Radiation mutants for mapping genes and markers in pericentromeric region of chromosome 3B of Norin 26 wheat
Soichi Yamano1, Hisashi Tsujimoto2, Takashi R. Endo1, and Shuhei Nasuda1
1 Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
2 Laboratory of Plant Genetics and Breeding Science, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
Corresponding author: Shuhei Nasuda
E-mail: nasushu@kais.kyoto-u.ac.jp
Bread wheat (Triticum aestivum L. 2n = 6x = 42, AABBDD) has a large genome (17 gigabases (Gb)), of which 80% is composed of repetitive sequences (Smith and Flavell 1975). Such plants with large genomes as wheat, maize (2.5 Gb) and barley (5 Gb) show gradual increase of recombination from the centromeres to telomeres (Lukaszewski and Curtis 1993; Kunzel et al. 2000; Anderson et al. 2003; Saintenac et al. 2009). Mapping studies in other species indicated that recombination suppression in proximal region is general feature in plants and animals (see review by Jones 1987). On chromosome 3B of wheat, for which genome study is most advanced, a detailed survey showed that crossover frequency per physical distance (cM/Mb) within bins C-3BS1-0.33 and C-3BL2-0.22 were only 0.006 and 0.012, respectively, in striking contrast to the highest value of 0.85 in bin 3BS8-0.78-0.87 (Saintenac et al. 2009). Thus genes located on recombination-poor region are inaccessible by map-based cloning approaches. One of the methods to achieve the high resolution mapping in the recombination-depleted region of genome is the radiation hybrid (RH) mapping (Goss and Harris 1975; Riera-Lizarazu et al. 2007). RH method was recently applied to construct the high-resolution RH map of chromosome 1D of wheat (Kalavacharla et al. 2006) and also used in physical mapping of chromosome 3B (Paux et al. 2008). This RH mapping approach allows ordering molecular markers previously unordered within a chromosomal bin and does not need allelic polymorphism. In addition, RH maps with the required resolution can be produced by altering the dosage of radiation. The objective of this study is to develop chromosome structural mutants induced by irradiation for mapping the Igc1 gene in pericentromeric region of chromosome 3B of Norin 26 wheat (Yamano et al., in preparation). Igc1 is known as the suppressor to Gc3-C1, one of the gametocidal (Gc) genes in wheat (Tsujimoto and Tsunewaki 1985). We here report development of chromosome structural mutants of chromosome 3B of Norin 26 wheat induced by irradiation for mapping genes in centromeric region (Fig. 1). The parental plants, Triticum aestivum cv. ‘Norin 26’ (N26) and T. aestivum cv. ‘Chinese Spring’ 3B-3C substitution line (CS-3C”(3B”)), which has a pair of the 3C chromosomes derived from Aegilops triuncialis substituting for chromosome 3B, were grown in the field located at the Department of Agriculture, Kyoto University, Kyoto, Japan in the year 2009. Each line was planted in a pot. The whole spike of N26 at efflorescence was irradiated with 15 Gy of X-rays at the radiation facility in Tottori University, Japan. Then, the irradiated pollen of N26 (N26IR) was used as male parent to cross to CS-3C”(3B”). The progeny of the cross was expected to have the 3B chromosome from N26IR monosomically and no 3B from CS. In total, 390 F1 seed was obtained. All 390 seed was sawn on a filter paper soaked in water, and germinated plants were cultivated in a plant incubator. We could finally recover 199 F1 progeny, which accounted for 51.0% out of total progeny (Table 1). A large portion of seed (160 seed) could not germinate, six plants were dead during early stages of development, and 15 were dead after rhizogenesis without shoot.
Total genomic DNA was extracted from frozen leaves harvested 4 weeks after seeding, using the DNeasy Plant Mini Kit (QIAGEN), according to the manufacturer’s protocol. A total of 102 insertion site-based polymorphism markers (ISBP, Paux et al. 2006) were selected from each contig assigned to bins C-3BS1-0.33 (51 ISBP markers) and C-3BL2-0.22 (51 ISBP markers) of chromosome 3B for physical mapping (The complete list of the markers will be provided elsewhere (Yamano et al., in preparation). PCR reactions were carried out in 15 µl of 0.5 units of Taq polymerase, 0.5 pmol for primer, 25 mmol for MgCl2, 2.5 mmol for each dNTP and 25 ng for template DNA. ISBP markers were amplified under the following touchdown method; 95 oC for 5 min, 10 cycles of 95 oC for 30 sec, 65 oC for 30 sec decreasing by 0.5 oC per cycle and 72 oC for 30 sec, and 30 cycles of 95 oC for 30 sec, 60 oC for 30 sec and 72 oC for 30 sec, and 72 oC for 7 min. PCR products of ISBP markers were visualized on the 1.5% agarose gel stained by ethidium bromide. As positive and negative controls, total genomic DNA of N26 and CS-3C”(3B”), respectively, were used as templates in PCR. Scoring presence and absence of the ISBP markers, we found that 122 F1 progeny (61.9% of the recovered and 31.3% of total F1 progeny) had at least one breakpoint along the chromosome 3B of N26 (Table 2). Out of the 122 chromosome 3B deletion lines, 45, 42 and 35 plants had breakpoints in short arm of 3B, long arm of 3B and both arms, respectively. In total, 592 breakpoints were found in bin C-3BS1-0.33 or C-3BL2-0.22 through the analysis of 122 chromosome deletion lines with 102 markers. The number of markers deleted in individual plant ranged from 0 to 64. Ninety-six of 102 tested ISBP markers were absent at least one deletion line. The average marker retention frequency in total 199 F1 progeny was 95.75%. This value is higher than that reported in previous report (74%) in the RH panel of chromosome 1D of wheat (Kalavacharla et al. 2006). These differences may reflect the marker locations; while we selected markers from pericentromeric region of 3B, which occupies a quarter of total 3B and might include indispensable chromosomal region to be transmitted to next generation, Kalavacharla et al. (2006) evenly picked up markers along chromosome 1D.
The radiation mutants produced in this study inherited chromosome 3C monosomically, which induces the Gc effect in gametogenesis (Endo 1978). Depending on the presence or absence of the Igc1 gene, the suppressor of the Gc action of chromosome 3C (Tsujimoto and Tsunewaki 1985), the radiation mutants will differ in seed-fertility; fertile in the presence of Igc1 and semi-sterile in the absence of Igc1. In future we will analyze the location of the Igc1 gene by the RH mapping approach.
References
Anderson LK, Doyleb GG, Brighama B, Cartera J, Hookera KD, Laia A, Ricea M and Stack SM (2003) High-resolution crossover maps for each bivalent of Zea mays using recombination nodules. Genetics 165: 849-865.
Endo TR and Katayama Y (1978) Finding of a selectively retained chromosome of Aegilops caudata L. in common wheat. Wheat Inf Serv 47/48: 32–35.
Goss SJ and Harris H (1975) New method for mapping genes in human chromosomes. Nature 255: 680-684.
Jones GH (1987) Chiasmata. Academic Press, Orlando, FL.
Kalavacharla V, Hossain K, Gu Y, Riera-Lizarazu O, Vales MI, Bhamidimarri S, Gonzalez-Hernandez JL, Maan SS and Kianian SF (2006) High-resolution radiation hybrid map of wheat chromosome 1D. Genetics 173: 1089-1099.
Kunzel G, Korzun L and Meister A (2000) Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints. Genetics 154: 397-412.
Lukaszewski AJ and Curtis CA (1993) Physical distribution of recombination in B-genome chromosomes of tetraploid wheat. Theor Appl Genet 86: 121-127.
Paux E, Roger D, Badaeva E, Gay G, Bernard M, Sourdille P and Feuillet C (2006) Characterizing the composition and evolution of homoeologous genomes in hexaploid wheat through BAC-end sequencing on chromosome 3B. Plant J 48: 463-74.
Paux E, Sourdille P, Salse J, Saintenac C, Choulet F, Leroy P, Korol A, Michalak M, Kianian S, Spielmeyer W, Lagudah E, Somers D, Kilian A, Alaux M, Vautrin S, Berges H, Eversole K, Appels R, Safar J, Simkova H, Dolezel J, Bernard M and Feuillet C (2008) A physical map of the 1–gigabase bread wheat chromosome 3B. Science 322: 101-104.
Riera-Lizarazu O, Vales MI and Kianian SF (2008) Radiation hybrid (RH) and HAPPY mapping in plants. Cytogenet Genome Res 120: 233-240.
Saintenac C, Falque M, Martin OC, Paux E, Feuillet C and Sourdille P (2009) Detailed recombination studies along chromosome 3B provide new insights on crossover distribution in wheat (Triticum aestivum L.). Genetics 181: 393-403.
Smith DB and Flavell RB (1975) Characterization of wheat genome by renaturation kinetics. Chromosoma 50: 223-242.
Tsujimoto H and Tsunewaki K (1985) Gametocidal genes in wheat and its relatives. II. Suppressor of the chromosome 3C gametocidal gene of Aegilops triuncialis. Can J Genet Cytol 27: 178-185.