J Appl Genet 50(1), 2009, pp. 25–28
Short communication
Isolation and chromosomal distribution of a novel Ty1-copia-like sequence from Secale, which enables identification of wheat–Secale africanum introgression lines J. Jia1, Z. Yang1, G. Li1, Ch. Liu1, M. Lei1, T. Zhang1, J. Zhou1, Z. Ren1,2 1 2
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan, China Key Laboratory for Plant Genetics and Breeding, Sichuan Agricultural University, Ya’an, Sichuan, China
Abstract. A repetitive sequence of 411 bp, named pSaO5411, was identified in the Secale africanum genome (Ra) by random amplified polymorphic DNA (RAPD) analysis of wheat and wheat–S. africanum amphiploids. GenBank BLAST search revealed that the sequence of pSaO5411 was highly homologous to a part of a Ty1-copia retrotransposon. Fluorescence in situ hybridization (FISH) analyses indicated that pSaO5411 was significantly hybridized to S. africanum chromosomes of a wheat–S. africanum amphiploid, and it was dispersed along the Secale chromosome arms except the terminal regions. Basing on the sequence of pSaO5411, a pair of sequence-characterized amplified region (SCAR) primers were designed, and the resultant SCAR marker was able to target both cultivated rye and the wild Secale species, which also enabled to identify effectively the S. africanum chromatin introduced into the wheat genome. Keywords: FISH, molecular marker, Secale africanum, Ty1-copia, retrotransposon.
A grass genome typically consists of a large number of repetitive DNA families, which are likely to be or to have evolved from retrotransposons (Belyayev et al. 2001). Particularly, the long-terminal repeat (LTR) retrotransposons of the Ty1-copia and Ty3-gypsy groups and non-LTR retrotransposons or LINE are found in large quantities in cereal genomes (Heslop-Harrison et al. 1997; Presting et al. 1998; Kumar and Bennetzen 1999). The distribution of the LTR retrotransposon family has been routinely suggested to be limited in some species, genera or tribes. Several LTR retrotransposons have been isolated from members of the tribe Triticeae, including Secale (Ko et al. 2002; Ito et al. 2004). Most of these LTR sequences were used to study the evolution and biodiversity of related genomes (Kumar et al. 1997), but few of them were applied as molecular markers to assist breeding programs.
The genus Secale contains cultivated rye (Secale cereale L.) and several wild species (Cuadrado and Jouve 1997), which have many agronomically important characters, such as high yield, wide adaptability, and resistance to many pests and diseases, and thus offer a potential to increase the genetic variability and introduce desirable genes for wheat improvement (Zeller and Hsam 1983; Jiang et al. 1994). At present, researches are mainly focused on cultivated Secale and its introgression lines. After several wheat –Secale africanum amphiploids were produced (Jiang et al. 1992), a program for introducing the novel genes from S. africanum to common wheat was set up by crossing the amphiploids with cultivated wheat (Yang et al. 2005). It is thus essential to establish a series of reliable molecular markers to screen a large number of wheat– S. africanum introgression lines effectively. Since McIntyre
Received: April 21, 2008. Accepted: June 20, 2008. Correspondence: Z. Yang, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, Sichuan, China; e-mail:
[email protected]
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et al. (1990) first reported the molecular marker pSc119.1 specific to and ubiquitous on rye chromosomes, many rye-derived molecular markers combined with other techniques, such as C-banding and fluorescence in situ hybridization (FISH), have been applied to the identification of wheat–rye derivatives (Mukai et al. 1993; Ko et al. 2002). However, few of these reports involved the wild Secale (Cuadrado and Jouve 1995; Liu et al. 2008). In this study, a new repetitive DNA sequence belonging to a Ty1- copia family was isolated from S. africanum by random amplified polymorphic DNA (RAPD) analysis, and its chromosomal distribution was identified by fluorescence in situ hybridization (FISH). In addition, the sequence of this diagnostic marker was deter-
mined, to enable tracing Secale chromatin in wheat background. To produce the Secale-specific PCR marker, a total of 240 RAPD primers were used in this study to amplify the wild and cultivated Secale species. Triticum aestivum lines Chinese Spring (CS), Mianyang11 (MY11), Anyuepaideng, T. carthlicum, and a T. uratu–Aegilops tauschii amphiploid were included as controls. We found that one primer OPO5 (5’-CCCAGTCACT-3’) gives rise to a specific DNA band of about 400 bp from all materials containing rye chromatin, including cultivated rye (S. cereale), wild rye (S. africanum, S. sylvestre and S. vavilovii), and hexaploid triticale Fenzhi-1, while it cannot amplify the DNA sample from the tested common wheat cultivars (Figure 1a). Subsequently, the Secale-spe-
Figure 1. PCR and FISH pattern of pSaO5411: (a) RAPD analysis with primers O5-F and O5-R in rye and wheat: M = marker (DL4500); 1 = Triticum aestivum cv. Chinese Spring; 2 = T. aestivum cv. Mianyang11; 3 = T. aestivum cv. Anyuepaideng; 4 = T. carthlicum; 5 = T. uratu–Aegilops tauschii amphiploid; 6 = Fenzhi–1 triticale; 7 = Secale africanum; 8 = S. sylvestre; 9 = S. strictum subsp. anatolicum; 10 = S. vavilovii; 11 = S. cereale cv. Jingzhou; (b) FISH pattern of metaphase chromosomes on S. cereale cv. Jingzhou, using pSaO5411 as a probe; (c) FISH pattern of metaphase chromosomes on a T. turgidum cv. Jianyangailanmai–S. africanum amphiploid, using pSaO5411 as a probe; Arrows indicate the wheat–S. africanum translocated chromosomes; (d) SCAR-PCR with primers O5-F and O5-R in wheat, rye and wheat–Secale derivatives: M = marker; 1 = S. africanum; 2–8 = CSDA1R to CSDA7R; 9 = Triticum aestivum cv. Anyuepaideng; 10 = T. aestivum cv. Anyuepaideng–S. africanum amphiploid; 11 = T. carthlicum; 12 = T. carthlicum–S. africanum amphiploid; 13 = T. turgidum cv. Jianyangailanmai; 14 = T. turgidum cv. Jianyangailanmai–S. africanum amphiploid; 15–30 = BC1F7 derivatives of a T. durum–S. africanum amphiploid (15 = L2; 16 = L4; 17-24 = L5-L12; 25 = L14; 26 = L15; 27–30 = L17-L20).
A Secale specific Ty1-copia like sequence
cific RAPD band was cloned and sequenced. The full length of the 411-bp sequence was obtained and designated as pSaO5411 (GenBank accession No. EU360795). The nucleotide sequence of pSaO5411 shows 51.3% AT content. BLAST search in the NCBI GenBank revealed that pSaO5411 has 83% sequence identity to a retrotransposon Wis-2p-copia of Triticum turgidum. BLASTX analysis revealed that the deduced amino acid sequence of pSaO5411 is significantly homologous to the retrotransposon protein-coding domain of Ty1-copia subclass in Oryza sativa. This shows that the present sequence is a part of a Ty1-copia retrotransposon, and that the RAPD analysis was a powerful approach to isolate the retrotransposon sequences in Triticeae plants with large repetitive genomes. In order to determine the chromosomal distribution of the isolated Ty1-copia like sequence, pSaO5411 was labeled with fluorescein-14-dUTP by nick translation, and the probe was used to hybridize the mitotic metaphases of S. cereale cv. Jingzhou and of a T. turgidum cv. Jianyangailanmai–S. africanum amphiploid by FISH according to the protocols from Liu et al. (2008). The whole chromosomal arms except telomeric regions in the S. cereale cv. Jingzhou genome (Figure 1b) and in 14 S. africanum chromosomes in the amphiploid genome, showed strong hybridization signals dispersed across the chromosome arms, while quite faint hybridization occurred in a few wheat chromosomes in the amphiploid (Figure 1c). In addition, FISH also detected one wheat–Secale chromosome translocation in the amphiploid (Figure 1c). Thus in this study, the FISH using Ty1-copia like sequences pSaO5411 as a probe revealed that the signals were distributed on whole arms except their terminal regions on both S. cereale cv. Jingzhou chromosomes and S. africanum chromosomes (Figure 1b–c). This differs from the previously published data on chromosomal distribution of cultivated rye Ty1-copia retrotransposons, in which Francki (2001) isolated a Ty1-copia retrotransposon only distributed in the centromeric heterochromatin region of rye chromosomes, and Pearce et al. (1997) reported that rye Ty1-copia retrotransposon sequences are absent from both centromeric regions and the terminal heterochromatin in the rye chromosomes. Consequently, we regarded pSaO5411 as a novel Ty1-copia like sequence on the basis of its unique distribution on Secale chromosomes. Basing on the nucleotide sequence of pSaO5411, a pair of primers O5-F (5’-CCCA
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GTCACTACAACGAGAGT-3’) and O5-R (5’-GCTACAAGAGCTTCGTGCAG-3’) was designed to test the validity of the molecular marker in wheat–S. africanum introgression lines. The PCR amplification using primers O5-F and O5-R showed that the target band of 393 bp appeared in S. africanum and in a set of Chinese Spring–Imperial rye addition lines (Figure 1d, lanes 1–8). Furthermore, O5-F and O5-R were used to amplify a T. aestivum cv. Anyuepaideng–S. africanum amphiploid, a T. carthlicum–S. africanum amphiploid, a T. turgidum cv. Jianyangailanmai–S. africanum amphiploid, and their corresponding wheat parents. The results suggested that the target band amplified in all amphiploids, but it was absent in their wheat parents (Figure 1d, lane 9–14). Meanwhile, we used O5-F and O5-R to amplify BC1F7 derivatives (L1–L20) from wheat crossed with a Triticum durum –S. africanum amphiploid. Their amplification indicated that a 393-bp DNA band was present in all plants except L2, L4, L5, L6, L19 and L20 (Figure 1d, lanes 15–30), which is consistent with the results from Liu et al. (2008). Therefore, we can expect that the diagnostic PCR marker and FISH for the sequence of pSaO5411 can detect the Secale chromatin introduced into wheat background, especially in the early generations of wheat–Secale hybridization. Acknowledgements. We are grateful for financial support from the National Natural Science Foundation of China (grants no.30671288,30730065 and 30871518), Program for New Century Excellent Talents in University (grant no. NCET-06-0810), and Young Scholars Foundation from the Science and Technology Committee of Sichuan, China (grant no. 2008-31-371). REFERENCES Belyayev A, Raskina O, Nevo E, 2001. Chromosomal distribution of reverse transcriptase-containing retroelements in two Triticeae species. Chromosome Res 9: 129–136. Cuadrado A, Jouve N, 1997. Distribution of highly repeated DNA sequences in species of the genus Secale. Genome 40: 309–317. Cuadrado A, Jouve N, 1995. Fluorescent in situ hybridization and C-banding analyses of highly repetitive DNA sequences in the heterochromatin of rye (Secale montanum Guss.) and wheat incorporating S. montanum chromosome segments. Genome 38: 795–802. Francki MG, 2001. Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye (Secale cereale L.). Genome 44: 266–274.
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