Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones

Abstract

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 bacterial /binary bacterial artificial chromosome (BAC/BIBAC) 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 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific 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 restriction fragment length polymorphism (RFLP)-derived BAC/BIBAC clones as probes to apply genome-wide analysis 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.

Keywords: BAC-FISH; RFLP genetic map; cytogenetic map; sunflower.

Figure 1

An entire metaphase cell with 34 chromosomes counterstained with DAPI (blue). (A, B) FISH of a BAC clone 59A24 generated a specific signal (arrow) on chromosome Ha16; (C) FISH image of BAC clone 135J2 corresponding to RFLP marker 7D5 on Ha17 showing a strong dispersed pattern on the pericentromeric region of almost all sunflower chromosomes, and reflecting the distribution of repetitive DNA; (D) With 100× blocking DNA applied, clones 470I10 (Ha15) produced hybridization signals (arrow) on one pair of chromosome with high background due to nonspecific binding of the probe; (E-F) Two BAC clones, 401C5 (E) and 367P3 (F), identified with RFLP markers from same linkage group LG15, were FISH-mapped on two different morphologic chromosomes based on two independent in situ hybridizations. The clone 401C5 was localized on a metacentric chromosome and 367P3 on a satellite chromosome. (G) 84K7 from LG18 was located on one submetacentric chromosome; (H, I) BAC clones 367P3 from LG15 and 84K7 from LG18 were cohybridized on one submetacentric chromosome Ha09. 367P3 (green) was located proximal to the centromere and 84K4 (red) distal to the centromere. Insets in H and I: magnified images for two chromosomes showing two-color FISH with two probes. The bars indicate 5 μm.

Figure 2

Identification of individual chromosomes and comparison with the linkage map (Jan et al. 1998). The RFLP genetic map with RFLP markers (A) was used to select BAC clones to identify their corresponding chromosomes (B) by FISH. Numbering of the chromosomes follows Tang et al. (2002). Ha12^t and Ha14^t indicate numbering of the chromosomes is tentative. The RFLP markers are showed on the left of each chromosome in bold, and the names of corresponding BAC clones are on the right. The red signals were digoxigenin-labeled probes detected by antidigoxigenin-rhodamine, and the green signals were biotin-labeled probes detected by avidin-fluorescein. All slides were counterstained with DAPI (blue). The bars indicate 5 μm for chromosomes and 50 cM for the linkage maps. Note that the length of chromosomes cannot be compared in the figure as they were derived from different cells.

Figure 3

Partial linkage map of SSR-LG 6 and SSR-LG15 (Tang et al. 2002) with two linked RFLP markers (Jan et al. 1998), based on the analysis of an F₂ population from the cross of CMS HA89 x RHA280. Markers labeled “STS” were RFLP-derived markers. Markers labeled “ORS” were SSR markers. The distances are in cM.