Study on the Translocation of Wheat- E. elongatum by Fluorescence in Situ Hybridization

Transcription

1 Available online at Procedia Environmental Sciences 12 (2012 ) International Conference on Environmental Science and Engineering (ICESE 2011) Study on the Translocation of Wheat- E. elongatum by Fluorescence in Situ Hybridization Min Qu Department of Bioengineering, Harbin University of Commerce, Harbin, China, qumin777@126.com Abstract Elytrigia Desv. is the third gene source. It has always been as highly valued as it used to be in wheat s breeding and genetic improvement. By using wheat chromosome engineering and establishing its heterology translocation, it will be a effective way which the excellent properties are improved into wheat. Some species from Aegilops have gametocidal gene. Variety types of chromosome structural variation can be induced high frequency by the not fully gametocidal function, and chromosome translocation and deletion will be produced. This variation are high frequency, directionally, stability. In this paper, by using ChineseSpring- Ae.cylindrical-2C disomic addition to cross with ChineseSpring- E. elongatum 7E (2n=14EE), the translocation and deletion of ChinesesSpring-E. elongatum 7E were induced. And by using Fluorescence In Situ Hybridization technique, the materials with translocation and deletion were identified. It showed that it is a important method which useful genes are transferred into wheat by using wheat and some translocation and deletion of it s kindred genus by Fluorescence In Situ Hybridization Published by Elsevier by Elsevier B.V. Selection Ltd. Selection and/or peer-review and/or peer-review under responsibility under responsibility of National University of [name of Singapore. organizer] Open access under CC BY-NC-ND license. Keywords:Fluorescence In Situ Hybridization; Gametocidal chromosome; E. elongatum; Translocation 1. Introduction Wheat group that does not have AABBDD these three genomes of species is the third-grade gene source of wheat [1]. This foreign gene, which was urgently needed for wheat s high efficient and sustainable development, is key genes. More than 100 high-qualities, stress resistance, disease resistance, insect resistance of the foreign gene were transferred into wheat, which played an important role for improvement. Wheat were improved by these foreign genes and put into production, and had a significant long-term economic benefit [2]. By using kindred genera of wheat, China had successfully used Elytrigia Desv Published by Elsevier B.V. Selection and/or peer-review under responsibility of National University of Singapore. Open access under CC BY-NC-ND license. doi: /j.proenv

2 Min Qu / Procedia Environmental Sciences 12 ( 2012 ) Aegilops L. Haynaldia Schur Agropyron Caertn. Leymus Hochst etc.. Elytrigia Desv has always been as highly valued as it used to be in wheat's breeding and genetic improvement. Elytrigia Desv, perennial wild plants of Triticeae Dumort, has many good properties: tillering ability, rich roots, cold and drought resistance, barrenness tolerance, high saline-alkali tolerance, large spike and high yielding, high protein content and good baking quality as well as ability against a variety of diseases, all of which are immune to and even high resistant to stripe rust, leaf rust, stem rust, powdery mildew, yellow dwarf virus, stripe mosaic disease and bunt. It showed that it is an important method which exogenous genetic materials are transferred into wheat by using wheat chromosome engineering to establish its heterogonous translocation. Endo [3] found some species of Aegilops. When these monomers with gametocidal genes were attached to common wheat with different genotypes, they had complete or incomplete gametocidal role. Variety types of chromosome structural variation can be induced high frequently by not fully gametocidal function, and chromosome translocation and deletion will be produced. This variation are high frequent, directional, stable. In this paper, by using Chinese Spring- Ae.cylindrical-2C disomic addition line to cross with Chinese Spring- E. elongatum 7E (2n=14EE), the translocation and deletion of ChinesesSpring-E. elongatum 7E were induced. By using Fluorescence in Situ Hybridization technique, the materials with translocation and deletion were identified. 2. Materials and Methods Materials and Disposal The materials used in the study are Chinese Spring- E. elongatum 7E (E.elongatum, 2n=14 EE) 2n 44 AABBDD 2E disomic addition line, Chinese Spring- Ae.cylindrical 2n 44 AABBDD 2C disomic addition line, diploid agropyron elongatum and Chinese Spring comparison which was provided by Professor Li Jilin from Genetics Lab, Life and Environment Science College, Harbin Normal University. By sowing in the fields separately and using Chinese Spring- Ae.cylindrical-2C disomic addition line to cross with Chinese Spring- E. elongatum 7E (2n=14EE); After sowing Hybridism F1, backcross and selfing respectively, F2 B1F2 sowing Crossing as normal. Chinese Spring comparison was sowing in the field simultaneously. During 9-11 A.M. and 4-6 P.M in F1 booting, took pollen mother cells of panicles in the field during meiosis, chose anthers in the mid-meiotic by microscopic examination. Chromosome Preparation Technique of Pollen Mother Cells and Root Tip Cells Chromosome in meiosis of F1 pollen mother cell adopt conventional tabletting method, obtained anther materials when in meiosis of F1 anther panicle, were fixed with acetic carmine Kano and stained by aceto-carmine. Chromosome root tip cells used conventional tabletting method. Fluorescence In Situ Hybridization The Extraction of Plant Genomic DNA Extract total genomic DNA from Agropyron elongatum and Chinese Spring using CTAB method. Measured purity and concentration of the extracted DNA by spectrophotometer till it achieved the requirements, packaged and stored at Probe Mark Labled probe using nick translation. Took the total DNA100ul of diploid agropyron elongatum, broke the total DNA using ultrasonic mill for seconds, and then observed it by electrophoresis. Took the total DNA1ug of diploid agropyron elongatum, and marked it according to GIBCOBRL kit. Place the probe at Labled probe using biotin. Keep it for at least one year under the conditions in TE buffer and In Situ Hybridization In this study, took genomic DNA of diploid Agropyron elongatum (2n = 14EE) as probe, biotin as tagging,

3 1222 Min Qu / Procedia Environmental Sciences 12 ( 2012 ) Chinese Spring genomic DNA as blocking, did in situ hybridization. Did In situ hybridization by Zhi Ma s [5] method. Detected and observed the chromosome after in situ hybridization by using Qumin s method, and then carried out image processing. 3. Results and Analysis Detection of Genomic DNA Figure 1 The electrophoretogram of probe and blocking genome total DNA 1. 2uL genome total DNA of Chinese-Spring (taken out RNA) 2. 2uL genome total DNA of E. elongatum (taken out RNA) 3. Marker: DL2000 Figure 2 The electrophoretogram of cracked probe and blocking genome total DNA 1.1uL Spring genome total DNA (taken out RNA), high pressure broken 1min 30s 2. 1uL genome total DNA of E. elongatum (taken out RNA), ultrasonic broken 1min 30s 3. Marker DL2000 Extracted a large amount of DNA by CTAB, after RnaseA treatment, both concentration and purity met the requirements (Figure 1) by UV detection, the purity OD260/280 value reached , concentration above 0.4ug/ul. Broke the total DNA using ultrasonic mill for 1 minute or 90 seconds, length of DNA fragments was around bp (Figure 2). Detection of Probe Labeling Efficiency Detect Agropyron elongatum chromosome using total DNA probe of Agropyron elongatum which is marked by biotin, resulted in clear green hybridization signals on all 14 chromosomes of Agropyron elongatum, which meant the success of probe mark (Figure 3). Figure 3 The test of E. elongatum crossing (The marking were successful by clear blue signal)

4 Min Qu / Procedia Environmental Sciences 12 ( 2012 ) F1 Meiotic of Pollen Mother Cells and In Situ Hybridizationof Root Tip Cells. Figure 4 The result of Fluorescence In Situ Hybridization in CS-7E" CS-2C F 1 PMC Line 7-11, F 1 PMC Met, the arrow showed the GISH crossing signal of translocation chromosome Took CS-7E " CS-2C" F1 meiotic cells at every stage and root tip cells of seeds as materials for in situ hybridization, Chinese Spring chromosomes in the G excitation light, PI fluorescence emerged red; in the B excitation light, FITC fluorescence showed green fluorescent. Agropyron elongatum chromosome through the comparison of green FITC fluorescence and red PI, the composited chromosome fluorescence appeared yellow or yellow-green fluorescent. Compared with P1, un-hybridized chromosome emerged orange-red fluorescence under the same excitation light, That is, Agropyron elongatum chromosomes or chromosome fragments with a hybridization signal emerged yellow or yellow-green, wheat chromosomes displayed orange and red (Figure 4,5) Figure 5 The result of Fluorescence In Situ Hybridization in CS-7E" CS-2C F 1 root tip cell Line 7-39, metaphase of root tip cell, the arrow showed the GISH crossing signal of T7E 7B translocation chromosome 4. Discussion In situ hybridization of wheat was that clone repeated DNA sequences of wheat and its kindred genus, took advantage of these specific or repeated DNA sequence as a probe, combined with fluorescence techniques, used reactions of organisms to different fluorescent dye-labeler, did identification and positioning to a gene, a chromosome fragment, a chromosome, or even a gene. Schawarzacher et al [7] successfully did

5 1224 Min Qu / Procedia Environmental Sciences 12 ( 2012 ) fluorescence in situ hybridization by using foreign genomic DNA as a probe, identified all the chromosomes of rye, and identified H. chilense of wheat and the 1BL/HS translocation of barley. Friebe et al [8] analyzed chromosome composition of wheat - Thinopyrum intermedium partial double diploid TAF-46 using sequenced banding in situ hybridization, and identified additional six pairs of Thinopyrum intermedium chromosomes. Jiang [9] identified translocation of wheat and rye by using in situ hybridization technique. Chen Peidu et al. [10] detected H. villosa chromosomes of wheat by GISH technique. Yang Zujun et al. [11] identified perennial villosa chromatin which is improved into wheat by GISH technology. Qumin et al. identified translocation of Chinese Spring and diploid Agropyron elongatum (1E) by using Fluorescence In Situ Hybridization technique. Therefore, it is an important method which useful genes are transferred into wheat by using wheat, amphidiploid, additional system, substitution system and some translocation and deletion of it s kindred genus by Fluorescence In Situ Hybridization. Acknowledgement The research was supported by the Nature Science Foundation of Heilongjiang Province (C200602), the technology foundation of Harbin Science and Technology Bureau (2005AFXXJ027), the technology foundation of Heilongjiang Education Bureau ( ). References [1] Dong Yuchen, Genepools of Common Wheat [J]. Acta Tritical Crops. 2000, 20(3), P [2] Li Lihui, Yang Xinming, Li Xiuquan, et al. Study and Utilization of Wild Relatives of Wheat in China [J]. Review of China Agricultural Science and Technology, 2000, 2(6), P73-76 [3] T.R.Endo, Gametocidal chromosome of three Aegilops species in common wheat [J]. Can. J. Genet. Cy tol. 1982, 24, P [4] T.R.Endo, Gametocidal chromosomal and their induction of chrosome mutations in wheat [J]. Japanese Journal of Genetics, 1990, 65, P [5] Ma Youzhi, M. Tomita,N. Nakata, et al.analysis of the Chromosomes Composition of Common Wheat- Agropyronintermedium Partial Amphiploid, Yuan Zhong 2 by in situ hybridization [J]. Acta Genetic Sinica, 1997, 24(4), P [6] Qu Min, Zhang Yanming, Zhang Xueting, et al. Study on Chromosome Aberration of Chinese Spring-Elytriga 1E Disomic Addition Induced by Gametocidal Chromosome 2C [J]. Food Science, 2007, 28(10), P [7] T. K. Schawarzacher, Anamthawat-Jónsson,G.E., Harrison,et al. Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat [J]. Theor Appl Genet. 1992, 84, P [8] B. Friebe, Y. Mukal, B.S. Gill, et al. C-banding and in situ hybridization analyses of Agropyron intermedium,a partial wheat Ag. Intermedium ampliploid, and six derived chromosome addition lines [J]. Theor Appl Genet. 1992, 84, P [9] J.M. Jiang, B.S. Gill, Sequential chromosome banding and in situ hybridization analysis [J]. Genome, 1993, 36, P [10] Chen Peidu, Zhou Bo, Qi Lili. Identification of Wheat-Haynaldia villosa Amphiploid, Addition, Substitution and Translocation Lines by in situ Hybridization Using Biotin-labelled Genomic DNA as a Probe [J]. Acta Genetic Sinica, 1995, 22(5), P [11] Yang Zujun, Feng Juan, Ren Zhenglong, et al. Identification of Dasypyrum breviaristatum chromatin in wheat background by in situ hybridization [J]. Southwest China Journal of Agricultural Sciences, 2005, 18(5), P