|Discrimination of repetitive sequences polymorphism in Secale cereale by GISH-banding
Jian-Ping ZHOU1, Zu-Jun YANG1*, Guang-Rong LI1, Cheng LIU1 and Zheng-Long REN1, 2*
1Shcool of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
2State Key Laboratory for Plant Genetics and breeding, Sichuan Agricultural University，Ya’an 625014, China
Received: 3 Dec. 2006 Accepted: 13 Feb. 2007
Handling editor: Zheng Meng
Abstract Genomic in situ hybridization banding (GISH-banding) technique, slightly modified from conventional GISH, probed with the Chinese native rye (Secale cereale L.) DNA, was presented, which enabled to visualize the individual rye chromosomes and create a universal reference karyotype of S. cereale chromosome 1R to 7R. The present GISH-banding approach was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aestivum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S. cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye, while the GISH-banding pattern of S. vavilovii was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of AT-riched heterochromatin are discussed.
Key words AT-riched heterochromatin, GISH-banding, karyotype, repetitive sequences, Secale cereale.
This work was supported by the National Natural Science Foundation of China (30671288 and 30671336)
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It has been reported that over 85% of the DNA in cereal genomes consists of repetitive families, and their similarity-dissimilarity may reflect evolutionary distances between species and these repetitive DNA sequences bring on the major differences between genomes (Flavell et al., 1993, Bennetzen, 1996). Based on the divergence of repetitive DNA, genomic in situ hybridization (GISH) with blocking DNA has been widely used for a direct characterization of the genomes and chromosomes in interspecific hybrid plants, allopolyploid species, and interspecific introgression lines (Schwarzacher, 2003). Rather than distinguishing different genomes in hybrids or allopolyploids, karyotyping techniques to differentiate the individual chromosomes within a genome was rather important. It was prominent that the conventional GISH may combine with the other methods, such as meiotic pairing analysis, morphogenetic and biochemical markers, chromosome C-banding and the multi-color FISH based on the distribution of special highly repetitive DNA sequences (Kato et al., 2005).
Fluorescent banding were occasionally observed on chromosomes when GISH, effective methods, so-called GISH-banding technique, were practically developed for discriminating the individual chromosomes from alien genome by modifying the conventional GISH on the treatment of probes or blocking DNA. Kuiper et al (1997) firstly reported that GISH banding patterns was coincidence with Giemsa C-banding patterns in genus Alstroemeria using GISH with blocking DNA. Another considerable approach of simultaneous genomic in situ hybridization with probe preannealing (SP-GISH) developed by Belyayev and Raskina (1998), was used to develop and construct GISH banding karyotype of Aegilops speltoides, and to study the evolutionary dynamics of repetitive sequences in Aegilops (Belyayev et al. 2001).
In this study, applying the modified method of genomic in situ hybridization, we developed a GISH-banding technique, which make it possible to build a universal reference karyotype of cultivated rye. The method also used to screen several Secale accessions to analyze the repetitive sequence polymorphism.
In the somatic mitosis metaphase cells of hexaploid triticale line Fenzhi-1, GISH with Jingzhou rye DNA as a probe and wheat MY11 DNA as blocking DNA revealed that not only 14 chromosomes of rye were strongly hybridized along the entire chromosome length but also stronger signals were observed terminal or sub-telomeric region (Figure 1.A). Because of the coincidence of hybridization signals and C-banding patterns this phenomenon is referred to as GISH-banding. Based on the GISH-banding, a universal reference karyotype of rye has been created (Figure 1.B). The chromosomes can be divided into two groups according to terminal or sub-telomeric banding position: a group consisting of five chromosomes with one or two bandings in every arm (chromosomes 1R, 3R, 4R, 5R, 7R) and the other group consisting of two chromosomes with one or two bandings in only one chromosome arm (2R, 6R). Chromosome 1R specially possess of a banding and nucleolus organization region with no hybrid signal in short arm. Chromosome 2R has only one banding in long arm. The signal banding in long arm of chromosome 3R was weaker than that of chromosome 4R. Chromosome 5R has a strongest signal banding in short arm and two bandings in long arm. Chromosome 6R possess of two bandings in long arm. Chromosome 7R has one banding in short arm and one banding in sub-telomeric region of long arm. The same GISH-banding karyotype was obtained in the somatic mitosis metaphase cells of S. cereale cv. Jingzhou rye using the same probe.
GISH-banding karyotype of rye also allowed us to distinguish the chromosomes or segments. As observed in Figure 1.C and Figure 1.D, the two 1RS/1BL translocation chromosomes of wheat cultivar ‘‘Chuannong 17’’and a chromosome 1R of wheat-1R monosomic addition line were identified by GISH-banding with Jingzhou rye DNA as a probe.
In addition, GISH-banding provided a simple and convenient way to study evolutionary distance in species of Secale genus or subspecies. Using labeled DNA of Jingzhou rye as probe to hybridize with the somatic mitosis metaphase cells of S. cereale subsp. Afghanicum and S. vavilovii, the hybridization signals presented resemble GISH-banding of the chromosomes of hexaploid triticale line Fenzhi-1, suggesting that S. cereale subsp. afghanicum was close to S. cereale cv. Jingzhou rye (Figure 1.E, F), and no signal in telomeric heterochromatin of S. vavilovii chromosomes, suggesting that S. vavilovii was distinct from S. cereale cv. Jingzhou rye (Figure 1.G), respectively.
Commonly, GISH extensively was used to discriminating the alien chromosomes and analyzing genome organization in interspecific hybrids, allopolyploid species and interspecific introgression lines, such as S. cereale (Schwarzacher et al., 1992), Thinopyrum intermedium (Chen et al., 2003), Dasypyrum (Minelli et al., 2005) and Hordeum (Pickering et al., 2000). However, GISH is not easy to distinguish the alien individual chromosomes. In the present study, we have successfully developed GISH-banding technique according to the GISH procedure. It will be useful to not only construct universal reference karyotype of rye to distinguish the alien chromatin and characterize the specific chromosomes simultaneously but also study the materials relationship of genus Secale or subspecies. In our study, seeding root tips digested with 0.2M HCl for 24h at room temperature were used to chromosome spreads and the probe was produced from total genomic DNA from S. cereale cv.Chinese rye, which was sheared to 500-1000bp, before labeled with digoxigenin-11-dUTP by nick translation. It was found in our experiments that fourteen rye chromosomes in hexaploid triticale line Fenzhi-1 displayed brighter fluorescence in the terminal or sub-telomeric regions than in the rest of the chromosomes, which is similar to the Giemsa-C banding patterns, and permits to distinguish each 7 chromosomes pairs. In fact, the phenomena of GISH-banding were occasionally observed in previous GISH studies, such as the GISH-banding figures of Dasypyrum chromosomes (Minelli et al., 2005) and Alstroemeria chromosomes (Kuipers et al., 1997). In the present paper, GISH-banding can be detected stably in rye chromosomes, especially in prophase chromosomes (Figure 1.F).
In most GISH studies, a uniform distribution of the fluorescently labeled probe DNA along the target chromosomes has been reported. However, our experiments with GISH studies of rye and triticale revealed positive (strongly hybridization signals) bands in telomeric and subtelomeric heterochromatic regions of rye chromosomes when total rye genomic DNA was used as a probe. The GISH banding pattern obtained coincided with the Giemsa C-banding and DAPI patterns which fluorescence was brighter at the GISH bands (data not shown), suggesting the presence of AT-riched DNA sequences in these chromosome regions. In other word, GISH banding discriminated AT-riched heterochromatin in rye.
Coincidence of GISH banding patterns and Giemsa C-banding patterns has so far been reported not only for genus Alstroemeria using GISH with blocking DNA (Kuipers et al., 1997) but also for specific repetitive sequences, such as a tandem repeat from Allium fistulosum, which was found to occur in major heterochromatic blocks (Irifune et al., 1995). However, Kuipers et al. (1997) found that the concurrent occurrence on the chromosomes of Alstroemeria aurea × Alstroemeria ligtu hybrid of negative/positive GISH banding pattern was taken as an indication that the molecular organization of the C-band regions of these species is different. Moreover, in comparable studies of rye, fluorescence in situ hybridization (FISH) of the highly repetitive sequences pSc119.2, pSc74, pSc34, pSc200 and pSc250 showed a striking correspondence with the C-banding pattern (Cuadrado & Jouve, 1995; Vershinin et al., 1995). It was taken account of the above mentions, we can know that the GISH banding pattern did not coincide completely with the Giemsa C-banding and GISH banding displayed some AT-riched highly repetitive DNA classes. In present paper, GISH with a probe DNA of Jingzhou rye did not produce signals in telomeric heterochromatin of S. vavilovii chromosomes (Figure 1.G) while prominent signal in Jingzhou rye. This result strongly supported that GISH banding was significant different from C-banding and indicated that the difference GISH banding patterns has been ascribed to rapidly evolving repetitive sequences including those AT-riched repetitive sequences that account for genome differentiation between S. cereale cv.Jingzhou rye and S. vavilovii.
In conclusion, GISH-banding technique was been successfully developed by the present study. And the prominent GISH banding, as observed on rye chromosomes, is of fundamental significance and would allow identifying the chromosomes or segments and a more accurate characterization of the constitutive heterochromatin.
Materials and methods
The following materials were used for preparations of chromosome spreads: S. cereale subsp.afghanicum (PI 618662) and S. vavilovii (PI 618682) were kindly supplied by National Plant Germplasm System, USDA. S. cereale cv. Jingzhou rye, a Chinese native accession, was collected from Hubei province, China. Wheat cultivar ‘‘Chuannong 17’’ with two 1RS/1BL translocation chromosomes and a wheat-1R monosomic addition line (2n = 43, AABBDD1R), which rye chromatin originated from R3 (Weining rye) collected from Guizhou province, China. Hexaploid triticale line Fenzhi-1 (2n=42, AABBRR) was created by Prof. Xian-Zhi Zhang, Sichuan Agriculture Univ., China. Fenzhi-1 was derived from the hybridization between T. aestivum variety, MY11, and Jingzhou rye.
The following two specials were isolated Genome DNA: S. cereale cv. Jingzhou rye. T. aestivum variety, MY11 was obtained from Sichuan province, China.
Chromosome preparation; GISH Banding
Seeds were germinated on moist filter paper at 23°C in the dark. The root tips were collected and transferred to ice water for 24h to accumulate metaphases and then fixed in 3:1 (v/v) 100% ethanol:acetic acid for 10–12 h at room temperature and stored at −20°C for at least 24 h. Seeding root tips was digested in 0.2M HCL for 24h at room temperature, and then chromosome slide was prepared using the conventional acetocarmine procedure.
The total genomic DNA was extracted from young leaves of S. cereale cv. Jingzhou rye and wheat MY11 using the CTAB method (Kidwell & Osborn, 1992). The DNA was sonicated to 500bp-1000bp and then was labeled with digoxigenin-11-dUTP by nick translation based on the protocols of the manufacturer (Roche Diagnostics Indianapolis, IN). Hybridization was carried out according to Jiang et al. (1996). The detection of digoxigenin was carried out with fluorescein-conjugated anti-digoxigenin Fab fragment (Roche Diagnostics). The slides were finally mounted in vectashield antifade solution (Vector Laboratoris, Burlingame, CA) with the 0.25 μg/ml propidium iodide (PI). Slides were examined on an Olympus BX-51 fluorescence microscope. Photographs were taken with a cooled CCD camera system (DP70).
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Figure. 1. Genomic in situ hybridization banding of Secale cereale mitotic chromosomes by the Secale cereale probe (yellow-green). Orange-red fluorescence shows DNA counterstained with propidium iodide (PI).
(A) GISH on metaphase chromosomes of Hexaploid triticale line Fenzhi-1 using Secale cereale Jinzhou rye DNA as probe. 14 chromosomes of Secale cereale showed strong signals (Arrows indicated).
(B) GISH-banding karyotype of Secale cereale according to Figuer 1.A.
(C) Identification of chromonsome arm 1RS from wheat cultivar ‘‘Chuannong 17’’ using GISH-banding. Two 1RS arms show strong GISH-banding signal (Arrows indicated).
(D) Identification of chromonsome 1R from wheat-1R monosomic addition line using GISH-banding. One 1R show strong GISH-banding signal (Arrow indicated).
(E) GISH-banding on metaphase chromosomes of Secale cereale subsp.afghanicum. The chromosomes present resemble GISH-banding of the chromosomes of hexaploid triticale line Fenzhi-1.
(F) GISH-banding on prophase chromosomes of Secale cereale subsp.afghanicum. Prominent GISH-banding was showed distinctly on prophase chromosomes.
(G) GISH-banding on metaphase chromosomes of Secale vavilovii. The telomeric heterochromatin of S. vavilovii chromosomes display no signal.