification of Gossypium genus. Notablely, the two 5S loci in E 4 is a great discovery in Gossypium genus, suggesting special evolutional implication in species of E genome.
Phylogenetic implication for Australia diploid species (G genome). The numbers of 45S rDNA were revealed three and four in G 3 and G 1 , respectively, although the two G-genome species have similar morphological traits [47]. It is a certainty for G genome in terms of the taxonomy, which had been well studied [40,47]. Therefore, the origin might account for the difference in the rDNA numbers between two species. According to the cpDNA analysis, the chloroplast genome of G 1 was similar with that of G. sturtianum, a morphologically distant C-genome species, suggesting that G 1 could have a reticulate history with G. sturtianum.
In summary, the current study has clarified systematically the interrelationship of the rDNA distribution among 25 Gossypium species and one variant covering AD, A, B, D, E, F and G genomes. And the corresponding phylogenetic implications have been revealed for the evolution of Gossypium genus. Further study is needed to investigate the more precise rDNA patterns on meiotic pachytene chromosomes between with the development of cotton FISH techniques [48].