Index INTRODUCTION MATERIALS AND METHODS Plant materials and clones SSR markers identification DNA probes preparation Chromosome preparation and FISH The integration of genetic map and chromosomal map RESULTS Examination of G. thurberi (D1), G. trilobum (D8) and D subgenome of G. barbadense (Db) with chromosome-specific SSR markers Individual chromosome assignments in D1, D8 and Db by BAC-FISH Comparison of the relative chromosomal positions of BAC clones DISCUSSION References

INTRODUCTION

The genus Gossypium, which may be a model crop for cytogenetic, genomic and evolutionary biology researches, comprises 52 species spreading over many tropical and subtropical regions (Wendel et al., 2010). It composes of 4 cultivated species and 48 wild species which were classified into 8 diploid (2n = 2x = 26) genomes, i.e. A, B, C, D, E, F, G and K, and one allotetraploid (2n = 4x = 52) genome, i.e. AD (Percival et al., 1999). D genome includes 13 species and has been more thoroughly collected and better studied than other genomes such as B, C, E, F, G and K (Wendel et al., 2010). The 13 species had received considerable phylogenetic attention and were divided into lineages corresponding precisely to the 6 taxonomic subsections (Wendel and Cronn, 2003). Among them, subsection Houzingenia consists of G. thurberi (D₁) and G. trilobum (D₈). G. thurberi was thought to be one of the D subgenome donors of the tetraploid cotton (AD) (Beasley, 1942). It is considered that G. thurberi can tolerate mild frost via defoliation as one of northern species (Huang et al., 2003). It had been employed to create the triple hybrid which was used to introgress high fiber strength into G. hirsutum which is the most important natural fibers for sustaining the economic textiles industry (Paterson et al., 2009; Beasley, 1942). G. trilobum, sister species to G. thurberi, was reported highly resistant to Verticillium and Fusarium wilt, drought, short-term frost (–7~10°C) and frost (Huang et al., 2003). The previous research revealed that male sterile cytoplasm and restorer factor (Rf₂) were found in G. trilobum (Stewart, 1992). And then this D₈ cytoplasmic male sterile (CMS-D₈) and its restorer were developed by introducing its cytoplasm and gametophytic nuclear gene Rf₂ into G. hirsutum (Stewart, 1992; Zhang and Stewart, 2001a, b). G. barbadense is the second largest cultivated species in the world with its better fiber quality but lower yield potential than G. hirsutum. Because G. hirsutum and G. barbadense are all allotetraploid (AD), they can be hybridized easily as to improve fiber length, fineness and strength to G. hirsutum or to overcome the low yield capability to G. barbadense (Lacape et al., 2005). Therefore, G. thurberi, G. trilobum and G. barbadense are the important reservoirs of useful genes which can be introgressed into the cultivated G. hirsutum, aiming to broaden its existing narrow genetic resources and to enrich existing varieties with desired agronomic traits. However, as fewer earlier research reports on the cytological and karyotypic data of these three species, it is very important to identify their individual chromosome.

Early individual chromosome identification was applied widely in cotton by analyzing chromosomal relative lengths, arm ratios, and nuclear organization regions (NORs) in the mitotic or meiotic metaphase (Wang and Li, 1990). However, this method has some limitations both genetically and veritably for many small and similar chromosomes in cotton. The unambiguous identification of individual chromosome is almost impossible based only on their morphology without suitable molecular cytogenetic markers. And then the fluorescence in situ hybridization (FISH) is undoubtedly versatile and accurate for chromosome identification by positioning repetitive and single copy DNA sequences along the chromosomes with respect to centromeres, telomeres and heterochromatic regions (Jiang and Gill, 2006; Dou et al., 2009). In early days, repeat-based FISH probes were usually used for chromosome identification (Jiang and Gill, 2006). Later, chromosome-specific markers were developed and applied in more and more plants for the individual chromosome recognition with large-insert genomic clones such as BACs (Uozu et al., 1997; Dong et al., 2000; Kim et al., 2002, 2005; Hasterok et al., 2006; Wang et al., 2008; Wai et al., 2010; Xiong et al., 2010). All these studies expressed BAC-FISH a powerful tool in plant molecular cytogenetic researches such as karyotyping, chromosome identification and physical mapping (Jiang and Gill, 2006).

Individual chromosome of G. hirsutum has been assigned with a set of chromosome-specific BACs which was constructed by translocation (Wang et al., 2007). Also, the nomenclature and standard karyotype of G. arboreum chromosomes were reported according to the homology of the A genome chromosomes in G. arboreum and the A-subgenome (A_h) chromosomes in the G. hirsutum (Wang et al., 2008). The recognition of chromosome with molecular cytogenetic markers is thought the fundamental study for cotton genomic research and breeding (Wendel et al., 2009). However, as the possible ancestor species of allotetraploid, G. thurberi and G. trilobum have not had any genetic linkage maps and BAC libraries for chromosome identification. Genetic linkage maps and BAC libraries in G. barbadense had been constructed (Chen et al., 2007; Rong et al., 2004; Guo et al., 2007) but the orientation of genetic linkage maps and the identification of individual chromosome have not been reported yet.

The main objectives of the present study were to establish a set of chromosome-specific molecular cytological markers and assign individual chromosome for G. thurberi (D₁), G. trilobum (D₈) and D subgenome of G. barbadense (D_b). The signal positions of each marker on the corresponding chromosomes among different genomes were compared in order to identify the relationship of chromosomal collinearity. The orientation of linkage group in D_b was estimated in the study using the D genome centromere-specific clone. Also, the physical location of rDNA was revealed. The individual chromosome assignment and rDNA locations of the two D genomes and the D subgenome described herein provided a platform for cytogenetic, genomic and polyploidy evolution analysis which does not currently exist in diploid D genome wild species and G. barbadense.

MATERIALS AND METHODS

Plant materials and clones

An accession of G. thurberi, D₁-36, another accession of G. trilobum, D₈-7 and a cultivar of G. barbadense, Pima 90-53, were used in this study. They were all introduced originally from Crop Germplasm Research Unit, Southern Plains Agricultural Research Center, USDA-ARS in College Station, Texas State, USA. Plants of the two wild accessions (D₁-36 and D₈-7) are growing perennially in National Wild Cotton Plantation in Sanya City, Hainan Island, sponsored by Cotton Research Institute of Chinese Academy of Agricultural Sciences (CRI-CAAS) in Anyang City, Henan Province, China. These two accessions are also conserved in pots in greenhouse at CRI-CAAS. The cultivar Pima 90-53 is also kept in the medium-term storage facilities for the National Cotton Germplasm Collection at CRI-CAAS.

Four types of probes were used in this study including 45S rDNA, 5S rDNA, BAC clone 150D24 and a set of D_h chromosome-specific BAC clones (Table 1). The 45S and 5S rDNA derived from Arabidopsis thaliana were kindly provided by Professor Yunchun Song, Wuhan University, China. The BAC clone 150D24 which contains centromere-specific repeats in D subgenome and D genome of Gossypium was screened from the Pima 90-53 BAC library (Wu et al., 2010) and was used to indicate the centromere position. The D_h chromosome-specific BAC clones (Table 1) used in the identification of individual chromosome were kindly provided by Professor Tianzhen Zhang, Nanjing Agricultural University, China (Wang et al., 2007). SSR primers specific to single chromosomes (Table 1) were derived from the Cotton Marker Database (http://www.cottonmarker.org/) and were synthesized by Sunbiotech Co., Ltd. in Beijing.

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Table 1 Chromosome-specific SSR markers and their corresponding BAC clones in D subgenome of G. hisutum (Dh)

SSR markers identification

The total genomic DNAs (gDNAs) from G. thurberi, G. trilobum and G. barbadense were extracted from young leaves using modified CTAB method (Paterson and Wendel, 1993). PCR amplifications with 13 pairs of SSR primers on a Takara PCR Thermal Cycler Dice and product eletrophoresis were basically performed as previously described (Zhang et al., 2002).

DNA probes preparation

The probes 45S, 5S rDNA and BAC DNA were isolated using a standard alkaline extraction (Sambrook and Russell, 2002). 45S rDNA and BAC clone 150D24 were labeled by standard Dig-nick translation reactions, whereas 5S rDNA and a set of D_h subgenome chromosome-specific BAC clones (Table 1) were labeled with Biotin-nick translation reactions, according to the instructions of the manufacturer (Roche Diagnostics, USA).

Chromosome preparation and FISH

Preparation of mitotic chromosomes and the FISH procedure were conducted following the modified protocols (Wang et al., 2001). Biotin-labeled and digoxigenin-labeled probes were detected by avidin-fluorescein (green) and anti-digoxigenin-rhodamine (red) (Roche Diagnostics, USA), respectively. Chromosomes were counterstained by 4’,6-diamidino-2- phenylindole (DAPI) in the antifade VECTASHIELD solution (Vector Laboratories, Burlingame, CA). For the probe-cocktail mixture, we used gDNA as block DNA instead of Cot-1 DNA. The dose of block DNA was 200 times of the chromosome-specific BAC. The hybridization signals were observed using a fluorescence microscope (Leica MRA2) with a charge-coupled device (CCD) camera (Zeiss) and arranged by Adobe Photoshop 7.0.

The integration of genetic map and chromosomal map

The relative chromosomal positions of BAC clones according FISH signal patterns in D₁, D₈ and D_b can be compared with their SSR markers on genetic linkage maps. The positions of these SSR markers to each D_b linkage group were obtained according to the linkage map (Guo et al., 2008) and the location of centromere. The short arm is indicated by convention positioned on top.

RESULTS

Examination of G. thurberi (D₁), G. trilobum (D₈) and D subgenome of G. barbadense (D_b) with chromosome-specific SSR markers

The 13 D_h chromosome-specific BAC clones (Table 1) were used to identify individual chromosomes of D₁, D₈ and D_b. These BAC clones were screened using 13 pairs of chromosome-specific SSR markers derived from D_h (Wang et al., 2007). The 13 SSR markers were examined to be all existent in D₁, D₈ and D_b (Fig. 1, a–c). The size of the most amplified fragments was found accordantly in the two D genomes and the D subgenome, showing a relatively good concordance of their inter-genomic relationships. Therefore, these results were thought worthy to be used next in subsequence researches.

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Fig. 1 Examination of G. thurberi (a), G. trilobum (b) and G. barbadense (c) with the Dh chromosome-specific SSR markers. M: DNA marker; Lane 1-13: Fragments of the 13 SSR markers corresponding Dh01-13 BACs respectively; Blue arrows indicate fragments size.

Individual chromosome assignments in D₁, D₈ and D_b by BAC-FISH

We have conducted a successful individual chromosome assignment in D₁, D₈ and D_b with 45S rDNA, 5S rDNA, the D genome centromere-specific BAC clone and the set of D_h chromosome-specific BACs as FISH probes. The results showed that the set of D_h BAC clones was located on the corresponding chromosomes and chromosomal arms in D₁, D₈ and D_b (Fig. 2, Fig. 3 and Fig. 4). Nine hybridization loci were found nearby to centromere regions (Fig. 2, a, b, c, f, g, i, k, l and m; Fig. 3, a, b, c, f, g, i, k, l and m; Fig. 4, a, b, c, f, g, i, k, l and m), two loci near to terminals (Fig. 2, d and j; Fig. 3, d and j; Fig. 4, d and j) and two loci at the middle part of the long arms (Fig. 2, e and h; Fig. 3, e and h; Fig. 4, e and h).

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Fig. 2 Identification of individual chromosome by dual-color FISH with multiple probes in G. thurberi (D1). a–m: FISH images on the corresponding chromosome 01-13 of G. thurberi with Dh01-13 chromosome-specific BAC clones, respectively (green fluorescence signals marked with green arrows); 5S rDNA: green fluorescence signals without arrow near the centromere of the short arm of chromosomes 09; 45S rDNA: red fluorescence signals at the terminal of the short arm of chromosomes 03, 07, 09, and 11; centromere clone 150D24: red fluorescence signals at the intercalary chromosomes. n: Eight red 45S rDNA (white arrows) and two green 5S rDNA signals (green arrows) in G. thurberi metaphase chromosomes. Marked chromosomes were enlarged at the top-right corner with the short arm on the top. Bar = 5 μm.

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Fig. 3 Identification of individual chromosome by dual-color FISH with multiple probes in G. trilobum (D8). a–m: FISH images on the corresponding chromosome 01-13 of G. trilobum with Dh01-13 chromosome-specific BAC clones, respectively (green fluorescence signals marked with green arrows); 5S rDNA: green fluorescence signals without arrow near the centromere of the short arm of chromosomes 09; 45S rDNA: red fluorescence signals at the terminal of the short arm of chromosomes 03, 07, 09, and 11; centromere clone 150D24: red fluorescence signals at the intercalary chromosomes. n: Eight red 45S rDNA (white arrows) and two green 5S rDNA signals (green arrows) in G. trilobum metaphase chromosomes. Marked chromosomes were enlarged at the top-right corner with the short arm on the top. Bar = 5 μm.

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Fig. 4 Identification of chromosomes (Db01-13) by dual-color FISH with multiple probes in D subgenome of G. barbadense (Db). a–m: 13 FISH images on the corresponding chromosome Db01-13 of G. barbadense with Dh01-13 chromosome-specific BAC clones, respectively (green fluorescence signals marked with green arrows); 5S rDNA: green fluorescence signals without arrow near the centromere of the short arm of chromosomes Db09; 45S rDNA: red fluorescence signals at the terminal of the short arm of chromosomes Db07 and Db09; centromere clone 150D24: red fluorescence signals at the intercalary chromosomes. n: Six red 45S rDNA (white arrows) and four green 5S rDNA signals (green arrows) in G. barbadense metaphase chromosomes. Marked chromosomes were enlarged at the top-right corner with the short arm on the top. Bar = 5 μm.

Based on the individual chromosome assignment, the number and chromosomal locations of rDNA in D₁, D₈ and D_b were confirmed (Fig. 2, c, g, i, k and n; Fig. 3, c, g, i, k and n; Fig. 4, g, i and n; Table 2). Four 45S rDNA loci were all located at the end of the short arm of chromosomes 03, 07, 09 and 11, whereas one 5S rDNA locus was intercalary at the short arm of chromosomes 09 (Fig. 2, c, g, i and k; Fig. 3, c, g, i and k). Among them, the rDNA signal on D₈ chromosome 11 showed weak performance and could not be detected sometimes. In G. barbadense (A_b and D_b together), three 45S rDNA loci and two 5S rDNA loci were revealed (Fig. 4n). Among these, in D_b, two 45S rDNA loci were identified at the terminal of the short arm of chromosomes D_b07 and D_b09, whereas one 5S rDNA locus was distributed near to the centromere of the short arm of chromosome D_b09 (Fig. 4, g and i). Position comparison of the 45S and 5S rDNA in D₁, D₈ and D_b expressed that most of rDNA had been marked at the corresponding locations, indicating the conserved collinearity relationship in the two D genomes and the D subgenome. Also, the synteny relationship between 45S and 5S rDNA loci had been confirmed as they were found located at the short arm of chromosome 09 in the two D genomes and the D subgenome (Table 2).

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Table 2 Number and chromosome position of rDNA loci in two D genomes (D1 and D8) and D subgenome (Db)

Comparison of the relative chromosomal positions of BAC clones

The relative chromosomal positions of BAC clones according to FISH signal patterns in D₁, D₈ and D_b (Fig. 2, Fig. 3 and Fig. 4.) had been compared. The relative positions of these BAC clones between the two D genomes and the D subgenome expressed identical, suggesting the collinearity of chromosomal locations (Fig. 5, B–D). The positions of the SSR markers to the linkage groups were estimated according to the orientation of chromosome (Fig. 5, A–D). Some positions of SSR markers (Chr. 01, 02, 04, and 10) in the genetic map that was derived from the reported cotton genetic map (Guo et al., 2008) had been found reversed according to the location of the corresponding BAC-FISH signals. Although most of the positions between BAC clones in D₁, D₈ and D_b, as well as SSR markers showed a consistent trend (Fig. 5, A–D), one difference was found that BAC clone 59B08 was marked near the centromere of the short arm of chromosome 11 in D_b, D₁ and D₈, whilst the corresponding SSR marker NAU694 was located at the top half of D_b linkage maps.

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Fig. 5 The integration of genetic map and chromosomal map. A: The SSR marker positions to each Db linkage map (Guo et al., 2008). B, C and D: FISH images of chromosome-specific BACs for Db Chromosome Db01-13, D1 Chromosome 01-13 and D8 Chromosome 01-13, respectively, The correspond FISH images were depicted according to those in Fig. 4, Fig. 2 and Fig. 3, respectively. The orientations of chromosomes and linkage group were showed with the shorter arm on the top.

DISCUSSION

The assignments based on chromosome “bridge” and genetic linkage group had been used to both identify chromosomes and integrate chromosomes and genetic maps in sorghum (Kim et al., 2002, 2005), potato (Dong et al., 2000; Tang et al., 2008, 2009), Medicago truncatula (Kulikova et al., 2001), Brassica oleracea (Howell et al., 2002; Kim et al., 2009), and cotton (Wang et al., 2007, 2008). It is more accurate and simpler to communicate among different varieties and accessions in Gossypium, as well as among different research institutes, than the previously reported recognitions mainly based on chromosome length (Dong et al., 2000). In Gossypium, Wang et al. (2007, 2008) reported the individual chromosome assignments in G. hirsutum and G. arboretum but they did not use marker to distinguish the chromosomal centromere location and arms. In present study, we have successfully used D genome centromere-specific BAC clones to locate the relative chromosomal positions of centromere and the individual chromosome-specific BAC clones. The BAC clones signals on the short arm of chromosomes 01, 02, 04, 07, 08, 09 and 12 in previous assignments (Wang et al., 2007) were more accurately inversed to the long arms of corresponding chromosomes in our experiments.

In addition, referencing to the positions of some SSR markers linked to the genetic linkage groups, in present study we have found one discrepancy in chromosome 11 whilst most of the SSR markers and their BAC clones have the corresponding positions. The corrections of positions of molecular markers to genetic maps had been reported in many other plant integrated maps (Howell et al., 2002; Islam-Faridi et al., 2002; Kim et al., 2002, 2005; Wang et al., 2007, 2008). For example, the FISH markers in sorghum marked near the ends of chromosomes, while their corresponding genetic markers were near the middle of linkage group (Kim et al., 2002; Wang et al., 2007).

Ribosomal DNA (rDNA) is a useful chromosomal landmark for tracking the plant genome evolution. The fluorescent in situ hybridization with rDNA probe (rDNA-FISH) is a visualized method in the location of rDNA. The analysis of the rDNA loci at the intragenus or intergenome level provided evidences for taxonomic research (Bogunic et al., 2011; Hamon et al., 2009; Rosato et al., 2008), species evolution (Chung et al., 2008) and the research of rDNA evolution and function (Nguyen et al., 2010). Although there are conserved loci in interspecies, there are discrepancies between different species. It is found that the closer of the interspecies relationship, the more conserved expression in the rDNA loci (Chung et al., 2008). In cotton, the research on rDNA-FISH has been mainly focused on the number of rDNA loci in the related species (Hanson et al., 1996) or chromosome location in G. hirsutum and G. arboreum (Price et al., 1990; Crane et al., 1993; Wang et al., 2008). The researches on the chromosome location of rDNA in other cotton species and interspecies relationship are less reported for their less translocation materials. In present study, we investigated and compared the chromosomal locations of the rDNA in D₁, D₈ and D_b (Table 2). The results showed that the rDNA loci on chromosomes 07 (D_b07), 09 (D_b09) in the two D genomes and the D subgenome are conserved, while the loci are accordantly with that of G. hirsutum (Price et al., 1990; Crane et al., 1993; Ji et al., 1999), suggesting the two rDNA loci possibly conserved in D genome (or D subgenome). As well, the rDNA loci on chromosomes 03 and 11 in D₁ and D₈ are expressed conserved but the FISH signal on D₈ chromosome 11 was too weak to be detected sometimes. And, the two 45S rDNA loci have not been detected on D_b chromosome 03 and 11. These undetected 45S rDNA genes show that the variability between D genome and D subgenome, suggesting some rearrangement events occurred in the course of evolution. The variability in the position of the rDNA loci resulted from rearrangement events has been found in the different rice species (Shishido et al., 2000), wheat species (Dubcovsky and Dvořák, 1995) and genus Brassica (Fukui et al., 1998) and so on. The rearrangement events might be explained with known mechanisms, such as chromosomal translocations and transpositional mobility, although these mechanisms seem to be not competent (Shishido et al., 2000). It is anticipated that the conserved and variable rDNA loci presented in the current study will provide evidences for the mechanism of rDNA variability.

Corresponding orders (collinearity) among different species will contribute to deduce the shared ancestry of genes and to study less-well-understood species (Tang et al., 2008). The allotetraploid cotton derived from the union of A genome and D genome species 1–2 million years ago (Wendel and Cronn, 2003). Most of the previous comparative mapping studies between D genome and D subgenome of allotetraploid cotton (D_t) were based on the genetic markers. The genetic mapping comparisons between D genome and D_t have showed collinearity (Rong et al., 2004) and locus order differences (Brubaker et al., 1999), providing insights into cotton polyploidy evolution. The result of integrating markers between genetic linkage map and physical map indicated that the chromosomal collinearity existing within D₁ and D₈ and also between the two and D_b. The comparative analysis based on FISH have detected extensive collinearity between A. thaliana and B. rapa (Kim et al., 2009), Oryza and Sorghum (Hass-Jacobus et al., 2006), Oryza intragenus (Teranishi et al., 2008) and Musa intragenus (Lescot et al., 2008). As can be predicated that the chromosomal collinearity will leverage the insights into the evolutionary process of polyploidy and genome sequencing.

We deeply thank Dr. Tianzhen Zhang (Nanjing Agricultural University, China) for providing the set of chromosome-specific BAC clones, Yunchen Song (Wuhan University, China) for supplying the 45S and 5S rDNA. This work was sponsored by a grant from the National Natural Science Foundation of China (No. 31071466), National High-tech R&D Program of China (863 Program) (No. 2003AA207051), Essential Science Research Funds to National Non-profit Institutes of China (No. SJA0901).