Background

Cotton (Gossypium spp.) is the most important fiber crop in the world and one of the most important oilseed crops. Within the genus Gossypium, two cultivated allotetraploid species, G. hirsutum L. and G. barbadense L., account for 90% and 8% of the world cotton production respectively [1]. The construction of a molecular genetic map is a foundation in genetic dissection of economically important traits, marker-assisted selection (MAS), and map-based cloning. It provides new insights into genome structure and chromosomal architecture of the cotton genome. However, the allotetraploid (2n = 4x = 46) species has a large genome size of ~ 2246 Mb [2], which has hindered the development of a high-density map.

To explore the cotton genome structure and to identify quantitative trait loci (QTLs) for agronomically important traits which can facilitate MAS in cotton, several genetic maps have been constructed including high-density interspecific [3–14, 15]. Because of these characteristics, microsatellites have become the most favoured genetic markers for plant breeding and genetics such as genetic diversity assessment, genetic map construction, QTL map**, and marker aided selection, etc [16]. In general, SSRs are identified from either genomic DNA or cDNA sequences. The usual source sequences for SSRs have included SSR-enriched library clones, expressed sequence tags (ESTs), and bacterial artificial chromosome end sequences [17–19]. However, conventional SSR marker development (from enriched libraries) is costly and time consuming [20]. In recent years, with the rapid increase of ESTs in public databases and the advent of bioinformatics tools, SSR marker development has become easier and more cost-effective. Mining SSRs from ESTs is becoming popular for SSR development.

Thanks to global efforts, 11938 SSR markers have been released (CMD website, http://www.cottonmarker.org) up to 2009. Among these SSRs, more than half are EST-SSRs. However, compared to the huge ESTs tank of cotton, only ~25% of the cotton ESTs is applied to SSR development. Cotton ESTs are still a valuable resource for SSR marker development, especially for gene-derived SSRs. It is worth mentioning that the genetic map constructed by Guo et al. contains 71.96% functional marker loci, of which 87.11% are EST-SSR loci [7]. High-density genetic maps of EST-SSR markers are an essential tool for enhanced genome analysis. They represent the transcript part of the genome and can be links between genetic and physical maps [21]. Moreover, as EST-SSRs target coding regions of the genome, they may be useful in association with genes of known function to facilitate the dissection of complex traits [22].

With an endless effort to construct a high-density genetic map of cotton in our laboratory, we have tried RAPDs, SRAPs, and SSRs when no sufficient easy-to-use markers such as SSRs in cotton were available [5, 23]. In the last 5 years, the cotton EST project and genome sequencing project generated a great number of sequences that could promote the marker development, and thousands of EST-SSRs and BAC-end SSRs have been developed. Benefited from these projects, we constructed an SSR-based genetic map using SSRs available in 2008 [27–29], and a final map with 2316 loci map was constructed. This sequence-based, high-density map allowed us to detect segregation distortion regions within the whole genome, to identify gene distribution on chromosomes and homologs between chromosomes.

Results

Marker development

A total of 1831 new EST-SSRs were developed from the assembled cotton ESTs in the TIGR database http://www.tigr.org based on the criteria of marker development (see Materials and Methods): 346 from G. arboretum (HAU231-HAU576), 293 from G. raimondii (HAU577-HAU869), and 1192 from G. hirsutum (HAU870-HAU2061).

The 131182 ESTs released by Yuxian Zhu were clustered and assembled into 46296 unique sequences, consisting of 10691 contigs and 35605 singletons. A total of 1047 unique EST-SSRs (HAU2062-HAU3108) were developed from these sequences.

The 10979 ESTs from develo** fiber of G. barbadense acc. 3-79 generated in our laboratory were clustered and assembled into 5852 unique sequences, consisting of 1492 contigs and 4360 singletons. A total of 299 novel EST-SSRs (HAU3109-HAU3407) were developed from these sequences.

All the marker primers, sequence ID, sequences, motifs, estimated product size, and BLASTX results are listed in Additional file 1.

Maker polymorphism

Of the 700 Gh-prefixed gSSRs derived from G. hirsutum, 134 SSRs (19.1%) showed polymorphism and generated 172 polymorphic loci with an average of 1.28 loci/SSR. Among the 1554 and 754 NAU-prefixed EST-SSRs derived from G. raimondii and G. hirsutum, respectively, and 578 gSSRs from BAC sequences of G. hirsutum, 439 (24.7%), 109 (14.5%), and 68 (11.8%) SSRs were polymorphic, and they generated 537, 131, and 71 loci, respectively, with an average of 1.22 loci/SSR, 1.20 loci/SSR, and 1.0 loci/SSR, respectively.

For these EST-SSRs from the assembled ESTs in TIGR, 28 (8.1%), 33 (13.8%), and 233 (19.5%) SSRs were polymorphic for EST-SSRs from G. arboretum, G. raimondii, and G. hirsutum, respectively, and they generated 29, 36, and 236 loci, respectively. One hundred and sixty-eight SSRs (16.0%) of the 1047 EST-SSRs (HAU2062-HAU3108) were polymorphic and 199 loci were produced; 42 SSRs (14.0%) were polymorphic from the 299 EST-SSRs (HAU3109-HAU3407) and 47 loci were produced.

Map construction and overview

A total of 1458 loci obtained in this study, adding to the 1026 loci published by Zhang et al. [27–29], a total of 2521 loci were applied to map construction. After calculation, 2316 loci including 2311 SSR loci and 5 gene-derived loci were mapped on 26 cotton chromosomes; the total length of this map was 4418.9 cM with an average of 1.91 cM between adjacent markers (see Additional file 2 Figure 1, 2, 3, 4, 5, 6, 7).

Figure 1
figure 1

The genetic map of Chr01/Chr15 homoeologous chromosome. The interspecific genetic map was constructed using the BC1 population [(Emian22 × 3-79) × Emian22]. Duplicated loci are in bold. Map distances are given in centimorgans (cM). Markers showing segregation distortion are underlined and indicated by asterisks (*P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.001; ***** P < 0.0005; ****** P < 0.0001 for markers skewed toward the 'Emian22' allele or degree symbols (°P < 0.05; °°P < 0.01; °°°P < 0.005; °°°°P < 0.001; °°°°°P < 0.0005; °°°°°°P < 0.0001 for markers skewed toward the heterozygous allele). Segregation distortion regions (SDRs) are named as 'Chromosome + No. SDR', for example, SDR2.1 refers to the first SDR on Chr02.

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Figure 2
figure 2

The genetic maps of Chr02/Chr14 and Chr03/Chr17 homoeologous chromosome. All legends are same as described for Figure 1.

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Figure 3
figure 3

The genetic maps of Chr04/Chr22 and Chr05/Chr19 homoeologous chromosome. All legends are same as described for Figure 1.

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Figure 4
figure 4

The genetic maps of Chr06/Chr25 and Chr07/Chr16 homoeologous chromosome. All legends are same as described for Figure 1.

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Figure 5
figure 5

The genetic maps of Chr08/Chr24 and Chr09/Chr23 homoeologous chromosome. All legends are same as described for Figure 1.

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Figure 6
figure 6

The genetic maps of Chr10/Chr20 and Chr11/Chr21 homoeologous chromosome. All legends are same as described for Figure 1.

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Figure 7
figure 7

The genetic maps of Chr12/Chr26 and Chr13/Chr18 homoeologous chromosome. All legends are same as described for Figure 1.

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The chromosome with most loci was Chr19 (134 loci); Chr02 and Chr04 (53 loci) were the chromosomes with the fewest loci. Eighty-nine loci were on each chromosome on average, with 1043 and 1273 loci on At and Dt subgenomes, respectively. More loci were distributed on the Dt subgenome mainly because the NAU-prefixed EST-SSRs were from G. raimondii (D5), the progenitor of the Dt subgenome of tetraploid cotton [7].

The longest chromosome was Chr21 (265.9 cM) and the shortest was Chr14 (102.2 cM); the average chromosome length was 169.96 cM. The total lengths of the At and Dt subgenomes were 2250.1 cM and 2168.8 cM, respectively, which was the result of more loci on the Dt subgenome to increase recombinants.

The biggest average distance between markers was on Chr02 (2.78 cM) and the least was on Chr14 (1.12 cM). The average distances for At and Dt subgenomes were 2.16 and 1.70 cM, respectively, which also benefited from the more loci on the Dt subgenome. The biggest gap between markers was 23.2 cM on Chr03; there were a total of 35 gaps >10 cM with 15 on At and 20 on Dt subgenome, respectively.

SSRs were not evenly distributed on the cotton chromosomes with more gSSRs and EST-SSRs on the Dt subgenome. More gSSRs were on Chr11, Chr19, and Chr21, and more EST-SSRs on Chr05, Chr11, Chr15, Chr19, Chr21, Chr24, and Chr26. gSSRs and EST-SSRs were also differently distributed on each chromosome; they were similar on Chr02, Chr04, Chr11, Chr19, and Chr20 (difference < 5%), but dramatically different on Chr05, Chr15, Chr18, and Chr26 (difference > 50%).

For different genome-derived SSRs, the A genome-derived SSRs mostly targeted Chr05 and Chr15, and of course preferentially targeted the At subgenome; D genome-derived SSRs mostly targeted Chr05, Chr11, Chr19, Chr21, and Chr26, and also preferentially targeted the Dt subgenome; the AD genome-derived SSRs mostly targeted Chr11, Chr19, and Chr21, but preferentially targeted the Dt subgenome.

Segregation distortion

Among the 2521 polymorphic loci, 423 loci (16.8%) including one gene-specific locus showed segregation distortion (P < 0.05) with 139 loci segregating toward the 'Emian22' allele and 284 loci toward the heterozygous allele. For SSR loci, 15.0% of gSSRs and 18.2% EST-SSRs were distorted, respectively.

A total of 323 distorted loci, accounting for 12.8% of the mapped loci, were mapped on cotton chromosomes with 74.9% segregating toward the heterozygous allele (Figure 1, 2, 3, 4, 5, 6, 7). These segregation distorted loci were unevenly distributed on the 26 cotton chromosomes with 3-51 loci on each chromosome (see Additional file 2). More distorted loci were located on the Dt subgenome than on the At subgenome (195 versus 128). The most distorted loci were on Chr02, Chr16, and Chr18 (> 50% of loci were distorted), and the least on Chr05, Chr08, Chr20, and Chr25 (< 5% of loci were distorted). A total of 21 segregation distortion regions (SDRs) were found on the 26 cotton chromosomes with 8 SDRs on the At subgenome and 13 on the Dt subgenome. More SDRs were found on Chr02, Chr16, and Chr18, the chromosomes with the most distorted loci. The distorted loci showed a phenomenon in which loci skewing toward the same allele appeared on the same chromosomes or within the same SDRs (e.g., Chr02, Chr13, Chr16, and Chr18; Figure 2, 4, 7). Interestingly, adjacent markers in some SDRs showed the same degree of segregation (SDR5.1, SDR7.1, SDR18.1, SDR18.2, SDR24.1, and SDR24.2).

Annotation and functional characteristics of sequences containing SSRs

In addition to the 1261 EST-SSRs and 5 gene-derived markers, 367 gSSR loci had homologous sequences to cotton ESTs by BLASTN with E ≤ 1 e-15 (see Additional file 3), which indicated that they were transcribed sequences. Thus, a total of 1633 loci of this map (70.6%) were functional markers. The BLASTX results of these transcribed loci showed that 809 loci sequences had no hits to protein; 976, 302, and 54 loci sequences (total 57.5%) had hits to known gene products, hypothetical proteins, and unknown genes, respectively (see Additional file 4).

These sequences containing SSRs totally targeted 38 items of molecular function with 1236 sequences involved in, 85 items of biology process with 2123 sequences involved in and 23 items of cell component with 2273 sequences involved in. However, sequences on different chromosomes targeted different Gene Ontology (GO) catalogs: Chr05, Chr11, Chr21, and Chr26 targeted more molecular functions; Chr03, Chr05, Chr11, Chr16, Chr19, and Chr21 targeted more biology processes; and Chr05, Chr11, and Chr26 targeted more cell components. Most chromosomes targeted more molecular functions and biology processes than cell components, and some chromosomes targeted more special catalogs: Chr03 and Chr21 predominately targeted biology processes and Chr05, Chr11, and Chr26 predominately targeted cell components (Figure 8).

Figure 8
figure 8

Chromosome distribution of GO catalogs.

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A correlation analysis between gSSRs, EST-SSRs, total loci and GO catalogs showed that GO catalogs were highly correlated to EST-SSR and total loci; gSSR was highly correlated only to the 'biology process'. The results agreed with the concept that functional SSR sequences were mainly derived from EST-SSRs (Table 1).

Table 1 Correlation between GO catalogs and gSSRs, EST-SSRs and total loci
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On level 2 of the GO classification, 'binding' (48.95%) and 'catalytic activity' (30.87) dominated the molecular function; 'metabolic process' (27.82%) and 'cellular process' (27.69%) dominated 50% of the biology process; and 'cell part' (32.86%), 'cell' (32.86%), and 'organelle' (25.27%) predominated the cellular component (see Additional file 5).

On level 3 of the GO classification, 'nucleic acid binding', 'ion binding', 'transferase activity', 'protein binding', 'nucleotide binding', and 'hydrolase activity' predominated in nearly 70% of the molecular function; 'cellular metabolic process', 'primary metabolic process', 'macromolecule metabolic process', and 'biosynthetic process' took more than 50% of the biology process; and 'intracellular', 'intracellular part', 'intracellular organelle', and 'membrane-bounded organelle' were major items of cell component (see Additional file 6).

When taking individual chromosomes into account, we found that some GO items were found only in some chromosomes, for example, Chr08 occupied many special GO items of biology process. Also, some chromosomes dominated some GO items, for example, Chr05 and Chr26 dominated the four major items of cell component (see Additional file 7).

Discussion

High-density genetic maps are becoming increasingly important in theoretical and applied genetic research [5, 7, 31–33] and even less (4.13%-7.9%) among G. hirsutum germplasms [12, 13, 34, 35]. Due to the low polymorphism and the large genome size in cotton, the only way to construct a high-density genetic linkage map is to apply more markers. In light of this, we developed new SSRs from ESTs annotated to proteins and newly released ESTs including novel ESTs from G. barbadense acc. 3-79 developed in our laboratory. As a result, 3177 new EST-SSRs were developed.

These EST-SSRs showed a lower polymorphism ratio (8.1%-19.5%) when compared with other researches [7, 31–33]. Among the NAU-prefixed EST-SSRs and gSSRs, only half of the polymorphism was detected compared to the results of Guo et al. [7, 25]. Although our population and the population described by Guo et al. [7] are both interspecific populations, a different polymorphism was found between the two populations, which might result from different materials or different genoty** methods (denatured polyacrylamide gel vs. non-denatured polyacrylamide gel).

The 6185 SSR primers developed in this study generated 1458 loci, and with our previous data [23]. Twice as many loci segregated toward the heterozygous allele than the 'Emian22' allele, and EST-SSRs showed more segregation distortion than gSSRs did. For the mapped loci, 12.8% of them were mapped on cotton chromosomes with 74.9% segregating toward the heterozygous allele. The loci segregated toward the heterozygous allele with a high frequency because the heterozygous allele could not be distinguished from the '3-79' allele in the BC1 population. Three chromosomes (Chr02, Chr16, and Chr18) showed extreme segregation distortion in that >50% of loci were distorted, among which 99.9% of the distorted loci segregated toward the heterozygous allele (Figures 2, 4, 7). Other maps have also proved that these chromosomes show more segregation distortion [3, 6, 7]. The extremely distorted chromosomes indicated to us that segregation distortion loci exist on these chromosomes. Faris et al. [36] and Kumar et al. [37] used reciprocal backcross populations to identify segregation distortion loci in Aegilops tauschii and tetraploid wheat, respectively, which provided us with an example for identifying segregation distortion loci in cotton. The loci skewing toward the same allele clustered on the same chromosomes or within the same SDRs indicated that genetic hitchhiking effects occur in cotton.

More than 8000 EST-SSRs were used in our map** population; however, only 1261 EST-SSRs were mapped. Although gSSRs derived from genomic sequences, their mother sequences can also be transcribed or translated. By blasting the gSSR sequences to cotton ESTs, 367 gSSR sequences were matched to cotton ESTs. As a result, 1633 loci of this map (70.6%) including five gene-derived markers were functional markers, which was fewer than those reported by Guo et al. [7]. By blasting to the protein database, 1332 loci were derived from translated sequences. Functional annotation of these loci sequences revealed that some chromosomes preferentially targeted certain GO catalogs, specifically, Chr03 and Chr21 mostly targeted the biology process; some GO items were found only on some chromosomes, that is, many special GO items of the biology process were detected only on Chr08. These results indicated that some chromosomes in cotton perform special functions. What's more, because these ESTs were mainly developed from develo** fibers, this map can also be used to identify fiber-related genes and to detect expressed QTLs (eQTLs) for fiber quality.

Conclusions

A total of 3177 new EST-SSRs were developed to enrich our interspecific BC1 genetic linkage map. The final map included 2316 loci on the 26 cotton chromosomes, 4418.9 cM in total length and 1.91 cM in average distance between adjacent markers. Segregation distorted chromosomes were identified, which is a guide to identify segregation distortion loci in cotton and to understand the mechanism of segregation distortion in interspecific cross between G. hirsutum and G. barbadense in cotton. SSR sequences were functionally annotated, which is helpful to identify functions of cotton chromosomes and to detect eQTLs for fiber quality. This map can be compared and integrated with other cotton maps to construct a consensus map in cotton.

Methods

Plant materials

The map** population used in this study is the BC1 population [(Emian22 × 3-79) × Emian22] including 141 individuals which had been used to construct a 917-locus map [40]. A preliminary sequencing of the cDNA library produced 887 high-quality ESTs that were used to construct the transcript map of develo** fiber [40] and also to isolate EST-SSRs [41]. After that, an additional 10090 high quality ESTs were obtained. These new sequences combined with the previous sequences were explored in the Serafer pipeline to develop SSR markers. The SSR markers that were the same as all the previously developed SSRs were excluded from the results, and this was finished as of August 2008.

All the SSR primers were named with a prefix HAU and synthesized by Sunbiotech Co. Ltd. (Bei**g).

Marker analysis

PCR, electrophoresis and silver staining were performed as previously described [23].

Map construction

The map** data for each parent were scored as the BC1 data according to the definition of JoinMap 3.0 [42], and the linkage map was constructed using a logarithm of odds (LOD) threshold of 5.0 and a maximum recombination fraction of 0.4. Map distances in centi-Morgans (cM) were calculated using the Kosambi map** function [43]. The resulting linkage map was drawn using MapChart 2.2 software [44]. Linkage groups were assigned to corresponding chromosomes by mapped SSRs http://www.cottonmarker.org. Homoeologous chromosomes were identified by duplicated loci as described in previous reports [2, 3, 7].

Segregation distortion

For each segregating marker, a χ2 analysis was performed to test for deviation from the 1:1 expected segregation ratio. A region with at least three adjacent loci showing significant segregation distortion (P < 0.05) was defined as the segregation distorted region (SDR) [45].

Annotation and functional classification of sequences containing SSRs

Sequences corresponding to gSSRs were indentified to be transcribed sequences by BLASTN to cotton ESTs with E ≤ 1 e-15 and were annotated using BLASTX (NCBI, Bethesda, MD, USA). The BLASTX results were classified into three groups: known gene products, hypothetical proteins, and unknown genes.

Functional annotation of ESTs was based on GO annotation [46], and performed with BLAST2GO [47, 48]. When running BLAST2GO, BLASTX DB was set to NCBI non-redundant DB and expectation value threshold was set at 1.0E-3, whereas high-scoring segment pairs (HSPs) length cutoff was set at 15. The top 20 BLAST hits were retained, then go-map** was run, and an annotation step with default parameters was performed. Furthermore, InterPro Scan was performed and InteProScan GOs was merged to annotation. Finally, the 'goslim_plant.obo' ontology subset was used to achieve specific GO terms.