CN102943073A - Molecular markers having haynaldia villosa 6VS chromosome specificity and use thereof - Google Patents

Molecular markers having haynaldia villosa 6VS chromosome specificity and use thereof Download PDF

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Publication number
CN102943073A
CN102943073A CN2012102947149A CN201210294714A CN102943073A CN 102943073 A CN102943073 A CN 102943073A CN 2012102947149 A CN2012102947149 A CN 2012102947149A CN 201210294714 A CN201210294714 A CN 201210294714A CN 102943073 A CN102943073 A CN 102943073A
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wheat
chromosome
haynaldia villosa
molecular markers
t6al
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何华纲
别同德
朱姗颖
胡向均
李晶晶
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Jiangsu University
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Jiangsu University
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Abstract

The invention discloses molecular markers having haynaldia villosa 6VS chromosome specificity and a use thereof, and relates to the technical field of molecular biology and genetic breeding science. The molecular markers can specifically track a haynaldia villosa 6VS chromosome. The molecular markers as primers are designed according to an EST sequence BE585684 of a short arm of a wheat sixth-part homologous group and have sequences of 6VS-SSDF: 5'-CCAACTCTAGCTGACCGCAGACTA-3' and 6VS-SSDR: 5'-ATTCCATGGTGTAATAGCTCCAACTAC-3'. Through PCR, the molecular markers are amplified into a specific DNA band having the length of 350bp. The molecular markers can fast and effectively track a haynaldia villosa 6VS chromosome and wheat powdery mildew resistance controlled by the haynaldia villosa 6VS chromosome, and has strong practical values in wheat powdery mildew-resistant molecular marker-assistant selective breeding of wheat.

Description

Molecule marker 6VS-SSD that cluster hair wheat 6VS karyomit(e) is special and uses thereof
Technical field
The present invention relates to molecular biology and genetic breeding science and technology field, be specifically related to chromosomal molecule marker of special tracking cluster hair wheat 6VS and using method thereof.
Background technology
Wheat is one of global Three major grain crops, also is the food crop of China's gross output first, is one of important determinative that ensures China's grain security.Wheat often can be subject to the impact of biology or abiotic stress in its growing process.Wheat powdery mildew is a kind of worldwide wheat diseases, improves along with China's farm irrigation facility in recent years and the water and fertilizer condition raising, and its harm increases the weight of year by year.The wheat-haynaldia villosa T6AL.6VS translocation line of breeding by distant hybirdization and chromosome engineering technique means all shows high resistance or immunity, its mildew-resistance gene to the Powdery Mildew physiological strain of having found at present Pm21Being positioned on the cluster hair wheat 6V the short arm of a chromosome (6VS), is present, gene that resistance is the strongest the widest to the anti-spectrum of wheat powdery mildew.The T6AL.6VS translocation line is extensively utilized in the wheat anti-powdery mildew breeding of new variety in recent years, has bred at present more than 20 powdery-mildew-resistance wheat new variety, and popularizing area is above 5,000 ten thousand mu.
The T6AL.6VS translocation line is to have replaced the chromosomal galianconism of common wheat 6A (6AS) by cluster hair wheat 6V the short arm of a chromosome (6VS) in common wheat genetic background, thereby forms the T6AL.6VS translocation chromosome.Because cluster hair wheat and common wheat sibship are far away, cause the two that Chromosome recombination does not occur, the T6AL.6VS translocation chromosome appears among the offspring with the form of transposition all the time.At present, find to utilize the change that occurs the chromosome translocation form in new variety that the T6AL.6VS translocation line cultivates or the new lines.Therefore, the proterties of cluster hair wheat 6VS control and the dna sequence dna that is loaded with thereof all appear among the offspring in the collaborative mode of transmitting.
At present, part Study has been announced part and the upper anti-powdery mildew gene Pm 21 linked related molecular marker OPT17 of 6VS 1400, OPT17 1900, SCAR 1400, SCAR 1265, NAU/XiBao15 902And NAU/XiBao16 1586But in general, these are marked at and all have unsettled shortcoming in the marker assisted selection breeding, and a reason is determined by labeling pattern, such as RAPD mark OPT17 1400And OPT17 1900Next is that the PCR reproducibility of results is poor to be caused because flag sequence long (mostly approach or surpass 1000bp), PCR reaction system and reaction conditions require harshly.These shortcomings all are unfavorable for the application of marker assisted selection technology in the wheat breeding practice.
EST(expressed sequence tag, expressed sequence tag) refers to random choose clone from the cDNA library of plant different tissue sources, to the check order short cDNA sequence of acquisition of its 5 ' and 3 ' end.EST-STS(sequence-tagged site, STS) mark is that direct basis has been positioned the est sequence design primer on the Physical map, genome or cDNA are increased, therefrom seek chain with goal gene or have the EST mark of chromosome specific.Because the EST-STS mark is directed to the expressing gene sequence, conservative property is higher, is particularly conducive to the difference that defines between the species.Therefore, the genomics principle is utilized common wheat EST collection of illustrative plates based on the comparison, can develop equally the specific chromosomal specific molecular marker of common wheat affinity species cluster hair wheat.
The present invention just is based under the above-mentioned thought guidance, utilize the comparative genomics principle, design take the EST-STS mark of round pcr as the basis according to the est sequence on the 6th homoeologous group galianconism on the common wheat bin-map collection of illustrative plates, screening cluster hair wheat 6V the short arm of a chromosome (6VS) specific molecular marker, the mildew-resistance proterties that is used for rapid detection and tracking common wheat genetic background T6AL.6VS translocation chromosome and control thereof is for the breeding of wheat anti-powdery mildew molecular marker assisted selection provides novel, practical, efficient molecule marker.
Summary of the invention
The purpose of this invention is to provide chromosomal molecule marker of a kind of special tracking cluster hair wheat 6VS and using method thereof, it utilizes the comparative genomics principle, est sequence according to the 6th homoeologous group galianconism on the common wheat bin-map collection of illustrative plates, design is take the EST-STS labeled primer of round pcr as the basis, screening cluster hair wheat 6VS chromosome specific molecule marker is for the powdery-mildew-resistance wheat-application of cluster hair wheat T6AL.6VS translocation line in the wheat anti-powdery mildew rearing new variety provides a kind of novel, practical, efficient Molecular Marker Assisted Selection Technology.
In order to solve the existing problem of background technology, the present invention is by the following technical solutions: it is that design obtains according to the est sequence BE585684 of common wheat bin-Map collection of illustrative plates the 6th homoeologous group galianconism, the DNA band of one section about 350bp that labeled primer amplifies is positioned at cluster hair wheat 6VS karyomit(e), can follow the trail of specifically the resist powdery mildew of wheat proterties of 6VS karyomit(e) and control thereof, the labeled primer sequence is:
6VS-SSDF：5’-CCAACTCTAGCTGACCGCAGACTA-3’ （SEQ ID NO.1），
6VS-SSDR：5’-ATTCCATGGTGTAATAGCTCCAACTAC-3’ （SEQ ID NO.2）。
The invention also discloses the purposes of the special molecule marker 6VS-SSD of described cluster hair wheat 6VS karyomit(e) in following the trail of wheat-haynaldia villosa T6AL.6VS translocation chromosome.
Follow the trail of the method for wheat-haynaldia villosa T6AL.6VS transposition dyeing with the special molecule marker 6VS-SSD of described cluster hair wheat 6VS karyomit(e), with this molecule marker primer detected materials to be carried out pcr amplification, amplify the material of 350bp band, for containing the material of wheat-haynaldia villosa T6AL.6VS translocation chromosome.
The detailed directions of molecule marker of the present invention is:
(1) PCR reaction system: contain about 10-20ng template DNA in the 10 μ L reaction systems, 1 * PCR buffer, 1.5mmol L -1MgCl 2, 200mmol L -1DNTP, two primer final concentrations respectively are 0.2 μ mol L -1, 0.5U TaqArchaeal dna polymerase is with sterile distilled water postreaction system to 10 μ L;
(2) PCR response procedures: 94 ℃ of denaturations 3 minutes; 94 ℃ of sex change 20 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 32 circulations 30 seconds; 72 ℃ were extended 5 minutes; 4 ℃ of preservations;
(3) the PCR product detects: pcr amplification product is electrophoretic separation on 8% non-denaturing polyacrylamide gel, dyes with argentation.
The present invention is parent when carrying out the tracking of mildew-resistance proterties utilizing the T6AL.6VS transposition, and every material that amplifies the 350bp band all contains wheat-haynaldia villosa T6AL.6VS translocation chromosome, and to Powdery Mildew performance high resistance; Every material that can not amplify the 350bp band does not have the T6AL.6VS translocation chromosome, corresponding powder mildew resistance of losing by the control of 6VS karyomit(e).
The present invention has following beneficial effect:
1, molecule marker 6VS-SSD of the present invention high specificity when identifying cluster hair wheat 6VS karyomit(e): the molecule marker OPT17 that has developed 1400And OPT17 1900Non-6VS specific markers all when only having the polymorphism with 6VS between the parent, just can utilize, and when using 6VS-SSD, no matter relates to which kind of parent, as long as the 350bp band occurs, just can determine to contain in the material 6VS karyomit(e).
2,6VS chromosome specific EST-STS mark of the present invention is compared the SCAR mark and is easier to identify " false negative " that pcr amplification unsuccessfully causes: the codominant marker of being labeled as of the present invention, in common wheat genetic background, no matter whether has the T6AL.6VS translocation chromosome, can amplify two background bands that are about 500bp, can be used for judging whether pcr amplification is successful, gets rid of " false negative ".
3, molecule marker good stability of the present invention, be the breeding practice utilization easily: mark of the present invention is because target stripe is shorter, 350bp only, less demanding to the dna profiling quality during pcr amplification, in the dna profiling of Partial digestion, still can effectively amplify target product, and the extension time of PCR reaction only need 30 seconds, be conducive to improve the efficient of marker assisted selection.
Description of drawings
Fig. 1: the pcr amplification result of molecule marker 6VS-SSD.M:DNA marker DL2000; 1: cluster hair wheat; 2: common wheat; 3: wheat-haynaldia villosa translocation line T6AL.6VS; 4 ~ 12: the segregating population of peaceful wheat No. 9 and the cross combination of T6AL.6VS translocation line.Arrow is depicted as the specific band of 350bp.
Embodiment
The specific embodiment of the invention is by the following technical solutions: the est sequence BE585684 according to common wheat bin-Map collection of illustrative plates the 6th homoeologous group designs a pair of labeled primer, utilize the DNA band of one section 350bp that round pcr amplifies, this band can be positioned at cluster hair wheat 6VS karyomit(e), can follow the trail of specifically the resist powdery mildew of wheat proterties of 6VS karyomit(e) and control thereof.
Embodiment:
1, the design of primer is with synthetic
Information according to the website http://wheat.pw.usda.gov of U.S. farming section, search is positioned the est sequence of common wheat the 6th homoeologous group, utilize Primer Premier 5.0 softwares to carry out design of primers, primer is synthetic by Shanghai Jierui Biology Engineering Co., Ltd.
2, the screening of cluster hair wheat 6VS chromosome specific EST-STS mark
(1) pcr amplification: (be public germplasm materials take cluster hair wheat, common wheat, wheat-haynaldia villosa translocation line T6AL.6VS as material, provided by cytogenetics institute of Agricultural University Of Nanjing), extract the laggard performing PCR amplification of genomic dna, contain about 10-20ng template DNA in the 10 μ L PCR reaction systems, 1 * PCR buffer, 1.5mmol L -1MgCl2,200mmol L -1DNTP, two primer final concentrations respectively are 0.2 μ mol L -1, 0.5U Taq archaeal dna polymerase is with sterile distilled water postreaction system to 10 μ L; The PCR response procedures is: 94 ℃ of denaturations 3 minutes; 94 ℃ of sex change 20 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 32 circulations 30 seconds; 72 ℃ were extended 5 minutes; 4 ℃ of preservations.
(2) detection of PCR product: pcr amplification product carries out electrophoretic separation at 8% non-denaturing polyacrylamide gel (29:1), adopts argentation dyeing.
(3) PCR interpretation of result: the present invention screens a molecule marker 6VS-SSD that cluster hair wheat 6VS karyomit(e) is special, and labeled primer is that concrete sequence is according to the est sequence BE585684 design that is positioned wheat the 6th homoeologous group galianconism:
6VS-SSDF：5’-CCAACTCTAGCTGACCGCAGACTA-3’ （SEQ ID NO.1），
6VS-SSDR：5’-ATTCCATGGTGTAATAGCTCCAACTAC-3’ （SEQ ID NO.2）。
Carry out pcr amplification with this labeled primer, can in common wheat, increase two and be about the DNA band of 500bp, amplification is to a DNA band that is about 350bp in cluster hair wheat, and in wheat-haynaldia villosa translocation line T6AL.6VS, two treaty 500bp bands can increase, a treaty 350bp band (see among Fig. 11 ~ 3) also can increase, show that the 350bp band can be positioned cluster hair wheat 6VS karyomit(e), therefore, molecule marker 6VS-SSD can specificity follow the trail of cluster hair wheat 6VS karyomit(e) in Wheat Background.
3, the application of cluster hair wheat 6VS chromosome specific mark 6VS-SSD in the molecular marker assisted selection breeding
The cluster hair wheat 6VS chromosome specific labeled primer 6VS-SSD that utilization screens, (public to peaceful wheat No. 9, cultivated by the Jiangsu Province Agriculture Science Institute) (public with mildew-resistance T6AL.6VS translocation line, by cytogenetics institute of Agricultural University Of Nanjing initiative and provide) segregating population of cross combination identifies, identify in conjunction with powder mildew resistance, the result is presented at all in the examination material, and all materials with 350bp band all show high mildew-resistance; The material of all disappearances 350bp band all shows high sense Powdery Mildew (see among Fig. 14 ~ 12).This result shows that the EST-STS mark 6VS-SSD of the present invention's exploitation can effectively follow the trail of the cluster hair wheat 6VS karyomit(e) in the Wheat Background, has potential using value in the molecular marker assisted selection breeding.
SEQUENCE LISTING
＜110〉Jiangsu University
＜120〉special molecule marker 6VS-SSD of cluster hair wheat 6VS karyomit(e) and uses thereof
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 1
ccaactctag ctgaccgcag acta 24
<210> 2
<211> 27
<212> DNA
＜213〉artificial sequence
<400> 2
attccatggt gtaatagctc caactac 27

Claims (3)

1. molecule marker 6VS-SSD that cluster hair wheat 6VS karyomit(e) is special, it is characterized in that: the sequence of this molecule marker primer is:
6VS-SSDF：5’-CCAACTCTAGCTGACCGCAGACTA-3’，
6VS-SSDR：5’-ATTCCATGGTGTAATAGCTCCAACTAC-3’。
2. the purposes of the special molecule marker 6VS-SSD of cluster hair wheat 6VS karyomit(e) claimed in claim 1 in following the trail of wheat-haynaldia villosa T6AL.6VS translocation chromosome.
3. method of following the trail of wheat-haynaldia villosa T6AL.6VS transposition dyeing with the special molecule marker 6VS-SSD of cluster hair wheat 6VS karyomit(e) claimed in claim 1, with this molecule marker primer detected materials to be carried out pcr amplification, amplify the material of 350bp band, for containing the material of wheat-haynaldia villosa T6AL.6VS translocation chromosome.
CN2012102947149A 2012-08-20 2012-08-20 Molecular markers having haynaldia villosa 6VS chromosome specificity and use thereof Pending CN102943073A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104278028A (en) * 2014-09-16 2015-01-14 天津师范大学 Sequence of dasypyrum villosum 6VS DNA permeating into powdery mildew resistant near-isogenic line of wheat and application
CN104877996A (en) * 2015-05-12 2015-09-02 江苏大学 Haynaldia villosa's 6VS chromosome specific molecular marker 6VS-BH1 and application thereof
CN112301142A (en) * 2019-07-30 2021-02-02 山东省农业科学院作物研究所 Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof
CN113528695A (en) * 2021-06-23 2021-10-22 淮阴师范学院 Haynaldia villosa 2VL chromosome specific RFLP-STS molecular marker and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAO等: "A sequence-specific PCR marker linked with pm21 distinguishes chromosomes 6AS,6BS,6DS of triticum aestivum and 6VS of haynaldia villosa", 《PLANT BREEDING》 *
王振英等: "簇毛麦6VS 上4 个新分子标记的鉴定及与抗白粉病基因Pm21 的连锁", 《作物学报》 *
陈升位等: "簇毛麦6V染色体短臂特异性EST 标记的开发及缺失定位", 《麦类作物学报》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104278028A (en) * 2014-09-16 2015-01-14 天津师范大学 Sequence of dasypyrum villosum 6VS DNA permeating into powdery mildew resistant near-isogenic line of wheat and application
CN104877996A (en) * 2015-05-12 2015-09-02 江苏大学 Haynaldia villosa's 6VS chromosome specific molecular marker 6VS-BH1 and application thereof
CN104877996B (en) * 2015-05-12 2019-05-31 江苏大学 Haynaldia villosa 6VS chromosome-specific molecule marks 6VS-BH1 and application thereof
CN112301142A (en) * 2019-07-30 2021-02-02 山东省农业科学院作物研究所 Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof
CN112301142B (en) * 2019-07-30 2021-11-02 山东省农业科学院作物研究所 Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof
CN113528695A (en) * 2021-06-23 2021-10-22 淮阴师范学院 Haynaldia villosa 2VL chromosome specific RFLP-STS molecular marker and application thereof

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Application publication date: 20130227