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Isolation of expressed sequences from a specific chromosome of Thinopyrum intermedium infected by BYDV Shu-Mei Jiang, Wei-Bo Yin, Jun Hu, Rui Shi, Ruo-Nan Zhou, Yu-Hong Chen, Guang-He Zhou, Richard R.-C. Wang, Li-Ying Song, and Zan-Min Hu
Abstract: To map important ESTs to specific chromosomes and (or) chromosomal regions is difficult in hexaploid wheat because of its large genome size and serious interference of homoeologous sequences. Large-scale EST sequencing and subsequent chromosome localization are both laborious and time-consuming. The wheat alien addition line TAi-27 contains a pair of chromosomes of Thinopyrum intermedium (Host) Barkworth & D.R. Dewey that carry the resistance gene against barley yellow dwarf virus. In this research, we developed a modified technique based on chromosome microdissection and hybridization-specific amplification to isolate expressed sequences from the alien chromosome of TAi-27 by hybridization between the DNA of the microdissected alien chromosome and cDNA of Th. intermedium infected by barley yellow dwarf virus. Twelve clones were selected, sequenced, and analyzed. Three of them were unknown genes without any hit in the GenBank database and the other nine were highly homologous with ESTs of wheat, barley, and (or) other plants in Gramineae induced by abiotic or biotic stress. The method used in this research to isolate expressed sequences from a specific chromosome has the following advantages: (i) the obtained expressed sequences are larger in size and have 3’ end information and (ii) the operation is less complicated. It would be an efficient improved method for genomics and functional genomics research of polyploid plants, especially for EST development and mapping. The obtained expressed sequence data are also informative in understanding the resistance genes on the alien chromosome of TAi-27. Key words: TAi-27, Thinopyrum intermedium, chromosome microdissection, hybridization-specific amplification (HSA), expressed sequences, barley yellow dwarf virus (BYDV). Re´sume´ : Il est difficile de situer des EST importants sur des chromosomes ou re´gions chromosomiques spe´cifiques chez le ble´ hexaploı¨de en raison de la grande taille de son ge´nome et de se´rieuses interfe´rences cause´es par des se´quences home´ologues. Le se´quenc¸age d’EST a` grande e´chelle et leur localisation chromosomique sont laborieux et prennent beaucoup de temps. La ligne´e d’addition chromosomique exotique TAi-27 contient une paire de chromosomes du Thinopyrum intermedium (Host) Barkworth & D.R. Dewey qui porte le ge`ne de re´sistance contre le virus de la jaunisse nanisante de l’orge. Dans ce travail, les auteurs ont de´veloppe´ une technique modifie´e base´e sur la microdissection chromosomique et l’amplification spe´cifique des se´quences hybride´es (« hybridization specific amplification ») pour isoler des se´quences exprime´es a` partir du chromosome exotique dans TAi-27. Un tel isolement est re´alise´ par hybridation entre l’ADN du chromosome exotique, obtenu par microdissection, et l’ADNc du T. intermedium infecte´ par le virus de la jaunisse nanisante de l’orge. Douze clones ont e´te´ se´lectionne´s, se´quence´s et analyse´s. Trois de ceux-ci correspondaient a` des ge`nes inconnus (sans homologue dans GenBank) et les neuf autres pre´sentaient une forte homologie avec des EST induits par des stress abiotiques ou biotiques chez le ble´, l’orge ou d’autres espe`ces de gramine´es. La me´thode employe´e dans ce travail pour isoler des se´quences exprime´es a` partir d’un chromosome spe´cifique pre´sente les avantages suivants : (i) les se´quences exprime´es obtenues sont de grande taille et fournissent de l’information sur l’extre´mite´ 3’ et (ii) l’ope´ration est moins complique´e. Il s’agirait d’une me´thode ame´liore´e efficace pour des travaux en ge´nomique ou en ge´nomique fonctionnelle chez les plantes polyploı¨des, particulie`rement pour le de´veloppement d’EST et la cartographie. Les donne´es obtenues sur les se´quences ex-
Received 26 July 2008. Accepted 1 November 2008. Published on the NRC Research Press Web site at genome.nrc.ca on 18 December 2008. Corresponding Editor: G. Scoles. S.-M. Jiang,1 W.-B. Yin, J. Hu, Y.-H. Chen, and Z.-M. Hu.2 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, P.R. China. R. Shi. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, P.R. China; Forest Biotechnology Group, North Carolina State University, Campus Box 7247, Raleigh, NC 27695-7247, USA. R.-N. Zhou and L.-Y. Song. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, P.R. China; Graduate University of Chinese Academy of Sciences, Beijing 100049, P.R. China. G.-H. Zhou. Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100000, P.R. China. R.R.-C. Wang. USDA-ARS, FRRL, Utah State University, Logan, UT 84322-6300, USA. 1Present
address: School of Biology and Environmental Science, University College Dublin, Beifield, Dublin 4, Ireland. author (e-mail:
[email protected]).
2Corresponding
Genome 52: 68–76 (2009)
doi:10.1139/G08-108
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Jiang et al.
69 prime´es sont e´galement utiles pour la compre´hension des ge`nes de re´sistance situe´s sur le chromosome exotique pre´sent chez TAi-27. Mots-cle´s : TAi-27, Thinopyrum intermedium, microdissection chromosomique, amplification spe´cifique de se´quences hybride´es (HSA), se´quences exprime´es, virus de la jaunisse nanisante de l’orge (BYDV).
______________________________________________________________________________________ Introduction Expressed sequence tag (EST) analysis is one of the ways to clone genes controlling desirable traits in plant (Rounsley and Linx 1998; Sasaki 1998). At present, most ESTs registered in GenBank databases are from cDNA libraries constructed from different tissues at different developmental stages. EST identification and mapping are laborious and time-consuming, especially for polyploid plants. Although many important genes have been located on specific chromosome and (or) specific regions of chromosomes by traditional genetics and modern molecular biotechniques (Spielmeyer et al. 2000; Pen˜uela et al. 2002), isolating expressed sequences directly from specific chromosomes or specific chromosome segments would be more advantageous. There are several reports on the direct isolation of expressed sequences from specific chromosome and (or) specific chromosomal regions, most of which are on expressed sequences isolated from human chromosomes or chromosome segments using microdissected chromosome DNA as probes to screen expressed sequences from cDNA libraries (Su et al. 1994; Zhang et al. 1997) or using microdissection-mediated cDNA capture (Gracia et al. 1997; Yokoyama et al. 1997; Kim et al. 2001). Our group recently reported a method to rapidly isolate expressed sequences from a specific chromosome by combining chromosome microdissection and hybridization-specific amplification (HSA) techniques (Zhou et al. 2008). Barley yellow dwarf virus (BYDV) causes serious yield losses in all cereals worldwide. Resistance genes against BYDV have not been found in common wheat but are available in barley, oat, and some wild Gramineae species. Until now, no resistance genes against BYDV were cloned, but several polypeptide markers and PCR markers linked with BYDV resistance genes have been reported (Holloway and Heath 1992; Paltridge et al. 1998; Wang et al. 2002) and are being used routinely in wheat breeding (Ayala et al. 2001; Stoutjesdijk et al. 2001; Xin et al. 2001; Ayala-Navarrete et al. 2007) . Wheat – Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (syn. Agropyron intermedium (Host) P. Beauv.) alien addition line TAi-27 is one of 14 alien addition lines carrying a pair of chromosomes from Th. intermedium in common wheat (He et al. 1998). Resistance gene(s) to BYDV in TAi-27 is (are) located on the alien chromosomes (Zhang et al. 1991; Han et al. 1998; Tian et al. 2000). Tian et al. (2000) showed that TAi-27 possessed two pairs of St genome chromosomes, one being a disomic addition and the other substituted for a pair of wheat chromosomes. Liu et al. (2001) provided evidence that TAi-27 has one group 2 and one group 7 alien chromosome. Furthermore, Zhang et al. (2001) showed that a group 2 St genome chromosome derived from partial amphiploid
Zhong 4 awnless, the same source for TAi-27, is responsible for BYDV resistance. Therefore, it appears that the BYDV resistance bearing chromosome in TAi-27 is the same group 2 chromosome as in addition line Z1 (Larkin et al. 1995; Han et al. 2003). We previously obtained several resistancerelated ESTs from the alien chromosome of TAi-27 by combining the techniques of chromosome microdissection, homology-based cloning, and subtractive library screening (Jiang et al. 2004, 2005). In this research, we modified the method to isolate expressed sequences from a specific chromosome by combining chromosome microdissection and HSA techniques. This modified method resulted in the cloning of 12 expressed sequences from the alien chromosome in TAi-27 following BYDV infection. It is an improved method for genomic and functional genomics research of polyploid plants. The data obtained in this research could enhance our understanding of the resistance genes on the alien chromosome of TAi-27.
Materials and methods Materials Germplasm used in this research included the wheat–Thinopyrum alien addition line TAi-27 (AABBDD plus a pair of St genome chromosomes derived from Th. intermedium, 2n = 44) along with its two parents, Th. intermedium (E1E1E2E2StSt with the St genome originated from Pseudoroegneria, 2n = 42) (Zhang et al. 2000) and 3B-2 (Triticum aestivum L., AABBDD, 2n = 42, the maternal parent). All materials were kindly supplied by Prof. Menyuan He of Northeast Normal University and Prof. Xiangqi Zhang of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Chromosome microdissection and DNA amplification of the alien chromosome in TAi-27 The chromosome microdissection and DNA amplification of the alien chromosome in TAi-27 were performed according to the procedures described by Jiang et al. (2004, 2005). In brief, the alien chromosome in TAi-27 was identified by its smallest size (Fig. 1) and then microdissected using a glass needle fixed on the arm of a Leitz micro-operation instrument on an inverted phase-contrast microscope (Olympus 1M, Japan). The microdissected chromosome was digested with 20 mL of proteinase K solution (19 ng/mL in 1 T4 ligase buffer) and then 0.02 U of Sau3AI (Promega) in an Eppendorf tube. Subsequently, the chromosomal DNA was amplified using Sau3AI linker adaptor mediated PCR (LA-PCR). The adaptor sequence (adaptor 1) is given below. The digested chromosomal DNA was linked with Sau3AI adaptor (adaptor 1, 2 mL, 5 ng/mL) using T4 DNA 1igase (0.5 mL, 3 U/mL) (Promega) in a total volume of Published by NRC Research Press
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Fig. 1. A modified procedure to isolate expressed sequences from a specific chromosome.
24.5 mL. Two rounds of PCR were performed. The first round of PCR was carried out in the same tube by adding 10 mL of 10 Taq buffer, 6 mL of 25 mmol/L MgCl2, 2 mL of 10 mmol/L dNTPs, 1 mL of 19 mer primer (50 ng/ mL), 2 U of Taq DNA polymerase (Promega), and doubledistilled water in a total volume of 100 mL. After denaturing at 94 8C for 5 min, amplification was performed with 35 cycles of 1 min at 94 8C, 1.5 min at 50 8C, and 3 min at 72 8C followed by a final 15 min extension at 72 8C. The second round of PCR was done under the same conditions described above except that only a 2 mL product from the first round of PCR was used as the template.
mRNA isolation and cDNA synthesis Leaves of Th. intermedium and 3B-2, at the three-leaf stage, were infected with the GAV strain of BYDV carried by Schizaphis graminum Rondani to induce resistance gene expression. Leaves were sampled once every 2 h six times on the day of inoculation and then once a day until 3B-2 turned yellow and ceased growing 3 weeks later. The samples collected were processed to isolate total RNA by using a TRIzol kit (Gibco BRL). The dT-Adaptor primer (5’GTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGTTTTTTTTTTTTTTTT-3’) that contains the oligo dT region and the special sequence designed from the Published by NRC Research Press
Jiang et al.
sequence used by Diatchenko et al. (1996) for suppression subtractive hybridization (SSH) was used to synthesize the single-strand cDNA using the procedure of the Takara RNA PCR kit (AMV) (version 2.1) (TaKaRa, Japan). After RNase digestion, the single-strand cDNA was purified by phenol– chloroform extraction and resuspended in distilled water. Generation of expressed sequences from the alien chromosome in TAi-27 oligonucleotides Adaptors Adaptor 1 (Sau3AI linker adaptors): 5’-GATCCTGAGCTCGAATTCGACCC-3’ (23 mer), 3’-GACTCGAGCTTAAGCTGGG-5’ (19 mer); adaptor 2: 5’TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3’, 3’-CCTCCCGCCACTAG-5’. Adaptor 2 was designed based on the sequences used for SSH by Diatchenko et al. (1996) with a slight modification by changing the blunt end into an asymmetric end so that it is recognizable to Sau3AI. PCR primers 19-mer primer: 5’-GGGTCGAATTCGAGCTCAG-3’; P1: 5’-GTAATACGACTCACTATAGGGC-3’; P2: 5’-TGTAGC GTGAAGACGACAGAA-3’; PN1: 5’-TCGAGCGGCCGC CCGGGCAGGT-3’; PN2: 5’-AGGGCGTGGTGCGGAG GGCGGT-3’. We designed two pairs of primers according to the sequences of the oligo dT-Adaptor primer and adaptor 2. P1 and PN1 correspond to the oligo dT-Adaptor primer and P2 and PN2 correspond to adaptor 2. Hybridization between alien chromosome DNA in TAi-27 and cDNA of Th. intermedium induced by BYDV Fifty microlitres of the second-round PCR products of the alien chromosome DNA was purified by phenol–chloroform extraction, resuspended in 10 mL of distilled water, and then digested with 10 U of Sau3AI for 3 h in a 50 mL volume. Finally, the reaction was inactivated at 80 8C for 20 min. After another phenol–chloroform purification, the digested alien chromosome DNA above was suspended in 3 mL of distilled water (~500 ng/mL) and linked with adaptor 2 by adding 1 mL (10 mmol/L) of prepared adaptor 2 and 1 U of of T4 ligase (New England Biolabs, USA). The ligation was performed at 16 8C for 16 h in a 10 mL volume and finally inactivated at 80 8C for 20 min. A 100 excess of Cot 1 DNA of TAi-27, made using the procedure described by Lecerf et al. (2001), was added to the denatured alien chromosome DNA of TAi-27 with adaptor 2 and annealed for 10 h at 68 8C. Subsequently, 3 mL of single-strand cDNA (~500 ng) of Th. intermedium infected by BYDV and 7 mL of alien chromosome DNA (~1000 ng) of TAi-27 annealed with Cot 1 DNA of TAi-27 were hybridized at 68 8C for an additional 10 h. The final hybridization solution was diluted 50–100 times with dilution buffer (20 mmol/L HEPES (pH 8.3), 50 mmol/L NaCl, and 0.2 mmol/L EDTA). Suppression PCR amplification Two rounds of suppression PCR were performed by using PCR conditions described for the Clontech PCR-Select cDNA subtraction kit. In brief, the first-round PCR amplification was carried out in a 25 mL mixture containing 1 mL
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of diluted hybridization sample, 10 mL of PCR Premix (TaKaRa, Japan), and 1 mL each of P1 and P2 (5 mmol/L). After the 7 min filling in of the ends at 75 8C, the PCR was performed with 30 cycles of 30 s at 94 8C, 30 s at 68 8C, and 90 s at 72 8C followed by a 7 min final extension at 68 8C. The PCR product was diluted in a ratio of 1:10 with distilled water. Subsequently, the second-round suppression PCR (nested PCR) was carried out in a 25 mL solution with 1 mL of the diluted first-round suppression PCR product described above, 10 mL of PCR Premix, and 1 mL each of PN1 and PN2 (5 mmol/L). The second-round suppression PCR was performed under the same conditions described above except that it was performed for 12 cycles and without the 7 min filling in of the ends at 75 8C. Cloning and analysis of expressed sequences from the alien chromosome of TAi-27 Products from the secondary suppression PCR were cloned into pUCmT-Vector (Sagon, Shanghai, China). Recombinant plasmids were isolated with the alkaline minipreparation method (Sambrook et al. 1989). Inserts of 100 randomly selected recombinant plasmids were released by PCR amplification using primers PN1 and PN2 and dot-blotted onto nylon membranes (Hybond+, Amersham, USA). Dot hybridization was carried out by using 1–2 mg of cDNA of Th. intermedium and 3B-2 (induced by BYDV) digested by Sau3AI and labeled by digoxygenin (DIG) as the probe. Two parallel membranes were prepared to hybridize with the two probes, respectively. Hybridization and detection were performed following the instructions of the DIG DNA labeling and detection kit (Roche, USA). Data analysis The inserts were sequenced by the Bio-Asia Company (Shanghai, China). The DNA sequences obtained in this research were compared with those in the GenBank database using the Blast program at the GenBank/EMBL web sites.
Results Procedures to isolate expressed sequences from the alien chromosome of TAi-27 The procedures of this method are shown in Fig. 1. The strategy of this method is based on that described by Zhou et al. (2008) but distinctly improved. As shown in Fig. 1, following LA-PCR of microdissected chromosomes, amplified microdissected DNAs were digested by Sau3AI, linked with adaptor 2, and annealed with excessive Cot 1 DNA of TAi-27. The single-strand cDNA of Th. intermedium infected by BYDV was synthesized using the oligo dT-Adaptor primer containing the oligo dT region and the specific sequence designed from the sequence used for SSH by Diatchenko et al. (1996). The single-strand cDNA was hybridized with the chromosome DNA with adaptor 2 annealed with Cot 1 DNA of TAi-27 for 10 h. Two kinds of hybrid chains were generated: DNA–DNA hybrid chains and DNA–cDNA hybrid chains. Finally, two rounds of PCR amplification were performed to select the hybridized fraction of the samples. Only the DNA–cDNA hybrid chains, which resulted from different samples with the adaptor 2 and the Published by NRC Research Press
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oligo dT-Adaptor primer sequences, respectively, could be exponentially amplified.
Genome Vol. 52, 2009 Fig. 2. Mitotic metaphase chromosomes of a root tip cell of TAi 27. Arrows indicate the additional chromosomes.
Hybridization between alien chromosome DNA of TAi-27 and cDNA of Th. intermedium induced by BYDV and suppression application The alien chromosome (Fig. 2) was microdissected and its DNA was obtained using Sau3AI LA-PCR and linked with adaptor 2. The alien chromosome DNA with adaptor 2 was hybridized with single-strand cDNA of Th. intermedium induced by BYDV. After hybridization, the hybridized fragments between alien chromosome DNA of TAi-27 and BYDV-induced cDNA of Th. intermedium were amplified by two rounds of suppression PCR using single primer PN1 or PN2 followed by double primers PN1 and PN2. There were no evident PCR products when single primer PN1 or PN2 was used, whereas PCR products ranging from approximately 250 to 750 bp were obtained when double primers PN1 and PN2 were used (Fig. 3), indicating that hybridization and suppression were successful in the experiment. Cloning of expressed sequences from the alien chromosome and screening of sequences upregulated by BYDV induction PCR products amplified with PN1 and PN2 were cloned into PUCmT vector. About 150 recombinant clones were obtained. The inserts of recombinant clones were released by PCR using PN1 and PN2 as the primers; the molecular mass of most cloned inserts ranged from 200 to 500 bp (data not shown). For screening out expressed sequences upregulated by BYDV induction, the plasmid DNAs of 100 randomly selected recombinant clones were extracted, transferred onto nylon membranes, and hybridized with BYDVinduced and DIG-labeled cDNA of Th. intermedium and 3B-2 as the probe. Differences in the intensity of hybridization signals were observed for some clones (Fig. 4). The inserts of 12 clones that showed positive or stronger signals when hybridizing with the Th. intermedium cDNA but possessing no or weaker signals with 3B-2 cDNA were selected, sequenced, named, registered in GenBank, and analyzed with a Blast search in the GenBank/EMBL database. The results are given in Table 1. Those sequences possessing a polyA tail as well as PN1 and PN2 primer sequences, an indication that they were expressed sequences of the alien chromosome, were successfully isolated. Of the 12 recombinant clones, three (WTI 8, WTI 9, and WTI 10) had no hits in the GenBank/EMBL database; the other nine sequences were highly homologous (identities >91%) with ESTs of wheat and (or) other Gramineae plants subjected to abiotic stress. Six (WTI 1, WTI 2, WTI 3, WTI 11, WTI 12, and WTJ 19) of the nine were also highly homologous (identities >92%) with ESTs from wheat and (or) other Gramineae plants inoculated with pathogens. That is, excluding the three unknown expressed sequences, all expressed sequences of the alien chromosome isolated in this study are highly homologous with ESTs induced by biotic and (or) abiotic stress. One of the expressed sequences, WTJ 19, is identical to a sequence previously isolated from the alien chromosome of TAi-27 by our group (Jiang et al. 2004). According to the sequences of these 12 inserts, we de-
Fig. 3. Electrophoresis pattern of two rounds of suppressing PCR with the hybridized DNA–cDNA between microdissected alien chromosome DNA of TAi-27 and cDNA of Th. intermedium as the template. Lane 1, primary amplification with primers P1 and P2; lane 2, molecular mass marker DL2000; lane 3, secondary amplification with primers PN1 and PN2; lanes 4 and 5, amplification with single primer PN1 and PN2, respectively.
signed nested primers for each one to recover the obtained sequences from cDNA of Th. intermedium. All of them can be amplified from cDNA of Th. intermedium (data not shown). It suggested that the sequences that we obtained should be the cDNA and the ESTs of Th. intermedium. Published by NRC Research Press
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Fig. 4. Screening of expressed sequences upregulated by BYDV induction with dot blotting of plasmid DNAs of recombinant clones as the template and BYDV-induced and DIG-labeled cDNA of (a) Thinopyrum intermedium and (b) 3B-2 as the probe.
Discussion EST analysis has sped up gene discovery in all organisms, irrespective of their genome size (Adams et al. 1991; Hillier et al. 1996; Covitz et al. 1998; Ewing et al. 1999; Fernandes et al. 2002; Shoemaker et al. 2002; Van der Hoeven et al. 2002). EST identification and localization are laborious and time-consuming, especially for large-genome polyploid plants owing to serious interference of homoeologous sequences in different but related genomes. Construction of single-chromosome or chromosome region EST libraries could circumvent this obstacle to isolating targeted expressed sequences located on specific chromosomes and (or) specific chromosomal regions. Several methods to isolate chromosome-expressed sequences (CES) have been reported. Zhou and Hu (2007) reviewed the strategy to isolate CES by the probe screening method, the hybrid selection method, and the microdissection-mediated cDNA capture method. Zhou et al. (2008) reported a method that rapidly clones CES from a specific chromosome by combining the chromosome microdissection and HSA techniques. In the probe screening method, the hybrid selection method, and the microdissection-mediated cDNA capture method, DNA and (or) cDNA library construction, numerous cloning operations and hybrid selection, and dot and (or) in situ hybridization must be done. Furthermore, disadvantages such as false positives and losing lowabundance cDNA are not avoidable (Zhou et al. 2008). In Zhou et al.’s (2008) method, the shortcome is that the obtained ESTs are smaller in size. In this research, we modified Zhou et al.’s (2008) method to clone the CES from a specific chromosome of a plant. According to the strategy shown in Fig. 1, the microdissected chromosome DNA is amplified by using Sau3AI LA-PCR so that the amplified DNA should be larger in size than that obtained by degenerated oligonucleotide-primed PCR (Zhou et al. 2000). The amplified DNAs have the Sau3AI site that also exists in adaptor 2; thus, it is easily linked with adaptor 2. The single-strand cDNA is synthesized with oligo dT-Adaptor primer containing polyT and a
sequence for SSH and directly used to hybridize with microdissected chromosome DNA. Only the DNA fragments hybridized between microdissected chromosome DNA and cDNA can be amplified by primers P1 and P2 and nested primers PN1 and PN2. The amplified DNA should contain a polyA tail and be derived from the microdissected chromosome of TAi-27. Our results proved that the strategy is correctly working. The insert size is obviously larger than those obtained by Zhou et al. (2008). All 12 sequenced fragments are ESTs with a polyA tail, one (WTJ19) of which has been previously located on the alien chromosome of TAi-27 (Jiang et al. 2004). Compared with Zhou et al.’s (2008) method, the modified method in this research has following advantages: (i) the obtained ESTs are larger in size and have polyA information, (ii) because Cot 1 DNA was used to competitively hybridize repetitive elements existing in chromosome DNA, a higher hybridization frequency resulted from eliminating the influence of redundant sequences on hybridization between chromosome DNA and cDNA, and (iii) the operation is less complicated by using Sau3AI LA-PCR. In this method, CES can be obtained without the DNA and (or) cDNA library construction as well as dot and (or) in situ hybridization. Furthermore, only singlestrand cDNA is used and the 3’ end of sequences can be read directly. Previous studies have shown that TAi-27 expressed resistance against BYDV in the field (Zhang et al. 1991; He et al. 1998; Tian et al. 2000; Jiang et al. 2004). Research on molecular markers and special probes of alien addition lines has been carried out since the wheat–Thinopyrum alien addition lines were created (Zhang et al. 1991, 2000; Jiang et al. 2004, 2005). Jiang et al. (2004) obtained nine ESTs from a subtractive library of TAi-27 infested by S. graminum carrying GAV strain of BYDV of which seven were located on the alien chromosome. Four resistance gene analogs possessing the NBS-LRR conserved domains were cloned from Th. intermedium, one of which was located on an alien chromosome of TAi-27 (Jiang et al. 2005). In this paper, we obtained 12 expressed sequences from the alien chromosome after BYDV infection. Although we do not know the relaPublished by NRC Research Press
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Table 1. Expressed sequences isolated from the microdissected alien chromosome of wheat – Thinopyrum intermedium alien addition line TAi-27. GenBank accession No. EC997001
Name WTI 1
Length (bp) 362 .
WTI 2
162 . . 229 . 293 . . .
EC997002
496 . . 254 . . . 528 519 369 718 . . 533 . . 384
EC997006
WTI 3 WTI 4
WTI 6
WTI 7
WTI WTI WTI WTI
8 9 10 11
WTI 12
WTJ 19
. .
EC997003 EC997004
EC997007
EC997008 EC997009 EC997010 EC997011
EC997012
BQ788526
Homology comparison with the data in the GenBank/EMBL database: identities Wheat ESTs in response to powdery mildow infection: 356/357 EST from Triticum aestivum (dormant seed with cold treatment after water absorption): 345/ 346 EST from Triticum aestivum (608 degrees per day after pollination): 139/139 EST from wheat – Fusarium graminearum infected spike cDNA library: 139/139 EST from Triticum aestivum (root of desiccated seed): 138/139 EST from Triticum aestivum and Fusarium graminearum inoculated wheat heads: 193/195 EST from abiotic stress in wheat: 188/202 EST from wheat vernalized crown: 272/280 EST from DuPont Wheat (leaf 7-day-old etiolated seedling): 254/265 EST from stress response genes in common wheat: 228/231 EST from abiotic stress in wheat (cold-acclimated and salt-stressed wheat cultivar Norstar): 189/196 EST from Triticum aestivum: 474/482 EST from Triticum aestivum grain (174 degrees per day after pollination): 472/482 EST from Triticum aestivum (root of desiccated seed): 470/488 EST from Triticum monococcum: 195/227 EST from Triticum monococcum early reproductive apex cDNA library: 169/192 EST from shoot of 4-day-old etiolated wheat seedling library: 148/162 EST from Hordeum vulgare subsp. spontaneum: 148/162 Unknown Unknown Unknown ESTs from creeping bentgrass inoculation with the dollar spot fungus: 457/488 EST from drought-stressed tall fescue: 441/475 EST from Lolium infected by powdery mildew: 442/478 Oryza sativa (japonica cultivar group) Os02g0754300, chromosome 2: 429/496 EST from Avena sativa etiolated leaf: 475/482 EST from Colonial bentgrass after inoculation with the dollar spot fungus: 479/519 EST from the additional chromosome of wheat–Thinopyrum alien addition line TAi-27: 384/ 384 EST from wheat inoculated with leaf rust pathogen Puccinia triticina: 327/332 EST from SSH cDNA library from CI14106 (wheat line) genotype cold hardened: 316/326
tionship between the 12 expressed sequences (this paper), the four resistance gene analogs from Th. intermedium (Jiang et al. 2005), or the seven ESTs from the alien chromosome of TAi-27 (Jiang et al. 2004) obtained by our group and the tolerance to BYDV, these genes may eventually lead to an understanding of the mechanism of BYDV resistance in Th. intermedium and TAi-27. Defense response genes belong to a broad class of genes involved in plant defense, including hypersensitive response genes, pathogenesis-related genes, genes for the flavonoid metabolic pathway, genes encoding proline/glycine-rich proteins, ion channel regulators, lipoxygenase, lectin, and others (Li et al. 1999). Of the 12 expressed sequences that are highly expressed in Th. intermedium but weakly or unexpressed in 3B-2, three (WTI 8, WTI 9, and WTI 10) cannot be grouped into the classes mentioned above for lack of information. The remaining nine are homologous with ESTs from wheat, barley, and (or) Gramineae plants induced by abiotic or biotic stress. This suggests that Th. intermedium had many genes expressed upon infection by BYDV, most of which are commonly expressed genes induced by other abiotic and (or) biotic stresses. WTI 8, WTI 9, and WTI 10
may be specifically expressed genes induced by BYDV; thus, they need to be further investigated. In conclusion, a modified efficient way to clone expressed sequences from a specific chromosome of a plant was established in this research and it would be a useful method for genomic and functional genomics research of polyploid plants. Twelve CES from the alien chromosome in TAi-27 were cloned, sequenced, and analyzed. These CES should be helpful in gaining an understanding of the resistance mechanism of, and to clone resistance genes from, the alien chromosome in TAi-27 and Th. intermedium. Investigation of the biological function of expressed sequences or genes in the alien chromosome of TAi-27 obtained in this and previous research needs to be done in the future.
Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 30270708), Chinese Academy of Sciences (No. KZCX1-SW-19), and Ministry of Science and Technology of China (Nos. Z2002-B-004 and Published by NRC Research Press
Jiang et al.
JY03-B-23). We thank Prof. Menyuan He of Northeast Normal University and Prof. Xiangqi Zhang of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for kindly providing plant materials.
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