CN1322133C

CN1322133C - Wheat anti-powdery mildew gene Pm21 linked codorminant PCR marker and its usage

Info

Publication number: CN1322133C
Application number: CNB2005100409917A
Authority: CN
Inventors: 陈雅平; 陈佩度; 王秀娥; 张守忠; 王苏玲; 周波
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2005-07-12
Filing date: 2005-07-12
Publication date: 2007-06-20
Anticipated expiration: 2025-07-12
Also published as: CN1737151A

Abstract

The present invention discloses a mark linked with a wheat anti-powdery mildew gene Pm21, which can be used for the molecular marker-assisted selection of the disease resistance and the breeding of wheat powdery mildew and can be simultaneously used for identifying the purity of common wheat seeds. According to sequence designing primers NAU/XiBao16F and NAU/XiBao16R of resistant gene analogs Ta-LRR2, which are obtained by the inhibiting subtractive hybridization of a Yangmai number 5 and a translocation line, a mark NAU/XiBao16 is linked with an anti-powdery mildew gene Pm21 of a translocation line 6VS/6AL by a series of material verification, and two specific belts can be simultaneously amplified from chromosomes of common wheat 6AS and of protoplast wheat 6VS. The mark NAU/XiBao16 can be used for the molecular marker-assisted selection of the disease resistance and the breeding of wheat powdery mildew and can be also used for identifying the purity of common wheat seeds, particularly identifying materials created by cell engineering and chromosome engineering, which relates to the variation of a sixth isogenous group chromosome 6AS.

Description

One with wheat anti-powdery mildew gene Pm 21 linked codominance PCR mark and using method thereof

One, technical field

The invention discloses and wheat anti-powdery mildew gene Pm 21 linked mark, can be used for wheat powdery mildew breeding for disease resistance molecular marker assisted selection; This mark can be used to differentiate the common wheat seed purity simultaneously, especially is used for the material that common wheat the 6th homology group karyomit(e) 6AS morphs that relates to that identification of cell engineering and chromosome engineering create.

Two, technical background

Wheat (T.aestivum L) has critical role as global important crops in agriculture production, but it is produced and but to be subjected to a lot of biologies and to coerce influence with abiotic stress.The wheat powdery mildew (Ergsiphe greminis f.sp.tritici) that is caused by obligatory parasitism fungi wheat powdery mildew (Erysiphegraminis DC) is China and endangers one of disease that is on the rise in many national Wheat Production in the world, and, the trend that constantly increases the weight of is arranged in recent years along with the improvement of fertilizer and water condition.

Common wheat (T.aestivum L) has 21 pairs of karyomit(e)s, every pair comprises two the same karyomit(e)s, these 21 karyomit(e)s are typically expressed as 1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 3D, 4A, 4B, 4D, 5A, 5B, 5D, 6A, 6B, 6D, 7A, 7B, 7D, and common wheat is directly represented (Fig. 8) with AABBDD usually.Cluster hair wheat (Haynaldia villosa L) has 7 pairs of karyomit(e)s, and every pair comprises two the same karyomit(e)s, and these 7 karyomit(e)s are respectively 1V, 2V, 3V, 4V, 5V, 6V, 7V, and cluster hair wheat is represented (Fig. 9) with VV usually.Every karyomit(e) of common wheat and cluster hair wheat comprises long-armed and galianconism again, represents with L and S respectively, comprises long-armed 6VL and galianconism 6VS such as cluster hair wheat 6V karyomit(e).

The original wheat breed major part of China all contains the anti-source of Pm8, but along with the continuous variation of powdery mildew microspecies, Pm8 has lost resistance gradually, seeks and utilizes new mildew-resistance ospc gene significant for the security of China's Wheat Production.Agricultural University Of Nanjing's cytogenetics just be devoted to the excellent gene the cluster hair wheat (Haynaldia villosa L) is changed over to common wheat by the chromosome engineering technology from the beginning of the eighties in last century, what obtained high mildew-resistance relates to cluster hair wheat 6V chromosome translocation system and adds disappearance system that (document sees reference: 1, Chen P D, Qi L L, Zhou B, et al.Development and molecular cytogenetic analysis ofwheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew.TheorAppl Genet, 1995,91:1125-1128.2, L.L.Qi, S.L.Wang, P.D.Chen, D.J.Liu, B.S.Gill 1998Identification and physical mapping of three Haynaldia villosa chromosome-6V deletion lines TAG97:1042-1046).Wherein wheat-haynaldia villosa translocation line 6VS/6AL replaces into cluster hair wheat 6V the short arm of a chromosome 6VS to common wheat 6A the short arm of a chromosome 6AS exactly, forms a translocation chromosome, and this karyomit(e) is made up of 6AL and 6VS, represents this translocation chromosome with 6VS/6AL.The wheat-haynaldia villosa translocation line 6VS/6AL that isozygotys compares with common wheat, and two 6A karyomit(e)s have become two 6VS/6AL karyomit(e)s (Figure 11); HeterozygosisWheat-haynaldia villosa translocation line 6VS/6AL compares with common wheat, has only a 6A karyomit(e) to become 6VS/6AL karyomit(e), also preserves a 6A (6AS/6AL) karyomit(e) (Figure 12).Further research through molecular cytogenetics, powdery mildew resistance gene Pm 21 in the cluster hair wheat is located in the cluster hair wheat the short arm of a chromosome 6VSFL0.58 section that (document sees reference: L.L.Qi, S.L.Wang, P.D.Chen, D.J.Liu, B.S.Gill 1998 Identification and physical mapping ofthree Haynaldia villosa chromosome-6V deletion lines TAG 97:1042-1046).Because 6VS karyomit(e) and common wheat karyomit(e) parent source relation are distant, and be difficult with common wheat karyomit(e) generation exchange, also finds the phenomenon with common wheat karyomit(e) generation exchange at present.All carry 6VS karyomit(e) so carry the material of Pm21 gene at present.Pm21 is the widest gene of the strongest to powder mildew resistance so far, anti-spectrum, fungus strain or microspecies and 120 European physiological strains that can resist existing all the white powder germs of China, in order in wheat breeding, to utilize this disease-resistant gene resource better, are task of top priority by the improved seeds that gene pyramiding or genetically modified mode import different economical characters with it.But in same colony, detect a plurality of polymeric genes simultaneously, traditional conventional breeding method is difficult to competent, therefore find with the closely linked molecule marker of target gene and just become extremely important, and molecule marker also is absolutely necessary for the clone of this gene and conversion.

Found the molecule marker of some and mildew-resistance Pm21 gene-correlation in the wheat: (document sees reference: Qi LL for RAPD and SCAR, Cao MS, Chen PD, Li WL, Liu DJ (1996) .Identification, mapping, and appli-cation ofpolymorphic DNA associated with resistance gene Pm21 of wheat.Genome 39,191-197Liu ZY, Sun QX, Ni ZF et al (1999) .Development of SCAR markers linked with Pm21 gene conferringresistance to the powdery mildew in common wheat.Plant Breeding 118,215-219).The RAPD mark is convenient but very unstable in these marks, though the SCAR mark convenient and stable but be a dominant marker, can not distinguish this karyomit(e) of 6AS/6VS and be in and isozygoty or heterozygous state.

When utilizing the Pm21 gene to carry out the mildew-resistance breeding, the parent that breeding units is used is the wheat-haynaldia villosa translocation line (6VS/6AL) that Agricultural University Of Nanjing's cytogenetics is provided mostly, there have been several kinds to pass through variety certification, also had a lot of strains to be in the authorization process.Because 6VS karyomit(e) and 6AS karyomit(e) do not exchange, and all carry 6VS/6AL karyomit(e) so carry the plant of Pm21 gene in the seed.No matter in the breed breeding process or kind in the production use, it is all very important that this karyomit(e) of 6VS/6AL is in homozygotic state, otherwise offspring's powder mildew resistance will be separated, and this utilizes this material to carry out the target of breeding as the parent exactly.In addition, its purity will obtain guaranteeing when kind was used on producing, for the high powdery-mildew-resistance wheat kind of utilizing wheat-haynaldia villosa translocation line (6VS/6AL) to come out as parent's seed selection, the ratio that the seed that can utilize 6VS/6AL karyomit(e) to be in homozygotic state accounts for is as an important indicator of seed purity.But, utilize SCAR ₁₄₀₀And SCAR ₁₂₆₅When carrying out molecular marker assisted selection or identification of seed purity, can only whether contain 6VS/6AL karyomit(e) in the clear and definite material, and this karyomit(e) is to be in to isozygoty or heterozygous state can't be learnt.So, seek a kind of not only to specialization of 6VS but also can distinguish it and isozygoty or the mark of heterozygous state, thereby determine Pm21 gene pure or heterozygous state, not only can improve molecular marker assisted selection efficient, and when detecting seed purity, also can bring into play very big effect.

The mark that can differentiate the 6th crowd of karyomit(e) 6AS and 6VS simultaneously in a PCR reaction of PCR-based is attempted to seek in this laboratory in the process of clone Pm21 gene, be used for differentiating the common wheat seed purity.

Three, summary of the invention

Technical problem the object of the present invention is to provide the molecule marker chain with Pm21 of PCR-based, and in a PCR reaction, differentiate common wheat the 6th homology group karyomit(e) 6AS in specialization ground simultaneously by the banding pattern that amplifies with this primer, thereby determine isozygotying or heterozygous state of Pm21 gene, this primer can also be used for identifying the common wheat seed purity.This mark not only can be used for improving molecular marker assisted selection efficient, and is detecting seed purity and identifying and also can bring into play very big effect when relating to the material that the 6th homology group karyomit(e) 6AS morphs.

Technical scheme

The present invention and Pm21 are chain and can differentiate and the mark of common wheat seed purity it is characterized in that this primer is that stencil design obtains with disease-resistant gene analogue Ta-LRR2 sequence.Its sequence is: NAU/XiBao16F:5 '-GTAGACAGTGCCGCTATTTC-3 ' and NAU/XiBao16R:5 '-CAACTCATGTGTCCAGTTCA-3 '.

The fragment of the 1586bp that this mark amplifies from cluster hair wheat is positioned at 6VS karyomit(e) apart from kinetochore FL0.58 section, and the special band of 1882bp that amplifies from common wheat is positioned at the last distal section apart from kinetochore FL0.34 of 6AS.

The using method of above-mentioned mark:

(1) with material DNA to be identified as template, carry out PCR with NAU/XiBao16F and NAU/XiBao16R as primer, the PCR reagent set becomes: dna profiling 2 μ L, 2.5 μ L 10 * PCR buffer, 1.8 μ LMgCl2,2 μ LdNTP, each 0.5 μ L of left and right sides primer, 0.25 μ L Taq DNA polymerase, 16 μ L d.d H ₂O;

The PCR program is: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change 30 seconds, 56 ℃ of annealing 40 seconds, 72 ℃ were extended 32 circulations 2 minutes; 72 ℃ were extended 10 minutes; 4 ℃ of preservations, the PCR product is electrophoretic separation on 39: 1 glue of the polyacrylamide of non-sex change, dyes with argentation.

(2) mark identifies that the result of Pm21 gene is: the individual plant that can amplify the special band of 1586bp fragment carries the Pm21 gene, and its Pm21 gene is positioned on the cluster hair wheat 6VS karyomit(e); What can not amplify the special band of 902bp fragment does not then carry the Pm21 gene.

(3) mark identifies that the disease-resistant karyomit(e) 6VS/6AL of individual plant isozygotys or the result of heterozygous state is:

Can amplify the plant of 1507bp, 1886bp and 1586bp three bands, its disease-resistant karyomit(e) 6VS/6AL is in heterozygous state: the 1507bp band comes from common wheat BBDD genome, and these karyomit(e)s all are paired existence; The band of 1886bp is from karyomit(e) 6AS, and the band of 1586bp shows to have karyomit(e) 6AS/6AL and disease-resistant karyomit(e) 6VS/6AL in this individual plant simultaneously that disease-resistant karyomit(e) 6VS/6AL can not exist in pairs, is in heterozygous state from 6VS.

The disease-resistant karyomit(e) 6VS/6AL that can amplify the plant of 1507bp and 1586bp two bands is in homozygotic state: the band amplification from the chromosomal 1886bp of 6AS is not come out, 6AS karyomit(e) does not just exist, so 6VS/6AL karyomit(e) exists in pairs, is in homozygotic state.

This marker detection is carried the method for Pm21 gene seed purity: random choose single seed from seed to be detected at first, utilize mark to identify that single seeded 6VS/6AL karyomit(e) isozygotys or heterozygous state, add up seed proportion in detecting the seed total amount that 6VS/6AL karyomit(e) is in homozygotic state again, be the seed purity index.

The present invention is advantage and beneficial effect compared with the prior art:

1, molecule marker NAU/XiBao16 of the present invention and Pm21 gene is chain tightr.The sharp Pm21 gene of band that comes out is positioned at 6VS karyomit(e) simultaneously apart from kinetochore FL0.58 section because primer NAU/XiBao16F and NAU/XiBao16R increase in cluster hair wheat, and 6VS and common wheat karyomit(e) do not exchange, so NAU/XiBao16 can be used as the molecule marker of powdery mildew resistance gene Pm21 and uses, and analyzing sequence that this primer amplification comes out finds to contain in its amino acid that pushes over disease-resistant gene conserved domain leucine and repeats (LRRs), and generally all being form with gene family, disease-resistant gene is present on the karyomit(e) that (document sees reference: Baker Barbara, Zambryski Patricia, Staskawicz Brian, Dinesh-Kumar SP (1997) .Sig-naling inPlant-Microbe Interactions.Science 276,726-733.Geffroy V, Sicard D, De Oliveira JC et al. (1999) .Identification of an ancestral resistance gene cluster involved in the coevolution process betweenPhaseolus vulgaris and its fungal pathogen Colletotrichum lindemuthianum.Mol Plant-Microbe Interact12,774-784.), therefore the NAU/XiBao16 gene fragment that increases out may be exactly the part of Pm21 gene family, so this mark and Pm21 gene linkage degree are than mark SCAR ₁₄₀₀And SCAR ₁₂₆₅Higher with Pm21 gene linkage degree.

2, molecule marker NAU/XiBao16 of the present invention is more efficient when identifying common wheat 6AS and cluster hair wheat 6VS karyomit(e).Molecule marker NAU/XiBao16 can amplify two bands that vary in size on 6AS and 6VS, this two band can specificity be followed the trail of this two karyomit(e)s.In research in the past, often need to determine to contain in the material which bar in these two karyomit(e)s, and in a PCR reaction, just can reach these two chromosomal purposes of discriminating with molecule marker NAU/XiBao16 with several marks.When carrying out the mildew-resistance breeding as the parent with wheat-haynaldia villosa translocation line (6VS/6AL), carry out molecular marker assisted selection with NAU/XiBao16, not only can whether contain 6VS/6AL in the expert evidence, can also identify whether 6VS/6AL karyomit(e) is in homozygotic state, can improve the efficiency of selection of breeding greatly like this.

3, molecule marker NAU/XiBao16 of the present invention is identifying that disease-resistant karyomit(e) 6VS/6AL isozygotys or more convenient and quicker, economy during heterozygous state.Use SCAR ₁₄₀₀And SCAR ₁₂₆₅During these two molecule markers,, can not distinguish 6VS/6AL and be in and isozygoty or heterozygous state as long as contain 6VS karyomit(e) and just can amplify corresponding special band in the material.Whether 6VS/6AL karyomit(e) was in homozygotic state in the expert evidence in the past, adopt tip of a root karyomit(e) mitosis metaphase C-banding technique or hybridization in situ technique to carry out often, not only experimental technique is loaded down with trivial details for these two kinds of technology, and cost is very high, has limitation so use when a large amount of expert evidence.If use NAU/XiBao16 to carry out molecular marker assisted selection, can easily identify with wheat-haynaldia villosa translocation line (6VS/6AL) and common wheat to be that whether 6VS/6AL karyomit(e) is homozygotic state in parent's the progeny material quickly.

4, when creating breeding material, can differentiate the material that common wheat the 6th homology group karyomit(e) 6AS morphs with the NAU/XiBao16 mark with cell engineering or chromosome engineering.

Four, description of drawings

Fig. 1: with raise wheat No. 5, translocation line (6VS/6AL), cluster hair wheat DNA as template, carry out pcr amplification with the primer NAU/XiBao16 among the present invention, contain the band that cluster hair wheat and translocation line can both amplify a common molecular weight 1586bp, and common wheat is raised wheat and lacks this band for No. 5, therefore infers that this 1586bp band is positioned on the 6VS.

Fig. 2: with the common wheat China spring add

cluster hair wheat

1V, 2V, 3V, 4V, 5V, 6V, chromosomal 7 the material DNA of 7V and cluster hair wheat 6V chromosome substitution respectively the chromosomal substitution line DNA of common wheat 6A be template, carry out pcr amplification with the primer NAU/XiBao16 among the present invention, the result shows that the material that has only 6V karyomit(e) to exist can amplify the band of the 1586bp the same with cluster hair wheat, illustrates that the band of this 1586bp is positioned on the cluster hair wheat 6V.

Fig. 3: with common wheat 6V add disappearance be del6V#2S-1DNA as template, carry out pcr amplification with the primer NAU/XiBao16 among the present invention, the result shows the still existence of 1586bp band on the 6V, the band on the 6VS is between kinetochore and FL0.58 section.

Fig. 4: with cover nullisomic limbs (N1A/T1D, N1A/T1B, N1B/T1A, a N1B/T1D of common wheat China spring, N1D/T1A, N1D/T1B, N2A/T2B, N2A/T2D, N2B/T2A, N2B/T2D, N3A/T3B, N3A/T3D, N3B/T3A, N3B/T3D, N4A/T4B, N4A/T4B, N4B/T4A, N4B/T4D, N4D/T4A, N4D/T4B, N5A/T5B, N5A/T5D, N5B/T5A, N5B/T5D, N5D/T5A, N5D/T5B, N6A/T6B, N6A/T6D, N6B/T6A, N6B/T6D, N6D/T6A, N6D/T6B, N7A/T7B, N7A/T7D, N7B/T7A, N7B/T7D, N7D/T7A, DNA N7D/T7B) are that template is carried out PCR, carry out pcr amplification with the primer NAU/XiBao16 among the present invention, the result shows that the material that lacks 6A has lacked the band of a 1886bp, can infer that according to the result band of 1886bp is positioned on the 6A karyomit(e).

Fig. 5: with raising wheat No. 5, cluster hair wheat, hard bunch of wheat (AABBVV) (hard bunch of wheat is by durum wheat AABB and cluster hair wheat VV synthetic), one grained wheat, intend this inferior that holder goatweed, Triticum tauschii, carry is it possible that tie up wheat, duckbill wheat and 6VS/6AL translocation line and 6VS/6DL translocation line DNA be template not, carry out pcr amplification with the primer NAU/XiBao16 among the present invention, the result shows and raises wheat No. 5, the 6DL/6VS translocation line, durum wheat-cluster hair wheat double diploid, one grained wheat, carry is it possible that tie up and all have this band of 1886bp in wheat and the duckbill wheat, but do not contain the genomic wheat crops of AA at 6VS/6AL translocation line and other; Cluster hair wheat, intend but not existing in this inferior you holder goatweed and Triticum tauschii, this band that further specifies 1886bp is positioned on the 6AS karyomit(e).

Fig. 6: with China spring disappearance be DNA as template, carry out pcr amplification with the primer NAU/XiBao16 among the present invention, disappearance is the band that band that 6AS-1 amplifies has lacked a 1886bp, the 1886bp band is positioned at FL0.35 karyomit(e) far-end.

Fig. 7: with raising No. 5 * wheat-haynaldia villosa of wheat translocation line (6VS/6AL) F ₂The individual plant DNA of colony is a template, carries out the result of pcr amplification with the primer NAU/XiBao16 among the present invention.Whole mildew-resistances are identified in the material field of containing the 1586bp band, and the material that does not contain the 1586bp band is all susceptible.The translocation line that has 1886bp band and 1586bp band simultaneously is the heterozygosis transposition; The material that has only 1586bp band and do not have a 1886bp band is the translocation line that isozygotys.

Fig. 8: common wheat karyomit(e) diagrammatic sketch

Fig. 9: cluster hair wheat karyomit(e) diagrammatic sketch

Figure 10: its karyomit(e) of wheat-haynaldia villosa translocation line 6VS/6AL 6VS/6AL is the diagrammatic sketch when isozygotying

Figure 11: the diagrammatic sketch when its karyomit(e) of wheat-haynaldia villosa translocation line 6VS/6AL 6VS/6AL is heterozygosis

Figure 12: with the translocation line first chain cDNA is nucleotide sequence and the deduced amino acid that template is carried out the total length Ta-LRR2 gene that RT-PCR amplification obtains.The initial son of expression and the terminator of red part.

Figure 13: Ta-LRR2 gene deduced amino acid and paddy rice, Arabidopis thaliana and corn LRR aminopeptidase gene acid sequence comparative result.Last line expression position, LRR unit, the black matrix subdivision is represented the unitary amino-acid residue of disease-resistant gene conserved domain LRR, the unitary number of numeral LRR.The common structure in LRR unit is: axxaxaxx, and wherein a represents any fats amino-acid residue such as I, F L, M, A or V; X represents any residue.Asterisk is represented relatively identical with the similar amino acid in back of four species LRR aminopeptidase gene acid sequences respectively with stain.

Five, embodiment

1, screening is positioned at the disease-resistant gene analogue on the 6VS karyomit(e)

Mildew-resistance 6VS/6AL translocation line with raise wheat and backcrossed for 7 generations for No. 5, obtain background and raise No. 5 approaching translocation lines of wheat, this translocation line and parent Yang Mai are cultivated in the basin alms bowl for No. 5, and isolate, in case extraneous living contaminants with the cover that transparent film is made.Plant is long during to two leaf stage, with the Powdery Mildew mixed strains translocation line is shaken off method and inoculates, and induces 6h and 12h respectively, induces without the white powder germ and raise wheat No. 5.The translocation line blade of getting then after inducing is not raised No. 5 blades extractions of wheat RNA with having inductive, making up with translocation line blade RNA respectively is Tester, forward inhibition subtractive hybridization (SSH) library that to raise No. 5 RNA of wheat be Driver and to raise No. 5 RNA of wheat be Tester, translocation line blade RNA is the reverse SSH library of Driver, with reverse library cDNA is probe screening forward SSH library, obtain hybridization signal, the positive colony that expression amount difference is arranged, The sequencing results shows that one of them is disease-resistant gene analogue (Figure 13), this gene fragment may be positioned on the 6VS karyomit(e) as the candidate gene of Pm21.

2, develop the molecule marker of PCR-based according to the disease-resistant gene analogue of screening

Make probe search wheat expressed sequence tag (EST) database (http://www.graingenes.org) with the above-mentioned sequence fragment that obtains, obtain three height homologous ESTs sequences with it, by forming full length sequence behind the electronic splicing, and according to the sequence of this splicing Primer3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) design primer NAU/XiBao16F:5 '-GTAGACAGTGCCGCTATTTC-3 ' and NAU/XiBao16R:5 '-CAACTCATGTGTCCAGTTCA-3 ', with translocation line cDNA is that template increases and obtained full-length gene (Figure 12), and be Ta-LRR2 with this unnamed gene, have disease-resistant gene conserved domain (Figure 13) according to this sequence deduced amino acid.

It is (public to raise wheat No. 5 (AABBDD) with common wheat, draw the institute of agricultural sciences of in Yangzhou, going to river), (6VS/6AL) is (public for the wheat-haynaldia villosa translocation line, Agricultural University Of Nanjing cytogenetics laboratory externally provides), the DNA of cluster hair wheat (VV) is template, is that primer carries out pcr amplification with NAU/XiBao16F and NAU/XiBao16R.PCR reagent is formed: dna profiling 2 μ L (50-100ng), 2.5 μ L10 * PCR buffer, 1.8 μ LMgCl ₂, 2 μ LdNTP (2.5mmoL/L), each 0.5 μ L left and right sides primer (10mmol/L), 0.25 μ L Taq DNA polymerase (5u/ μ L), 16 μ L d.d H ₂O.The PCR program is: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change 30 seconds, 56 ℃ of annealing 40 seconds, 72 ℃ were extended 33 circulations 2 minutes; 72 ℃ were extended 10 minutes; 4 ℃ of preservations.The PCR product shows that it is the band of 1586bp that translocation line (6VS/6AL) and cluster hair wheat have a common molecular weight, lacks this band (Fig. 1) for No. 5 and raise wheat through 8% gather after third uncommon acid amides (39: 1) the gel electrophoresis silver dyes.(public to add cluster hair wheat 1V-7V chromosomal interpolation system and substitution line in the common wheat China spring respectively, see Sears, E.R.1953.Addition of the genome ofHaynaldia villosa to Triticum aestivum.Am.J.Bot.40:168-174.) DNA is a template, carry out the PCR and the electrophoresis of the same terms, the result shows that it is the special band of 1586bp that the material of 6V karyomit(e) existence can amplify molecular weight, illustrates that this band of 1586bp obtains (Fig. 2) from the 6VS amplification.Further adding disappearance with wheat-haynaldia villosa 6V is that (del6V#2S-1 sees reference document: Lili Qi, Mingshu Cao, Peidu Chen, Wanlong Li and Dajun Liu.Identification, mapping, andapplication of polymorphic DNA associated with resistance gene Pm21 of wheat.Genome39:191-197,1998) DNA carries out PCR as template, still can amplify this band (Fig. 3), the disappearance breakpoint result of study of adding disappearance system in conjunction with wheat-haynaldia villosa 6V shows that this 1586bp product on the 6VS is positioned at the section of 6VS apart from kinetochore FL0.58, because it is still disease-resistant that identify in the del6V#2S-1 field, so Pm21 and 1586bp band are positioned at the same zone of karyomit(e) 6VS.

42 nullisomic limbs systems with China spring are (public, document sees reference: KM Devos, ME Sorrells, JA Anderson, TE Miller, SM Reader, AJ Lukaszewski, J Dubcovsky, PJ Sharp, J Faris, MD Gale.1999.Chromosomeaberrations in wheat nullisomic-tetrasomic and ditelosomic lines.Cereal Research Communications27:231-239) DNA be template, carry out the PCR and the electrophoresis of the same terms, lack the band (Fig. 4) that the band that amplifies in the material of 6A has lacked a 1886bp.The result shows that the band of the 1886bp that increases out from common wheat is on the 6A karyomit(e).With raising wheat No. 5, cluster hair wheat, hard bunch of wheat (AABBVV) (hard bunch of wheat is by durum wheat AABB and cluster hair wheat VV synthetic), one grained wheat, intend this inferior that holder goatweed, Triticum tauschii, carry is it possible that tie up wheat, duckbill wheat and 6VS/6AL translocation line and 6VS/6DL translocation line DNA are template, carry out pcr amplification with the primer NAU/XiBao16 among the present invention, the result shows and raises wheat No. 5, the 6DL/6VS translocation line, durum wheat-cluster hair wheat double diploid, one grained wheat, carry is it possible that tie up and all have this band of 1886bp in wheat and the duckbill wheat, but do not contain the genomic wheat crops of AA at 6VS/6AL translocation line and other: cluster hair wheat, intend but not existing in this inferior that holder goatweed and the Triticum tauschii, this band that further specifies 1886bp is positioned on the 6AS karyomit(e).

With the chromosomal excalation of China spring 6A system (is chromosome deletion by the part of karyomit(e) end, public, document Endo TR sees reference, Gill BS.The deletion stocks of common wheat.Heredity 87:295-307,1996) DNA is a template, carry out identical PCR, product is electrophoresis on 8% poly-third uncommon acid amides (39: the 1) gel.The result show with China spring deletant 6AS-1 be template amplification to the PCR product lacked the band of 1886bp.According to the collection of illustrative plates of disappearance system, this band of 1886bp is positioned at the last karyomit(e) far-end (Fig. 5) apart from kinetochore FL0.34 of 6AS.

3, detect No. 5 (public the kind) * wheat-haynaldia villosa translocation line F that raise wheat with mark NAU/XiBao16 ₂Colony

With NAU/XiBao16F and NAU/XiBao16R is primer, F2 generation 33 the individual plant DNA that raise No. 5 * wheat-haynaldia villosa of wheat translocation line are that template is carried out PCR, the result shows that the field disease-resistant individual plant 9-33 of evaluation can both amplify the special band on the 6VS, the field identifies that susceptible individual plant 1-8 can not amplify the special band (Fig. 7) on the 6VS, and anti-susceptible ratio was near 3: 1.Can explanation amplify special band on the 6VS with anti-susceptible directly related, and this primer can use as the closely linked molecule marker of Pm21 gene.

The amplification banding pattern of disease-resistant individual plant can be divided into two types again, one class can amplify three bands, as individual plant 19-33, this three band is respectively on 6AS (1886bp), 6VS (1586bp) and the BBDD karyomit(e) (1509bp) from big to small, and the individual plant that amplifies this banding pattern only carries a 6VS/6AL translocation chromosome; Another kind ofly can only amplify two bands, as 9-18, this two band is respectively that the band on the 6AS does not amplify on 6VS (1586bp) and the BBDD karyomit(e) (1509bp) from big to small, and the individual plant that can amplify this banding pattern carries two 6VS/6AL translocation chromosomes.This result shows the NAU/XiBao16 mark can identify not only whether the F2 that raises No. 5 * wheat-haynaldia villosa of wheat translocation line carries the 6VS/6AL translocation chromosome for each individual plant, and can also identify this chromosomal isozygotying or heterozygous state.

Claims

1, with wheat anti-powdery mildew gene Pm 21 linked labeled primer, its sequence is:

NAU/XiBao16F:5 '-GTAGACAGTGCCGCTATTTC-3 ' and NAU/XiBao16R:5 '-CAACTCATGTGTCCAGTTCA-3 '

2, the described and chain labeled primer of Pm21 according to claim 1, it is characterized in that: the fragment of the 1586bp that this primer amplifies from cluster hair wheat is positioned at 6VS karyomit(e) apart from kinetochore FL0.58 section, the band of the 1886bp that amplifies from common wheat can specially be followed the trail of 6AS with changing, and is positioned on the 6AS chromosome arm apart from kinetochore FL0.34 far-end.

3, the usage of claim 1 or 2 described and wheat anti-powdery mildew gene Pm 21 linked labeled primers:

1) material DNA to be identified carries out PCR with NAU/XiBao16F and NAU/XiBao16R as primer as template,

The PCR reagent set becomes: dna profiling 2 μ L, 2.5 μ L10 * PCR buffer, 1.85 μ LMgCl ₂, 2 μ LdNTP, each 0.5 μ L of left and right sides primer, 0.25 μ L Taq DNA polymerase, 16 μ L d.d H ₂O;

The PCR program is: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change 30 seconds, 56 ℃ of annealing 40 seconds, 72 ℃ were extended 33 circulations 2 minutes; 72 ℃ were extended 10 minutes; 4 ℃ of preservations, the PCR product is electrophoretic separation on 39: 1 glue of the polyacrylamide of non-sex change, is dyeing with argentation;

2) labeled primer identifies that the result of Pm21 gene is: the individual plant that can amplify the special band of 1586bp fragment carries the Pm21 gene, and its Pm21 gene is positioned on the cluster hair wheat 6VS karyomit(e); What can not amplify the special band of 1586bp fragment does not then carry the Pm21 gene.

4, according to the usage of the described and wheat anti-powdery mildew gene Pm 21 linked labeled primer of claim 3, it is characterized in that the disease-resistant karyomit(e) 6VS/6AL that can amplify the plant of 1886bp, 1509bp and 1586bp three bands is in heterozygous state: the 1509bp band is that common wheat BBDD genome increases out; The band of 1886bp is from karyomit(e) 6AS; The band of 1586bp is from disease-resistant karyomit(e) 6VS; Show to have karyomit(e) 6AS/6AL and disease-resistant karyomit(e) 6VS/6AL in this individual plant simultaneously that disease-resistant karyomit(e) 6VS/6AL does not exist in pairs, is in heterozygous state;

The disease-resistant karyomit(e) 6VS/6AL that can amplify the plant of 1586p and 1509bp two bands is in homozygotic state: the band amplification from the chromosomal 1886bp of 6AS is not come out, 6AS karyomit(e) does not just exist, so 6VS/6AL karyomit(e) exists in pairs, is in homozygotic state; Isozygoty or heterozygous state in order to the disease-resistant karyomit(e) 6VS/6AL that identifies individual plant.

5, the usage of and wheat anti-powdery mildew gene Pm 21 linked labeled primer according to claim 3 is characterized in that,

Detect the method for carrying Pm21 gene seed purity with this labeled primer: random choose single seed from seed to be detected at first, utilize labeled primer to identify that single seeded 6VS/6AL karyomit(e) isozygotys or heterozygous state, add up seed proportion in detecting the seed total amount that 6VS/6AL karyomit(e) is in homozygotic state again, be the index of seed purity.