CN109022607A - A kind of breeding method of how anti-gene pyramiding soybean and the application of how anti-gene pyramiding soybean - Google Patents
A kind of breeding method of how anti-gene pyramiding soybean and the application of how anti-gene pyramiding soybean Download PDFInfo
- Publication number
- CN109022607A CN109022607A CN201810870691.9A CN201810870691A CN109022607A CN 109022607 A CN109022607 A CN 109022607A CN 201810870691 A CN201810870691 A CN 201810870691A CN 109022607 A CN109022607 A CN 109022607A
- Authority
- CN
- China
- Prior art keywords
- soybean
- rhg4
- scn11
- detection
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Botany (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Environmental Sciences (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
Abstract
The present invention provides a kind of breeding method of how anti-gene pyramiding soybean excellent germplasm and the application of how anti-gene pyramiding soybean excellent germplasm, belongs to soybean breeder field.The breeding method of mostly anti-gene pyramiding soybean excellent germplasm hybridizes anti-northeast mosaic virus soybean and salt tolerant soybean；By F1Hybridize with anti-soybean cyst nematode soybean；Extract F2The genomic DNA of group uses the Markers for Detection of Rhg4, SALT3 and SCN11 respectively；Select the F of 2 Rhg4, SALT3 and SCN11 molecular labeling homozygosis2Single plant and 3 dual anti-single-plant propagation respectively obtain F at plant4、F5And F6Group；By F5And F6Group is detected using the molecule labelling method, selects the plant of molecular labeling resistance type for how anti-gene pyramiding soybean excellent germplasm.Breeding obtains providing resistant material with three anti-genotype and the excellent strain of economical character for soybean breeder.
Description
Technical field
The invention belongs to soybean breeder fields, and in particular to the breeding method of a kind of how anti-gene pyramiding soybean and mostly anti-
The application of gene pyramiding soybean.
Background technique
Soybean cyst nematode (Soybean cystnematode, SCN), soybean Mosaic (Soybean
Mosaic virus, SMV) it is the serious plant disease for endangering world soybean production.Wherein, soybean cyst nematode is soil-borne disease,
And increase year by year in the soybean producing region occurring area of China, cultivate disease-resistant variety, the non-host crop of efficent rotation is to prevent and treat the disease
Most economical effective measures (Chen S Y, PorterP M, Reese C D, Stienstra W C.Crop sequence
effects on soybean cyst nematode and soybean and cornyields.Crop Sci,2001,41:
1843–1849.).Soybean Mosaic is distributed in the major beans producing region in the world, disease incidence 30%~60%, when morbidity not
Only plant strain growth is had an impact, can also make kind of skin generate mottled, therefore, seriously affect soybean yields and exterior quality (king
Army forever, Dong Fangyang, Wang Xiuqiang wait the positioning Acta Genetica Sinica of 5 mosaic virus strain resistant genes of soybean, 2004,31 (1):
87-90.).Now to the prevention and treatment of soybean Mosaic mainly for popular strain, genetic improvement is carried out to variety resistance,
Cultivate and promote disease-resistant variety.
Multi-parent strain excellent genes are carried out polymerization and added up, innovation obtains height by Li Candong etc. by step cross breeding method
" closing rich 53 ", (Li Candong, Guo Tai, Wang Zhixin wait excellent genes polymerization utilization and high oil for oily high yield, disease-resistant new soybean varieties
Soybean " closes the innovation agricultural journal of rich 53 ", 2014,4 (10): 5-8.).Maroof S etc. is resisted SMV with marker assisted selection
Property gene Rsv1, Rsv3 and Rsv4, are aggregated in the same kind (MaroofS, Jeong S C, Gunduz by composite-crossing
I,et al.Pyramiding ofsoybean mosaic virus resistance genes by marker-assisted
selection.Crop Science,2008,48(2):517-526.).King is just equal greatly with (rich No. 1 of Qihuang 1 × section) × (big
Hemp × Nan Nong 1138-2) offspring carried out label auxiliary RSC14Q, RSC8, RSC43 resistant genes be homozygosis polymerization
(Bai Li, Li Haichao, Ma Ying wait heredity and assignment of genes gene mapping soybean of the soybean to soybean mosaic virus SC-11 strain resistance to material
Science, 2009,28 (1): 1-6.).
Although there are many research of multiple gene polymerization in soybean, yet there are no anti-soybean cyst nematode, anti-soybean
Mosaic virus and the trait related gene of salt resistance carry out pyramiding breeding and obtain the report of homozygous line.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of breeding method of how anti-gene pyramiding soybean and how anti-bases
Because polymerizeing the application of soybean, the method anti-soybean cyst nematode while capable of obtaining with inheritance stability resists big Tofu pudding
The soybean germplasm of leaf disease viral disease and salt resistance.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of breeding methods of how anti-gene pyramiding soybean excellent germplasm, comprising the following steps:
(1) anti-northeast mosaic virus soybean and salt tolerant soybean are hybridized, obtains F1；
(2) F1 is hybridized with anti-soybean cyst nematode soybean, obtains F2；
(3) with the F of extraction2The genomic DNA of group is template, uses the molecule of Rhg4, SALT3 and SCN11 respectively
Label carries out PCR amplification, identification, and wherein the PCR product of Rhg4 carries out digestion, obtained enzyme using restriction enzyme BsrBI
The PCR product for cutting product, SALT3PCR product and SCN11 carries out electrophoresis, and the long 364bp of the PCR product of SALT3 is resistance type,
The long 564bp of the PCR product of SALT3 is responsive type, while the sample for generating 364bp and 564bp band is heterozygous；SCN11's
The long 980bp of PCR product is resistance type, and the long 1070bp of PCR product is responsive type, while generating the sample of 980bp and 1070bp band
This is heterozygous；The segment of the digestion products 132bp and 273bp of Rhg4 are resistance type, and the digestion products 369bp of Rhg4 is sensitivity
Type, while the sample for generating the band of 132bp, 273bp and 369bp is heterozygous；
(4) the F2 plant for selecting Rhg4, SALT3 and SCN11 molecular labeling homozygosis and dual anti-single-plant propagation are obtained at plant
F3Plant；
It is heterozygous that the dual anti-single plant, which is Rhg4 Markers for Detection, and the Markers for Detection of SALT3 and SCN11 are anti-
Property type；
(5) by the F3Plant detects according to molecule labelling method in step (3), according to testing result according to step (4)
Propagation method carry out breeding three generations, respectively obtain F4、F5And F6Group；
(6) by F5And F6Group selects point of Rhg4, SALT3 and SCN11 using molecule labelling method detection in step (3)
The plant of son label resistance type is how anti-gene pyramiding soybean excellent germplasm.
Preferably, anti-northeast mosaic virus soybean germplasm is middle product 95-5383 in the step (1).
Preferably, salt tolerant soybean is soybean varieties iron rich No. 8 in the step (1).
Preferably, anti-soybean cyst nematode kind is middle yellow 57 in the step (2).
Preferably, the Markers for Detection of Rhg4 with primer is Rhg4-389 in the step (3)；
The forward primer of Rhg4-389 has such as SEQ ID No.1 in sequence table；
The reverse primer of Rhg4-389 has such as SEQ ID No.2 in sequence table.
Preferably, the response procedures of the Markers for Detection of Rhg4 are as follows:
| It is denaturalized for the first time | 95 DEG C, 5min |
| Denaturation | 94 DEG C, 30s |
| Annealing | 56 DEG C, 40s |
| Extend | 72 DEG C, 50s |
| Recurring number | 38 |
| Finally extend | 72 DEG C, 8min |
Preferably, 10 μ L:PCR product of endonuclease reaction system, 5 μ L, 3U restriction endonuclease BsrBI, 1.5 μ LNEB buffers；Digestion
Program: 37 DEG C of constant temperature 30min.
Preferably, the Markers for Detection primer of SALT3 is 08067 in the step (3)；
08067 forward primer F2 has such as SEQ ID No.3 in sequence table；
08067 reverse primer R has such as SEQ ID No.4 in sequence table；
08067 reverse primer R3 has such as SEQ ID No.5 in sequence table.
Preferably, the response procedures of the Markers for Detection of SALT3 are as follows:
| It is denaturalized for the first time | 95 DEG C, 5min |
| Denaturation | 95 DEG C, 30s |
| Annealing | 57 DEG C, 30s |
| Extend | 72 DEG C, 1min |
| Recurring number | 34 |
| Finally extend | 72 DEG C, 8min |
Preferably, the Markers for Detection of SCN11 with primer is SCN11 in the step (3)；
The forward primer of SCN11 has such as SEQ ID No.6 in sequence table；
The reverse primer of SCN11 has such as SEQ ID No.7 in sequence table.
Preferably, the response procedures of the Markers for Detection of SCN11 are as follows:
Inspection of the how anti-gene pyramiding soybean excellent germplasm obtained the present invention provides the method breeding in different generations
Application in survey.
Inspection of the how anti-gene pyramiding soybean excellent germplasm obtained the present invention provides the method breeding in germ plasm resource
Application in survey.
The present invention provides a kind of breeding method of how anti-gene pyramiding soybean excellent germplasm, the present invention passes through molecular labeling
Assisted Selection selects polymerization Rhg4, and the germplasm of SALT3 and SCN11 gene, having cultivated has three anti-genes and economical character
Excellent germplasm provides resistant material for soybean breeder.
Detailed description of the invention
Fig. 1 is middle yellow 57 × [the middle rich 8] F of product 95-5383 × iron2The distribution of group's genotype, wherein Fig. 1-a is by individually marking
It scores other to F2Group identified, the single plant number filtered out；Fig. 1-b is the excellent equipotential base of carrying at least one filtered out
The germplasm of cause；
Fig. 2 is to utilize molecular labeling centering Huang 57 × [selection of middle product 95-5383 × iron rich 8], how anti-gene pyramiding material
The generation process of material；
Specific embodiment
The present invention provides a kind of breeding methods of how anti-gene pyramiding soybean excellent germplasm, comprising the following steps:
(1) by anti-northeast mosaic virus soybean strain and salt tolerant soybean strain cross, F is obtained1；
(2) by the F1Hybridize with anti-soybean cyst nematode Heterodera glycines soybean, obtains F2Group；
(3) with the F of extraction2The genomic DNA of group is template, uses the molecule of Rhg4, SALT3 and SCN11 respectively
Label carries out PCR amplification, and wherein the PCR product of Rhg4 carries out digestion using restriction endonuclease, and obtained digestion products, SALT3PCR are produced
The PCR product of object and SCN11 carry out electrophoresis, and the long 364bp of the PCR product of SALT3 is resistance type, and the PCR product of SALT3 is long
564bp is responsive type, while the sample for generating 364bp and 564bp band is heterozygous；The long 980bp of the PCR product of SCN11 is
Resistance type, the long 1070bp of PCR product are responsive type, while the sample for generating 980bp and 1070bp band is heterozygous；Rhg4's
The segment of digestion products 132bp and 273bp are resistance type, and the digestion products 369bp of Rhg4 is responsive type, at the same generate 132bp,
The sample of the band of 273bp and 369bp is heterozygous；
(4) F of 2 plants of Rhg4, SALT3 and SCN11 molecular labeling homozygosis is selected2Plant and 3 dual anti-single-plant propagation strains
Row, obtains F3Plant；
It is heterozygous that the dual anti-single plant, which is Rhg4 Markers for Detection, and the Markers for Detection of SALT3 and SCN11 are anti-
Property type；
(5) by the F3Plant detects according to molecule labelling method in step (3), according to testing result according to step (4)
Propagation method carry out breeding three generations, respectively obtain F4、F5And F6Group；
(6) by F5And F6Group selects point of Rhg4, SALT3 and SCN11 using molecule labelling method detection in step (3)
The plant of son label resistance type is how anti-gene pyramiding soybean excellent germplasm.
The present invention hybridizes anti-northeast mosaic virus soybean and salt tolerant soybean, obtains F1。
In the present invention, the preferably middle product 95-5383 of the anti-northeast mosaic virus soybean.The salt tolerant soybean is preferred
For iron rich 8.Middle product 95-5383 and iron rich 8 are originated from soybean gene resource seminar of the Chinese Academy of Agricultural Sciences.Middle yellow 57 once with
This for material delivered article (in Institute of Crop Science, Chinese Academy of Agricultural Science soybean rising star --- the middle rural area yellow 57 [J]
Science and technology, 2011,5:6.).Middle product 95-5383 once delivered article (Zheng C M, Chang R Z, Qiu L as material
J,et al.Identification and characterization ofa RAPD/SCAR marker linked to a
resistance gene for soybean mosaic virus in soybean[J].Euphytica,2003,132:
199-210.).Iron rich 8 was once published an article (Guan R X, Qu Y, Guo Y, et al.Salinity as material
tolerance in soybean is modulated by natural variation in GmSALT3[J].The
Plant Journal,2014,80:937-950.).Applicant promises to undertake that Huang 57, middle product 95-5383, rich 8 germplasm of iron are equal in parent
It can freely obtain.
In the present invention, the method for the hybridization is not particularly limited, using hybridizing method known in the art.
Obtain F1Afterwards, the present invention is by the F1Hybridize with anti-soybean cyst nematode Heterodera glycines homozygosis soybean, obtains F2Group.It is described anti-
The preferably middle Huang 57 of soybean cyst nematode Heterodera glycines homozygosis soybean.The method of the hybridization is not particularly limited, and use is known in the art
Hybridizing method.
Obtain F2After group, the present invention extracts F2The genomic DNA of group uses the molecule of Rhg4, SALT3 and SCN11 respectively
Label detection, obtains resistance type, responsive type and heterozygous F according to testing result2Plant.
The present invention is not particularly limited the method for extracting DNA, using DNA extraction method known in the art,
For example, CTAB method, RNA isolation kit etc..The method that the molecular detecting method preferably uses PCR amplification carries out.Point of the Rhg4
Son label detection is preferably Rhg4-389 with primer；The forward primer of Rhg4-389 has such as SEQ ID No.1 in sequence table；
The reverse primer of Rhg4-389 has such as SEQ ID No.2 in sequence table.The response procedures of the Markers for Detection of the Rhg4
It is preferred that are as follows:
The Rhg4 passes through PCR amplification, and obtained PCR product carries out endonuclease reaction.The 10 μ L of endonuclease reaction system:
5 μ L of PCR product, 3U restriction endonuclease, 1.5 μ LNEB buffers；The program of digestion: 37 DEG C of heat preservation 30min.
In the present invention, the Markers for Detection of the SALT3 is preferably 08067 with primer；08067 forward primer F2
With SEQ ID No.3 in such as sequence table；08067 reverse primer R has such as SEQ ID No.4 in sequence table, 08067 it is anti-
Have to primer R3 such as SEQ ID No.5 in sequence table.The primer preferably entrusts the raw work biotechnology service in Shanghai to have
Limit company synthesizes to obtain.The response procedures of the Markers for Detection of the SALT3 are preferred are as follows:
| It is denaturalized for the first time | 95 DEG C, 5min |
| Denaturation | 95 DEG C, 30s |
| Annealing | 57 DEG C, 30s |
| Extend | 72 DEG C, 1min |
| Recurring number | 34 |
| Finally extend | 72 DEG C, 8min |
.The reaction system of the Markers for Detection of the SALT3 is removed with the reaction system of the Markers for Detection of Rhg4 draws
The type of object, other components are consistent.
In the present invention, the Markers for Detection of the SCN11 is preferably SCN11 with primer；The forward primer of SCN11 has
Just like SEQ ID No.6 in sequence table；The reverse primer of SCN11 has such as SEQ ID No.7 in sequence table.The SCN11's
The response procedures of Markers for Detection are preferred are as follows:
.The reaction system of the Markers for Detection of the SCN11 is removed with the reaction system of the Markers for Detection of Rhg4 draws
The type of object, other components are consistent.The source of Markers for Detection primer is not particularly limited, and using this field, institute is ripe
The synthetic method known.
After obtaining PCR product, the PCR product is preferably carried out electrophoresis by the present invention, obtains electrophorogram.The electrophoresis is used
Gel is the Ago-Gel that mass concentration is 1%.The voltage of the electrophoresis is preferably 120V.The time 20min of electrophoresis.
Obtain resistance type, responsive type and heterozygous F2After plant, the present invention is to 2 plants of Rhg4, SALT3 and SCN11 molecule marks
Remember homozygous F2Plant and 3 dual anti-single-plant propagation obtain F at plant3Plant；The dual anti-single plant is the inspection of Rhg4 molecular labeling
Surveying is heterozygous, and the Markers for Detection of SALT3 and SCN11 are resistance type.
In the present invention, it is plantation that the method for being multiplied into plant, which is specifically divided as unit of selected single plant, by continuous
Mostly for Single-plant selection until the character stable and consistent of strain.
Obtain F3After plant, the present invention is by the F3Plant detects according to the molecule labelling method, presses according to testing result
Breeding three generations is carried out according to the propagation method, respectively obtains F4、F5And F6Group；By F5 and F6Group uses the molecular labeling
Method detection, selects the plant of the molecular labeling resistance type of Rhg4, SALT3 and SCN11 for excellent kind of how anti-gene pyramiding soybean
Matter.
Inspection of the how anti-gene pyramiding soybean excellent germplasm obtained the present invention provides the method breeding in different generations
Application in survey.
Inspection of the how anti-gene pyramiding soybean excellent germplasm obtained the present invention provides the method breeding in germ plasm resource
Application in survey.
Breeding method and how anti-base below with reference to embodiment to a kind of how anti-gene pyramiding soybean provided by the invention
Because the application of polymerization soybean is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1 materials and methods
1.1 test material
Parent used in this test has 3, including middle yellow 57, highly resistance soybean cyst nematode, with middle product 661 for parent's breeding
No. 3 strains (SMV3) of anti-northeast mosaic virus in product 95-5383, with salt tolerance soybean varieties iron rich 8.It is handed over by three
Construct middle yellow 57 × [the middle rich 8] F of product 95-5383 × iron2For segregating population, altogether include 438 single plants, in 2015 plantation in
Shunyi experiment station of the Chinese Academy of Agricultural Sciences.It is by molecular markers for identification and repeatedly southern numerous, cultivate the soybean for polymerizeing how anti-gene
New lines.
1.2 DNA are extracted and detection
The extraction purification of genomic DNA uses CTAB method.Each kind acquires the tender fresh blade of 5 childrens from different single plants,
Blade is cut into 1cm with punch2It is cooling to be put into liquid nitrogen for size.Extracting genome DNA is carried out after drawing a design sufficiently.Add after extraction
Enter ddH2200 μ L of O is placed in 4 DEG C of sufficiently dissolution precipitatings, and is put in -20 DEG C and saves backup.
Agarose concentration is 0.8%, and electrophoretic buffer is 1 × TAE, and voltage is set as 135V, electrophoresis time 15min,
EB dyeing, after Bio-RAD gel imaging observes identification extraction quality.With the A260/ of ultraviolet specrophotometer measurement DNA sample
A280 ratio meets PCR requirement substantially between 1.8 to 2.2.
The selection and synthesis of 1.3 primers
The label Rhg4-389 developed by Rhg4 is chosen, the label 08067 developed by SALT3, and it is chain with SCN11
SCAR mark.All primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.See Table 1 for details for primer sequence information.
Table 1 Rhg4, SALT3, SCN11 mark information
The foundation of 1.4 PCR reaction systems
PCR is using genomic DNA as template, the unified reaction system for using 20 μ L, including 100ng genomic DNA, 10 ×
The 1.5 μ L of dNTPs of PCR buffer 2 μ L, 2mmol/L, each 0.3 μ L and 1U Taq polymerase of 10 μm of ol/L upstream and downstream primers
(Quan Shijin Bioisystech Co., Ltd).Wherein the primer proportion of salt tolerant label 08067 is F2:R:R3=2:1.5:2, and PCR exists
It is carried out in the PCR amplification instrument of ABI (Applied Biosystems, the U.S.) company, response procedures and agarose testing conditions are shown in
Table 2.
2 PCR response procedures of table and agarose gel electrophoresis condition
Wherein, label Rhg4-389 need to carry out digestion after PCR amplification, 10 μ L:PCR product of endonuclease reaction system, 5 μ L,
3U restriction endonuclease BsrBI, 1.5 μ LNEB buffers.Endonuclease reaction system is put into 37 DEG C of insulating boxs or water-bath, is taken after 30min
Out, digestion products are detected using 1.5% agarose gel electrophoresis.
2 results and analysis
2.1 hand over F using target gene Markers for Detection three2Group
It is handed over by three and formulates multiple gene polymerization material, middle yellow 57 × [middle rich 8] the F2 separation group of product 95-5383 × iron of building
Body shares 438 single plants.Plantation is in Shunyi experiment station of the Chinese Academy of Agricultural Sciences within 2015.Middle yellow 57 are hybridized by Hartwig with Shanxi 1265
Breeding, highly resistance soybean cyst nematode, middle product 95-5383 are with middle product 661 for parent's breeding, the blood with Williams 82
Edge, the strain of anti-soybean Mosaic northeast 3, soybean varieties iron rich 8 have salt tolerance.
Utilize the label of the CAPS label Rhg4-389 of soybean cyst nematode Heterodera glycines gene Rhg4, soybean salt-tolerance gene GmSALT3
The 08067 and relevant linked marker SCN11 of soybean mosaic virus, centering Huang 57 × [the middle rich 8] F of product 95-5383 × iron2Separation
437 single plants of group are analyzed.
As a result as shown in Figure 1.Fig. 1-a: by single marking respectively to F2Group identified, the single plant number filtered out；
Fig. 1-b: the germplasm of the excellent allele of carrying at least one filtered out.
Qualification result (Fig. 1-a) display singly marked, the SCN resistance allele single plant in the site Rhg4-389 account for
20.59%, the single plant of the salt tolerant allele in 08067 site has the single plant of the SMV resistance banding pattern in the site 16.48%, SCN11 most
It is more, account for 92.91%.Chi-square Test shows the disease-resistant homozygous genotype, heterozygous genotypes, susceptible homozygous base in the site Rhg4-389
Because the single plant number of type meets 1:2:1 (X2=4.56, P=0.10 > 0.05).The equipotential in two sites of label 08067 and SCN11
Gene Isolation does not meet ratio distribution.
Based on molecular labeling centering Huang 57 × [the middle rich 8] F of product 95-5383 × iron2Single plant qualification result (Fig. 1-b), screening
Single plant 97, dual anti-type (have and only 2 kinds of excellent allelotypes) out, accounts for F2The 22.20% of single plant sum, including polymerization are anti-
Single plant 4 of soybean cyst nematode Heterodera glycines and salt tolerant, polymerize resistance to by single plant 59 of simultaneous anti-soybean cyst nematode Heterodera glycines and soybean Mosaic
Salt and the single plant of anti-soybean Mosaic 34.Filtering out three anti-type (having 3 kinds of resistant genotypes) single plants has 17, polymerization
Anti- soybean cyst nematode Heterodera glycines, salt tolerant and anti-soybean mosaic virus account for the 3.9% of F2 single plant sum.
Fig. 2 using molecular labeling centering Huang 57 × [selection of middle product 95-5383 × iron rich 8], A:M are DL2000, three
Label is respectively to F5It is identified for single plant.08067 detection 3 is salt density value type, remaining is salt-resistant type；Rhg4-389 detects 3
Feel SCN type, remaining SCN resistance type；SCN11 detection 2 is sense SMV type, remaining is SMV resistance type.
In F2After molecular marker screening (Fig. 2), since natural calamity has only retained three anti-single plant 2 and dual anti-(SCN
For heterozygosis resistance, salt tolerant and SMV are homozygous resistant) single plant 3,2015 Hainan are multiplied into plant, using 3 molecular labelings to 38
A single plant identified, totally 19 three anti-single plants, and (wherein 6 parts of sites Rhg4 are heterozygous to 19 dual anti-single plants, and 13 parts are sensitivity
Type).Successively in Hainan (2015 or 2016) and Beijing (2016,2017) breeding, 2016,2017 in Beijing to erect type single plant
It is selected.The Shunyi of Beijing in 2017 obtains the strain of 11 erect types, wherein strain 1-5 be it is three anti-(i.e. SMV resistance, salt tolerant and
SCN resistance), strain 6-10 is dual anti-type (SMV resistance and salt tolerant, SCN are heterozygous genotypes).
Embodiment 2
The detection of germ plasm resource
Material: foreign countries introduce 43 parts of soybean germplasm.
Method is the same as embodiment 1.
As a result with analysis:
Screening of the table 3 using 3 molecular labelings to 43 parts of soybean germplasms
Genotype mirror is carried out to 43 parts of soybean germplasms using 3 relevant to SCN, SMV resistance and soybean salt-tolerance labels
Fixed, the germplasm for having polymerize 3 sites as the result is shown has 8 parts, and the germplasm for having polymerize 2 sites has 18 parts, with single characteristic
Germplasm has 17 parts.
Embodiment 3
The detection of germ plasm resource
Material: 37 parts of domestic improved variety.
Method is the same as embodiment 1.
As a result with analysis:
Screening of the table 4 using 3 molecular labelings to 37 parts of soybean germplasms
Genotype mirror is carried out to 37 parts of soybean germplasms using 3 relevant to SCN, SMV resistance and soybean salt-tolerance labels
Fixed, the germplasm for having polymerize 3 sites as the result is shown only has 1 part, and the germplasm for having polymerize two sites of salt tolerant and SMV resistance has 1 part, tool
There is the germplasm of single characteristic there are 22 parts.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>application of a kind of breeding method of how anti-gene pyramiding soybean and how anti-gene pyramiding soybean
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cctccaagcc ttccacctcg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cctccaagcc ttccacctcg 20
<210> 3
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gcgggagtaa tgttatcgg 19
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gtcgtatctt gggagaggag 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cgattagctc caccaaccct 20
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ttcacgtggc cctcctatc 19
<210> 7
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cgccgcaaac tcacaggac 19
Claims (9)
1. a kind of breeding method of how anti-gene pyramiding soybean excellent germplasm, which comprises the following steps:
(1) anti-northeast mosaic virus soybean and salt tolerant soybean are hybridized, obtains F1；
(2) by the F1Hybridize with anti-soybean cyst nematode soybean, obtains F2Group；
(3) with the F of extraction2The genomic DNA of group is template, respectively with the molecular labeling of Rhg4, SALT3 and SCN11 into
Row PCR amplification, wherein the PCR product of Rhg4 using restriction endonuclease carry out digestion, obtained digestion products, SALT3PCR product and
The PCR product of SCN11 carries out electrophoresis, and the long 364bp of the PCR product of SALT3 is resistance type, and the long 564bp of the PCR product of SALT3 is
Responsive type, while the sample for generating 364bp and 564bp band is heterozygous；The long 980bp of the PCR product of SCN11 is resistance type,
The long 1070bp of PCR product is sensibility, while the sample for generating 980bp and 1070bp band is heterozygous；The digestion of Rhg4 produces
The segment of object 132bp and 273bp are resistance type, and the digestion products 369bp of Rhg4 is responsive type, while generating 132bp, 273bp
Sample with the band of 369bp is heterozygous；
(4) Rhg4, SALT3 and SCN11 molecular labeling are homozygous F2Plant and dual anti-single-plant propagation obtain F at plant3；
The dual anti-single plant is that Rhg4 Markers for Detection result is heterozygous, the Markers for Detection result of SALT3 and SCN11
For homozygous resistant type；
(5) the F3 plant is detected according to molecule labelling method in step (3), according to testing result according to the numerous of step (4)
It grows method and carries out breeding three generations, respectively obtain F4、F5And F6Group；
(6) by F5And F6Group is equal using molecule labelling method detection, the molecular labeling of Rhg4, SALT3 and SCN11 in step (3)
Plant for resistance type is how anti-gene pyramiding soybean excellent germplasm.
2. breeding method according to claim 1, which is characterized in that anti-northeast mosaic virus is big in the step (1)
Beans are middle product 95-5383；
Salt tolerant soybean is soybean salt-tolerance kind iron rich No. 8 in the step (1).
3. breeding method according to claim 1, which is characterized in that anti-soybean cyst nematode product in the step (2)
Huang 57 in kind.
4. breeding method according to claim 1, which is characterized in that the Markers for Detection of Rhg4 in the step (3)
It is Rhg4-389 with primer；
The forward primer of Rhg4-389 has such as SEQ ID No.1 in sequence table；
The reverse primer of Rhg4-389 has such as SEQ ID No.2 in sequence table；
The response procedures of the Markers for Detection of Rhg4-389 are as follows:
5. breeding method according to claim 1 or 4, it is characterised in that label Rhg4-389 needs after PCR amplification
BsrBI carries out digestion；10 μ L:PCR product of endonuclease reaction system, 5 μ L, 3U restriction endonuclease, 1.5 μ LNEB buffers；The program of digestion:
37 DEG C of constant temperature 30min.
6. breeding method according to claim 1, which is characterized in that the Markers for Detection of SALT3 in the step (3)
It is 08067 with primer；
08067 forward primer has such as SEQ ID No.3 in sequence table；
08067 reverse primer has such as SEQ ID No.4 in sequence table；
The response procedures of the Markers for Detection of the SALT3 are as follows:
7. breeding method according to claim 1, which is characterized in that the Markers for Detection of SCN11 in the step (3)
It is SCN11 with primer；
The forward primer of SCN11 has such as SEQ ID No.5 in sequence table；
The reverse primer of SCN11 has such as SEQ ID No.6 in sequence table；
The response procedures of the Markers for Detection of SCN11 are as follows:
8. the how anti-gene pyramiding soybean excellent germplasm that claim 1~7 any one the method breeding obtains is in not contemporaneity
Application in the detection in generation.
9. the how anti-gene pyramiding soybean excellent germplasm that claim 1~7 any one the method breeding obtains is provided in germplasm
Application in the detection in source.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810870691.9A CN109022607A (en) | 2018-08-02 | 2018-08-02 | A kind of breeding method of how anti-gene pyramiding soybean and the application of how anti-gene pyramiding soybean |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810870691.9A CN109022607A (en) | 2018-08-02 | 2018-08-02 | A kind of breeding method of how anti-gene pyramiding soybean and the application of how anti-gene pyramiding soybean |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109022607A true CN109022607A (en) | 2018-12-18 |
Family
ID=64648667
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810870691.9A Pending CN109022607A (en) | 2018-08-02 | 2018-08-02 | A kind of breeding method of how anti-gene pyramiding soybean and the application of how anti-gene pyramiding soybean |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109022607A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102808021A (en) * | 2012-07-03 | 2012-12-05 | 南京农业大学 | Method for polymerizing and screening anti-soybean mosaic virus genes for soybean |
| CN104774937A (en) * | 2015-04-03 | 2015-07-15 | 中国农业科学院作物科学研究所 | Method for aided detection of heterodera glycines resistance and special primers for detection of heterodera glycines resistance |
| CN106029883A (en) * | 2014-02-17 | 2016-10-12 | 国立研究开发法人国际农林水产业研究中心 | Salt-tolerance-controlling gene qNaCl3 on soybean chromosome 3, and use thereof |
-
2018
- 2018-08-02 CN CN201810870691.9A patent/CN109022607A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102808021A (en) * | 2012-07-03 | 2012-12-05 | 南京农业大学 | Method for polymerizing and screening anti-soybean mosaic virus genes for soybean |
| CN106029883A (en) * | 2014-02-17 | 2016-10-12 | 国立研究开发法人国际农林水产业研究中心 | Salt-tolerance-controlling gene qNaCl3 on soybean chromosome 3, and use thereof |
| CN104774937A (en) * | 2015-04-03 | 2015-07-15 | 中国农业科学院作物科学研究所 | Method for aided detection of heterodera glycines resistance and special primers for detection of heterodera glycines resistance |
Non-Patent Citations (3)
| Title |
|---|
| YING-HUI LI等: "Dissecting the Genetic Basis of Resistance to Soybean Cyst Nematode Combining Linkage and Association Mapping", 《PLANT GENOME》 * |
| 叶俊华等: "大豆引进种质抗胞囊线虫病、抗花叶病毒病和耐盐基因型鉴定及优异等位基因聚合种质筛选", 《作物学报》 * |
| 张维耀: "优良基因聚合利用及大粒大豆绥农50的选育", 《黑龙江农业科学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Aneja et al. | Sequence-related amplified polymorphism (SRAP) molecular marker system and its applications in crop improvement | |
| Li et al. | The origin of cultivated citrus as inferred from internal transcribed spacer and chloroplast DNA sequence and amplified fragment length polymorphism fingerprints | |
| Simonetti et al. | Quantitative trait loci influencing free-threshing habit in tetraploid wheats | |
| CN103305510A (en) | Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof | |
| CN104894124B (en) | The ISSR SCAR marks and its authentication method of the fragrant green vegetables in Wujiang can be identified | |
| CN109628627A (en) | The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm | |
| CN106555001A (en) | A kind of molecular labeling of rice blast resistant gene and its application | |
| KR101409012B1 (en) | Specific primers for Pines distinction, analysis kit using its primers, and Pines distinciton method using thereof | |
| CN109321671A (en) | Wheat stripe rust resisting ospc gene QYr.sicau-1B-1 SSR molecular marker and application | |
| Ranade et al. | Gene tagging with random amplified polymorphic DNA (RAPD) markers for molecular breeding in plants | |
| CN102747070A (en) | Two CAPs markers tightly linked with muskmelon anti-powdery mildew gene Pm-AN and its application method | |
| CN103320427B (en) | Method for assisting in identifying resistance of soybeans to soybean mosaic viruses | |
| CN104789648B (en) | Identify molecular labeling and its application of the section haplotypes of rice CMS restoring genes Rf 1 | |
| CN103789419A (en) | Co-dominance tag primer group for identifying allele type of rice photo-thermo-sensitive genic male-sterile gene p/tms12-1, and applications thereof | |
| CN106460063A (en) | SNP combination for Chinese cabbage genetic resources diversity analysis and molecular breeding and application thereof | |
| CN115976248B (en) | Method for specifically identifying populus euphratica pie plants and kit used by method | |
| CN112159861B (en) | SNP marker linked with cucumber watermelon mosaic virus resistant gene wmv, kit and method thereof | |
| CN104762298A (en) | Rice seedling-stage salt-tolerant gene qST11 and molecular marker method thereof | |
| CN109022607A (en) | A kind of breeding method of how anti-gene pyramiding soybean and the application of how anti-gene pyramiding soybean | |
| Ishikawa et al. | Genetic erosion from modern varieties into traditional upland rice cultivars (Oryza sativa L.) in northern Thailand | |
| CN108085406A (en) | A kind of identification method in upland cotton precocity molecular breeding correlation SSR marker site | |
| CN112159862A (en) | SNP marker linked with cucumber mosaic virus resistance gene cmv as well as kit and method thereof | |
| CN110257553A (en) | A kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm | |
| CN111004857A (en) | Molecular marker primer of major QTL (quantitative trait locus) site of soybean branch number and application of molecular marker primer | |
| CN109652579A (en) | The codominant marker and its detection method of rice blast resistant gene Pi2 and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |