CN107354215A - A kind of corn molecule auxiliary breeding means - Google Patents
A kind of corn molecule auxiliary breeding means Download PDFInfo
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Abstract
The invention discloses a kind of corn molecule auxiliary breeding means.Corn molecule auxiliary breeding means disclosed by the invention, including：AA genotype or AB maize genotypes first are hybridized with BB maize genotypes second, obtain first-filial generation F1；By first-filial generation F1It is returned with corn second, backcrossing number is 38 times, and backcrossing every time selects AB maize genotypes to be returned with corn second, obtain backcross progeny, the selfing of AB maize genotypes is selected in backcross progeny, obtains self progeny, selects AA maize genotypes to obtain purpose corn inbred line in self progeny；AB maize genotypes have long tasseling stage, long loose powder phase and/or long spinning phase, disease-resistant and degeneration-resistant characteristic under long-day conditions in backcross progeny.It is demonstrated experimentally that corn inbred lines disease-resistant, degeneration-resistant and with superior yield character potentiality can be obtained using the corn breeding method of the present invention.
Description
Technical field
The present invention relates in biological technical field, a kind of corn molecule auxiliary breeding means.
Background technology
Reproduction related gene directly determines the various characters related to reproduction of biology, also determine biology in modes of reproduction
On to the adaptability of local environment.The flowering time of higher plant is the important character related to reproduction, and it directly limits plant
The regional condition such as latitude of thing existence.In addition, florescence is also the character closely related with cereal crops yield.
The approach in regulation and control corn florescence mainly has (1) Photoperiod pathway (2) hormone pathway (3) autonomous pathway.Corn is opened
Flowering traits mainly include tasseling stage, loose powder phase and spinning phase, belong to quantitative inheritance character.Corn is influenceed to bloom the gene of character
More, research is more difficult, be cloned at present participate in corn bloom process gene it is seldom.Corn Dwarf8 genes and wheat
" green revolution " gene Reduced height-1 (Rht-B1 and Rht-D1) and arabidopsis Gibberellin
Insensitive (GAI) is homologous, is initially found related to control plant height, and may influence the variation of flowering time.Then
Thornsberry etc. (2001) clones Dwarf8, and proves that it is related to corn florescence.
Corn stalk rot disease (also known as bacterial wilt), it is a kind of fungi soil-borne disease for seriously endangering Maize Production, global beautiful
There is morbidity in rice plantation producing region.After being found first in the U.S. from 1914, worldwide occur in succession.Corn stalk rot disease
One of corn Major Diseases in world wide are turned into.The brocade flood of summer in 1962 waits to be reported China's corn stalk rot disease first,
Hereafter stem rot is increasingly becoming the Major Diseases for endangering China's Maize Production.
Stem rot all brings pole to endanger to corn yield and quality.22, U.S. state and Ontario, Canada statistical result
It has been shown that, the corn yield loss as caused by stem rot in 2013 is 1.83 hundred million bushels (Mueller and Wise, 2014).
China, this disease occurs to existing 16 provinces, municipalities and autonomous regions in the whole nation in 2000, corn underproduction caused by stem rot according to investigations reaches
25%, the general incidence of disease is serious up to more than 50% between 10-20%.
Majority research shows that corn has quantitative inheritance characteristic to the resistance of base rot disease, by multiple quantitative trait locus controls
System, based on additive gene effect, there is also certain dominant effect.Meanwhile also there is researcher to think that corn resists to stem rot
Property is regulated and controled by single key-gene.From the point of view of current result of study, the resistance locus in multiple researchs is all located in No. 1 dye
Colour solid and No. 10 chromosomes.In the research to other diseases, this two chromosomes are also repeatedly detected depositing for disease-resistant QTL
.This explanation, this two chromosomes have important researching value with the disease-resistant hot spot region of corn.
The research of corn stalk rot disease largely all rests on positioning stage at present.Resistance locus in multiple researchs is all determined
Positioned at No. 1 chromosome and No. 10 chromosomes.In the research to other diseases, this two chromosomes are also repeatedly detected disease-resistant
QTL presence.The gene being cloned into is seldom.So far, the gene Rcg1 of only one anti-anthrax stem rot is cloned into.
In addition, a disease-resistant QTL-qRfg2 is by finely positioning and proves that it is related to auxin.
Soil depletion is to influence the principal element that corn yield is unstable in world wide.The arable land soil that 1,400,000,000 hectares of the world
In earth, 22.5% soil is by nutrient condition of serious stress of soil, and only about 10.0% soil is without nutrient stress or mild stress.It is beautiful
In rice production, applied nitrogen is one of important measures of corn yield increasing, but both sides in production practices be present.When
High yield developed regions nitrogenous fertilizer is excessively applied, and not only causes utilization rate of nitrogen fertilizer to decline, and production cost improves, but also is likely to cause
Groundwater azotate is exceeded；Second, low yield under-developed area, often due to nitrogen application is insufficient, yield level is relatively low.In view of nitrogenous fertilizer
Further reduced using the space of volume increase, and many qualities and environmental problem caused by applied nitrogen, pass through science of heredity etc.
The efficient corn variety of means seed selection nitrogen is only the basic solution route for solving grain-production safety and environmental protection.
Moll etc. has done the definition of classics, i.e. crop yield under the conditions of unit applying Namount to nitrogen efficiency.Nitrogen efficiently can be with
It is divided into two parts, one is the efficiency that root system of plant absorbs nitrogen from soil, and two be nitrogen assimilation utilization ratio in plant.
It is main that Moll etc. thinks that nitrogen use efficiency serves under the conditions of low nitrogen, and nitrogen absorption efficiency plays main work under the conditions of high nitrogen
With.Under the conditions of Lafitte and Edmeades thinks low nitrogen, nitrogen use efficiency and the correlation of yield are more preferable.The results such as meter Guo Hua
It has been shown that, absorbed under the conditions of high nitrogen and both utilization ratios are laid equal stress on.And meter Guo Hua etc. thinks to cause nitrogen absorption efficiency and nitrogen profit
With the different achievements in research of efficiency relative importance be probably due to without genotype in different environments caused by.
The nitrogen of corn is efficiently a complicated process.The physiological Mechanism that nitrogen efficient maize absorbs at present includes：(1) it is good
Root system configuration (form and spatial distribution) and Root Characteristics；(2) physiological metabolism of good root system is active (respiration etc.)；
(3) aerial part possesses merit；(4) single cycle promotes absorption of the root system to nitrogen in seedling stage plant body.And corn is efficient
Included using the physiological mechanism of nitrogen：(1) there are the N metabolic key enzymes of high activity；(2) interaction between nutrient
And the influence of some materials；(3) feedback effect of storage capacity；(4) vacuole storage NO3 -Make full use of and aerial part nitrogen is waved
The reduction of hair；(5) nitrogen to seed to retransfer ability strong.Most of important economical characters such as yield, quality of crop and
Resistance is all the quantitative character controlled by multiple quantitative character gene locus therefors (quantitative trait loci, QTL).
Current many results of study show that the nitrogen efficiency of crop is also to be controlled by multiple quantitative trait locis.
Counted according to Second National soil survey information, on the premise of coastal tidal is not included, China's salinized soil area
For 34,870,000 hectares, about 500,000,000 mu, the area that can be developed is up to 200,000,000 mu.The salt tolerant alkali ability of crop is improved, for exploitation
Marginal land, grain yield is improved, is all had great importance.Cultivated maize is big to field water demand at present, to saline and alkaline adaptation
Ability is relatively low.Seedling stage is more sensitive to salt stress, its limit salinity (salt that the suppressed yield of plant strain growth referred to declines
Concentration) only about 1.7dsm-1, about 0.1%NaCl, from science of heredity, the salt tolerance of plant is the number by controlled by multiple genes
Character is measured, Genetic Mechanisms are complicated, and easily influenced by environmental conditions.Positioning and clone's salt tolerant are for illustrating plant salt tolerance mechanism
Important meaning is respectively provided with Breeding Application.At present for salt tolerance of crop QTL grind make internal disorder or usurp in existing remarkable break-throughs, it is such as small
Wheat, barley, soybean, rice etc., wherein rice research system the most.But delay for the QTL progress of salt tolerance of corn
Slowly.
The content of the invention
The invention provides a kind of corn molecule auxiliary breeding means.
Corn molecule auxiliary breeding means provided by the invention, including：
M1) corn first and corn second are hybridized, obtain first-filial generation F1, the corn first is AA maize genotypes or AB bases
Because of type corn；The corn second is BB maize genotypes；Two chromosomes of the AA maize genotypes are free of in sequence table
DNA fragmentation shown in the 89-5210 positions of sequence 1；Two chromosomes of the BB maize genotypes contain sequence in ordered list
DNA fragmentation shown in the 89-5210 positions of row 1；
M2) by the first-filial generation F1Corn second backcrossing with as recurrent parent, obtains backcross progeny, returns every time
Hand over and select AB maize genotypes to be returned with the corn second；The selfing of AB maize genotypes is selected in the backcross progeny, is obtained
To self progeny, AA maize genotypes are selected to obtain purpose corn inbred line in the self progeny；
The item chromosome of the AB maize genotypes contains the DNA fragmentation shown in the 89-5210 positions of sequence 1, another
DNA fragmentation shown in the 89-5210 positions of article chromosome without sequence 1；
AB maize genotypes have following R1 in the backcross progeny), R2) and/or feature R3)：
R1) long tasseling stage, long loose powder phase and/or long spinning phase under long-day conditions；
R2) disease resistance；
R3) resistance.
AB maize genotypes have above-mentioned compared with the BB maize genotypes in the backcross progeny in the backcross progeny
R1), R2) and/or feature R3).
In the above method, the disease resistance can be disease caused by anti-Fusarium graminearum (Fusarium graminearum).
In the above method, the resistance can be low nitrogen resisting stress and/or high-salt stress.
Nitrogen content in the environment of the Low nitrogen stress concretely 3.13kg N/ mus.
N in the environment of the Low nitrogen stress can apply fertilizer at twice, and each institute's nitrogen fertilizer application can be the 50% of total nitrogenous fertilizer.
Salt content in the environment of the high-salt stress concretely 1.25 ‰ (mass percent).
In the above method, M2) in backcrossing number can be more than or equal to 3 times.M2 backcrossing number concretely 3-8 times, such as 4 in)
It is secondary or 5 times.
In the above method, AB maize genotypes are selected to treat selection corn gene using primer pair in backcross process
Group DNA enters performing PCR amplification and detected, and determines whether the corn to be measured is AB maize genotypes according to pcr amplification product；
DNA fragmentation shown in the 89-5210 positions of the primer pair energy specific recognition sequence 1 or thereon downstream sequence.
In the above method, the primer pair can be：The primer pair of TE1C-FP and Primer4RP composition, Primer4FP with
The primer pair of Primer4RP composition, the primer pair of Primer2FP and Primer2RP compositions, Primer1FP with
The primer pair of Primer1RP compositions, the primer pair of Primer3FP and Primer3RP compositions, Primer3FP and TE1C-RP are formed
Primer pair, and/or, the primer pair of the TE1C-FP and the TE1C-RP composition；
The TE1C-FP is the DNA molecular shown in sequence 3 in sequence table；
The TE1C-RP is the DNA molecular shown in sequence 4 in sequence table；
The Primer1FP is the DNA molecular shown in sequence 5 in sequence table；
The Primer1RP is the DNA molecular shown in sequence 6 in sequence table；
The Primer2FP is the DNA molecular shown in sequence 7 in sequence table；
The Primer2RP is the DNA molecular shown in sequence 8 in sequence table；
The Primer3FP is the DNA molecular shown in sequence 9 in sequence table；
The Primer3RP is the DNA molecular shown in sequence 10 in sequence table；
The Primer4FP is the DNA molecular shown in sequence 11 in sequence table；
The Primer4RP is the DNA molecular shown in sequence 12 in sequence table.
In the above method, methods described may additionally include the AB maize genotypes obtained in backcross process to selection according to such as
Lower P1), P2) and/or P3) further selected：
P1) Phenotypic Selection：Corn is inoculated with using Fusarium graminearum (Fusarium graminearum), selects disease resistance strong
Plant；
P2) restructuring selection：Using molecular labeling SSR47, SSR93, STS01, STS378, STS450, STS444, STS02,
SSR261, SSR164, SSR172 and STS03 select the plant consistent with each molecular labeling result of the recurrent parent；
The sequence of two primers of the SSR47 is respectively sequence 13 and 14 in sequence table；
The sequence of two primers of the SSR93 is respectively sequence 15 and 16 in sequence table；
The sequence of two primers of the STS01 is respectively sequence 17 and 18 in sequence table；
The sequence of two primers of the STS378 is respectively sequence 19 and 20 in sequence table；
The sequence of two primers of the STS450 is respectively sequence 21 and 22 in sequence table；
The sequence of two primers of the STS444 is respectively sequence 23 and 24 in sequence table；
The sequence of two primers of the STS02 is respectively sequence 25 and 26 in sequence table；
The sequence of two primers of the SSR261 is respectively sequence 27 and 28 in sequence table；
The sequence of two primers of the SSR164 is respectively sequence 29 and 30 in sequence table；
The sequence of two primers of the SSR172 is respectively sequence 31 and 32 in sequence table；
The sequence of two primers of the STS03 is respectively sequence 33 and 34 in sequence table；
P3) Foreground selection：Select to carry on the back using GoldenGate 6KSNP (Illumina, San Diego, CA, USA) chip
The high plant of scape response rate.
Wherein, the Phenotypic Selection, the selection time of the restructuring selection and the Foreground selection and selection number are equal
It can carry out according to specific needs.The Phenotypic Selection specifically can be in third backcross generation (BC3F1) and/or four generation (BC of backcrossing4F1) enter
OK.The restructuring selection and the Foreground selection can be carried out in backcrossing advanced lines.The restructuring selection specifically can be in backcrossing four
Carried out to five generations or more advanced lines.The Foreground selection can specifically enter backcrossing highest generation (i.e. the offspring of last time backcrossing)
OK, such as five generation (BC of backcrossing5F1)。
In the above method, the corn first concretely CIMBL157, GEMS23, GEMS14,1145, Zhong69,
CIMBL40, CIMBL151, CIMBL53 or CML454.
The corn second concretely Chang7-2, Zheng58, A5302 or F349.
The 89-5210 positions of sequence 1 or the DNA molecular shown in its any fragment or the 89- containing sequence 1 in sequence table
The DNA molecular of 5210 falls within protection scope of the present invention.
DNA molecular shown in the 89-5210 positions of sequence 1 or its any fragment or the 89-5210 positions containing sequence 1
Application of the DNA molecular in corn breeding falls within protection scope of the present invention.
In the present invention, the sunshine-duration of the long-day can be 14-16 hours.
DNA molecular of the present invention according to the 89-5210 positions of sequence 1 establishes a kind of corn marker assisted selection side
Method, two chromosomes corn (AA genotype) without the DNA molecular is contained as donor parents, two chromosomes should
In the backcross progeny that the corn (BB genotype) of DNA molecular assembles as recurrent parent, AB maize genotypes are (in two chromosomes
DNA molecular shown in one article of 89-5210 position containing sequence 1, the DNA shown in another article of 89-5210 position without sequence 1
Molecule) Correlated Yield Characters, under the conditions of low nitrogen, the Correlated Yield Characters of AB maize genotypes --- spike length, tassel row number, grain
It is long, grain is thick, area is all remarkably higher than BB maize genotypes, and compared with BB genotype AB maize genotypes correlation with yield
Shape --- fringe is thick, girth also has increased trend；Under high salt conditions, the Correlated Yield Characters of AB maize genotypes --- fringe
It is long, fringe is thick, row grain number, grain length, grain are wide, area, girth and 100-grain weight are all remarkably higher than BB maize genotypes, and with BB genotype
Compared to the Correlated Yield Characters of AB maize genotypes --- tassel row number and grain thickness also have increased trend.Illustrate, above-mentioned backcrossing
The ability of AB maize genotypes high salt tolerance and Low nitrogen stress is higher than BB maize genotypes in offspring；Under long-day conditions, with BB
Genotype is compared, Flowering-related traits (tasseling stage, loose powder phase and spinning phase are equal) increase of AB maize genotypes, and in short-day
Under the conditions of two kinds of genotype the tasseling stage of corn, loose powder phase and spinning phase without significant changes.Furthermore it is also possible to by returning
Select the strong plant of disease resistance further to obtain disease-resistant plant during friendship, genetic background can also be obtained by Foreground selection
And the plant that other characters are similar to recurrent parent.In addition, obtained backcross progeny is selfed, obtained AA genotype
Homozygous corn inbred line, the corn inbred line have disease-resistant, degeneration-resistant and superior yield character potentiality, can further with other
Corn hybridization prepares the cenospecies commonly used in production, so as to by above-mentioned Correlated Yield Characters, Flowering-related traits and disease resistance trait
For in production, realizing the improvement of corn.
Brief description of the drawings
Fig. 1 is molecule assist-breeding florescence, anti-Fusarium graminearum stem rot and resistance to low nitrogen and salt stress corn flow chart.
It is donor parents genotype that AA, which represents selection site, and AB is heterozygous genotypes, and it is recurrent parent genotype that BB, which represents selection site,.
Fig. 2 is ZmCCT difference haplotype backcross progenies BC3F1(Beijing) and BC4F1(Hainan) Characteristics in florescence investigates column
Figure, (-/-) are BB genotypes, (- /+) it is AB genotypes.H3, H4, H5, H6, H7, H12, H13, H14 and H15 points
Not Biao Shi CIMBL157, GEMS23, GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML454 with
Chang7-2 offspring.*P<0.05,**P<0.01；NS, difference is not notable；The data of "/" or so are respectively corresponding gene in figure
The investigation plant number of type strain.
Fig. 3 is different backcross progeny BC3F1Disease index investigates block diagram, and (-/-) is BB genotypes, (- /+) it is AB
Genotype.H3, H4, H5, H6, H7, H12, H13, H14 and H15 represent respectively CIMBL157, GEMS23, GEMS14,
1145th, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML454 and Chang7-2 offspring.*P<0.05,**P<
0.01；NS, difference is not notable；The data of "/" or so are respectively the investigation plant number of corresponding gene type strain in figure.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, it is
Conventional method.Material used, reagent, instrument etc., unless otherwise specified, are commercially obtained in following embodiments.
Quantitative test in following examples, it is respectively provided with and repeats to test three times, results averaged.
Fusarium graminearum (Fusarium graminearum) in following embodiments be recorded in document (Yang, Q., Yin,
G.,Guo,Y.,Zhang,D.,Chen,S.,&Xu,M.(2010).A major QTL for resistance to
Gibberella stalk rot in maize.Theoretical and applied genetics,121(4),673-
687.) in, the public can obtain the biomaterial at applicant, and the biomaterial is only attached most importance to the related experiment institute of duplicate invention
With can not be used as other purposes.
Corn (Zea mays L.) self-mating system Chang7-2 in following embodiments, it is intolerant to Low nitrogen stress and salt density value
Kind, be recorded in document " Yang, X., Gao, S., Xu, S., Zhang, Z., Prasanna, B.M., Li, L., Li, J.,
Yan,J.(2011).Characterization of a global germplasm collection and its
potential utilization for analysis of complex quantitative traits in
Maize.Molecular Breeding, 28 (4), in 511-526 ", the public can obtain the biomaterial, the life at applicant
Thing material is only attached most importance to used in the related experiment of duplicate invention, can not be used as other purposes.
Corn (Zea mays L.) kind CIMBL157, GEMS23, GEMS14 in following embodiments, 1145,
Zhong69, CIMBL40, CIMBL151, CIMBL53 and CIMBL454 be recorded in document " Yang, X., Gao, S., Xu, S.,
Zhang,Z.,Prasanna,B.M.,Li,L.,Li,J.,Yan,J.(2011).Characterization of a global
germplasm collection and its potential utilization for analysis of complex
Quantitative traits in maize.Molecular Breeding, 28 (4), in 511-526 ", the public can be from application
These biomaterials are obtained at people, these biomaterials are only attached most importance to used in the related experiment of duplicate invention, can not be used as other use
Way uses.
Embodiment 1, corn molecular labeling are related to corn yield correlated traits, flowering time character and degeneration-resistant character
A kind of corn molecular labeling and a kind of breeding method of corn are present embodiments provided, corn molecular labeling is designated as
ZaMM, ZaMM are the DNA fragmentation shown in the 89-5210 positions of sequence 1 in sequence table.
First, corn molecular labeling
ZaMM co-exists in three kinds of situations in corn, and the first is that two chromosomes of Maize genome contain sequence 1
89-5210 positions shown in DNA fragmentation, second is that two articles of chromosomes of Maize genome do not contain the 89- of sequence 1
DNA fragmentation shown in 5210, the third is two articles of chromosomes, one article of 89-5210 position containing sequence 1 of Maize genome
Shown DNA fragmentation, the DNA fragmentation shown in another article of 89-5210 position without sequence 1, is the first situation by chromosome
The VDA genotypes of corn be BB genotype, the VDA genotypes by the corn that chromosome is second of situation are AA genes
Type, it is AB genotype that the genotype for the corn that chromosome is the third situation, which is scheduled,.
2nd, the cultivation of corn NIL and phenotypic evaluation
One) cultivation of corn NIL
Advanced lines NIL is built using the method for molecular labeling backcrossing assisted Selection, recurrent parent used is
Chang7-2, donor parents be respectively CIMBL157, GEMS23, GEMS14,1145, Zhong69, CIMBL40, CIMBL151,
CIMBL53 and CML454.
Genotype identification：Each recurrent parent and the genomic DNA of donor parents are extracted, is utilized respectively seven each bases of primer pair
Because a group DNA enters performing PCR amplification, shown after carrying out sequencing splicing to PCR primer, Chang7-2 contains the DNA pieces shown in sequence 1
Section, and without the DNA fragmentation shown in sequence 2, show, Chang7-2 is BB maize genotypes；Each donor parents are containing only sequence 2
Shown DNA fragmentation and without the DNA fragmentation shown in sequence 1, shows, each donor parents are AA maize genotypes.Sequence 1
With differing only in for sequence 2：Compared with sequence 1, sequence 2 lacks the 89-5210 positions of sequence 1.
Seven primer pairs used are respectively：TE1C-FP and Primer4RP, Primer4FP and Primer4RP,
Primer2FP and Primer2RP, Primer1FP and Primer1RP, Primer3FP and Primer3RP, Primer3FP with
TE1C-RP, TE1C-FP and TE1C-RP.The sequence of each primer is as follows：
TE1C-FP：5'-CACGTATGTGCATCCATCAG-3'(sequences 3)
TE1C-RP：5'-TTCTCAATCCAAGGTGCAGG-3'(sequences 4)
Primer1FP：5'-TCCATGAGGTTTTCCGCCTC-3'(sequences 5)
Primer1RP：5'-CTTCCCCGTGGATATGCTCC-3'(sequences 6)
Primer2FP：5'-TGGAGGAGGTGGCATAGTCA-3'(sequences 7)
Primer2RP：5'-GTTTGGCAAAGCGCATGGTA-3'(sequences 8)
Primer3FP：5'-AGCCATAAGTCGCGTCACAA-3'(sequences 9)
Primer3RP：5'-CGGTTCTTAGGGTACCGTCG-3'(sequences 10)
Primer4FP：5'-AGTCGGAAGTGTCAGCGTTT-3'(sequences 11)
Primer4RP：5'-CCACCTATCCCGCAGTTTGT-3'(sequences 12)
TE1C-FP and TE1C-RP PCR amplification conditions are：95℃3min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35
Circulation；72℃10min.The primer pair is only capable of obtaining the DNA molecular shown in sequence 2 under the PCR amplification conditions, and can not obtain
DNA molecular shown in sequence 1.
The specific building process (Fig. 1) of corn NIL is as follows：
1) donor parents are hybridized with recurrent parent respectively, obtains F1Generation, by donor parents be CIMBL157, GEMS23,
The F obtained when GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML4541In generation, is designated as 157- respectively
F1、23-F1、14-F1、1145-F1、69-F1、40-F1、151-F1、53-F1And 454-F1, each F1Generation is AB genotype；
2) by each F1In generation, is returned with recurrent parent respectively, obtains BC1F1；Be CIMBL157, GEMS23 by donor parents,
The BC obtained when GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML4541F1157- is designated as respectively
BC1F1、23-BC1F1、14-BC1F1、1145-BC1F1、69-BC1F1、40-BC1F1、151-BC1F1、53-BC1F1And 454-BC1F1；
To each BC1F1Foreground selection is carried out, obtains positive BC1F1, each positive BC1F1It is AB genotype, each BC1F1In non-AB genes
The genotype of type strain is BB genotype；
3) by each positive BC1F1It is returned respectively with recurrent parent, obtains BC2F1；Be CIMBL157 by donor parents,
The BC obtained when GEMS23, GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML4542F1Remember respectively
For 157-BC2F1、23-BC2F1、14-BC2F1、1145-BC2F1、69-BC2F1、40-BC2F1、151-BC2F1、53-BC2F1With
454-BC2F1；To each BC2F1Foreground selection is carried out, obtains positive BC2F1, each positive BC2F1It is AB genotype, each BC2F1In
The genotype of non-AB Genotype Strains be BB genotype；
4) by each positive BC2F1It is returned respectively with recurrent parent, obtains BC3F1, using Chang7-2 as recurrent parent,
CIMBL157, GEMS23, GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML454 are as donor
The BC that parent obtains3F1157-BC is designated as respectively3F1、23-BC3F1、14-BC3F1、1145-BC3F1、69-BC3F1、40-BC3F1、
151-BC3F1、53-BC3F1And 454-BC3F1；To each BC3F1Phenotypic Selection and foreground selection are carried out, obtains positive BC3F1, each sun
Property BC3F1It is AB genotype, each BC3F1In the genotype of non-AB Genotype Strains be BB genotype；
5) by each positive BC3F1It is returned respectively with recurrent parent, obtains BC4F1, using Chang7-2 as recurrent parent,
CIMBL157, GEMS23, GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML454 are as donor
The BC that parent obtains4F1157-BC is designated as respectively4F1、23-BC4F1、14-BC4F1、1145-BC4F1、69-BC4F1、40-BC4F1、
151-BC4F1、53-BC4F1And 454-BC4F1；To each BC4F1Carry out foreground selection and restructuring selects, obtain positive BC4F1；Prospect
Select obtained positive BC4F1It is AB genotype, each BC4F1In the genotype of non-AB Genotype Strains be BB genotype；
6) by each positive BC4F1It is returned respectively with recurrent parent, obtains BC5F1, using Chang7-2 as recurrent parent,
CIMBL157, GEMS23, GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 and CML454 are as donor
The BC that parent obtains5F1157-BC is designated as respectively5F1、23-BC5F1、14-BC5F1、1145-BC5F1、69-BC5F1、40-BC5F1、
151-BC5F1、53-BC5F1And 454-BC5F1；To BC5F1Foreground selection, restructuring selection, Phenotypic Selection and Foreground selection are carried out, is obtained
To positive BC5F1。
7) by positive BC5F1Selfing, obtains BC5F2, to utilizing above-mentioned seven couples of primer pair BC5F2Genomic DNA enter performing PCR
Amplification, the DNA fragmentation for selecting to comprise only shown in sequence 2 in pcr amplification product do not contain the plant of the DNA fragmentation shown in sequence 1,
Obtain the BC that genotype is AA5F2Improved inbred lines, the self-mating system further can give birth to other corn varieties or incross system
Cenospecies used in production.
Wherein, foreground selection genomic DNA is entered using the primer pair that TE1C-FP and TE1C-RP is formed performing PCR expand into
OK, the strain (i.e. AB Genotype Strains) containing purposeful amplified production is selected, the sequence of purpose amplified production is sequence in sequence table
Row 2.The strain without purposeful amplified production is BB Genotype Strains in each offspring.The PCR amplification bars of foreground selection primer
Part is：95℃3min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35 circulations；72℃10min.
Phenotypic Selection is carried out as follows：40 days left sides after Fusarium graminearum (Fusarium graminearum) inoculation
Right investigation character, investigate altogether three times, every minor tick one week.Last time is investigated, and corn basal part of stem need to be rived, and observes pathogen
Upgrowth situation, the comprehensive result investigated three times, determine the grade that corn is fallen ill.Six grades (0,1,2,3,4,5) are divided into, wherein 0
Level is most disease-resistant, and 5 grades most susceptible, selects 5 grades of AB genotypes.
Restructuring selection is carried out as follows：Restructuring selection molecular labeling dispersed distribution used is in No. 10 chromosomes
In 89.6-115.3Mb (according to B73RefGen_V4 reference sequences) region, the primer sequence of molecular labeling be shown in Table 1, SSR47,
SSR93, STS01, STS378, STS450, STS444, STS02, SSR261, SSR164, SSR172 and STS03 site difference
For 89.6,91.5,93.1,94.2,94.4,95.1,96.6,98.1,106,113 and 115.3Mb, annealing temperature is respectively 62,
60th, 60,60,60,60,60,60,60,58 and 58 DEG C.PCR amplifications carry out electricity on 1%, 2%, or 4% agarose gel
Swimming, with Universal Hood II (Bio-Rad, Hercules, USA) gel imaging system observed result.Selection and samsara parent
The consistent plant of this Chang7-2 each molecular labeling result.
Table 1, restructuring selection list of primers
Foreground selection is carried out using GoldenGate 6KSNP (Illumina, San Diego, CA, USA) chip, respectively
It is control using recurrent parent, selects background response rate highest individual plant.The highest background for the offspring that each donor parents obtain is returned
Multiple rate is shown in Table 2.
Table 2, background response rate
| Donor parents | Background response rate (%) |
| CIMBL157 | 95.13 |
| GEMS23 | 95.69 |
| GEMS14 | 95.59 |
| 1145 | 92.66 |
| Zhong69 | 95.29 |
| CIMBL40 | 96.47 |
| CIMBL151 | 92.17 |
| CIMBL53 | 96.26 |
| CML454 | 95.33 |
Two) identification of phenotype
1st, flowering time character
Investigate each backcross progeny BC3F1(Beijing, long-day conditions, sunshine-duration are 14-16 hours) and BC4F1(Sanya,
Short-day condition, sunshine-duration are 10-12 hours) in the tasseling stage of AB genotype and BB genotype, the loose powder phase and spinning phase (table
2nd, table 3 and Fig. 2), carry out T inspections.Wherein, tasseling stage refers to expose to tassel from sowing 1/2 number of days, and the loose powder phase refers to from broadcasting
Kind starts the number of days of loose powder to tassel, and the spinning phase refers to the number of days from sowing to female fringe filigree discharge 1-2cm.
Test result indicates that in the offspring of different donor parents, under long-day conditions, compared with BB genotype, remove
151-BC3F1Outside, the tasseling stage of AB maize genotypes, loose powder phase and spinning phase dramatically increase, and tasseling stage increases 3.1-6.1 days,
The loose powder phase increases 5.1-9.4 days, spinning phase increase 6.7-11.6 days (a-c in Fig. 2), and 151-BC3F1In AB genotype it is beautiful
Tasseling stage, loose powder phase and the spinning phase of rice are also respectively provided with increased trend relative to BB genotype；Under the conditions of short-day, two kinds
Tasseling stage, loose powder phase and spinning phase between genotype is without significant difference (d-f in Fig. 2).
Table 2, BC3F1For examination maize genotype and the flowering time character under the long-day
Table 3, BC4F1For examination maize genotype and the flowering time character under short-day
2nd, resistance trait
Soil is taken to bury the method for hindering root (referring to " the health cares cultivation step such as Song Zuoheng is ground to corn stalk rot disease control effect
Study carefully, the phases of Liaoning agricultural sciences .1993 the 05th "), the BC with Fusarium graminearum to each backcross progeny colony3F1Connect at bacterium
Reason, specific steps：After plant fusulus, apart from plant 5-10 centimeters cut-off parts capillary straight down
Root, soil is cast aside, numerous Fusarium graminearum (Fusarium graminearum) is expanded in 50-70 grams of embedment with corn kernel, and pours
Water moisturizing.Test site is Beijing and Shandong.
Disease resistance is investigated, disease index is calculated and carries out statistical analysis.It is specific as follows：With Fusarium graminearum artificial infection 45
After it, disease resistance is investigated for the first time, and three times, each investigation interval one week, investigation result is recorded as investigation altogether：R (disease-resistant type), S
(susceptible type), M (osculant).Observe for the first time, disease plant dry up early ageing, and stem is pinched in second of investigation, for the third time, by basal part of stem
Rive, observe mycelial growth state, comprehensive result three times, backcross progeny is evaluated using six grades of scoring systems (0,1,2,3,4,5)
Resistance, standards at different levels are as follows：
0 grade：Investigation result is R three times, and disease resistance is most strong；
1 grade：Preceding investigation result twice is R, and third time result is M；
2 grades：Preceding investigation result twice is R, and third time result is S；
3 grades：First time investigation result is R, and second of result is M, and third time result is S；
4 grades：First time investigation result is R, after twice investigation result be S；
5 grades：Investigation result is S three times, and disease resistance is most weak.
Disease index (Disease severity index, DSI) computational methods：
DSI=∑s [(series × corresponding plants number) × 100/ (5 × total plant number)]
Field design is RANDOMIZED BLOCK DESIGN, and every kind of backcross progeny sets three repetitions, and the DSI each repeated is independently counted
Calculate.
Experimental result is shown, in the statistical result (table 4 and Fig. 3) of two places, the disease index of AB maize genotypes is aobvious
Write less than BB genotype or with the trend for being less than BB genotype, compared with BB genotype, AB genotype refers in Pekinese's state of an illness
Number reduces 28.1-45.4, and the disease index in Shandong reduces 2.2-28.2.
Table 4, BC3F1For examination maize genotype and disease index
3rd, the Correlated Yield Characters under the conditions of low nitrogen and high-salt stress
(1) experimental method
By each backcross progeny BC3F1Plant, harvest under Low nitrogen stress (LN) and high-salt stress (HS) field condition for plant
After determine Correlated Yield Characters.Statistical analysis is carried out with the T methods of inspection.
(2) experiment condition
Low nitrogen field condition is located at Xinzheng experiment station of Agricultural University Of He'nan, fertilizer used：Nitrogenous fertilizer is urea (N content
46%), phosphate fertilizer is Diammonium phosphate (DAP) (P2O5Content 46%, N content 18%), potash fertilizer is potassium chloride (K2O content 59%).
Control treatment (CK) dose N is 14kg/ mus, and specific fertilization mode is as follows：Urea (23.63kg/ mus), di(2-ethylhexyl)phosphate
Ammonium (17.39kg/ mus), potassium chloride (8.47kg/ mus)；
For low nitrogen dose N by 3.13kg N/ mus (nitrogen is to apply the nitrogen in Diammonium phosphate (DAP)), specific fertilization mode is as follows：
Urea (0), Diammonium phosphate (DAP) (17.39kg/ mus), potassium chloride (8.47kg/ mus).The nitrogenous fertilizer (Diammonium phosphate (DAP)) of low nitrogen processing is for the first time
The 50% of nitrogen pool is applied, top dressing (Diammonium phosphate (DAP)) applies the 50% of nitrogen pool.
High salt conditions are located at the area along the Yangtze River in Jiangsu Province's Institute of agricultural sciences, and the average salt content of five test points is 1.25 ‰
(mass percent).
(3) measurement index
During harvest the every kind of genotype of every kind of donor parents offspring and recurrent parent and donor parents select at random 6 it is strong
Kang Guosui is used for investigating Correlated Yield Characters, is divided into two parts of panicled characters and grain portion character, with Uniscan M1 scanners
(Qing Hua Ziguang, Beijing) scans fruit ear, scan area 29.70cm × 42.00cm, 1600 × 1600dpi of scanning resolution.Measure
Panicled characters have：Spike length, tassel row number, row grain number, fringe are thick；Grain portion character is investigated after harvesting after one month, 65 before investigation
DEG C/48h is dried to constant weight, with SC-G species tests automatic analysis system (ten thousand is deep, Hangzhou) analysis grain portion character, the grain portion character of measure
Have that grain length, grain are wide, grain is thick, seed area, seed girth, 100-grain weight.Donor parents are GEMS14 BC3F1Each index knot
Fruit is as shown in table 5.
(4) experimental result
For each genotype, its indices in control treatment is normalized to 1, statistics Low nitrogen stress (LN)
With each panicled characters and grain portion character normalization result (i.e. with the multiple proportion of control treatment) under high-salt stress (HS).Donor
Parent is GEMS14 BC3F1Each index normalization after result it is as shown in table 6.
The Correlated Yield Characters of table 5, GEMS14 offspring under stress conditions
The Correlated Yield Characters normalization result of table 6, GEMS14 offspring under stress conditions
In table 5 and 6, area and girth are respectively seed area and seed girth.
T examines combination normalization result to show, each to supply examination corn under the conditions of low nitrogen, spike length, the fringe of AB maize genotypes
Line number, grain length, grain are thick, seed area is all remarkably higher than BB maize genotypes, and the AB maize genotypes compared with BB genotype
Fringe is thick, seed girth also has increased trend；It is each for examination corn under high salt conditions, spike length, the fringe of AB maize genotypes be thick,
Row grain number, grain length, grain are wide, seed area, seed girth and 100-grain weight are all remarkably higher than BB maize genotypes, and with BB genotype
Also there is increased trend compared to the tassel row number and grain thickness of AB maize genotypes.Show, AB maize genotypes low nitrogen resisting and high salt
The ability of stress is higher than BB maize genotypes.
3rd, other donor parents backcross progenies and phenotypic evaluation
The preparation of backcross progeny：
According to the construction method of corn NIL in step 1, using GEMS14 as donor parents, respectively with conduct
Zheng58, A5302 and F349 of recurrent parent assemble backcross progeny.Wherein, Zheng58 be recorded in document " Yang, X.,
Gao,S.,Xu,S.,Zhang,Z.,Prasanna,B.M.,Li,L.,Li,J.,Yan,J.(2011).Characterization
of a global germplasm collection and its potential utilization for analysis
Of complex quantitative traits in maize.Molecular Breeding, 28 (4), in 511-526 ".
F349 is recorded in document and " Xu little Wei, Xu Li, Dong Xin, waits the high oil of Seeds of First Post-flight and conventional corn self-mating system variability observation [J]
Nuclear agricultural science report, 2011,25 (2):In 0220-0225. ", A5302 is lucky single 53 male parent.
According in step 2 one) in genotype identification method, identify each donor parents and the genotype of recurrent parent, tie
Fruit shows that each donor parents are AA maize genotypes, and each recurrent parent is BB maize genotypes.
According to the building process of the corn NIL in step 2, each recurrent parent and corresponding donor parent are respectively obtained
This BC3F1。
Each BC is detected according to 3 method in step 2 two)3F1Phenotype under high salt and Low nitrogen stress, spike length, hundred
The result of weight index is as shown in table 7, then for each genotype, its indices in control treatment is designated as into 1, system
Meter Low nitrogen stress (LN) and spike length under high-salt stress (HS), 100-grain weight character normalization result (the multiple pass i.e. with compareing
System), as a result as shown in table 8.
The Correlated Yield Characters of table 7, each donor parents backcross progeny under stress conditions
The Correlated Yield Characters normalization result of table 8, each donor parents backcross progeny under stress conditions
As a result show, for each donor parents under the conditions of high salt and low nitrogen, the spike length and 100-grain weight of AB maize genotypes are aobvious
Work is higher than BB maize genotypes.Show, the ability of AB maize genotypes high salt tolerance and Low nitrogen stress is above BB maize genotypes.
<110>China Agricultural University
<120>A kind of corn molecule auxiliary breeding means
<160> 34
<170> PatentIn version 3.5
<210> 1
<211> 5351
<212> DNA
<213>Corn
<400> 1
cacgtatgtg catccatcag gaatcaatgg ccgctggccc cagcttcgaa ttttgctgct 60
gccttttaac aaagctatag ctagtagcgg ggatacccag tcggaagtgt cagcgtttcc 120
gacggggccg tcggaagtaa acttatttcc gacggccagg gtagcgccgt cggaaataaa 180
gcgaggtggg ccccgcagac ggccggccgc cggcgcctga cgccgtcaca gcttagttcc 240
gacggggccg tcggaaataa gcggccgtcg gaaataatta aaaaacagag aaacagaatt 300
tgtctgcttt ttgttttaca aacaatcaca cagattcaat ctgtacacaa tatccagttt 360
tcatccagaa tcacatacaa taccacatga aacacacaat aacacaagtt cacaagttaa 420
agcacaagtt cacaagttaa agcacaaatt cacaatgttc acaagttcac aagttcacat 480
gttcagaagt tcacaataac acaagttccc aataccacaa gttcacaagt ccatagtacg 540
agaaaaacac aagcagacag gctactcaaa atggaggagg ttgatcaaat ggatgtggtt 600
gatttgatct gctagctcct ccactgttag aagaccgtcc acaaaagaag cgaccgcatc 660
ttcagagtcc tcatttcccg gagttagcaa gtttcctgga gggacctaca acattcaaaa 720
aaatgttagt agttaaattc ttgttatgca acgtcaaaag aaaaagtcaa gtaatataac 780
ctgaggtgca ggtgtggccc aactaaattg tggagtcggt acaaactgcg ggataggtgg 840
aggaggtggc atagtcatcg ctgaaaaatc tggacgttgc cctagtgcta tttgctgaaa 900
gaaattgaaa gctatgttac gaggatatat tagaaaatat gtgctccaat tatagtagag 960
gctcacgagg atctaattac ctgcatcatc tcctgttgtc gtgcaaattg agcagcccag 1020
tactcttgtt gttgcttgtt atactcctcc tgccgcctca aagcttcctg catccggatc 1080
agctcggcgt ttccttgggt actagagcga ggaccacggc tagacgacct cgatggacga 1140
gacctttcag gcctcacctg agtcgagtcg ataacactac caaacatggg gtaactagaa 1200
aaaatagaaa gaagttaaac acgcaacaca tcatgttaga aaagatcaaa tcgacgaatt 1260
aataatagca aactcaccgt ccatgaggtt ttccgcctcc actagcatac acaacctgag 1320
gatcgatagg atgattcatc caatcatagt cttgcccatg ctccgacatc atggaagcac 1380
catatgaggc ctgacaaata ataaacaatg cagttatatt tttctcgact cttagaatag 1440
aagtgcattg taactagttg cgaaagtctc accacacgct cagcagctga ctgagaacac 1500
aactgttctg gatttgtagg atccgacccc ctgtgacctc taaggtaaac ctgaacatca 1560
ctcggcgtaa caccagaact agcttcctac aggcaagaga aaataacata aacacataga 1620
agattcatga tatgcacgac cataaattac acttaccatg cgctttgcca aacgaacgtg 1680
gccatcacca ccatatctgt ggtgaacatc atgcccacga tttgagctgt ttctcattga 1740
aacaccttta aatccatctg ttgcccaata tttgcataca gcccgccaac cagcctcatt 1800
tgatgccatc catggcaccg agtctgaaaa ctttatttcg atccaatgta ttgttagtac 1860
taacacttgg acatgaatta ggataaaaac aatttacctc aagatattct tcttcggtta 1920
agtatttgtt tgatgcctca cttttggaac gtggccgacg tccttcctgc cttaatatgt 1980
aatcagaagt gacctggatc cgagcataat acatctgatc cctaaccaag gtagtcgcgc 2040
tcttgttaaa aacgatccgt gcacgatcgt tcatacttcc atcttcaggc aacttgtaac 2100
gtttctgaaa aaaacaagaa acaagatttt tagcacattg tatagttgaa tcaattcaag 2160
ttgggctaga gaacataccc agaactcatg ccacacagca ccttgggcat tcctatgttc 2220
cgcattaaac gcgagcccgt actgatccca agacatacaa ggcacctcaa cgcctttctc 2280
catcactaca ccaggccaga gatatttgca tatacttccc aaaactttat tcacctgtgt 2340
gcgacgtcct tgaccttgaa aaagatgcat caatccaaga cctgcaacca cattgtacaa 2400
aagaagcatt aacaaatttc atataatgca tgaacaaata tttaacatta gaatcaactc 2460
actcgtcgcc acacggtacg atcaaaatat tatcctcctc acgagcagga actttaggag 2520
gaggcaccca atgtgttttc ctcgacttcc gtgcccgaga agaccctcca ccactagaag 2580
ctccagcatc actgctaggc catgagtccc atccgcctgc tgaccatcca ccctcaaact 2640
gaccaccacc ggaagaccac ccatctccac ctccacctcc agcatcatct tcctgctcat 2700
agtctgcatc ggccgcatcg taatcggccg catcctcacc ccttgcctcc tcctctcgag 2760
tctcgttaac atcctgcaca tgaaacactt taatcattag attcatataa acattactat 2820
attgaaataa aataataatt tagagtaatc acacctgtcc atgcatatca ggtggaagat 2880
gttccatcaa agcctgtcgt ctgttgctcg agagcgactc ggtaccctgg aacagacact 2940
cctgtgcttt gcttcttcct gaagaagaag aagagccctt cgtcatgccc ttcacaaaat 3000
tcttcaactt tcgagccgcc atcctgcata ttgaaaatta tatgtctttg ggcattttgt 3060
gatctggtgg taatacgtcg ctaatcaaat tcaaaagttc atttaagcag ttgatggaga 3120
atgcgaactt agacttgata gccataagtc gcgtcacaaa agcaagaatg gttactctcg 3180
tgtgttcgtg caacggctct tctgctgcct tgagaagttc aaagaatttt ttaacttccg 3240
gcggttgagg atcctcagga ttatctggaa acactatatc gggatcctct cttaaatcct 3300
gaaccatctc ctccattcgg tctaaatccg ggtcaaaatc gtcttgacct tctggagcat 3360
atccacgggg aagctcctca ccatgatgca cccaaacttc ataatttggc acgtagccat 3420
ttttgcaaag atgaccagac atctctttct tggtttgacg ttgataattt ttgcaaacac 3480
tgcatgggca ccacactcga ccatttcttg acgaagaaaa aacacgattg ataaaatctt 3540
cggttttctg tatccactca agcgaaggat cattttttcg ccagccttca tacatccagc 3600
gacgactatc acccatcgtt cgactaatta aagatcaaga caagttcatt aatttctaga 3660
tatattatga gccggtggtc ctacaattat aggaaaggat aggtcctaaa cccacctaag 3720
ggtgaacgac atatcacaga tttatgataa gtcaatgagt ttgaacgaaa tttcggcagc 3780
ataaccctgt tgttctccat tgcacgcccg taatggtgca acggagaaca agagggttat 3840
gctgtcgaaa tctcgctcaa actcattgac taatcataaa tcatatttga tatgtcgttc 3900
actctaaggc tgtccaaaaa gggacaattt cggtacaaca cactacttta ttaagcaatg 3960
tgttggaccg aaacgggtga cgcataagtt taaacctatt ttagtatagt atcgcattat 4020
actacaacat cacatcaaat tatactacag catcacatta tactacaaca tgacattaat 4080
aaagttaaaa attacctgaa ttgcccacag gagtgacgac tggcgaccgg tgtgaagctg 4140
cgtgcgcgcg ctcacgtgcg gccacgttcg ggcccgtgcg aacggccacg tgcgcgcgct 4200
cacgtgacaa gactcgacga cggcctcggg cttggggacg gctgcggcct cgggtaacgc 4260
ggcggccaag gcggggaggt cccggcggcc acgggcggcg ctggccaggg gcggggaggc 4320
cgcggcgtcc accgcggcgc tctggccagg ggcggcggcg gcggctctgg cgcgcgagcc 4380
ccccgggcgg cggcggccac gggcggcgct gtccaagggt ctggcgagcc ccccgggcgg 4440
cggtgcgcgg ctctggcgac ccgccggtgc acaccttccc gcaccacgag ttcgccgaga 4500
tccccgacgt ctccgacacg gcggcgctct acggcgcgca ggagctgcag taccgctcct 4560
tctgcttcga cccggaccgg ctggagcgcg tccgcgggct gacgcttgcc gacggcgccc 4620
tcggccgctg caccaccttc gaggcgctgt cggggctcgt gtggcgtctc tggcgtctcc 4680
ggcgagcctc cggggcggcg gcggcgcgct gtgcgggcgg cggcacggac gcgggcggca 4740
gcgcggacgc gggcggcggc gcggacgtgg gtgtgcgggc ggcggcgcgg acgtcgacga 4800
gggacgtggg cgcgctgtgc gggcgacggc ggcggacggg cggcggcggc gcggtgggtg 4860
tgcgggcgcg cggctgtgcg ggcgtgtgtg cgttgtgtgc gtccgtgcgc ggggtaacgg 4920
acgacgttag ggaaattgaa ttatttccga cggccaaggt ggtgggccgt cggaaataga 4980
cttatttccg acggctggcc gaaggccgtc ggaaataagg ctatttccga cggtacccta 5040
agaaccgtcg gaaacagtgt aggccgtcgg aaactggtcg ttttcctgta gtgagctacg 5100
gagatatcca tctatctcga cctccattgc cattgggtcg tcatgcttgt ggttcgtgct 5160
gcaggaatag atagacagat agatagatag cctcatattt gatttgattc tacggagata 5220
tccatctatc tcgacctcca ttgccattgg gtcgtcatgc ttgtggttcg tgctgcagga 5280
atagatagac agatagatag atagcctcat atttgatttg attcgggtac gcctgcacct 5340
tggattgaga a 5351
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<211> 229
<212> DNA
<213>Corn
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cacgtatgtg catccatcag gaatcaatgg ccgctggccc cagcttcgaa ttttgctgct 60
gccttttaac aaagctatag ctagtagcta cggagatatc catctatctc gacctccatt 120
gccattgggt cgtcatgctt gtggttcgtg ctgcaggaat agatagacag atagatagat 180
agcctcatat ttgatttgat tcgggtacgc ctgcaccttg gattgagaa 229
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
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cacgtatgtg catccatcag 20
<210> 4
<211> 20
<212> DNA
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ttctcaatcc aaggtgcagg 20
<210> 5
<211> 20
<212> DNA
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tccatgaggt tttccgcctc 20
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cttccccgtg gatatgctcc 20
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tggaggaggt ggcatagtca 20
<210> 8
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<212> DNA
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gtttggcaaa gcgcatggta 20
<210> 9
<211> 20
<212> DNA
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agccataagt cgcgtcacaa 20
<210> 10
<211> 20
<212> DNA
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cggttcttag ggtaccgtcg 20
<210> 11
<211> 20
<212> DNA
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agtcggaagt gtcagcgttt 20
<210> 12
<211> 20
<212> DNA
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ccacctatcc cgcagtttgt 20
<210> 13
<211> 20
<212> DNA
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cacacacatc cacaagacct 20
<210> 14
<211> 20
<212> DNA
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tccaagacaa gagcttcagc 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
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cgccgtacag actgctatga 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
cacatgctac gactgcgatg 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence
<400> 17
cctccggtac gcaccttact 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence
<400> 18
ccaaggtcaa cttcagccat 20
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence
<400> 19
tgcagcaggt tcatgtttat 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<400> 20
ttccaactta tcagcgacga 20
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence
<400> 21
aacgacgacg acctcatcag 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
caatacttac gatggtggcg 20
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence
<400> 23
actggatgga atggatggat 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<400> 24
gacgaataat gatggctgct 20
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence
<400> 25
atctacaacg gcacgctgat 20
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<400> 26
cctccagttc tagccagctt 20
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
ggagtatcaa tcttcgaggc 20
<210> 28
<211> 20
<212> DNA
<213>Artificial sequence
<400> 28
gtggtcaatg caattcagag 20
<210> 29
<211> 20
<212> DNA
<213>Artificial sequence
<400> 29
cacgcagtca tgtgaggtcc 20
<210> 30
<211> 20
<212> DNA
<213>Artificial sequence
<400> 30
ggaggcagac tcttggcgat 20
<210> 31
<211> 20
<212> DNA
<213>Artificial sequence
<400> 31
ccaatgtggt ctcagaaacg 20
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence
<400> 32
ccgaaaatga tgcagaatgt 20
<210> 33
<211> 23
<212> DNA
<213>Artificial sequence
<400> 33
cttgtatcat cagctagggc atg 23
<210> 34
<211> 20
<212> DNA
<213>Artificial sequence
<400> 34
gtgatctgaa cgccaacctc 20
Claims (10)
1. corn breeding method, including：
M1) corn first and corn second are hybridized, obtain first-filial generation F1, the corn first is AA maize genotypes or AB genotype
Corn；The corn second is BB maize genotypes；Two chromosomes of the AA maize genotypes are free of sequence 1 in sequence table
89-5210 positions shown in DNA fragmentation；Two chromosomes of the BB maize genotypes are containing sequence in ordered list 1
DNA fragmentation shown in 89-5210 positions；
M2) by the first-filial generation F1Corn second backcrossing with as recurrent parent, obtains backcross progeny, backcrossing is equal every time
AB maize genotypes are selected to be returned with the corn second；The selfing of AB maize genotypes is selected in the backcross progeny, is obtained certainly
Offspring is handed over, selects AA maize genotypes to obtain purpose corn inbred line in the self progeny；The AB maize genotypes
Item chromosome contain DNA fragmentation shown in the 89-5210 positions of sequence 1, another item chromosome is free of the 89- of sequence 1
DNA fragmentation shown in 5210；
AB maize genotypes have following R1 in the backcross progeny), R2) and/or feature R3)：
R1) long tasseling stage, long loose powder phase and/or long spinning phase under long-day conditions；
R2) disease resistance；
R3) resistance.
2. according to the method described in claim 1, it is characterised in that：The disease resistance is anti-Fusarium graminearum (Fusarium
Graminearum disease caused by).
3. method according to claim 1 or 2, it is characterised in that：The resistance is that low nitrogen resisting is coerced and/or high salt is coerced
Compel.
4. according to any described method in claim 1-3, it is characterised in that：M2 backcrossing number is more than or equal to 3 times in).
5. according to any described method in claim 1-4, it is characterised in that：AB maize genotypes are selected in backcross process
Using primer pair treat selection corn gene group DNA enter performing PCR amplification detected, institute is determined according to pcr amplification product
State whether corn to be measured is AB maize genotypes；
DNA fragmentation shown in the 89-5210 positions of the primer pair energy specific recognition sequence 1 or thereon downstream sequence.
6. according to the method for claim 5, it is characterised in that：The primer pair is：TE1C-FP and Primer4 RP are formed
Primer pair, the primer pair of Primer4 FP and the Primer4 RP composition, Primer2 FP and Primer2 RP compositions
Primer pair, the primer pair of Primer1 FP and Primer1RP compositions, the primer pair of Primer3 FP and Primer3 RP compositions,
The primer pair of Primer3 FP and TE1C-RP compositions, and/or, the primer pair of the TE1C-FP and TE1C-RP compositions；
The TE1C-FP is the DNA molecular shown in sequence 3 in sequence table；
The TE1C-RP is the DNA molecular shown in sequence 4 in sequence table；
The Primer1 FP are the DNA molecular shown in sequence 5 in sequence table；
The Primer1 RP are the DNA molecular shown in sequence 6 in sequence table；
The Primer2 FP are the DNA molecular shown in sequence 7 in sequence table；
The Primer2 RP are the DNA molecular shown in sequence 8 in sequence table；
The Primer3 FP are the DNA molecular shown in sequence 9 in sequence table；
The Primer3 RP are the DNA molecular shown in sequence 10 in sequence table；
The Primer4 FP are the DNA molecular shown in sequence 11 in sequence table；
The Primer4 RP are the DNA molecular shown in sequence 12 in sequence table.
7. according to any described method in claim 1-6, it is characterised in that：Methods described is additionally included in right in backcross process
Obtained AB maize genotypes are selected according to following P1), P2) and/or P3) further selected：
P1) Phenotypic Selection：Corn is inoculated with using Fusarium graminearum (Fusarium graminearum), selects the strong plant of disease resistance
Strain；
P2) restructuring selection：Using molecular labeling SSR47, SSR93, STS01, STS378, STS450, STS444, STS02,
SSR261, SSR164, SSR172 and STS03 select the plant consistent with each molecular labeling result of the recurrent parent；
The sequence of two primers of the SSR47 is respectively sequence 13 and 14 in sequence table；
The sequence of two primers of the SSR93 is respectively sequence 15 and 16 in sequence table；
The sequence of two primers of the STS01 is respectively sequence 17 and 18 in sequence table；
The sequence of two primers of the STS378 is respectively sequence 19 and 20 in sequence table；
The sequence of two primers of the STS450 is respectively sequence 21 and 22 in sequence table；
The sequence of two primers of the STS444 is respectively sequence 23 and 24 in sequence table；
The sequence of two primers of the STS02 is respectively sequence 25 and 26 in sequence table；
The sequence of two primers of the SSR261 is respectively sequence 27 and 28 in sequence table；
The sequence of two primers of the SSR164 is respectively sequence 29 and 30 in sequence table；
The sequence of two primers of the SSR172 is respectively sequence 31 and 32 in sequence table；
The sequence of two primers of the STS03 is respectively sequence 33 and 34 in sequence table；
P3) Foreground selection：Returned using GoldenGate 6KSNP (Illumina, San Diego, CA, USA) chip selection background
The high plant of multiple rate.
8. according to any described method in claim 1-7, it is characterised in that：The corn first be CIMBL157, GEMS23,
GEMS14,1145, Zhong69, CIMBL40, CIMBL151, CIMBL53 or CML454；
And/or the corn second is Chang7-2, Zheng58, A5302 or F349.
9.DNA molecules, the DNA molecular shown in 89-5210 positions or its any fragment for sequence 1 or the 89- containing sequence 1
The DNA molecular of 5210.
10. the DNA molecular shown in the 89-5210 positions of sequence 1 or its any fragment or the 89-5210 positions containing sequence 1
Application of the DNA molecular in corn breeding.
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