Completely Distinguishing Individual A-Genome Chromosomes and Their Karyotyping Analysis by Multiple Bacterial Artificial Chromosome-Fluorescence in Situ Hybridization
Wang, Kai, Guan, Bing, Guo, Wangzhen, Zhou, Baoliang, Hu, Yan, Zhu, Yichao, Zhang, Tianzhen, Genetics
Twenty bacterial artificial chromosome (BAC) clones that could produce bright signals and no or very low fluorescence in situ hybridization (FISH) background were identified from Gossypium arboreum cv. JLZM, and G. hirsutum accession (acc.) TM-1 and 0-613-2R. Combining with 45S and 5S rDNA, a 22-probe cocktail that could identify all 13 G. arboreum chromosomes simultaneously was developed. According to their homology with tetraploid cotton, the G. arboreum chromosomes were designated as A1-A13, and a standard karyotype analysis of G. arboreum was presented. These results demonstrated an application for multiple BAC-FISH in cotton cytogenetic studies and a technique to overcome the problem of simultaneous chromosome recognition in mitotic cotton cells.
COTTON is one of the most important natural fiber and edible oil crops in the world. Significant progress has been made in the development of geneticmaps for the purpose of gene and quantitative trait loci (QTL) mapping (Reinisch et al. 1994; Ulloa et al. 2002; Zhang et al. 2002; Mei et al. 2004; Nguyen et al. 2004; Rong et al. 2004; Song et al. 2005; Han et al. 2006; Guo et al. 2007). However, due to the large number and small size of the chromosomes and especially the absence of suitable cytogenetic markers such as bands, chromosome identification has lagged significantly behind the development of linkage mapping. Although many genetic materials such as translocation, aneuploid lines, and the nomenclature for the tetraploid cotton chromosomes have been developed (Brown 1980; Endrizzi et al. 1985), the routine and unambiguous identification of individual chromosomes, especially in mitotic cells, is almost impossible in cotton. The bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) technique has been developed and used in physical mapping (Hanson et al. 1995), and individual mitotic chromosomes were easily identified in cotton (Wang et al. 2006). Then one set of chromosome specific BACs was developed, and the comparison mapping between physical and genetic maps was conducted (Wang et al. 2007a,b). Therefore, BAC-FISH has shown a unique superiority for cotton chromosome identification and physical mapping.
The cottongenusGossypiumis composed of 45 diploid and 5 tetraploid species. Among them 4 are cultivated Gossypium species: G. hirsutum L. [n = 2x = 26, (AD)^sub 1^], G. barbadense L. [n = 2x = 26, (AD)^sub 2^], G. arboreum L. (n = x = 13, A^sub 2^), and G. herbaceum L. (n = x = 13, A^sub 1^). Tetraploid species (G. hirsutum L. and G. barbadense L.) dominate worldwide cotton production. G. arboreum, as an A-genome diploid cotton, has been domesticated and cultivated in China for almost 2000 years (Xiang and Shen 1989). Due to some of its superior agronomic traits, such as disease and insect resistance, high fiber strength, and excellent plasticity, which upland cotton cultivars lack, G. arboreum is still planted and is used worldwide as a germplasmresource in present-day cotton breeding programs. G. arboreum is generally regarded as one of the best exemplars of the A-subgenome progenitors (Endrizzi et al. 1985; Wendel et al. 1995). Therefore, the G. arboreum species is important for agricultural production and genomic and evolution research in cotton.
Here, we described the screening of one diploid cotton (G. arboreum) and two tetraploid cotton (G. hirsutum) BAC libraries to identify new chromosome-specific clones. Combining with previous BAC clones and 45S and 5S rDNA, a 22-probe cocktail was developed for the purpose of simultaneous chromosome identification and karyotyping in G. arboreum. This enabled us to identify all of the 13 G. arboreum chromosome pairs simultaneously. According to their homology with the A-subgenome (At) chromosomes in tetraploid cotton, the nomenclature and standard karyotype of G. arboreum chromosomes were developed.
MATERIALS AND METHODS
The BACs used in this study came from three genomic BAC libraries, of which two were constructed from the tetraploid cotton G. …