Rice (Oryza sativa) centromeric regions consist of complex DNA

Abstract

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.

Figure 1

FISH analysis of the rice centromeric DNA elements. The probes were biotinylated and hybridized in situ to rice chromosomes or DNA fibers. The probes were detected by fluorescein isothiocyanate-conjugated antibodies (green color) and the chromosomes were stained with propidium iodide (red color). Probe pRCS1 hybridized exclusively to the centromeric regions of the chromosomes from rice (A), rye (B), barley (C), sorghum (D), and maize (E). FISH signals also were detected in the centromeric regions of the acrocentric B chromosomes (arrows) from rye (B) and maize (E). Similarly, rice centromeric DNA families RCH2 (F), RCH1 (G), RCH3 (H), RCE1 (I), RCE2 (J), and RCS2 (K) all were located in the centromere of every rice chromosome. RCS2 showed major difference in size and intensity of FISH signals on different chromosomes (K). Two pairs of chromosomes with the strongest signals are indicated by arrows and the third pair with the weakest signals by arrowheads (K). The same metaphase cell (K) was washed under medium (L) and high (M) strigencies, and most signals are still discernible. (N) Fiber-FISH analysis using pRCS2 as a probe. The RCS2 family is organized into various sizes of uninterrupted arrays. The marked array between two arrows is 51 μm long and represents approximately 151-kb DNA. All bars are 10 μm.

Figure 2

Genomic organization of the RCS1 family. Rice genomic DNA was digested with Sau3AI (lane 1), DpnII (lane 2), HaeIII (lane 3), MspI (lane 4), HpaII (lane 5), SalI (lane 6), BamHI (lane 7), DraI (lane 8), EcoRI (lane 9), and HindIII (lane 10), and probed with pRCS1. One or few major and a number of minor hybridization bands were detected in most lanes.

Figure 3

Nucleotide sequence of element pRCS2. The 639-bp insert of clone pRCS2 contains four copies of a tandemly arranged repeat. The four members (A–D) range from 155 to 165 bp and share 84–91% sequence identity with one another. F represents the consensus sequence of the four members.

Figure 4

Genomic organization of the RCS2 family. Rice genomic DNA was digested with DpnII (lane 1), Sau3AI (lane 2), MspI (lane 3), HpaII (lane 4), SalI (lane 5), and HaeIII (lane 6), and probed with pRCS2. Five lanes showed ladder patterns, indicating a tandem repeat nature of this element in the rice genome.

Figure 5

Conservation of the RCH1 and RCE1 families in Gramineae species. Genomic DNA from sorghum (lane 1), maize (lane 2), sugar cane (lane 3), Ag. intermedium (lane 4), barley (lane 5), oats (lane 6), rye (lane 7), wheat (lane 8), Ae. squarrosa (lane 9), rice (lane 10), bamboo (lane 11), Pharus sp. (lane 12), J. effusus (lane 13), C. alternifolius (lane 14), and A. thaliana (lane 15) was digested with HindIII and probed with pRCH1 (A) and pRCE1 (B). The pRCH1 sequence is conserved across the species within the Gramineae family, whereas the pRCE1 sequence is present only in species within the Bambusoideae subfamily.