CN108624706A - Molecular labeling assists method of the quick breeding corn without tip of a leaf self-mating system - Google Patents
Molecular labeling assists method of the quick breeding corn without tip of a leaf self-mating system Download PDFInfo
- Publication number
- CN108624706A CN108624706A CN201810151638.3A CN201810151638A CN108624706A CN 108624706 A CN108624706 A CN 108624706A CN 201810151638 A CN201810151638 A CN 201810151638A CN 108624706 A CN108624706 A CN 108624706A
- Authority
- CN
- China
- Prior art keywords
- molecular labeling
- seq
- tip
- selection
- leaf
- 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.)
- Granted
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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides molecular labelings to assist method of the quick breeding corn without tip of a leaf self-mating system.The present invention assists foreground and background selection, selfing to obtain corn without tip of a leaf self-mating system using two SSR molecular markers with corn liguliss gene lg1 close linkages, by hybridization, three times backcrossing and molecular labeling.The method of the present invention is during backcross transformation by carrying out evaluation and screening to heterozygous genotypes Lg1lg1, remove the step of being selfed to backcross progeny from, compared with conventional breeding methods, the accuracy of selection can be not only improved, but also it is the time that can save the choosing of nearly half.Meanwhile the problem of period length is replied for traditional backcross transformation method background, the method for being combined the selection of molecular labeling auxiliary background, it is only necessary to be returned for 3 generations, be selfed for 1 generation, you can obtain genetic background substantially resume to recurrent parent without tip of a leaf Improved lines.
Description
Technical field
The present invention relates to molecular genetics fields, are selfed without the tip of a leaf more particularly to molecular labeling auxiliary quick breeding corn
The method of system, and in particular, to carry out assist-breeding using SSR molecular marker chain corn liguliss gene lg1.
Background technology
In recent years, with the resistance to dense planting of corn, the proposition of breeding of high photosynthetic efficiency target, plant type is compact, is rushed on blade etc. becomes
The important phenotypic character of corn breeding man concern.No tip of a leaf germplasm is due on the disappearance of tip of a leaf auricle, leaf sheath phimosis, upright blade
Punching, photosynthetic area is big, the efficiency of light energy utilization is high, is to carry out the breeding hybridized valuable source of resistance to dense planting.The corn having now been found that
No tip of a leaf character has dominant and two kinds recessive, and wherein lg1 genes are recessive liguliss genes, is located at Article 2 the short arm of a chromosome
End.
Using the method for backcross transformation, the orderly improvement of no tip of a leaf character lg1 genes may be implemented.Utilize traditional breeding
Method no tip of a leaf character lg1 channel genes to recurrent parent Breeding of Inbred Lines when, since the character is Recessive genes control,
Need to backcrossing 1 generation selfing, in the self progeny of separation selection continue to be returned without tip of a leaf plant, then be selfed, be returned, again from
Hand over, until being returned mostly almost the same with recurrent parent for genetic background, then be selfed for 2 generations, could acquired character stabilization without the tip of a leaf
Self-mating system.As it can be seen that utilizing conventional backcross transformation method, for the character of Recessive genes control, need using backcrossing, certainly
Alternate method is handed over, objective trait could accurately be chosen, choosing is that the time is long, efficiency is low.In addition, being educated using only conventional
It is more than generation to generally require backcrossing 5 for kind method, can just substantially resume to the genetic background of recurrent parent, the period is long.
Molecular mark, independent of Phenotypic Selection, i.e., not by environment, interaction of genes, gene prediction programs
Etc. factors influence, but directly genotype is selected, thus breeding efficiency can be greatly improved.Simple repeated sequence
(simple sequence repeats, SSR) be it is a kind of be widely present in it is on genome, by several nucleotide repeating units
The tandem repetitive sequence of composition.Due to its a large amount of distribution in the genome, polymorphism is high, and operating technology is simple, and expense is low
It is honest and clean, it is widely used in marker assisted selection.Therefore, the molecular labeling with liguliss gene close linkage is filtered out, profit
Corn liguliss gene is selected with molecular labeling, selects to accelerate recurrent parent something lost in combination with molecular labeling auxiliary background
No tip of a leaf corn inbred lines are had unique advantage by the reply speed for passing background.
Invention content
The purpose of the present invention is to provide using corn liguliss gene lg1 close linkages SSR molecular labelings, in conjunction with
The selection of molecular labeling auxiliary background carries out method of the corn without tip of a leaf inbred line breeding.
Applicant utilizes Maize genome database MaizeGDB (http://www.maizegdb.org/), selection with
Closer positioned at corn Article 2 the short arm of a chromosome end lg1 gene genetics distance, upstream and downstream section amounts to 9 SSR molecule marks
Note.Extract B73 without tip of a leaf mutant rla1 (corn EMS mutant library build and mutant Preliminary Identification, Agriculture of Anhui science,
2014,42 (11):3162-3165) and the genomic DNA of selfing based material (capital 2416 and capital 92), using above-mentioned molecular labeling,
Carry out the optimization and screening of PCR reaction conditions, it is final determine B73 without tip of a leaf mutant rla1 respectively with capital 2416 and capital 92
Between there is polymorphism, resolution ratio is good, and two closer SSR molecular labelings of distance lg1 gene genetics distance.
In Beijing base (spring sowing), by receptor self-mating system capital 2416 and capital 92 as female parent, it is mutated respectively without the tip of a leaf with B73
Body rla1 carries out hybridization and prepares F1.In Sanya base (autumn sowing) plantation F1 selfings, 2 corresponding F2 groups are harvested.Utilize rla1
× capital 2416, the F2 segregating populations in rla1 × capital 92, the linkage relationship of evaluation of markers and phenotype are screened with verifying the present invention
To 2 SSR markers can be used for the field molecular marker assisted selection of no tip of a leaf character.
The present invention provides the SSR molecular markers of corn liguliss gene lg1 close linkages, by nucleotide sequence such as SEQ
Primer pair PCR amplifications shown in ID NO.1-2 or SEQ ID NO.3-4 obtain.
Further, the present invention provides molecular labeling assist method of the quick breeding corn without tip of a leaf self-mating system, be
SSR molecular marker is carried out to heterozygous genotypes Lg1lg1 during backcross transformation and assists foreground selection, the SSR used in foreground selection
Molecular labeling is obtained by nucleotide sequence primer pair PCR amplification as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.
Method of the present invention, includes the following steps：
(1) acquisition of hybrid generation F1：Nonrecurrent parent does donor, recurrent parent does receptor hybridization and assembles F1 generation, harvests
The seed of F1 generation；The seed for planting F1 generation, obtains F1 generation plant；The nonrecurrent parent is the gene containing lg1 with nothing
The corn inbred line of tip of a leaf character；
(2) it is returned the acquisition in generation first time BC1；Utilize nucleotide sequence such as SEQ ID NO.1- 2 and SEQ ID NO.3-
Molecular labeling shown in 4 carries out molecular labeling to BC1 generations and assists foreground selection；
(3) it is returned the acquisition in second of BC2 generation；Utilize nucleotide sequence such as SEQ ID NO.1- 2 and SEQ ID NO.3-
Molecular labeling shown in 4 carries out molecular labeling to BC2 generations simultaneously and assists foreground selection；
(4) it is returned the acquisition in second of BC3 generation；Utilize nucleotide sequence such as SEQ ID NO.1- 2 and SEQ ID NO.3-
Molecular labeling shown in 4 carries out molecular labeling to BC3 generations simultaneously and assists foreground selection；
(5) BC3F1 being selected in by foreground selection is selfed to obtain BC3F2, BC3F1 is for group for plantation for single plant
Seed selects the plant without tip of a leaf character to continue to be selfed, that is, obtains genetic background and recurrent parent base in BC3F2 is for plant
This is consistent, while showing the new self-mating system without tip of a leaf character.
Carrying out foreground selection standard using molecular labeling auxiliary during step (2)-(3) backcross transformation is：Simultaneous selection
Nucleotide sequence molecular labeling as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4 carries out, nucleotide sequence such as SEQ ID
The molecular labeling of NO.1-2, amplified production size is 193/230bp, while nucleotide sequence is as shown in SEQ ID NO.3-4
When molecular labeling differentiates, amplified production size is 153/157bp, then corn to be measured has lg1 genes, can be used as foreground selection
Selected single plant.
It is using the standard of molecular labeling auxiliary progress foreground selection during step (4) backcross transformation：When selection nucleosides
When the molecular labeling of acid sequence such as SEQ ID NO.1-2 differentiates, if amplified production size is 193/230bp, and nucleotide is selected
Sequence molecular labeling as shown in SEQ ID NO.3-4 differentiates, if amplified production size is 153/157bp, corn tool to be measured
There are lg1 genes, can be used for the selfing of step (5).
Further include that the selection of molecular labeling auxiliary background is done to BC1 generations, method is after step (2) foreground selection：Respectively with
The DNA of nonrecurrent parent and recurrent parent is template, utilizes Chinese patent ZL's 201310751112.6 (CN104285776B)
40 pairs of SSR primers carry out PCR amplification respectively, and there are the primer of polymorphism, profits between nonrecurrent parent and recurrent parent for screening
The single plant being selected in the primer pair foreground selection filtered out is detected, the corresponding PCR amplification collection of illustrative plates obtained with recurrent parent into
Row compares, and chooses female parent of the preceding 10-15 single plant minimum with recurrent parent difference number of alleles as next step backcrossing.
Further include that the selection of molecular labeling auxiliary background is done to BC2 generations, method is after step (3) foreground selection：It utilizes
BC2, for the SSR primer pair different from recurrent parent amplification banding pattern, foreground is assisted to molecular labeling for the corresponding female parent BC1 of single plant
The selected single plant of selection carries out PCR amplification；It is compared with the corresponding PCR amplification collection of illustrative plates that recurrent parent obtains, selection and samsara
The single plant of parent's indifference heteroallele is as the female parent being further returned.
In the present invention, nonrecurrent parent is selected from rla1, and recurrent parent is capital 2416, capital 92.
In the embodiment of present aspect, there are polymorphic between nonrecurrent parent rla1 and recurrent parent capital 2416 for screening
The primer of property be respectively umc2007y4, bnlg1940k7, bnlg2291k4, bnlg161k8, bnlg1702k1,
umc1545y2、umc1125y3、bnlg240k1、phi080k15、 umc1506k12、umc1147y4、bnlg1671y17、
phi96100y1、umc1536k9、 bnlg1520k1、umc1489y3、bnlg490y4、umc1999y3、umc2115k3、
umc1429y7、bnlg249k2、umc2160k3、bnlg2235y5、phi233376y1、 umc2084w2、umc1231k4、
phi041y6、umc2163w3。
In the method for the present invention, nucleotide sequence molecular labeling as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4 is utilized
When carrying out auxiliary foreground selection, the PCR amplification condition that uses is 94 DEG C, 3min；94 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 30s,
Totally 30 cycles；72℃ 7min.
The present invention also provides application of the above-mentioned selection in corn germ plasm resource improvement.
The beneficial effects of the present invention are：The present invention, which utilizes, is suitable for specific improvement group, closely connects with lg1 genes
The codominant marker of lock carries out evaluation and screening during backcross transformation to heterozygous genotypes Lg1lg1, removes to backcrossing
The step of offspring is selfed can not only improve the accuracy of selection, but also can save nearly one compared with conventional breeding methods
Half choosing is the time.Meanwhile the problem of period length is replied for traditional backcross transformation method background, it is auxiliary to be combined molecular labeling
The method for helping Foreground selection, it is only necessary to be returned for 3 generations, be selfed for 1 generation, you can obtain the nothing that genetic background substantially resumes to recurrent parent
Tip of a leaf Improved lines.
Specific implementation mode
The content that following embodiment further illustrates the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Corn germ plasm resource used in the embodiment of the present invention comes from Corn Rearch Center, Beijing Farming ＆ Forestry Research Academy.If not
It specializes, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Screening of 1 corn of embodiment without tip of a leaf lg1 gene SSR molecular markers
1, Maize genome database MaizeGDB (http are utilized://www.maizegdb.org/), it selects and is located at jade
Rice Article 2 the short arm of a chromosome end lg1 gene genetics distance is closer, and upstream and downstream section amounts to 9 SSR molecular markers.
The position of 1 liguliss gene lg1 upstream and downstream of table label and primer sequence
Note：Position indicates hereditary position opposite in the IBM2 2008Neighbors Frame 2 on the websites MaizeGDB
It sets, wherein lg1 genes are 50.9
Genomic DNAs of the B73 without tip of a leaf mutant rla1 and selfing based material (capital 2416 and capital 92) is extracted, utilization is above-mentioned
Molecular labeling, carries out the optimization and screening of PCR reaction conditions, it is final determine two no tip of a leaf rla1 and have tip of a leaf capital 2416,
There is polymorphism, resolution ratio is good, and the closer SSR molecular marker of distance lg1 gene genetic distances between capital 92.The two SSR
Molecular labeling is isu488638 and umc1542, the primer pair sequence for two label isu488638 and umc1542 of PCR amplification
Row are respectively as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4.
2, two SSR molecular markers and corn are without tip of a leaf lg1 gene linkage relationship analyses
It is carried out respectively with the rla1 of no tip of a leaf by self-mating system capital 2416 and capital 92 as female parent in Beijing base (spring sowing)
F1 is prepared in hybridization.In Sanya base (autumn sowing) plantation F1 selfings, corresponding F2 groups are harvested.Utilize rla1 × capital 2416, rla1
The F2 segregating populations in × capital 92, the linkage relationship of analysis and phenotype.
With self-mating system rla1, capital 2416 and capital 92 and 2 F2 segregating populations, detection molecules mark isu488638 and
The linkage relationship of umc1542 and phenotype.
79 plants of the F2 segregating populations in rla1 × capital 2416, wherein 26 plants of field phenotypes are no tip of a leaf, 53 plants are to have the tip of a leaf,
81 plants of the F2 segregating populations in rla1 × capital 92, wherein 25 plants of field phenotypes are no tip of a leaf, 56 plants are to have the tip of a leaf.Extract genome
DNA is detected using isu488638 and umc1542.The results show that the two labels and lg1 gene close linkages, are used
When isu488638 carries out PCR detections, 160 plants of segregating populations (79 plants of the F2 segregating populations and rla1 in rla1 × capital 2416 ×
81 plants of the F2 segregating populations in capital 92) in there are 7 pnca gene types not to be inconsistent with phenotype；When being detected with umc1542, detached at 160 plants
There are 3 pnca gene types not to be inconsistent with phenotype and (refer to table 2) in group；It is carried out at the same time when with two labels of isu488638 and umc1542
When assisted Selection, genotype fits like a glove with phenotype, i.e., isu488638 genotype is 230/230 and umc1542 genotype is
157/157 strain number is 45 plants, and variable rate technology is no tip of a leaf；Isu488638 genotype be 193/230 or 193/193 and
The strain number that umc1542 genotype is 153/157 or 153/153 is 105 plants, and variable rate technology is to have the tip of a leaf, explanation
It is auxiliary that isu488638 and umc1542 can be used for the serial molecular labeling of the self-mating system backcross transformation without tip of a leaf character such as capital 2416, capital 92
Help selection.
2 isu488638 and umc1542 genotype of table and whether there is or not the comparisons of tip of a leaf phenotype
Selection and breeding of 2 corn of embodiment without tip of a leaf self-mating system
1, the acquisition of hybrid generation F1
The First Year summer, with corn inbred line rla1 (nonrecurrent parent, the no tip of a leaf) does donor, capital 2416 (is purchased from Beijing
City agricultural and forest science institute corn research center) (recurrent parent has the tip of a leaf) do receptor hybridization assemble F1In generation, harvests F1The seed in generation.
Winter in the same year Hainan first generation plants F1The seed in generation, obtains F1For plant.
2, it is returned the acquisition in a BC1 generation
(1) it is returned
Female parent is done with F1 generation plant, continues to be returned with recurrent parent capital 2416, harvests the seed in BC1 generations.Winter in the same year Hainan
The second generation, BC1 is for the seed of group for plantation, obtains BC1 for group.
(2) BC1 assists foreground selection for molecular labeling
To, for single plant, being examined first with objective trait selected marker by the tentatively selected BC1 of field Phenotypic Selection
It surveys, the molecular labeling of selected nucleotide sequence such as SEQ ID NO.1-2 differentiates, if amplified production size is 193/230bp, and
When selected nucleotide sequence molecular labeling as shown in SEQ ID NO.3-4 differentiates, if amplified production size is 153/157bp,
Then corn to be measured has lg1 genes, can be used as the selected single plant of foreground selection, carries out the molecular labeling auxiliary background choosing of next step
It selects.The present embodiment carries out assisted Selection using above-mentioned 2 molecular labelings simultaneously, and both comprehensive result is selected, to obtain
Most accurate the selection result.
The genomic DNA of sample is extracted using CTAB, concentration dilution to 200ng/ μ l.The molecular labeling primer of synthesis is with going out
The ddH of bacterium2O is diluted to final concentration of 10pM, and the DNA of sample to be tested is carried out PCR amplification.PCR reactions are 20 μ L systems, DNA
1 μ l of template, upstream and downstream mixed primer 1 μ l, MIX (containing 2 × Buffer) 10 μ l, ddH2O is 8 μ L.Response procedures：It is pre- to become
Property, 94 DEG C, 3min；PCR cycle 30 times (denaturation, 94 DEG C, 30 s；Annealing, 55 DEG C, 30s；Extend, 72 DEG C, 30s)；Finally prolong
It stretches, 72 DEG C, 7min.Amplified production is detected using 3730XL DNA analysis instrument.
(3) BC1 is selected for molecular labeling auxiliary background
Using rla1 and the DNA in capital 2416 as template, to 40 pairs of SSR primers (referring to Chinese patent ZL 2,013 1
0751112.6/CN (104285776B) carries out PCR amplification and electrophoresis detection, and screening has the primer of polymorphism between.
Compared by amplified production electrophoresis pattern, find umc2007y4, bnlg1940k7, bnlg2291k4, bnlg161k8,
bnlg1702k1、 umc1545y2、umc1125y3、bnlg240k1、phi080k15umc1506k12、 umc1147y4、
bnlg1671y17、phi96100y1、umc1536k9、bnlg1520k1、 umc1489y3、bnlg490y4、umc1999y3、
umc2115k3、umc1429y7、 bnlg249k2、umc2160k3、bnlg2235y5、phi233376y1、umc2084w2、
28 pairs of primers such as umc1231k4, phi041y6, umc2163w3 have differences on rla1 and capital 2416.
It is detected using 28 pairs of differential primers single plant selected to molecular labeling auxiliary foreground selection, is obtained with capital 2416
Correspondence PCR amplification collection of illustrative plates be compared.
According to amplification, the preceding 10-15 single plant minimum with 2416 difference number of alleles of capital is chosen as further
The female parent of backcrossing.
3, it is returned the acquisition in quadratic B C2 generations
(1) it is returned
It chooses and makees female parent with the minimum preceding 10-15 single plant of 2416 difference number of alleles of capital, using capital 2416 as male parent,
Assemble BC2 generations.
In summer next year, plantation BC2 is for the seed of group, and by head progeny row field planting, each head progeny row plants 50 plants.
(2) BC2 assists foreground selection for molecular labeling
It, will to by the tentatively selected single plant of field Phenotypic Selection, being detected first with objective trait selected marker
Nucleotide sequence molecular labeling amplified production as shown in SEQ ID NO.1-2 be 193/230bp while nucleotide sequence such as
Molecular labeling amplified production shown in SEQ ID NO.3-4 is that the single plant of 153/157bp is picked out, as entering for foreground selection
Menu strain carries out the molecular labeling auxiliary background selection of next step.
(3) BC2 is selected for molecular labeling auxiliary background
Using BC2 for the corresponding female parent BC1 of single plant for the SSR primer pair different from the amplification banding pattern of capital 2416, to molecule mark
The selected single plant of note auxiliary foreground selection carries out PCR amplification；It is compared with the corresponding PCR amplification collection of illustrative plates that capital 2416 obtains.Choosing
Take the single plant with 2416 indifference heteroallele of capital as the female parent being further returned.
4, it is returned the acquisition in BC3 generations three times
(1) it is returned
Make female parent with the single plant with 2416 indifference heteroallele of capital in BC2 generations, using capital 2416 as male parent, assembles
BC3F1 generations.
Seeds of the BC3F1 for group is planted, by head progeny row field planting, each head progeny row plants 50 plants.
(2) BC3 assists foreground selection for molecular labeling
To the single plant being selected in by field Phenotypic Selection, it is detected first with objective trait selected marker, by nucleosides
Acid sequence molecular labeling amplified production as shown in SEQ ID NO.1-2 is 193/230bp while nucleotide sequence such as SEQ
Molecular labeling amplified production shown in ID NO.3-4 is that the single plant of 153/157bp is picked out into next-generation.
5, the acquisition in BC3F2 generations
(1) it is selfed：The BC3F1 being selected in by foreground selection is selfed to obtain BC3F2 for single plant.Field planting
BC3F1 is for the seed of group, and by head progeny row field planting, each head progeny row plants 50 plants.
(2) BC3F2 is for objective trait Phenotypic Selection
It selects the plant without tip of a leaf character to continue to be selfed in BC3F2 is for plant, that is, obtains genetic background and capital 2416
It is almost the same, while showing the new self-mating system without tip of a leaf character.
Although having used general explanation, specific implementation mode and experiment above, the present invention is described in detail,
But it can make some to it on the basis of the present invention to modify or improve, this is apparent to those skilled in the art
's.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.
Sequence table
<110>Beijing City Agriculture and Forestry Institute
<120>Molecular labeling assists method of the quick breeding corn without tip of a leaf self-mating system
<130> KHP171114006.1
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
aggcaactcc tgtgtctgtg t 21
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
catgatcgcc cactcctt 18
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
taaagctatg atggcacttg caga 24
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
catatttgcc tttgcccttt tgta 24
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gccctgttat tggaacagtt tacg 24
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cgtcgtcttc gacatgtact tcac 24
<210> 7
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ttcttgcttg tctctagcag ctt 23
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
tgtggcgatg tccatgatt 19
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
tatcttcaga cccaaacatc gtcc 24
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
gtcgattgat ttcccgatgt taaa 24
<210> 11
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gagatcaccg gctagttaga gga 23
<210> 12
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
gtatggttgg gtacccgtct ttcta 25
<210> 13
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
catacgtaat cctacgcgac aaca 24
<210> 14
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
ttgtgaaaca aaaagaaagc acga 24
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
acatcagagc acaccactgc 20
<210> 16
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
tgtgggatgt ggtctctaac g 21
<210> 17
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
gcaaatgttt tcacacacac g 21
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
gtgcagaatg caggcaatag 20
Claims (10)
1. molecular labeling assists method of the quick breeding corn without tip of a leaf self-mating system, which is characterized in that during backcross transformation
SSR molecular marker is carried out to heterozygous genotypes Lg1lg1 and assists foreground selection, the SSR molecular marker used in foreground selection is by nucleosides
Acid sequence primer pair PCR amplification as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4 obtains.
2. the method as described in claim 1, which is characterized in that include the following steps：
(1) acquisition of hybrid generation F1：Nonrecurrent parent does donor, recurrent parent does receptor hybridization and assembles F1 generation, harvests F1 generation
Seed；The seed for planting F1 generation, obtains F1 generation plant；The nonrecurrent parent is the gene containing lg1 with no tip of a leaf
Character corn inbred line；
(2) it is returned the acquisition in generation first time BC1；Using nucleotide sequence as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4
Molecular labeling carries out molecular labeling to BC1 generations and assists foreground selection；
(3) it is returned the acquisition in second of BC2 generation；Using nucleotide sequence as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4
Molecular labeling carries out molecular labeling to BC2 generations and assists foreground selection；
(4) it is returned the acquisition in third time BC3 generations；Using nucleotide sequence as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4
Molecular labeling carries out molecular labeling to BC3 generations and assists foreground selection；
(5) BC3F1 being selected in by foreground selection is selfed to obtain BC3F2 for single plant, plants seeds of the BC3F2 for group,
It selects the plant without tip of a leaf character to continue to be selfed in BC3F2 is for plant, that is, obtains genetic background and recurrent parent basic one
It causes, while showing the new self-mating system without tip of a leaf character.
3. method as claimed in claim 2, which is characterized in that utilize molecular labeling during step (2)-(3) backcross transformation
Auxiliary carries out foreground selection standard：When the molecular labeling of selected nucleotide sequence such as SEQ ID NO.1-2 differentiates, if amplification production
Object size is 193/230bp；
And at the same time when selected nucleotide sequence molecular labeling as shown in SEQ ID NO.3-4 differentiates, if amplified production size
For 153/157bp；
Above-mentioned two situations are provided simultaneously with, then corn to be measured has lg1 genes, can be used as the selected single plant of foreground selection.
4. method as claimed in claim 2, which is characterized in that assisted using molecular labeling during step (4) backcross transformation
Carry out foreground selection standard be：When the molecular labeling of selected nucleotide sequence such as SEQ ID NO.1-2 differentiates, if amplified production
Size is 193/230bp；
And at the same time when selected nucleotide sequence molecular labeling as shown in SEQ ID NO.3-4 differentiates, if amplified production size
For 153/157bp；
Above-mentioned two situations are provided simultaneously with, then corn to be measured has lg1 genes, can be used for the selfing of step (5).
5. method as claimed in claim 2, which is characterized in that further include dividing BC1 generations after step (2) foreground selection
Son label auxiliary background selection, method are：Respectively using the DNA of nonrecurrent parent and recurrent parent as template, Chinese patent is utilized
40 pairs of SSR primers disclosed in CN104285776B carry out PCR amplification respectively, and screening is deposited between nonrecurrent parent and recurrent parent
In the primer of polymorphism, the single plant being selected in using the primer pair foreground selection filtered out is detected, is obtained with recurrent parent
Corresponding PCR amplification collection of illustrative plates is compared, and is chosen under being used as with the minimum preceding 10-15 single plant of recurrent parent difference number of alleles
The female parent of one step backcrossing.
6. method as claimed in claim 5, which is characterized in that further include dividing BC2 generations after step (3) foreground selection
Son label auxiliary background selection, method are：It is different from recurrent parent amplification banding pattern for single plant corresponding female parent BC1 generations using BC2
SSR primer pairs, the single plant selected to molecular labeling auxiliary foreground selection carry out PCR amplification；It is obtained with recurrent parent corresponding
PCR amplification collection of illustrative plates is compared, and chooses the single plant with recurrent parent indifference heteroallele as the female parent being further returned.
7. the method as described in claim 2-6 is any, which is characterized in that nonrecurrent parent is B73 without tip of a leaf mutant rla1,
Recurrent parent is capital 2416, capital 92.
8. method as claimed in claim 5, which is characterized in that screening is deposited between nonrecurrent parent and recurrent parent capital 2416
The primer of polymorphism be respectively umc2007y4, bnlg1940k7, bnlg2291k4, bnlg161k8, bnlg1702k1,
umc1545y2、umc1125y3、bnlg240k1、phi080k15、umc1506k12、umc1147y4、bnlg1671y17、
phi96100y1、umc1536k9、bnlg1520k1、umc1489y3、bnlg490y4、umc1999y3、umc2115k3、
umc1429y7、bnlg249k2、umc2160k3、bnlg2235y5、phi233376y1、umc2084w2、umc1231k4、
phi041y6、umc2163w3。
9. such as claim 1-6,8 any methods, which is characterized in that utilize nucleotide sequence such as SEQ ID NO.1-2
When carrying out auxiliary foreground selection with molecular labeling shown in SEQ ID NO.3-4, the PCR amplification condition that uses is 94 DEG C, 3min；94
DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 30s, totally 30 recycle；72℃7min.
10. application of any methods of claim 1-9 in corn germ plasm resource improvement.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810151638.3A CN108624706B (en) | 2018-02-14 | 2018-02-14 | Molecular marker assisted method for rapidly breeding maize leafless tongue inbred line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810151638.3A CN108624706B (en) | 2018-02-14 | 2018-02-14 | Molecular marker assisted method for rapidly breeding maize leafless tongue inbred line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108624706A true CN108624706A (en) | 2018-10-09 |
| CN108624706B CN108624706B (en) | 2021-04-27 |
Family
ID=63706082
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810151638.3A Active CN108624706B (en) | 2018-02-14 | 2018-02-14 | Molecular marker assisted method for rapidly breeding maize leafless tongue inbred line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108624706B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110777216A (en) * | 2019-11-27 | 2020-02-11 | 北京市农林科学院 | Method for identifying purity of Jingke waxy 2000 corn hybrid based on SNP marker |
| CN113999931A (en) * | 2021-09-03 | 2022-02-01 | 泰安市农业科学研究院 | Method for auxiliary breeding by using molecular markers related to Chinese cabbage flower colors |
| CN114667923A (en) * | 2022-03-31 | 2022-06-28 | 山东省农业科学院 | TuMV-resistant near-isogenic line of Chinese cabbage as well as construction method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101138313A (en) * | 2007-07-20 | 2008-03-12 | 山东大学 | Maize inbred line resistant to MRDV bred by using molecule making |
-
2018
- 2018-02-14 CN CN201810151638.3A patent/CN108624706B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101138313A (en) * | 2007-07-20 | 2008-03-12 | 山东大学 | Maize inbred line resistant to MRDV bred by using molecule making |
Non-Patent Citations (1)
| Title |
|---|
| 郭莹: "利用不同F2群体定位玉米株型性状的QTL", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110777216A (en) * | 2019-11-27 | 2020-02-11 | 北京市农林科学院 | Method for identifying purity of Jingke waxy 2000 corn hybrid based on SNP marker |
| CN110777216B (en) * | 2019-11-27 | 2022-07-01 | 北京市农林科学院 | Method for identifying purity of Jingke waxy 2000 corn hybrid based on SNP marker |
| CN113999931A (en) * | 2021-09-03 | 2022-02-01 | 泰安市农业科学研究院 | Method for auxiliary breeding by using molecular markers related to Chinese cabbage flower colors |
| CN114667923A (en) * | 2022-03-31 | 2022-06-28 | 山东省农业科学院 | TuMV-resistant near-isogenic line of Chinese cabbage as well as construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108624706B (en) | 2021-04-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10301644B2 (en) | Methods and compositions for Goss' Wilt resistance in corn | |
| Kordrostami et al. | Molecular markers in plants: concepts and applications | |
| Singh et al. | Evaluation of microsatellite markers for genetic diversity analysis among sugarcane species and commercial hybrids | |
| CN108779459A (en) | Cotton whole genome snp chip and its application | |
| CN112593007B (en) | SNP molecular marker linked with wheat grain length QTL and application thereof | |
| CN112795692B (en) | Molecular marker linked with corn plant height and application thereof | |
| CN108624706A (en) | Molecular labeling assists method of the quick breeding corn without tip of a leaf self-mating system | |
| US20170022574A1 (en) | Molecular markers associated with haploid induction in zea mays | |
| CN108476970B (en) | Molecular marker assisted method for quickly reducing included angle of corn leaves to improve Beijing agriculture 728 plant type | |
| BR112017026015B1 (en) | METHODS FOR SELECTING A CORN PLANT WITH RESISTANCE TO STALK ROT CAUSED BY ANTHRACNOSIS AND METHOD FOR INTROGRESSING A QTL ALLELE ASSOCIATED WITH RESISTANCE TO STALK ROT CAUSED BY ANTHRACNOSIS INTO A CORN PLANT | |
| US20240049666A1 (en) | Marker-assisted breeding in cannabis plants | |
| CN104862416B (en) | The locus associated with Fusarium ear mold resistance in corn | |
| CN104762298B (en) | A kind of rice seedling resistant gene of salt qST11 and its molecule labelling method | |
| CN107746895B (en) | Molecular marker for improving barley harvest index QTL site under low-phosphorus condition and application | |
| US10590491B2 (en) | Molecular markers associated with Mal de Rio Cuarto Virus in maize | |
| CN104846095B (en) | The locus associated with Fusarium ear mold resistance on No. 3 and No. 4 chromosomes of corn | |
| CN108103236B (en) | SSR molecular marker tightly linked with maize leafless tongue gene lg1 and application thereof | |
| CN110199871B (en) | Breeding method of high-lysine maize inbred line | |
| CN103220904B (en) | Association corn is to the locus of the resistance that Fijivirus belongs to | |
| US10172305B2 (en) | Diagnostic molecular markers for seed lot purity traits in soybeans | |
| CN108401895B (en) | Method for improving Beijing family 968 strain type by rapidly reducing corn leaf angle under assistance of molecular marker | |
| CN109197569A (en) | A kind of molecular breeding method improving Three-line rice stigma exposing ratio of sterile line | |
| US20190256933A1 (en) | Guayule Molecular Markers And Primers And Use Of Molecular Markers, Primers And Rate Of Apomixis For Guayule Identification, Characterization And Breeding | |
| Abebe et al. | Genetic Variations in CIMMYT and Ethiopian Maize (Zea mays L.) Inbred Lines as Determined by Microsatellite Markers | |
| CN104271769A (en) | Compositions and methods to select for reduced grain moisture in maize |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |