Molecular cytogenetic analysis of Triticum aestivum-Leymus racemosus reciprocal chromosomal translocation T7DS·5LrL/T5LrS·7DL

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In order to induce chromosome translocation between wheat chromosomes and chromosome 5Lr of Leymus racemosus, the microsporocytes during meiosis of T. aestivum-L. racemosus disomic addition line DA5Lr were irradiated by 60Co γ-rays 800 R (100 R/min).
    © Science China Press and Springer-Verlag Berlin Heidelberg 2010 csb.scichina.com www.springerlink.com *Corresponding authors (email: wanglinsheng1234@sohu.com; pdchen@njau.edu.cn) rces   SPECIAL TOPICS: Crop Genetics   April 2010 Vol.55 No.11: 1026  1031 doi: 10.1007/s11434-010-0105-7 Molecular cytogenetic analysis of Triticum aestivum -  Leymus    racemosus  reciprocal chromosomal translocation T7DS · 5LrL/T5LrS · 7DL WANG LinSheng 1,2* , CHEN PeiDu 2*  & WANG XiuE 2   1  College of Agronomy, Henan University of Science and Technology, Luoyang 471003, China; 2  State Key Laboratory of Crop Genetics and Germplasm Enhancement, Cytogenetics Institute, Nanjing Agricultural University, Nanjing 210095, China Received July 2, 2009; accepted November 2, 2009 In order to induce chromosome translocation between wheat chromosomes and chromosome 5Lr of  Leymus racemosus , the mi-crosporocytes during meiosis of   T. aestivum-L. racemosus  disomic addition line DA5Lr were irradiated by 60 Co  -rays   800 R (100 R/min). Before flowering, the treated spikes were emasculated and bagged. After 2–3 d, the emasculated flowerets were pollinated using pollens from T. aestivum  cv. Chinese Spring. One plant with two translocation chromosomes involved in both the long and short arm of 5Lr was detected in the M 1  by GISH. The plant was crossed with line DA5Lr, and its progenies with one 5Lr and two translocation chromosomes were analyzed for chromosome pairing behavior in their pollen mother cells (PMCs). A cross-shaped configuration at diplonema and Z-shaped or ring-shaped quadrivalent configuration at metaphase I were observed, indicating that the two translocation chromosomes were reciprocal translocation. Chromosome C-banding indicated that the wheat chromosomes involved in the reciprocal translocation belonged to A- or D-genome. Fluorescence in situ  hybridization using pSc119.2 and pAs1 as the probe found that only pAs1   signals were present in the wheat chromosome segments of the two translocation chromosomes. Combining these results, the reciprocal chromosomal translocation was designated as T7DS·5LrL/5LrS·7DL. The two transloca-tion chromosomes were found to be transmitted together in the gametes of heterozygous reciprocal translocation plants with the transmission ratios of 59.4% in the female gametes and 83.9% in the male gametes, revealing preferential pollen transmission. In the self-fertilized progenies of the heterozygous reciprocal translocation, a line with the homozygous translocation line with a pair of translocation chromosome T7DS·5LrL was identified. The T7DS·5Lr translocation line was highly resistant to wheat scab and can be used as a potential and new source in wheat improvement for scab resistance. Triticum aestivum ,  Leymus racemosus , reciprocal chromosomal translocation, microsporocyte, ionizing radiation Citation: Wang L S, Chen P D, Wang X E. Molecular cytogenetic analysis of Triticum aestivum -  Leymus racemosus  reciprocal chromosomal translocation T7DS • 5LrL/T5LrS • 7DL. Chinese Sci Bull, 2010, 55: 1026  1031, doi: 10.1007/s11434-010-0105-7   The transfer of agronomically useful alien genes from wild relatives by inducing wheat-alien chromosome translocation is an important strategy in the exploitation of the genetic resource of wheat [1–6].  Leymus racemosus (2 n  = 4X = 28), a distant relative species of wheat, has been identified to have many useful traits for wheat improvement, such as tolerances to drought, low temperature, salinity, and alka-linity, resistances to three types of rusts, and the high num- ber of seeds per spike and big kernel [7,8]. The most im- portant one are is that it is highly resistant to wheat scab disease ( Fusarium  Head Blight) [9] and has been regarded as a new scab-resistant source other than Sumai 3 and Wangshuibai, which are widely used in wheat breeding for scab resistance [10]. The transfer of useful genes from  L. racemosus into  T. aestivum  is important for broadening the genetic basis of genetic improvement of wheat. Three high scab-resistant addition lines of   T. aestivum -  L.racemosus , i.e., DALr2 (DA7Lr), DALr7 and DALr14 (DA5Lr), have been   WANG LinSheng et al  . Chinese Sci Bull   April (2010) Vol.55 No.11 1027   developed in the Cytogenetics Institute of Nanjing Agricul-tural University [11]. In addition, T. aestivum-L. racemosus  ditelosomic addition line 95G11(7Lr#1S) [12], ditelosomic substitution line 7Lr#1S(7A) [13] and several translocation lines [14–17] were also developed. These lines show high resistance to scab and play important roles in the genetic improvement of wheat. In the present paper, we report the development and characterization of a reciprocal chromosomal translocation by 60 Co  -rays irradiation of the microsporocytes of   T. aes-tivum-L. racemosus  disomic addition line DA5Lr. This is the first report of the reciprocal translocation between wheat and  L. racemosus chromosomes. 1 Materials and methods 1.1 Plant materials T. aestivum -  L. racemosus  disomic addition line DA5Lr was developed and preserved in Cytogenetics Institute, Nanjing Agricultural University, China (CINAU). Triticum   aestivum cv. ‘Chinese Spring’ was also preserved in CINAU. 1.2 Irradiation treatment The microsporocytes during meiosis of   T. aestivum-L. ra-cemosus  disomic addition line DA5Lr were irradiated by 60 Co  -rays   800 R (100 R/min). Before flowering, anthers of the treated plants were removed from the florets, and then the spikes were covered with paper bags immediately. After 2–3 d, the emasculated florets were pollinated with normal fresh matured pollens of common wheat variety “Chinese Spring”. Irradiation treatments were carried out at the Insti-tute for Application of Atomic Energy, Jiangsu Academy of Agricultural Sciences. 1.3 Cytological analysis The protocol for chromosome C-banding was described by Gill et al. [18]. The chromosome preparation of PMCs fol-lowed the protocol of Chen et al. [19]. Total genomic DNA of   L. racemosus  or plasmid DNA was labelled with fluo-rescein-12-dUTP or Cy Tm 3 dUTP by nick translation and used as probes. Fluorescence in situ  hybridization (FISH) analysis was conducted following Zhang’s protocol [20]. The fluorescein- or Cy Tm -labeled probes were counter-stained with propidium iodide or 4  ,6-diamidino-2-pheny- lindole in Vectashield, respectively. Signals were observed under an Olympus BX60 epifluorescence microscope. GISH images were captured with a SPOT CCD (charge- coupled device) camera. 2 Results and analysis 2.1 Inducement of interchanged chromosomes for T.  aestivum-L. racemosus  and their cytological behavior during meiosis By pollinating the emasculated spikes of the  -ray irradiated DA5Lr with the fresh pollen of “Chinese Spring”, 92 M 1 seeds were obtained. Among them, 79 germinated success-fully and their root tips were cut for cytogenetic analysis. GISH analysis indicated that one plant numbered as WLS6-7 (2 n  = 43) had two translocation chromosomes in-volving both the long arm and short arm of chromosome 5Lr (Figure 1(a)). According to the breakpoints and frag-ment sizes of the translocation chromosomes, it is postu-lated that this is a   reciprocal translocation between chro-mosomes of wheat and  L. racemosus.  In order to verify this hypothesis, WLS6-7 as the female parent was crossed with disomic addition line DA5Lr. The chromosome pairing be-havior of the PMCs of hybrid F 1,  which have one complete 5Lr and two translocation chromosomes, was investigated. The cross-shaped configuration at the diplonema stage of meiosis (Figure 2(a)), and the  Z  -shaped (Figure 2(b)) or ring-shaped (Figure 2(c)) quadrivalent configuration at metaphase I (MI) were observed to be constituted by two Figure 1  GISH of root cells of WLS6-7 (2 n  = 43) (a) and F 1  hybrid (2 n  = 44) of WLS6-7×DA5Lr at mitotic metaphase (b). The arrows indicate 2 translo-cation chromosomes; the arrowhead indicates one chromosome of 5Lr.  1028  WANG LinSheng, et al  . Chinese Sci Bull   April (2010) Vol.55 No.11 Figure 2  FISH of PMCs of F 1  hybrid for interchanged heterozygote WLS6-7 and T. aestivum-L. racemosus  disomic addition line DA5Lr at diplonema and MI of meiosis. (a) The arrows indicate configuration of cross-shaped association of interchanged heterozygote chromosomes at diplonema of meiosis; (b) the arrows indicate Z-shaped configuration of interchanged heterozygote chromosomes at MI of meiosis; (c) the arrows indicate ring-shaped configuration of interchanged heterozygote chromosomes at MI of meiosis. translocation chromosomes, one  L. racemosus  chromosome and one wheat chromosome (Figure 2). These are the typi- cal characteristics of association of heterozygous reciprocal translocation during meiosis, indicating these two transloca-tion chromosomes were reciprocal translocation chromo-somes between wheat and  L. racemosus . 2.2 Molecular cytogenetic characterization of the re-ciprocal translocation chromosomes C-banding and GISH of somatic chromosomes of the recip-rocal translocation showed that the breakpoint of transloca-tion was located in the centromere regions, indicating the reciprocal translocation chromosomes involved their whole arm. The involved wheat chromosome was not rich of C-bands, with only some C-bands in the short arm and rela-tive strong C-bands in the terminal region and sub-terminal region of the long arm (Figure 3). We speculated that the involved wheat chromosome was not from the B-genome chromosomes, which were the characteristic for their rich and strong C-band pattern. Zhang et al. [20] identified that the BAC-676D4 clone was only hybridized to A-genome chromosomes and could be used as an A-genome identifier. No hybridization signal was observed in the translocation chromosomes by FISH using BAC-676D4 as the probe (Figure 4(a)), indicating that none of the A-genome chro-mosome was involved. FISH using D-genome specific clone pAs1 as the probe indicated that the hybridization signal of pAs1 was present in the wheat segments of the reciprocal translocation chromosomes, indicating their D-genome identity. Clone pSc119.2 could produce FISH signals in wheat D-genome chromosomes except 1D, 6D or 7D. Further FISH using pSc119.2 as the probe also failed to observe any signal in the wheat segments, and hence we speculated that the chromosome involved in the transloca-tion was among chromosome 1D, 6D or 7D. According to the patterns of the pAs1 hybridization and C-banding, the chromosome was determined to be chromosome 7D. Therefore, the reciprocal translocation was designated as T7DS·5LrL/5LrS·T7DL. 2.3 Transmission of the reciprocal translocation chro-mosomes Reciprocal crosses between WLS6-7, a heterozygote recip-rocal translocation line, and Chinese spring were made. The chromosomes of somatic cells of the F 1  were identified by GISH. It was found that, when using WLS6-7 as the female parent, four types of female gametes were produced. The frequencies for gametes with T7DS·5LrL + T5LrS·7DL, T7DS·5LrL, or T5LrS·7DL and gamete without the translo-cation chromosome were 59.4%, 9.4%, 3.1% and 28.1%, respectively. While using WLS6-7 as the male parent, only translocation chromosomes T7DS·5LrL + T5LrS·7DL and T7DS·5LrL were recovered in the F 1 generation with fre-quencies of 83.9% and 16.1%, respectively (Table 1), and   WANG LinSheng et al  . Chinese Sci Bull   April (2010) Vol.55 No.11 1029   Figure 3  C-banding and GISH of the reciprocal translocation chromosomes T7DS·5LrL/5LrS·T7DL. From left to right: C-banded 7D, C-banded T7DS·5LrL, FISH T7DS·5LrL, C-banded 5LrS·T7DL, FISH 5LrS·T7DL and C-banded 5Lr. Figure 4  Bi-color FISH of the interchanged chromosomes of root cells at mitotic metaphase. (a) WLS6-7 heterozygote. Green Fluorescein was the FISH signal of pSc119.2 using Fluorescein-12-dUTP labeled. Orange Fluorescein was the FISH signal of total genomic DNA of  L. racemosus  using Cy Tm 3dUTP labeled. The arrow and arrowhead indicate the chromosome of T7DS·5LrL and T5LrS · 7DL, respectively. The wheat segment of translocation chromosome has no GISH signal of probe pSc119.2. (b) T7DS·5LrL''+ T5LrS·7DL'' homozygote. Green Fluorescein was the FISH signal of pAs1 using Fluo-rescein-12-dUTP labeled. Orange Fluorescein was the FISH signal of total genomic DNA of  L. racemosus  using Cy Tm 3 dUTP labeled. The wheat segment of translocation chromosome has the GISH signal of probe pAs1. The arrows indicate T7DS·5LrL, and the arrowheads indicate T5LrS·7DL. Table 1 Gametes transmission of heterozygote reciprocal translocation chromosomes Proportion of progenies (%)   Materials   Mode of transmission   No. of plants investigated   T7DS·5LrL + T5LrS·7DL   T7DS·5LrL   T5LrS·7DL   No alien   WLS6-7×CS   Female   32 59.4 9.4 3.1 28.1 CS×WLS6-7   Male   31 83.9 16.1 0 0 none of the F 1  were found to have chromosomes T5LrS·7DL or have no translocation chromosome, indicating that trans-mission of chromosome T7DS·5LrL+T5LrS·7DL and T5D S·5LrL through the male gamete is 83.9% and 16.1%, respec-tively. To summarize, the transmission rates of the reciprocal translocation chromosomes were higher than the expected rates either through male or female gametes. Those gametes with the reciprocal translocation chromosomes were more competitive in sexual reproduction and of typical preferential male gamete transmission. We also found that 7 types of plants with different chromosome constitutions were pro-duced by self-fertilizing of the heterozygous reciprocal translocation line, including plants with T7DS·5LrL '   + T5LrS·7DL '  , T7DS·5LrL ''   + T5LrS·7DL ''   (Figure 4(b)), T7DS·5LrL ''  +T5LrS·7DL '  , T7DS·5LrL '  + T5LrS·7DL ''  , T7D S·5LrL ''  , T7DS·5LrL '   and the plant of no alien translocation chromosome. The former two types of plants accounted for 70%. 3 Discussion Ionizing radiation has been widely used to introduce useful genes into common wheat. Since the first report of produc-tion of the wheat-  Aegielops umbellulata  translocation line with rust disease resistance using X-ray irradiation by Sears [22], various translocation lines between wheat and  Aegiel-ops ,  Haynaldia ,  Agropyron, Secale  and  Leymus , have been  1030  WANG LinSheng, et al  . Chinese Sci Bull   April (2010) Vol.55 No.11 developed [6,14–17]. Ionizing radiation is the most widely used method to induce chromosome translocation because it is highly effective and is not affected by the genetic rela-tionship between wheat and its related species as well as the genetic distance between the target gene and the centro-mere. Seeds, adult plants during meiosis [14], mature pollens [23–25], and mature female gametes [26] have been used as materials for irradiation to induce the chromosome translo-cation. In the present study, we used 60 Co  -ray to treat mi-crosporocytes of wheat-  L. racemosus  addition lines to in-duce chromosomal structural change. We found that the seed-set rate of the treated plants was 24.8% after being crossed with Chinese spring, and 79 out of 92 M 1  seeds could be germinated with a germination rate of 85.9%. Us-ing chromosome C-banding and GISH method, 12 of 79 M 1  plants containing chromosomal structure change of 5Lr were identified, including 6 translocations and 6 telosomics. The M 1 translocation plants had a high self-fertilizing seed-set rate. Except one plant which only produced 17 seeds, the others all produced more than 200 seeds. It is worth noting that one plant, WLS6-4, produced 1300 seeds, providing big enough population for selecting homozygous translocation lines in the next F 2  generation. These results suggested that the dosage used in the present research was suitable for microsporocytes radiation. We also tested 1200 R dose microsporocytes radiation on wheat-  L. racemosus  addition lines. However, we found that the treated plants could not head normally and those heading plants had a very low seed-set rate. In addition, most of the M 1  seeds were abnormal, both for roots and sprouts developments. This suggested that 1200 R had significant negative effect on plant growth. We also investigated the chromosome association of hete- rozygous reciprocal translocation line T7DS·5LrL/T5LrS·7 DL, and three types of chromosome pairing configuration were observed. The first one was that chromosome 7D as-sociated with the two translocation chromosomes forms a trivalent. The second was that chromosome 7D associated with T7DS·5LrL forms a bivalent, while T5LrS·7DL ap-pears as a univalent. The third was that chromosome 7D associated with T5LrS·7DL forms a bivalent, but T7DS·5LrL appears as univalent. We found that the first one was the most dominant. Most of the chromosome seg-regation of interchanged chromosomes at anaphase I was unbalanced with a segregation ratio of 2:1, with the T7DS·5LrL and T5LrS·7DL moving to one pole, while the other chromosome 7D moving to the opposite pole. The observation is consistent with Bie et al.’s result [27]. As a result, the female gametes with T7DS·5LrL + T5LrS·7DL and without any translocation chromosome were over-whelmingly dominant. Transmission analysis of gametes showed that heterozygous reciprocal translocation produced 4 types of female gametes, i.e. T7DS·5LrL + T5LrS·7DL, T7DS·5LrL, T5LrS·7DL and gamete without any transloca-tion. The transmission rate of the female gamete with T7DS·5LrL + T5LrS·7DL was 59.4%, which was much higher than that of gametes without alien translocation chromosome (28.1%), indicating the preferential transmis-sion of the reciprocal translocation chromosomes through the female gamete. When heterozygous reciprocal translo-cation was used as the male parent in the cross with Chinese spring, only two types of plants with T7DS·5LrL + T5LrS·7DL or T7DS·5LrL were obtained, and their propor-tion was 83.9% and 16.1%, respectively. This also indicated preferential pollen transmission of the translocation chro-mosomes involved in the long arm of 5Lr. The telosomics of the long arm of 5Lr also showed preferential pollen transmission. We speculated that there might be a preferen-tial pollen transmission gene on the long arm of 5Lr. We also identified a homozygous translocation with a pair of T7DS·5LrL. The scab resistance of line T7DS·5LrL was evaluated by artificial inoculation of Fusarium graminea-rum  at the early flowering stage. The line T7DS·5LrL, Chi-nese spring (S) and Sumai 3 (R) showed average percent-ages of infected spikelets of 2.8%, 43.8%, and 1.6%, re-spectively. This further confirmed that gene(s) conferred scab resistance of  L. racemosus  was located on the long arm of 5Lr, and the developed homozygous translocation line T7DS·5LrL could be a new source of scab resistance in wheat improvement. The work was supported by the National High-Tech Research and Deve- lopment Program of China ( Grant No. 2006AA10Z1F6  )  and Programme of  Introducing Talents of Discipline to Universities ( Grant No. B08025 ).   1   Zeller F J, Hsam S L. Broadening the genetic variability of cultivated wheat by utilizing rye chromatin. In: Proc the 6th Int Wheat Genet Symp, Kyoto, Japan, 1983. 161–171 2   Gale M D, Miller T E. The introduction of alien genetic variation into wheat. In: Lupton F G H, ed. Wheat Breeding: Its Scientific Basis. UK: Chapman and Hall, 1987. 173–210 3   Knott D R. Transferring alien genes to wheat. In: Heyne E G, ed. Wheat and Wheat Improvement. 2nd ed. Madison, WI: Am Soc Agron, 1987. 462–471 4   McIntosh R A. Alien sources of disease resistance in bread wheats. In: Sasakuma T, kinoshita T, eds. Proc of Dr. H Kihara Memorial Int Symp.on Cytoplasmic Engineering in Wheat. Nuclear and organellar genomes of wheat species. 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