Karyotype Analysis of Diploid and Spontaneously Occurring Tetraploid Blood Orange [Citrus sinensis (L.) Osbeck] Using Multicolor FISH With Repetitive DNA Sequences as Probes

Abstract

Blood orange [ Citrus sinensis (L.) Osbeck] has been increasingly appreciated by consumers worldwide owing to its brilliant red color, abundant anthocyanin and other health-promoting compounds. However, there is still relatively little known about its cytogenetic characteristics, probably because of the small size and similar morphology of metaphase chromosomes and the paucity of chromosomal landmarks. In our previous study, a naturally occurring tetraploid blood orange plant was obtained via seedling screening. Before this tetraploid germplasm can be manipulated into a citrus triploid seedless breeding program, it is of great importance to determine its chromosome characterization and composition. In the present study, an integrated karyotype of blood orange was constructed using sequential multicolor fluorescence in situ hybridization (FISH) with four satellite repeats, two ribosomal DNAs (rDNAs), a centromere-like repeat and an oligonucleotide of telomere repeat (TTTAGGG) 3 as probes. Satellite repeats were preferentially located at the terminal regions of the chromosomes of blood orange. Individual somatic chromosome pairs of blood orange were unambiguously identified by repetitive DNA-based multicolor FISH. These probes proved to be effective chromosomal landmarks. The karyotype was formulated as 2 n = 2 x = 18 = 16m+2sm (1sat) with the karyotype asymmetry degree belonging to 2B. The chromosomal distribution pattern of these repetitive DNAs in this spontaneously occurring tetraploid was identical to that of the diploid, but the tetraploid carried twice the number of hybridization sites as the diploid, indicating a possible pathway involving the spontaneous duplication of chromosome sets in nucellar cells. Our work may facilitate the molecular cytogenetic study of blood orange and provide chromosomal characterization for the future utilization of this tetraploid germplasm in the service of seedless breeding programs.

Integrated Karyotypes of Diploid and Tetraploid Carrizo Citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf.) as Determined by Sequential Multicolor Fluorescence in situ Hybridization With Tandemly Repeated DNA Sequences

“…Somatic chromosome preparation was made following our previously published protocol with minor optimizations (Deng et al, 2019a). Briefly, at least 20 fresh and fine roots (approximately 3-4 cm proximal to the root tip) were carefully dissected from plants with a tweezer on a sunny morning.…”

“…A digoxigenin-labeled oligonucleotide of (TTTAGGG) 3 (Nelson et al, 2014) was directly synthesized to map telomeric sequences. Details of DNA probe labeling via nick translation and sequential multicolor FISH targeting all repetitive elements followed our reported protocol without modification (Deng et al, 2019a). Three rounds of sequential FISH experiments were performed on the same well-spread mitotic metaphase chromosomes of diploid and tetraploid CC plants.…”

“…Probes and chromosomes were denatured together on a hot plate at 85 • C for 1.5 min and incubated in a 37 • C moist chamber at least 6 h or overnight. The procedure of FISH signal detection and posthybridization washes described by Deng et al (2019a) without modifications were used. The slides were mounted with Vectashield § antifade mounting medium containing 1.5 µg/mL 4 ,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, CA, United States).…”

“…(Cai et al, 2014), peanut (Arachis hypogaea) (Zhang et al, 2016), sweet potato (Sun et al, 2019), Oleaceae plants (Luo and Liu, 2019), Clementine mandarin (Deng et al, 2019b), and blood orange [C. sinensis (L.) Osb.] (Deng et al, 2019a). Elucidating chromosome constitution and the karyotype including the number, absolute and relative size, symmetry, and centromere position of the chromosome complement in nucleus cell of individual eukaryotic species, is a fundamental question in plant biology (Liang and Chen, 2015;Jiang, 2019).…”

“…In that study, the physical distribution of these repetitive DNAs on the same somatic metaphase chromosomes of Clementine mandarin and other several citrus cultivars were also analyzed (Deng et al, 2019b). Subsequently, a detailed karyotype of diploid blood orange was established by multicolor FISH with these excellent probes, and we did the same for its spontaneously occurring tetraploid plant (Deng et al, 2019a).…”