CN100526464C - Crested wheatgrass P genome specific sequence - Google Patents
Crested wheatgrass P genome specific sequence Download PDFInfo
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Abstract
the invention discloses an astopic sequence of crested wheatgrass P gene group, which is characterized by the following: adopting gene group DNA as mould to do PCR augmentation for RAPD primer; obtaining 1036bp nucleotide sequence; or using SCAR primer to do PCR augmentation to obtain 960bp nucleotide sequence; possessing a couple of oligonucleotide with sequence 3 and sequence 4 for SCAR primer; providing new astopic probe and molecular mark for P chromatin rapidly and exactly.
Description
Technical field
The present invention relates to crested wheatgrass P genome specific sequence.
Background technology
Wheat is human staple food crop, occupies critical role in agriculture production.At present, narrow wheat improvement is caused serious hindrance, wheat wild kindred plant is the huge gene pool of genetic improvement of wheat in the cultivated wheat hereditary basis.
There are 25 genus in wheat family, and nearly 400 kinds are occupied critical role in grass.The basic genome of Agropyron (Agropyron) plant is P group (Dong Yuchen, Zheng Diansheng. Chinese wheat genetic resource. Chinese agriculture press, 2000,174-177), be distributed in cool temperature zone plateau, Eurasia and sand ground, not only long-term arid, the abiotic stress that cold climate and soil depletion caused of adapting to, and main diseases such as the Powdery Mildew of wheat, yellow dwart, rust are had very strong resistance, in addition, wheatgrass has high yield plant type structures such as many small ears, many Xiao Hua.Therefore, wheatgrass P genome can be used as multiple excellent foreign gene donor, and is significant for genetic improvement of wheat.
(Li Lihui such as Li Lihui, Li Xiuquan, Li Pei. the establishment of wheat-wheatgrass alien addition line. Acta Genetica Sinica, 1997,24:154-159) breed the wheat-wheatgrass allos disomic addition line of a cover genetic stability in the world first, and obtained the confirmation of molecular cytogenetics.Heredity exchange between wheat ABD group and wheatgrass P group is created in being established as of wheat-wheatgrass addition line, and the excellent transgenosis that P karyomit(e) is carried provides important prerequisite in wheat.By the hybridization of wheat and wheatgrass addition line, obtained the higher wheat of a collection of using value-wheatgrass recombination system, very necessary to the chromatinic Genetic identification of P in the recombination system.
Genomic in situ hybridization (Genomic in situhybridization, method GISH), but this is often more effective for the evaluation of heterochromosome replacement and big fragment transposition are adopted in the heterochromatic evaluation of wheat external source usually.Because chromosome substitution and big fragment transposition may cause chain burden when importing excellent gene, so people more wish to obtain the transposition of heterochromatin small segment.Yet the evaluation difficulty for the transposition of heterochromatin small segment is a stubborn problem always.Along with the development and the application of Protocols in Molecular Biology, utilize genome high abundance distinguished sequence to be probe, further set up identification different genes group and chromosomal specific molecular marker, for new approach has been opened up in heterochromatic evaluation.Wheat-wheatgrass recombination system heterochromatin small segment transposition is carried out Genetic identification fast and accurately, pressed for chromosomal specific probe of P genome and P and molecule marker.
The research of wheat family plant different genes group distinguished sequence receives much attention always, the relation because the heredity that exists between the wheat family different genes group is developed, and genome specific sequence is for other genomes.The ideal distinguished sequence has specificity with respect to other most genomes.At present in the wheat kindred plant, clone the genomic distinguished sequence of R (Mao L from rye, Zhai WX, Hu H.Cloning and characterization of arepetitive sequence in rye (Secale cereal) .Plant Science, 1994,100:51-57; Zhou Jianping, Yang Zujun, Feng Juan. the separation and the evaluation of rye specific DNA tumor-necrosis factor glycoproteins. southwestern agriculture journal .2005,18 (5): 598-602); Clone the genomic distinguished sequence of H (Hueros G from barley, Loarce Y, FerreyE.A structure and evolutionary analysis of a dispersed repetitivesequence.Plant Mol Biol.1993,22:635-643; Anamthawat-Jonsson K, Heslop-harrison JS.Isolation and characterization of genome specific DNAsequences in Trticease species.Mol Gen Genet, 1993,240:151-158), set up the specific molecular marker (Huang Chaofeng of barley chromosome, Zhang Wenjun, the repercussions billows. the screening and the evaluation of barley 6H chromosome specific mark. Acta Genetica Sinica .2000,27 (8): 713-718); From long fringe couchgrass clone the genomic special tumor-necrosis factor glycoproteins of E (Li Hongjie. the separation of long fringe couchgrass (Thinopyrum elongatum) genome specific tumor-necrosis factor glycoproteins. Chinese Academy of Sciences's post-doctoral research work report, 2000); Clone the St genome specific sequence and set up the special PCR mark (Zhang Zengyan of 2Ai-2 karyomit(e) from middle couchgrass, Wang Lili, Xin Zhiyong. the foundation of the new mark of middle couchgrass karyomit(e) 2Ai-2 specific PCR and the clone of St genome specific sequence. Acta Genetica Sinica .2002,29 (7): 565-570); Clone the genomic distinguished sequence of D (Lagudahe S from aegilops tauschii, Appels R, Mcneild.The Nor-D3 of Triticum tauschii:Natural variation andgenetic linicage to markers in chromosome 5.Genome, 1991b, 34:387-395); Clone the genomic distinguished sequence of V (DePace C from cluster hair wheat, Deler V, Scarasciia M G T.Molecular and chromosomal characterization of repeated and single copy DNAsequences in the genome of Dasypyrum villosum.Hereditas, 1992,116:55-65; Li W L, Chen P D, Qi L L.Isolation, characterization and application of aspecies-specific repeated sequence from Haynaldia villosa.Theor Appl Genet, 1995,90:526-533), set up the genomic specific PCR mark (Liu Shoubin of cluster hair wheat V, the sunshine Tang Dynasty, You Mingshan. the foundation and the application of cluster hair wheat genome specificity PCR mark. Acta Genetica Sinica .2003,30 (4): 350-356); Clone the genomic distinguished sequence of C (Baldauf F from the shape of tail goatweed, Schubert V, Metzlaff M.Repeated DNA sequences of Aefilops markgra-fii (Geuter) Hammervar, markgrafii:cloning, sequencing and analysis of distribu-tion inPoaceaae species.Hereditas, 1992,116:71-78; Kong Xiuying, Zhou Ronghua, Dong Yuchen. the clone of shape of tail goatweed C genome specific tumor-necrosis factor glycoproteins. Science Bulletin, 1999a, 44 (8): 828-832); From intend this inferior you take off goatweed and clone the genomic distinguished sequence of S (Daud H M, and Gustafson J P.Molecular evidence for Triticum speltoides as a B-genome progenitor of wheat (Triticum aestivum) .Genome, 1996,39:543-548; Giorgi D, D ' Ovidio R, Tanzarella O A.Isolation and characterization of S genome specific sequencesfrom Aegilops sect.sitopis species.Genome, 2003,46:478-489); From rely grass clone the genomic distinguished sequence of Ns (Sigridur K B, and Kesara A.Isolation, characterization, and analysis of Leymus-specific DNA sequences.Genome, 2003,46:673-682).
Genome specific dna sequence dna and Study on Molecular Marker not only provide theoretical foundation for illustrating the botanical system evolution of wheat family, composition of genome and genome evolution relation, and provide the specific probe and molecule marker (the Orgaard M that can directly utilize for the chromatinic evaluation of wheat external source, Anamthawat-Jonsson K.Genomediscrimination by in situ hybridization in Icelandic species of Elymus andElytrigia (Poaceae:Triticeae) .Genome.2001,44:275-283; SchoenenbergerN, Felber F, Savova-Bianchi D.Introgression of wheat DNA markers from A, B and D genomes in early generation progeny of Aegilops cylindrical Host * Triticum aestivum L.hybrids.Theor Appl Genet.2005,111:1338-1346).
Yet, do not see special report as yet for the research of Agropyron P genome specific sequence and the chromosomal specific molecular marker of identification P.
Summary of the invention
The purpose of this invention is to provide a kind of crested wheatgrass P genome specific sequence.
Crested wheatgrass P genome specific sequence provided by the present invention is that the genomic dna with wheatgrass is a template, carries out the nucleotide sequence of the 1036bp that pcr amplification obtains with the RAPD primer, or carries out the nucleotide sequence of the 960bp that pcr amplification obtains with the SCAR primer; The nucleotide sequence of described RAPD primer is a sequence 1, a pair of oligonucleotide that described SCAR primer is made up of sequence 3 and sequence 4.
In actual applications, the template of described PCR can be the genomic dna of wheatgrass Z559, the genomic dna of wheatgrass Z804 or the genomic dna of wheatgrass Z1750.
Described crested wheatgrass P genome specific sequence, by sequence in the sequence table 2 from 5 ' the 75th sequence or sequence of forming to the 1034th deoxynucleotide in the sequence table 2 of end.
Because Agropyron P karyomit(e) exists heredity to reset phenomenon, bring difficulty for the research of the genomic evolution relation of P, the P genome specific sequence that the present invention obtains will provide new molecular biology foundation for illustrating the genomic evolution relation of wheat family phylogeny of plants and Agropyron plant P.The chromatinic detection of wheat external source P is a key of identifying wheat-wheatgrass recombination system, the a large amount of individual plant evaluation work of filial generation is very numerous and diverse, the P genome specific sequence of the present invention's screening and the P genome SCAR mark that obtains based on this sequence will provide new specific probe and molecule marker for wheat-chromatinic rapid detection of wheatgrass recombination system P and accurate the evaluation.
Description of drawings
Fig. 1 is the pcr amplification result of primer Opx-7 to Agropyron P genomic dna and the ABD of wheat family genomic dna
Fig. 2 is the pcr amplification result of SCAROPX-7 primer to wheatgrass and other different genes groups DNA of wheat family
Embodiment
Following experimental technique is ordinary method if no special instructions.
The other plant of the wheatgrass among the following embodiment, wheat and wheat family is all available from Institute of Crop Science, Chinese Academy of Agricultural Science germ plasm resource research centre.
The acquisition of embodiment 1, P genome specific sequence
Utilize RAPD primer Opx-7:5 '-GAGCGAGGCT-3 ' (sequence 1), Agropyron P genomic dna and the ABD of wheat family genomic dna are screened, separate and clone P genome specific amplified fragments.Wherein, the 20ul system of pcr amplification: 10 * PCRbuffer (contains 20mMol/L Mg
2+) 2ul
dNTP(10mM) 0.3ul
Primer (8uM) 0.7ul
Taq enzyme (2u/ul) 0.5ul
Template DNA (50ng/ul) 1ul
ddH
2O 15.5ul
PCR program: 94 ℃ of abundant sex change of 4min earlier; 94 ℃ of 30S sex change again, 37 ℃ of 45S renaturation, 72 ℃ of 1min extend, 45 circulations; Last 72 ℃ are extended 10min.4 ℃ of preservations.
1.2% sepharose, 100V constant voltage electrophoresis, ultraviolet gel imaging.
The pcr amplification result amplifies the special band that the ABD of wheat family genome does not have as shown in Figure 1 in diploid, tetraploid and hexaploid wheatgrass P genome, this stripe size is 1036bp.Among Fig. 1, L is 100bp DNA Ladder; P2, diploid wheatgrass Z804 (PP); P4, tetraploid wheatgrass Z559 (PPPP); P6, hexaploid wheatgrass Z1750 (PPPPPP); CS, common wheat China spring (AABBDD); Fu, common wheat Fukuho (AABBDD); A, einkorn wheat MO4 (AA); AB, durum wheat DR4 (AABB);-, blank (pure water); Arrow is depicted as P genome specific amplified band.
Cut the P genome specific DNA band of diploid wheatgrass Z804, tetraploid wheatgrass Z559 and hexaploid wheatgrass Z1750, and reclaim test kit Agarose Gel DNA Purification (precious biotechnology Dalian company limited) with DNA target DNA is reclaimed.Specific fragment is after reclaiming, use T-support agent box (precious biotechnology Dalian company limited) to connect through pMD-18Vector, connecting product is converted in the F-strain e. coli jm109, through blue hickie screening, the picking white colony extracts plasmid DNA, cut evaluation through pcr amplification and enzyme, determine to insert the clip size clone consistent check order (checking order) by Beijing an ancient unit of weight Yao great achievement biotech company with former recovery fragment.Sequencing result shows that the specific fragment length that increases is 1036bp from tetraploid wheatgrass Z559, have the nucleotide sequence of sequence 2 in the sequence table; The specific fragment length that increases from diploid wheatgrass Z804 is 1036bp, has the nucleotide sequence of sequence 2 in the sequence table; The specific fragment length that increases from hexaploid wheatgrass Z1750 is 1036bp, has the nucleotide sequence of sequence 2 in the sequence table.
Embodiment 2, crested wheatgrass P genome specific sequence checking
According to the sequencing result of P genome specific amplified fragments, optimize through the DNASTAR design software, design a pair of SCAROPX-7 primer: SCAROPX-7F:5 '-GCTACCATTCCAACTTCTACG-3 ' (sequence 3), corresponding to the 75-95nt of sequence 2; SCAROPX-7R:5 '-GCGAGGCTATGTTGGTTTTGA-3 ' (sequence 4) is with the 1014-1034nt reverse complemental of sequence 2.
With the SCAROPX-7 primer different genes group DNA of wheat family is increased (62 ℃ of annealing temperatures), amplification and deposition condition are with embodiment 1.The result shows, at diploid wheatgrass Z804, the length that all increases among tetraploid wheatgrass Z559 and the hexaploid wheatgrass Z1750 is the specific fragment (SCAR mark) of 960bp, and at wheat ABD genome and most wheat other genomes (C of family, D, E, AG, I, R.S.St, U, V, W does not obtain the specific fragment of this 960bp in Y), illustrates that the sequence that increases from the genomic dna of wheatgrass with the SCAROPX-7 primer is to wheat ABD genome and most wheat other genomes (C of family, D, E, AG, I, R.S.St, U, V, W, Y) performance specificity (Fig. 2) is a crested wheatgrass P genome specific sequence.Among Fig. 2, L is 100bp DNA Ladder; P2 is diploid wheatgrass Z804 (PP); P4 is tetraploid wheatgrass Z559 (PPPP); P6 is hexaploid wheatgrass Z1750 (PPPPPP); CS is common wheat China spring (AABBDD); Fu is common wheat Fukuho (AABBDD); A is einkorn wheat MO4 (AA); AB is durum wheat DR4 (AABB); C is shape of tail goatweed Ae14 (CC); D is aegilops tauschii Y93 (DD); E is a long fringe couchgrass (EE); AG is timopheevi wheat TI
1(AAGG); H is purple Herba Hordei Vulgaris Z2826 (HH); I is purple six ribs cultivation barley (II); M is tip awn goatweed Y258 (MM); Ns is new wheat straw R429 (NsNs); R is rye Z1383 (RR); S takes off goatweed Ae49 (SS) for intending this inferior that; St is false roegneria kamoji (StSt); U is umbellule goatweed Y39 (UU); V is cluster hair wheat Z1731 (VV); W is Australia wheat straw (WW); Y is middle roegneria kamoji (StStYY);-be blank (pure water).
Above-mentioned SCAR mark is checked order.Sequencing result shows that the specific fragment length that increases is 960bp from tetraploid wheatgrass Z559, by sequence in the sequence table 2 from 5 ' the 75th to the 1034th sequence that deoxynucleotide is formed of end; The specific fragment length that increases from diploid wheatgrass Z804 is 960bp, by sequence in the sequence table 2 from 5 ' the 75th to the 1034th sequence that deoxynucleotide is formed of end; The specific fragment length that increases from hexaploid wheatgrass Z1750 is 960bp, by sequence in the sequence table 2 from 5 ' the 75th to the 1034th sequence that deoxynucleotide is formed of end.
Sequence table
<160>4
<210>1
<211>10
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
<210>2
<211>1036
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
Claims (2)
1, a kind of crested wheatgrass P genome specific sequence, by sequence in the sequence table 2 from 5 ' the 75th to the 1034th sequence that deoxynucleotide is formed of end.
2, a kind of crested wheatgrass P genome specific sequence, its deoxynucleoside acid sequence is shown in the sequence in the sequence table 2.
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Families Citing this family (7)
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| CN103243164A (en) * | 2013-05-06 | 2013-08-14 | 西北农林科技大学 | Method for identifying psathyrostachys huashanica allogenic material in wheat genome |
| CN105441427B (en) * | 2014-08-07 | 2017-04-05 | 中国农业科学院作物科学研究所 | A kind of special tandem repetitive sequence in wheatgrass chromosome end and its application |
| CN105331605B (en) * | 2014-08-07 | 2017-07-25 | 中国农业科学院作物科学研究所 | A kind of Gypsy retrotransponsonses of P genome specifics and its application |
| CN105441428B (en) * | 2014-08-07 | 2017-07-21 | 中国农业科学院作物科学研究所 | A kind of special Gypsy retrotransponsonses sequence in wheatgrass centromere and its application |
| CN105368821B (en) * | 2014-08-07 | 2017-07-21 | 中国农业科学院作物科学研究所 | A kind of special Copia retrotransponsonses of nearly centric region of P genomes and its application |
| CN110055317B (en) * | 2019-04-29 | 2022-04-26 | 中国农业科学院作物科学研究所 | Method for discovering exogenous functional candidate genes in wheat distant hybridization progeny small-fragment translocation line |
| CN112280889A (en) * | 2020-11-10 | 2021-01-29 | 中国农业科学院作物科学研究所 | Specific oligonucleotide probe for wheatgrass chromosome and application thereof |
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| SU1450786A1 (en) * | 1986-05-08 | 1989-01-15 | Всесоюзный Научно-Исследовательский Институт Прикладной Молекулярной Биологии И Генетики | Method of determining perennial life of hybrid cereal crops based on agropyron |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| SU1450786A1 (en) * | 1986-05-08 | 1989-01-15 | Всесоюзный Научно-Исследовательский Институт Прикладной Молекулярной Биологии И Генетики | Method of determining perennial life of hybrid cereal crops based on agropyron |
Non-Patent Citations (4)
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