Development and utilization of new sequenced characterized amplified region markers specific for E genome of Thinopyrum

Abstract

Species containing E genome of Thinopyrum offered potential to increase the genetic variability and desirable characters for wheat improvement. However, E genome specific marker was rare. The objective of the present report was to develop and identify sequenced characterized amplified region (SCAR) markers that can be used in detecting E chromosome in wheat background for breeding purpose. Total 280 random amplified polymorphic DNA (RAPD) primers were amplified for seeking of E genome specific fragments by using the genomic DNA of Thinopyrum elongatum and wheat controls as templates. As a result, six RAPD fragments specific for E genome were found and cloned, and then were converted to SCAR markers. The usability of these markers was validated using a number of E-genome-containing species and wheat as controls. These markers were subsequently located on E chromosomes using specific PCR and fluorescence in situ hybridization (FISH). SCAR markers developed in this research could be used in molecular marker assisted selection of wheat breeding with Thinopyrum chromatin introgressions.

Keywords

Thinopyrum / Trititrigia / E genome / SCAR markers / FISH

Introduction

Thinopyrum indtermedium, an auto-allohexaploid, genomic formula is E^eE^eE^bE^bStSt where St genome origins from Pseduoroengeria, while E^e and E^b ( = J) are closely related to Th. elongatum and Th. bessarabicum respectively (Chen, 2005). Early cytogenetic data demonstrated the closeness of genome E^e and E^b (Dewey, 1984; Wang and Zhang., 1989). Herein, we designate E genome as the general name of E^e and E^b. The E genome has many outstanding agronomical characters that can be used in wheat breeding, such as salt tolerance (McGuire and Dvorak, 1981; Colmer et al., 2006), waterlogging tolerance (Taeb et al., 1993; McDonald et al., 2001), yellow dwarf resistance and three rust resistances (Sharma and Knott, 1966; Shukle et al., 1987; Sharma et al., 1989; Friebe et al., 1996; Ma et al., 2000; Yang and Ren, 2001; Zhang et al., 2005), Fusarium head blight resistance (Han and Fedak. 2003; Shen et al., 2004; Fu et al., 2012). From the last century, wheat breeders have been transferring the resistance genes from E-genome-containing species to wheat for breeding purpose, and in particularly, a number of wheat-Thinopyrum derivative lines have been bred (Sharma and Knott, 1966; Sun, 1981; Li et al., 1985; Sharma et al., 1989; Friebe et al., 1994; Zhang et al., 2005).

Repetitive DNA sequences are proved useful in molecular marker assisted selection (Yang et al., 2006b; Liu et al., 2008). Flavell et al. (1974) pointed out that more than 75% of Triticeae genome is repetitive DNA, therefore, different methods including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and restriction fragment length polymorphism (RFLP) have been used in isolating repetitive DNA sequences. RAPD has been commonly used as a simple, low-cost, and time-effective technique, and the SCAR markers converted from RAPD products are very useful in wheat molecular marker assistant breeding (Liu et al., 2008).

Wheat stripe rust and powdery mildew are unusually severe in China. It is urgent to find new resistant resources and to develop new resistant varieties including from wide hybridization between wheat and Triticeae species. Recently, we crossed Thinopyrum intermedium ssp. trichophorum to wheat, and the stripe rust and powdery mildew-immune wheat-Th. intermedium ssp. trichophorum partial amphiploids had been produced (Yang et al., 2006a). Further, we crossed the partial amphiploids to wheat and obtained a series of stripe rust and powdery mildew-immune wheat-Th. intermedium ssp. trichophorum introgressions which can be used as resistant resources in wheat breeding. With aim to produce powerful genome specific molecular markers for fast-identification of wheat-Thinopyrum introgressions, we established new SCAR and ISSR markers for St genome (Liu et al., 2007; Hu et al., 2008). It is also necessary to obtain E-genome molecular markers to detect E chromosome in wheat backgrounds. In the present study, we attempt to isolate E-genome specific DNA segments using RAPD technique, and then convert them to effective SCAR-PCR markers. The fluorescence in situ hybridization (FISH) was also used to verify the localization of the SCAR markers in wheat-Thinopyrum introgression lines.

Material and methods

Plant materials

Materials used in this study and their relevant information including genome composition, accession number, provenance and providers were all listed in Table 1.

DNA extraction and RAPD analysis

Total genomic DNA was prepared from young leaves using SDS protocol (Liu et al., 2006). RAPD amplification was performed in an Icycler thermalcycler (Bio-RAD Laboratories). Total reaction volume is 25 μL containing 10 mmol Tris-HCl (pH 8.3), 2.5 mmol MgCl₂, 200 mol of each dNTP, 50 ng template DNA, 0.2 U Taq polymerase (Takara, Japan), and 400 nmol primer. The cycling parameters are 94°C for 3 min to pre-denature; followed by 40 cycles of 94°C for 1 min, 38°C for 1 min, 72°C for 2 min; and then a final extension at 72°C for 10 min.

Cloning and sequencing of the specific RAPD product

The genome-specific RAPD products identified from PCR were excised from 1.0% agarose gels and purified by a gel extraction kit (Qiagen, Valencia, Calif.). The purified products were ligated into the pT7 Blue R-vector using T4 ligase, and then introduced into Escherichia coli DH5α, by heat shock transformation. Nucleotide sequencing was performed on a polyacrylamide gel with the ABI prism 377 sequencer (Perkin Elmer) as an automated fluorescent sequencing system. The BLAST program in the GeneBank database was used to search for sequence similarities with DNA (BLASTN).

Specific PCR primer and amplification

Based on the cloned sequence of RAPD products, six pair of specific PCR primers were designed by using software DNAMAN, and synthesized by SBS Biotech, Beijing, China. The PCR reaction with a 25 μL volume containing 50 ng genomic DNA, 0.2 μmol/L of each primer, 200 μmol/L of each dNTP, 1 × PCR buffer, 2.0 mmol/L MgCl₂ and 1U Taq polymerase (Takara, Japan), was performed at 5 min at 94°C; 45 cycles of the following program: 1min at 94°C, 1 min at 60-63°C, and 2 min at 72°C; with a final extension at 72°C for 10min. The PCR products were fractionated on 1% agarose gel.

Fluorescence in situ hybridization (FISH)

For FISH analysis, E-genome specific DNA and Pseduoroengeria spicata genomic DNA were labeled with digoxigenin-11-dUTP according to the manufacturer’s instruction (Roche Diagnostics, Indianapolis, IN). The DNAs were labeled with fluorescence-14-dUTP (Roche Diagnostics) by nick translation. The probes were diluted to a final concentration of 1 μg/mL in the hybridization solution and the hybridization mixture was prepared as described by Mukai et al. (1993). The digoxigenin labeled DNA signals were detected with fluorescein-conjugated antidigoxigenin antibody (Roche Diagnostics). The slides were finally mounted in Vectashield antifade solution (Vector Laboratories, Burlingame, CA) with (0.25 μg/ml) propidium iodide for only FITC detection. Microphotographs of GISH chromosomes were taken with an Olympus BX-51 microscope.

Results

Cloning and sequencing of Th. elongatum E genome specific DNA segments

Six random primers, Q10, D14, I4, E11, M4 and H11 were selected from a total of 280 primer sets tested. Using common wheat Chinese Spring, MY11, R57 and R25 as controls, six specific DNA bands about 900 bp, 1000 bp, 700 bp, 400 bp, 1000 bp and 900 bp were amplified from Th. elongatum, respectively, but not in wheat controls. These E genome-specific RAPD bands were cloned and sequenced. Their full length were 865 bp, 995 bp, 694 bp, 358 bp, 907 bp and 1033 bp, named 10Q₈₆₅ (GenBank accession No.EU331358), 14D₉₉₅ (EU331359), 4I₆₉₄ (EU331360), 11E₃₅₈ (EU331361), 4M₉₀₇ (EF566898) and 11H₁₀₃₃(EU483666), respectively. 10Q₈₆₅, 14D₉₉₅, 4I₆₉₄, 11E₃₅₈, 4M₉₀₇ and 11H₁₀₃₃ showed 50.8%, 52%, 45.4%, 45%, 50% and 48.4% GC content, respectively. NCBI BLASTN search showed that 14D₉₉₅, 11E₃₅₈ and 11H₁₀₃₃ had no any homology to sequences deposited in NCBI website, indicating they were new sequences. The nucleotide sites 182 to 862 of 10Q₈₆₅ had 77% homology with nucleotide sites 80832 to 80152 of Aegilops tauschii transposon “Jody” (AY534122S2); The nucleotide sites 4 to 692 of 4I₆₉₄ had 77% homology with nucleotide sites 93511 to 94215 of Triticum aestivum LTR retrotransposon “Latidu-1p” (DQ537335); The nucleotide sites 373 to 894 of 4M₉₀₇ had 86% homology with nucleotide sites 49252 to 48721 of Triticum monococcum LTR retrotransposon “Latidu” (AF459639); indicating that 10Q_865, 4I₆₉₄ and 4M₉₀₇ were probable transposon or retrotransposon.

Conversion of the RAPD products to specific PCR based markers

According to the nucleotide sequences of 10Q₈₆₅, 14D₉₉₅, 4I₆₉₄, 11E₃₅₈, 4M₉₀₇ and 11H₁₀₃₃, six pairs of primers were designed (Table 2). Specific PCR using these six primer pairs showed that corresponding target bands could be detected in all E-genome-containing species including diploid, hexaploid, octoploid and decaploid listed in Table 1, but could not detected in all wheat controls, suggesting that these primer pairs could be used in wheat breeding program for E chromosome detection. The PCR patterns of hexaploid and octoploid Trititrigia by using primer pair D14F and D14R were showed in Fig. 1 and Fig. 2, respectively.

To examine whether these markers were unique for E-genome, specific PCR were performed on Triticeae species with different genomes, such as Secale africanum, S. silvestre, S. cereale cv. Jingzhou rye, Aegilops tauschii, Dasypyrum breviaristatum, Th. intermedium, Th. elongatum, Ps. spicata, Ae. bicornis and Ae. crassa et al., as showed in control column of Table 1. PCR result showed that 10Q₈₆₅ existed in Th. elongatum, D. breviaristatum and D. villosum genomes. 4I₆₉₄ existed in Th. elongatum, D. breviaristatum, D. villosum and Australopyrum retrofractum genomes. 14D_995, 11E₃₅₈, 4M₉₀₇ and 11H₁₀₃₃ amplified only in Th. elongatum, which indicating they were E-genome unique markers.

Chromosomal location of the specific DNA bands by using PCR and FISH

Attempts were made to assign the six E-genome specific markers to individual Th. elongatum chromosome. PCR were performed on a set of Chinese spring- Th. elongatum additions using six pair of E-genome specific primers listed in Table 2. All the six primer pairs produced corresponding target bands in seven Chinese Spring-Th. elongatum additions, but absent in wheat controls, suggesting that these markers distributed on all the seven E chromosomes. Meanwhile, PCR were also performed on a set of wheat-Th. intermedium additions, as a result, only D14F, D14R and E11F, E11R could amplify target DNA bands in wheat-Th. intermedium additions (except Z3). The PCR pattern of D14F, D14R in wheat-Th. elongatum/Th. intermedium additions was showed in Fig. 3. Because wheat-Th. intermedium additions Z1-Z6 contain S-J^s or W-J^s chromosome (Z3 contains only S chromosome), D14F, D14R and E11F, E11R could amplify target bands from these lines, indicating that these two pair of primers could not be only used for detecting E chromosome, but also for detecting J^s chromosome, that is to say D14F, D14R and E11F, E11R had broader use than other primer pairs in detecting Th. intermedium chromatin in wheat backgrounds.

All six markers developed in this research could amplify wheat-Th. elongatum 1E-7E addition, however, it is unknown that whether they could be used to hybridize Th. elongatum chromosome or not by FISH. To investigate this, six probes were hybridized to mitotic metaphase chromosomes of Chinese spring-Th. elongatum amphiploid, respectively, as a result, only 4I₆₉₄ has significant signals on all Th. elongatum (Fig. 4A). This suggests that 4I₆₉₄ could be used to identify Th. elongatum chromosome by FISH in wheat background.

Screening of wheat-Th. intermedium ssp. trichophorum introgressions using E-genome SCAR markers established

Among the 72 wheat-Th. intermedium ssp. trichophorum introgression, 1908, Q155, Q156, Q160, Q161, Q168, et al., 27 plants in total amplified the corresponding target bands using all the six primer pairs, indicating that these six molecular markers could detect wheat-Th. intermedium ssp. trichophorum introgression efficiently, on the other word, these 27 lines contain E chromosome. To verify this, three wheat-Th. intermedium ssp. trichophorum introgressions, 1908, Q156 and Q168 were randomly selected for genomic in situ hybridization (GISH) analysis. GISH analysis using Ps. spicata genomic DNA as probe and with no blocking DNA used on mitotic metaphase chromosomes of 1908, Q156 and Q168 showed that the alien genomic composition of the three introgressions were 9St+ 3J^s + 4J, 8St+ 6J^s + 2J, and 5St+ 3J^s + 2J+ 3? (? = Th. intermedium chromosomes not determined), respectively (Fig. 4 B-4D). The GISH result confirmed the PCR result, suggesting that all the six SCAR markers developed in this study wre powerful tools for detecting E chromosome in wheat background.

Discussion

Retrotransposons or transposons have broad evolutionary impact on plant genome due to their prevalence and mobility (Brosius, 1991). A number of retrotransposons of wheat genomes are genome-specific, which provide chance to clone them for evolutional study or germplasm identification. Up to now, many approaches including RAPD, RFLP, AFLP and BAC library scanning were used to clone retrotransposons or transposons. Among them, RAPD is the most rapid and cheapest approach. Using RAPD technique, Ko et al. (2002) isolated Ty3-gypsy retrotransposon from rye, Yang et al. (2006b) cloned Sabrina-like retrotransposon from Dasypyrum species, Jia et al. (2009) isolated a Ty1-copia retrotransposon from S. africanum. In this study, we cloned six sequences from Th. elongatum using RAPD. The sequences of 14D995, 11E358 and 11H1033 were new, while 10Q865, 4I694 and 4M907 were transposon or retrotransposon. Therefore, 50% sequences cloned from RAPD were transposon or retrotransposon, indicating that RAPD was a good approach for cloning repetitive sequences in wheat and related genomes.

Wide hybridization has contributed to genetic improvement of polyploid wheat (Friebe et al., 1996). Cross amphiploid or partial amphiploid to wheat varieties was commonly used to generate wheat-alien additions, substitutions or translocations. Therefore, a mass of cross offspring need to be screened. Molecular marker assisted selection which is diffusely used in wheat breeding is one of the excellent approach and turns to be an international breeding trend (He et al., 2006). By far, molecular marker has been established in Secale (Liu et al., 2006 and 2008), Dasypyrum (Yang et al., 2006b) and Hordeum (Liu et al., 1996) et al. for breeding purpose. Though Thinopyrum species contribute a lot to wheat breeding, the markers established are less that other genera of Triticeae because of its complex genome (Liu et al., 2009).

Liu et al. (1998) established RAPD marker of Agropyron elongatum 1E and 3E, and You et al. (2002, 2003) obtained E genome specific RAPD and SSR markers. Chen et al. (2007) developed a set of resistance gene analog polymorphism (RGAP) markers for detecting Th. elongatum 1E to 7E chromosomes. Li et al. (2007) developed several cleaved amplified polymorphic sequence (CAPS) markers specific to E genome. Recently, Xu et al. (2012) established three SCAR markers, one is specific for chromosome 2E and 3E, the other two distributed on all E chromosomes. All the above markers are established only using Th. elongatum, wheat-Th. elongatum additions as material, but not be validated by using plentiful of E chromosomes-containing Thinopyrum species especially octoploid trititrigia. In the present research, we developed six E-genome SCAR markers which distributed on Th. elongatum 1E to 7E chromosomes. The experimental data showed that all the six markers can be used for detecting wheat-Th. intermedium ssp. trichophorum introgressions and could be used for identifying wheat-Thinopyrum hybrids which contain E-genome chromatin. In these six markers, 14D₉₉₅ and 11E₃₅₈ could be used for detecting both E and J^s chromosome in wheat background. This is the first report that molecular marker could detect both two chromosomes simultaneously. This report represents new tools which can be widely used in wheat breeding for attempting to identify individuals that contain Thinopyrum E-genome chromatins.

The ratio of informative markers acquirement is different by using different primers, however, clone genome specific fragment and then convert to SCAR marker is a good approach for marker development. Liu et al. (1998) developed three RAPD markers specific for 1E (marker OPE-05₁₃₀₀ and OPF-03₇₀₀) and 3E (marker OPF-15₄₀₀) chromosome from 26 RAPD primers tested, the ratio of informative markers acquirement is 11.5% (3/26). You et al. (2002) screened 100 RAPD primers, as a result, one specific RAPD fragment OPF3₁₂₉₁was cloned from Th. elongatum and then converted to SCAR marker, the ratio of informative markers acquirement is 1% (1/100). Xu et al. (2012) screened 36 RAPD primers, as a result, two specific RAPD fragment OPF3₁₄₀₇ and LW10₁₄₈₇ was cloned from Th. elongatum and then converted to SCAR marker, the ratio of informative markers acquirement is 5.6% (2/36). In this research, we established six SCAR markers based on the RAPD fragments cloned by screening 280 RAPD primers, the marker acquirement is 2.1% (6/280). It was worth mentioned that primer OPF3 was used without exception in the reports mention above, it seemed that the amplicons were totally different, for we have not detect polymorphic between Th. elongatum and Chinese spring, while the rest reports obtain a polymorphic fragment with the length of about 700 bp (Liu et al., 1998), 1291 bp (You et al., 2002) and 1407 bp (Xu et al., 2012), respectively. This might be the disadvantage of RAPD which are not stable among different studies. However, it is no doubt that the conversion of RAPD to SCAR marker is necessary to produce a specific amplification, which can be effectively used to marker assisted selection among different wheat background under different studies.

References

[1] Brosius J (1991). Retroposons—seeds of evolution. Science, 251(4995): 753

[2] Chen G Y, Dong P, Wei Y M, He K, Li W, Zheng Y L (2007). Development of Ee-chromosome-specific RGAP markers for Lophopyrum elongatum (Host) A. Love in wheat background by using resistance gene analog polymorphism. Acta Agron Sin, 33: 1782-1787

[3] Chen Q (2005). Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe—a landmark approach for Thinopyrum genome research. Cytogenet Genome Res, 109(1-3): 350-359

[4] Colmer T D, Flowers T J, Munns R (2006). Use of wild relatives to improve salt tolerance in wheat. J Exp Bot, 57(5): 1059-1078

[5] Dewey D R (1984). The genomic system of classification as a guide to intergeneric hybridization with the perennial Triticeae, in Gustafson JP (ed): Gene Manipulation in Plant Improvement. 16:209-279 (Plenum Press, New York)

[6] Flavell R B, Bennett M D, Smith J B, Smith D B (1974). Genome size and the proportion of repeated nucleotide sequence DNA in plants. Biochem Genet, 12(4): 257-269

[7] Friebe B, Jiang J, Knott D R, Gill B S (1994). Compensation indices of radiation-induced wheat-Agropyron elongatum translocations conferring resistance to leaf rust and stem rust. Crop Sci, 34(2): 400-404

[8] Friebe B, Jiang J, Raupp W J, McIntosh R A, Gill B S (1996). Characterization of wheat-alien translocations conferring resistance to diseases and pests: Current status. Euphytica, 91(1): 59-87

[9] Fu S L, Lv Z L, Qi B, Guo X, Li J, Liu B, Han F P (2012). Molecular cytogenetic characterization of wheat—Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat Fusarium Head Blight. J Genet Genomics, 39(2): 103-110

[10] Han F P, Fedak G (2003). Molecular characterization of partial amphiploids from Triticum durum × tetraploid Thinopyrum elongatum as novel sources of resistance to wheat Fusarium head blight. In: N.E. Pogna, M. Romano, E.A. Pogna, & G. Galterio(Eds.), Proc 10th Int Wheat Genet Symp III. Istituto Sperimentale per la Cerealicoltura, Rome, Italy, 1148-1150

[11] He Z H, Xia X C, Luo J, Xin Z Y, Kong X Y, Jing R L, Wu Z L, Li X P (2006). Trend analysis of international wheat breeding. Journal of Triticeae Crops, 26: 154-156

[12] Hu L J, Zeng Z X, Liu C, Yang Z J, Ren Z L (2008). Production and application of ISSR marker for St genome. Journal of Sichuan University, 45: 143-149

[13] Jia J Q, Yang Z J, Li G R, Liu C, Lei M P, Zhang T, Zhou J P, Ren Z L (2009). Isolation and chromosomal distribution of a Ty1-copia like sequences from Secale allows to identify the wheat-Secale africanum introgression lines. J Appl Genet, 50: 25-28

[14] Ko J M, Do G S, Suh D Y, Seo B B, Shin D C, Moon H P (2002). Identification and chromosomal organization of two rye genome-specific RAPD products useful as introgression markers in wheat. Genome, 45(1): 157-164

[15] Li X M, Lee B S, Mammadov A C, Koo B C, Mott I W, Wang R R C (2007). CAPS markers specific to Eb, Ee, and R genomes in the tribe Triticeae. Genome, 50(4): 400-411

[16] Li Z S, Rong S, Zhong G C, Chen S Y, Mu S M (1985). Wheat wide cross. Beijing: Science Press, 52-83

[17] Liu C, Li G R, Yang Z J, Feng J, Zhou J P, Ren Z L (2006). Isolation and application of specific DNA segments of rye genome. Acta Botanica Boreali-Occidentalia Sinica, 26: 2434-2438

[18] Liu C, Yang Z J, Jia J Q, Li G R, Zhou J P, Ren Z L (2009). Genomic distribution of a Long Terminal Repeat (LTR) Sabrina-like retrotransposon in Triticeae species. Cereal Res Commun, 37(3): 363-372

[19] Liu C, Yang Z J, Li G R, Zeng Z X, Zhang Y, Zhou J P, Liu Z H, Ren Z L (2008). Isolation of a new repetitive DNA sequence from Secale africanum enables targeting of Secale chromatin in wheat background. Euphytica, 159(1-2): 249-258

[20] Liu C, Yang Z J, Liu C, Li G R, Ren Z L (2007). Analysis of St-chromosome-containing triticeae polyploids using specific molecular markers. Yi Chuan, 29(10): 1271-1279

[21] Liu S B, Jia J Z, Wang H G, Kong L R, Zhou R H (1998). Special chromosome markers for E genome and DNA polymorphism between Agropyron elongatum (2n=14) and common wheat detected by RAPD marker. Acta Agron Sin, 24: 687-690

[22] Liu Z W, Biyashev R M, Saghai M M (1996). Development of simple sequence repeat DNA markers and their integration into a barley linkage map. Theor Appl Genet, 93(93): 869-876

[23] Ma J X, Zhou R H, Dong Y S, Jia J Z (2000). Control and inheritance of resistance to yellow rust in Triticum aestivum-Lophopyrum elongatum chromosome substitution lines. Euphytica, 111(1): 57-60

[24] McDonald M P, Galwey N W, Ellneskog-Staam P, Colmer T D (2001). Evaluation of Lophopyrum elongatum as a source of genetic diversity to increase the waterlogging tolerance of hexaploid wheat (Triticum aestivum). New Phytol, 151(2): 369-380

[25] McGuire G E, Dvorak J (1981). High salt tolerance potential in wheatgrasses. Crop Sci, 21(5): 702-705

[26] Mukai Y, Nakahara Y, Yamamoto M (1993). Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome, 36(3): 489-494

[27] Sharma D, Knott D R (1966). The transfer of leaf rust resistance from Agropyron to Triticum by irradiation. Can J Genet Cytol, 8: 137-143

[28] Sharma H C, Ohm H, Lister R, Foster J E, Shukle R H (1989). Response of wheatgrasses and wheat × wheatgrass hybrids to barley yellow dwarf virus. Theor Appl Genet, 77(3): 369-374

[29] Shen X R, Kong L R, Ohm H (2004). Fusarium head blight resistance in hexaploid wheat (Triticum aestivum)-Lophopyrum genetic lines and tagging of the alien chromatin by PCR markers. Theor Appl Genet, 108(5): 808-813

[30] Shukle R H, Lampe D J, Lister R M, Foster J E (1987). Aphid feeding behavior: relationship to barley yellow dwarf virus resistance in Agropyron species. Phytopathology, 77(5): 725-729

[31] Sun S C (1981). The approach and methods of breeding new varieties and new species from Agrotriticum hybrids. Acta Agron Sin, 7: 51-58

[32] Taeb M, Koebner R M D, Forster B P (1993). Genetic variation for waterlogging tolerance in the Triticeae and the chromosomal location of genes conferring waterlogging tolerance in Thinopyrum elongatum. Genome, 36(5): 825-830

[33] Wang R R C, Zhang X Y (1989). Geneome relationship between Thinopyrum bessarabicum and Th. elongatum: revisited. Genome, 32(5): 802-809

[34] Xu G H, Su W Y, Shu Y J, Cong W W, Wu L, Guo C H (2012). RAPD and ISSR-assisted identification and development of three new SCAR markers specific for the Thinopyrum elongatum E (Poaceae) genome. Genet Mol Res, 11(2): 1741-1751

[35] Yang Z J, Li G R, Chang Z J, Zhou J P, Ren Z L (2006a). Characterization of a partial amphiploid between Triticum aestivum cv. Chinese Spring and Thinopyrum intermedium ssp. trichophorum. Euphytica, 149(1-2): 11-17

[36] Yang Z J, Liu C, Feng J, Li G R, Zhou J P, Deng K J, Ren Z L (2006b). Studies on genome relationship and species-specific PCR marker for Dasypyrum breviaristatum in Triticeae. Hereditas, 143(2006): 47-54

[37] Yang Z J, Ren Z L (2001). Chromosomal distribution and genetic expression of Lophopyrum elongatum (Host) A. Love genes for adult plant resistance to stripe rust in wheat background. Genet Resour Crop Evol, 48(2): 183-187

[38] You M S, Li B Y, Tang Z H, Liu S B, Liu G T (2003). Development of specific SSR markers for E^e-genome of Thinopyrum ssp. by using wheat microsatellites. J Agric Biotechnol, 11: 577-581

[39] You M S, Li B Y, Tang Z H, Liu S B, Song J M, Mao S F, Liu G T (2002). Establishment of E-genome-specific RAPD and SCAR markers for Thinopyrum ssp. Journal of China Agricultural University, 7: 1-6

[40] Zhang W J, Lukaszewski A J, Kolmer J, Soria M A, Goyal S, Dubcovsky J (2005). Molecular characterization of durum and common wheat recombinant lines carrying leaf rust resistance (Lr19) and yellow pigment (Y) genes from Lophopyrum ponticum. Theor Appl Genet, 111(3): 573-582

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg