SUNFLOWER GERMPLASM DIVERSIFICATION AND CHARACTERIZATION UTILIZING WILD SUNFLOWER SPECIES, CYTOGENETICS, AND APPLIED GENOMICS
Location: Sunflower Research
Title: Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones
Submitted to: Genes, Genomes, Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 1, 2012
Publication Date: January 1, 2013
Citation: Feng, J., Liu, Z., Cai, X., Jan, C.C. 2013. Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones. Genes, Genomes, Genetics. 3(1):31-40.
Interpretive Summary: In the past decades, various genetic markers and the corresponding linkage maps have been developed in sunflower. These maps with almost full genome coverage allow for the cross-reference with each other and provide a dense genome-wide framework for sunflower research. Chromosome identification and development of a universal karyotype is highly desirable in sunflower. Meanwhile, an impressive series of technical and conceptual advances in molecular cytogenetics, such as BAC-FISH, have received considerable attention for the studies of the plant genome. However, the linkage groups have not been anchored to individual chromosomes due to the lack of proper genetic stocks and technical difficulties of chromosome identification in sunflower. Alignment of the linkage groups with individual chromosomes has become possible with the recently developed efficient FISH technique and the establishment of a BAC library and RFLP genetic map in sunflower. Previously, we developed an RFLP linkage map with 232 cDNA probes on 20 linkage groups, and constructed two BAC and BIBAC libraries from the inbred line HA89. Subsequently, a set of linkage group-specific BAC/BIBAC clones was identified from the libraries using the mapped cDNA-derived RFLP markers. In this study we have physically assigned those linkage group-specific BAC/BIBAC clones to individual chromosomes and integrate the genetic map with the cytogenetic map in sunflower. The resulting chromosome-specific clones will be valuable cytogenetic markers for molecular cytogenetic and genomic research in sunflower. The cytogenetic map will provide significant insights into a better understanding of the sunflower genomic structure and organization.
Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n=34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific BAC/BIBAC (bacterial /binary bacterial artificial chromosome) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (~100-150 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-FISH (fluorescence in situ hybridization) technique. Because most BAC/BIBAC clones contain large repetitive sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background signals. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with distinctive FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using RFLP-derived BAC/BIBAC clones as probes is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.