CN105821052A - Aegilops speltoides Tausch NAM-S1 gene and molecular marker and application thereof - Google Patents
Aegilops speltoides Tausch NAM-S1 gene and molecular marker and application thereof Download PDFInfo
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Abstract
The invention relates to an Aegilops speltoides Tausch NAM-S1 gene and a molecular marker and application thereof. The NAM-S1 gene is from a chromosome 6 of the Aegilops speltoides Tausch, and the nucleotide sequence of the NAM-S1 gene is shown in SEQ ID NO:1. The gene NAM-S1 plays an important role in wheat grain protein accumulation. The genetic distance between the gene NAM-S1 and the powdery mildew resistance gene Pm12 is 7.3 cM, and the NAM-S1 gene has the potential application value in high-protein wheat new-variety breeding. In addition, the invention further provides a molecular marker CauNAM-S1 and application for specifically detecting the NAM-S1 gene. The marker can specifically detecting whether the NAM-S1 gene exists in the wheat variety or not, can also detect whether the powdery mildew resistance gene Pm12 exists in wheat materials or not and can also be used as the molecular marker for wheat high protein and powdery mildew resistance screening.
Description
Technical field
The present invention relates to genetic engineering and biology field, specifically, relate to Ae.speltoides NAM-
S1 gene and molecular marker thereof and application.
Background technology
Semen Tritici aestivi is currently global first generalized grain crop, and the whole world there are about the population of 35%～40% as main
Food source, accounts for the 20% of whole world calorie total quantity consumed, whole world Semen Tritici aestivi annual production about 700,000,000 tons, it is provided that about 70,000,000 tons
Protein.Therefore, the protein content increasing wheat seed can not only improve the quality of Semen Tritici aestivi, and contributes to significantly carrying
The total amount of high edible protein.Wheat seed protein content (grain protein concentration, GPC) and composition thereof
Decide processing quality and the nutritional quality of Semen Tritici aestivi.The grain protein content of Semen Tritici aestivi is typical quantitative trait, and is easily subject to
Impact (Groos et al., 2003) to environmental condition.2006, Uauy etc. obtained control albumen by map based cloning and contains
The gene NAM-B1 of amount, this gene is positioned on Semen Tritici aestivi 6BS chromosome, a kind of NAC transcription factor of coding (Uauy et al.,
2006).Ae.speltoides contains disease-resistant, high-quality and other useful genes, and these excellent genes are when improveing Semen Tritici aestivi
Easily utilize.The S genome of Ae.speltoides (Aegilops speltoides) and the 1 B gene group of common wheat exist
There is in evolution nearer sibship, therefore speculate and also should be had on the 6SS of Ae.speltoides to control a seed
The gene of high protein content.
Powdery mildew is a kind of worldwide disease having a strong impact on yield and quality of wheat, all has in wheat belt, various countries and sends out
Raw.Recent two decades comes, and scope and area that wheat powdery mildew occurs constantly expand, and the extent of injury increases the weight of day by day, at present, and white wheat
Powder disease has become the important limiting factor affecting wheat yield.The pathogen of wheat powdery mildew is grass family dlumeria graminis Semen Tritici aestivi
Specialized form, belongs to Ascomycotina fungus, and this pathogenic bacteria has multiple biological strain, with host's interaction in different geographical ecological environmenies
During speed of mutation fast, the kind of some large area production applications or has lost resistance, therefore white lead disease at present
It is in the serious situation of pandemic at any time.Pesticide control serves certain effect to prevention and control powdery mildew, but can expend
Substantial amounts of human and financial resources, do not utilize ecological environment.Utilize modern biotechnology, constantly identify and excavate resisting in Semen Tritici aestivi
Powdery mildew gene or QTL site, pass through molecular gene engineering or molecular marker assisted selection by mildew-resistance gene or QTL site
Cultivate and promote disease-resistant variety, being that preventing and treating wheat powdery mildew is the safest, economically and efficiently approach.Powdery mildew resistance gene in wheat
Pm12 also is located on the 6SS of Semen Tritici aestivi-Ae.speltoides translocation line 6SS 6BL.So far, in existing document still
There is no Semen Tritici aestivi-Ae.speltoides translocation line controls the gene report of seed high protein content, do not know anti-white lead yet
Ospc gene Pm12 and the genetic distance of high protein content gene.
Summary of the invention
It is an object of the invention to provide Ae.speltoides NAM-S1 gene, and NAM-S1 gene is increasing Semen Tritici aestivi
Effect in grain protein content, and prove this gene and anti-white lead in Semen Tritici aestivi-Ae.speltoides translocation line
The genetic distance between ospc gene Pm12.
It is a further object of the present invention to provide Ae.speltoides NAM-S1 gene molecule marker CauNAM-S1 and
Whether application, utilizes in this labelling specific detection wheat breed whether there is NAM-S1 gene, be additionally operable to detect in wheat lines
Containing mildew-resistance gene Pm12, it is used simultaneously as Semen Tritici aestivi high protein and the molecular marker of mildew-resistance screening.
In order to realize the object of the invention, the Ae.speltoides NAM-S1 gene that the present invention provides, it is little for controlling
The gene of wheat grain protein content, utilizes this gene can improve the protein content of wheat seed.
The nucleotides sequence of Ae.speltoides NAM-S1 gene is classified as:
I) nucleotide sequence shown in SEQ ID NO:1；Or
Ii) nucleotide sequence shown in SEQ ID NO:1 be substituted, lack and/or increase one or more nucleotide and
Express the nucleotide sequence of identical function protein；Or
Iii) under strict conditions with sequence hybridization shown in SEQ ID NO:1 and express identical function protein nucleotide
Sequence, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Lower hybridization, and wash film with this solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express identical function protein
Nucleotide sequence.
The present invention also provides for by the albumen of above-mentioned Ae.speltoides NAM-S1 gene code, and its aminoacid sequence is such as
Shown in SEQ ID NO:2.
The present invention also provides for the expression cassette containing described Ae.speltoides NAM-S1 gene, recombinant vector, restructuring
Bacterium or transgenic cell line.
The present invention also provides for described Ae.speltoides NAM-S1 gene, containing the expression of described NAM-S1 gene
Box, recombinant vector, recombinant bacterium or transgenic cell tie up to improve the application in crop protein content.
The crop related in the present invention includes but not limited to Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), Herba bromi japonici, Ae.speltoides.
The expression vector carrying described genes of interest can turn by using Ti-plasmids, plant viral vector, direct DNA
The standard biologic technical methods such as change, microinjection, electroporation import (Weissbach, 1998, Method in plant cell
Plant Molecular Biology VIII, Academy Press, New York, the 411-463 page；Geiserson and
Corey, 1998, Plant Molecular Biology, 2nd Edition)。
Such as, can will pass through containing expression cassette, recombinant vector, recombinant bacterium or the transgenic cell line of described NAM-S1 gene
Agriculture bacillus mediated, pollen tube imports or the method for particle gun proceeds in crop, cultivates the genetically modified crops of high grain protein content.
The present invention also provides for the molecular marker CauNAM-S1 of described Ae.speltoides NAM-S1 gene, for spy
The primer of this molecular marker of specific PCR amplification is: forward primer F5'-ACCGCAGACGATGCCGGCT-3' and reverse primer
R5'-TCAGGGATTCCAGTTCACG-3'。
The present invention also provides for described molecular marker CauNAM-S1 NAM-S1 gene and/or Pm12 gene in detection crop
In application,
The present invention also provides for described molecular marker CauNAM-S1 at Semen Tritici aestivi high protein content and/or the molecule of mildew-resistance
Application in marker-assisted breeding.
The present invention also provides for described molecular marker CauNAM-S1 and has high protein content and mildew-resistance concurrently in screening and qualification
New variety of wheat in application.
The present invention also provides for for detecting NAM-S1 gene and/or the primer of Pm12 gene in crop, including forward primer
F5'-ACCGCAGACGATGCCGGCT-3' and reverse primer R5'-TCAGGGATTCCAGTTCACG-3'.
The present invention further provides the detection kit containing described primers F and R.
The present invention clones from Ae.speltoides and obtains a Semen Tritici aestivi NAM family gene, named NAM-S1,
NAM-S1 full length gene 1547bp, is made up of 3 exons and 2 introns, its open reading frame total length 1227bp, coding
408 aminoacid, it was predicted that its molecular weight is 43.6kDa, isoelectric point, IP is 9.007.
In Semen Tritici aestivi-Ae.speltoides 6SS 6BL translocation line, NAM-S1 gene improves seed after replacing NAM-B1
The content of corpuscular protein.According to NAM-S1 gene order, develop the molecular marker CauNAM-S1 of specific detection NAM-S1 gene,
In the segregating population of 6SS 6BL translocation line, prove that the genetic distance between this labelling and powdery mildew resistance gene in wheat Pm12 is
7.3cM.Therefore, the molecular marker utilizing CauNAM-S1 labelling and mildew-resistance gene Pm12 screens simultaneously, it is possible to obtain
Have the new variety of wheat of high protein and mildew-resistance concurrently.
The invention have the advantages that
(1) Semen Tritici aestivi is one of the most most important cereal crops, whole world Semen Tritici aestivi annual production about 700,000,000 tons, if Semen Tritici aestivi seed
Corpuscular protein content increases by 1%, is equivalent to whole world wheat protein content and increases by 7,000,000 tons.NAM-with common wheat 6BS chromosome
B1 gene is compared, and the NAM-S1 gene of the present invention is greatly improved Protein Content of Wheat Kernel, therefore, and NAM-S1 gene pairs
Significant in the edible protein supply increasing the whole world.
(2) the invention provides the molecular marker CauNAM-S1 labelling of specific detection NAM-S1 gene, this labelling is base
In the labelling of sepharose electrophoresis, method is relatively simple, and novel nucleic acid dye can be used to dye, more safe, practical.Right
In making full use of NAM-S1 gene, promote that Wheat Molecular Breeding is significant.
(3) the CauNAM-S1 labelling that the present invention provides can detect the gene NAM-controlling wheat seed protein content simultaneously
S1 and mildew-resistance gene Pm12, i.e. one labelling selects two kinds of character simultaneously, for improving the efficiency of Wheat Molecular Breeding, real
The pyramiding breeding of existing multiple characters has great importance.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection in the embodiment of the present invention 1 to the Ae.speltoides NAM-S1 gene that clone obtains
Result and genomic organization result；Wherein, the electrophoresis detection result of the NAM-S1 gene of a: clone, b:NAM-S1 gene is tied
Structure is analyzed；1-4 is respectively genomic DNA, 1kb DNA marker, DL2000DNA marker, cDNA.
Fig. 2 is the systematic evolution tree of the Ae.speltoides NAM-S1 gene built in the embodiment of the present invention 2.
Fig. 3 is that the embodiment of the present invention 3 Middle molecule labelling CauNAM-S1 is at the 10 anti-amplifications feeling in materials with 10；Its
In, S1-S10 represents sense powdery mildew wheat lines, and R1-R10 represents powdery-mildew-resistance wheat material.
Fig. 4 is the molecular marker linkage maps of NAM-S1 gene in the embodiment of the present invention 3；Wherein, the numerical value of left side mark
Represent the genetic distance (unit, cM) between adjacent sites.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, real below
Execute example all according to conventional laboratory conditions, such as Sambrook equimolecular Cloning: A Laboratory Manual (New York:Cold Spring
Harbor Laboratory Press, 1989) the operating technology code described in, or according to the experiment proposed by manufacturer
Condition；Raw materials used it is commercial goods.
The clone of embodiment 1 Ae.speltoides NAM-S1 gene
According on NCBI announce NAM-A1 (DQ869672), NAM-D1 (DQ869675), NAM-B1 (DQ869673),
NAM-B2 (DQ869676), the sequence information of NAM-D2 (DQ869677) gene, the primer of design amplification NAM-S1 gene
NAMS1ORF1, obtains the complete genome of NAM-S1 gene and cDNA sequence with high-fidelity enzyme PCR after expanding.PCR reaction system
For: template DNA 3ul, each 1ul of forward and reverse primer, dNTP (10mM) 0.4ul, high-fidelity Taq archaeal dna polymerase (5U/ μ l)
0.2ul, 10 × PCR reaction buffer 2ul, adds ddH2O mixes after supplying 20ul.PCR reaction condition is: 94 DEG C of 3min；94℃
30s, 58 DEG C of 30s, 72 DEG C of 2min, totally 33 circulations；72 DEG C of 7min, 16 DEG C are terminated reaction.After PCR reaction terminates, with 1% agar
Sugar electrophoresis detection amplification.Electrophoresis result shows, the size of NAM-S1 genomic DNA and cDNA is respectively 1.5kb (in Fig. 1 a
Swimming lane 1) and 1.2kb (swimming lane 4 in Fig. 1 a) left and right.
Table 1 primer sequence information
The phylogenetic analysis of embodiment 2 Ae.speltoides NAM-S1 gene
Download the NAM family gene of other species from NCBI GenBank, utilize MEGA 6.0 Software on Drawing to intend this inferior
The encoding proteins of your de-goatweed NAM-S1 gene and the systematic evolution tree of the encoding proteins of homology NAM gene in other species.
Result shows (Fig. 2), and the NAM albumen of separate sources divide into 4 classes (Group).Wherein NAM-S1 belongs to Group II, additionally
Include NAM-B1 (6B chromosome), NAM-B2 (2B chromosome) and NAM-A1 (6A chromosome), NAM-D2 (2D chromosome) and
AetNAM-D1 (6D chromosome), HvNAM-1 and HvNAM-2 (H gene group) of Fructus Hordei Vulgaris, carry is it possible that tie up NAM-G (the G gene of Semen Tritici aestivi
Group).Group II can be divided into two subclass further, and two subclass are derived from Semen Tritici aestivi the 6th and the second homology group respectively
Part NAM albumen, wherein from HvNAM-1, NAM-B1, NAM-A1, NAM-D1, NAM-V1 and NAM-G1 position of the 6th homology group
In first subclass (Subgroup I), NAM-B2, NAM-D2 and the HvNAM-2 from the second homology group is positioned at second subclass
(Subgroup II).NAM-S1 albumen and derive from the part NAM albumen of Semen Tritici aestivi the 6th homology group with being in the in cladogram
One subclass, shows that NAM-S1 gene source is in No. 6 chromosomes of Ae.speltoides.
In Group II, derive from NAM-A1, NAM-B1, NAM-B2, NAM-D1, the NAM-in Guard cell
D2 has been proved to have acceleration aging, improves the biological function of the content of protein, ferrum, zinc in seed.From cladogram
From the point of view of, derive from the NAM-S1 gene of Ae.speltoides with the gene of known function with being divided into a monoid (Fig. 2),
Can speculate that NAM-S1 gene may also have relevant function, i.e. accelerate aging, improve protein in seed, ferrum, the containing of zinc
Amount.
Embodiment 3 Ae.speltoides NAM-S1 gene molecule marker is developed
According to above sequence alignment result, design the labelling CauNAM-S1 for specific detection NAM-S1 gene, be used for
The primer of specific PCR amplification label CauNAM-S1 is shown in Table 1.
Select powdery mildew resistance gene in wheat Pm12F2In segregating population, 10 disease-resistant and 10 susceptible offsprings, have carried out NAM-
The Markers for Detection of S1 gene.Result is as it is shown on figure 3, this result shows, NAM-S1 gene and Pm12 gene genetic distance are relatively
Closely, at F2In segregating population, NAM-S1 gene and Pm12 gene there occurs exchange.
In order to determine the genetic distance of NAM-S1 and Pm12 gene, the present invention utilizes CauNAM-S1 at the F of Pm122(n=
430) segregating population detects.With the heredity between Mapmaker EXP 3.0 computed in software labelling and disease-resistant gene away from
From, LOD value is 3.0.Mapdraw V2.1 software is utilized to incorporate barc1169, cau127, barc198, Pm12 and wmc105
Molecular marker, depicts the genetic linkage maps (Fig. 4) of NAM-S1 gene.Result shows, the labelling nearest with NAM-S1 gene
For wmc105, being secondly Pm12, wherein the genetic distance between NAM-S1 gene and Pm12 is 7.3cM.
Embodiment 4 utilizes the NAM-S1 gene in the different wheat lines of molecular marker CauNAM-S1 detection
F to altogether 6 Pm122:370 individual plants of family have carried out the mensuration of grain protein content (GPC) and anti-white lead
Sick qualification, and the banding pattern of Pm12 is determined by molecular marker screening, result is as shown in table 2,.
Table 2 Pm12 segregating population GPC adds up
To each F2:3After family's based material carries out Disease Resistance Identification, add up disease-resistant, the susceptible and average egg of heterozygosis individual plant respectively
White matter content, statistical result is as shown in table 3, and disease-resistant, susceptible and heterozygosis individual plant average GPC is 20.54%, 17.56% respectively
With 19.97%, statistical result showed, the average GPC content of disease-resistant material is higher than heterozygosis and susceptible material, according to NAM-S1 and
The genetic distance of Pm12 gene, initial guess NAM-S1 is the gene having function.In 6SS 6BL translocation line, NAM-S1 gene
Importing can improve the GPC content of 6SS 6BL translocation line material.
Table 3 Pm12 segregating population GPC adds up
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
List of references
1、Groos,C.,Robert,N.,Bervas,E.and Charmet,G.(2003)Genetic analysis of
grain protein-content,grain yield and thousand-kernel weight in bread
wheat.Theor Appl Genet,106,1032-1040.
2、Cao,A.,Xing,L.,Wang,X.,Yang,X.,Wang,W.,Sun,Y.,Qian,C.,Ni,J.,Chen,
Y.and Liu,D.(2011)Serine/threonine kinase gene Stpk-V,a key member of powdery
mildew resistance gene Pm21,confers powdery mildew resistance in
wheat.Proceedings of the National Academy of Sciences,108,7727-7732.
3、Uauy,C.,Distelfeld,A.,Fahima,T.,Blechl,A.and Dubcovsky,J.(2006)A
NAC Gene Regulating Senescence Improves Grain Protein,Zinc,and Iron Content
in Wheat.Science,314,1298-1301.
4、A.(2006)The genus Dasypyrum––part 1.The taxonomy and
relationships within Dasypyrum and with Triticeae species.Euphytica,152,429-
440.
5、Blanco,A.,Resta,P.,Simeone,R.,Parmar,S.,Shewry,P.R.,Sabelli,P.and
Lafiandra,D.(1991)Chromosomal location of seed storage protein genes in the
genome ofDasypyrum villosum(L.)Candargy.Theoretical and Applied Genetics,82,
358-362.
6、Ma,J.,Zhou,R.,Dong,Y.,Wang,L.,Wang,X.and Jia,J.(2001)Molecular
mapping and detection of the yellow rust resistance gene Yr26in wheat
transferred from Triticum turgidum L.using microsatellite markers.Euphytica,
120,219-226.
Claims (10)
1. Ae.speltoides NAM-S1 gene, it is characterised in that its nucleotides sequence is classified as:
I) nucleotide sequence shown in SEQ ID NO:1；Or
Ii) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and expression
The nucleotide sequence of identical function protein；Or
Iii) under strict conditions with sequence hybridization shown in SEQ ID NO:1 and express identical function protein nucleotides sequence
Row, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Hybridization, and wash film with this solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express the nucleoside of identical function protein
Acid sequence.
2. by the albumen of Ae.speltoides NAM-S1 gene code described in claim 1, it is characterised in that its aminoacid
Sequence is as shown in SEQ ID NO:2.
3. contain expression cassette, recombinant vector or the recombinant bacterium of Ae.speltoides NAM-S1 gene described in claim 1.
4. Ae.speltoides NAM-S1 gene described in claim 1, or expression cassette, recombinant vector described in claim 2
Or recombinant bacterium is in the application improved in crop protein content, the most described crop is Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), Herba bromi japonici, to intend this inferior you
De-goatweed.
Application the most according to claim 4, it is characterised in that by the expression cassette described in claim 3, recombinant vector or weight
Group bacterium, is imported by agriculture bacillus mediated, pollen tube or the method for particle gun proceeds in crop, cultivates turning of high grain protein content
Gene crops.
6. the molecular marker CauNAM-S1 of Ae.speltoides NAM-S1 gene described in claim 1, it is characterised in that
The primer expanding this molecular marker for specific PCR is: forward primer F5'-ACCGCAGACGATGCCGGCT-3' is with reverse
Primer R5'-TCAGGGATTCCAGTTCACG-3'.
7. the application in NAM-S1 gene and/or Pm12 gene, the preferably institute in detection crop of molecular marker described in claim 6
Stating crop is Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), Herba bromi japonici, Ae.speltoides.
8. molecular marker described in claim 6 is in the molecular mark of Semen Tritici aestivi high protein content and/or mildew-resistance
Application.
9. molecular marker described in claim 6 in screening or identifies answering in high protein content and/or powdery-mildew-resistance wheat kind
With.
10. for detecting NAM-S1 gene and/or the primer of Pm12 gene in crop, it is characterised in that include forward primer
F5'-ACCGCAGACGATGCCGGCT-3' and reverse primer R5'-TCAGGGATTCCAGTTCACG-3'.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106434950A (en) * | 2016-10-26 | 2017-02-22 | 中国科学院成都生物研究所 | Primer, kit and method for detecting low grain protein content of wheat, and applications of method |
| CN108570517A (en) * | 2018-06-12 | 2018-09-25 | 江苏省农业科学院 | The relevant specific primer of low with the peaceful wheat of weak-gluten wheat No. 9 albumen and its application |
| CN110511940A (en) * | 2019-08-09 | 2019-11-29 | 江苏大学 | Ae.speltoides Pm6SS-1 gene and its molecular labeling and application |
| 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 |
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| CN104498490A (en) * | 2014-12-24 | 2015-04-08 | 江苏省农业科学院 | Molecular marker tightly linked with low-protein-content QTL of wheat grains and application of molecular marker |
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| CN104498490A (en) * | 2014-12-24 | 2015-04-08 | 江苏省农业科学院 | Molecular marker tightly linked with low-protein-content QTL of wheat grains and application of molecular marker |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434950A (en) * | 2016-10-26 | 2017-02-22 | 中国科学院成都生物研究所 | Primer, kit and method for detecting low grain protein content of wheat, and applications of method |
| CN106434950B (en) * | 2016-10-26 | 2019-07-05 | 中国科学院成都生物研究所 | A kind of primer, kit, detection method and its application detecting the low grain protein content of wheat |
| CN108570517A (en) * | 2018-06-12 | 2018-09-25 | 江苏省农业科学院 | The relevant specific primer of low with the peaceful wheat of weak-gluten wheat No. 9 albumen and its application |
| CN108570517B (en) * | 2018-06-12 | 2021-10-01 | 江苏省农业科学院 | Specific primer related to Ning-Mai No. 9 low protein of weak gluten wheat and application of specific primer |
| 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 |
| CN110511940A (en) * | 2019-08-09 | 2019-11-29 | 江苏大学 | Ae.speltoides Pm6SS-1 gene and its molecular labeling and application |
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| CN105821052B (en) | 2019-05-10 |
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