CN113557955B - Haploid induction line genetic purification method based on reproductive isolation traits - Google Patents

Haploid induction line genetic purification method based on reproductive isolation traits Download PDF

Info

Publication number
CN113557955B
CN113557955B CN202110812561.1A CN202110812561A CN113557955B CN 113557955 B CN113557955 B CN 113557955B CN 202110812561 A CN202110812561 A CN 202110812561A CN 113557955 B CN113557955 B CN 113557955B
Authority
CN
China
Prior art keywords
gene
haploid
plant
reproductive isolation
sequence
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.)
Active
Application number
CN202110812561.1A
Other languages
Chinese (zh)
Other versions
CN113557955A (en
Inventor
陈绍江
郭树威
焦炎炎
刘晨旭
陈明
钟裕
陈琛
祁晓龙
程德荷
李金龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Agricultural University
Original Assignee
China Agricultural University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by China Agricultural University filed Critical China Agricultural University
Priority to CN202110812561.1A priority Critical patent/CN113557955B/en
Publication of CN113557955A publication Critical patent/CN113557955A/en
Application granted granted Critical
Publication of CN113557955B publication Critical patent/CN113557955B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/021—Methods of breeding using interspecific crosses, i.e. interspecies crosses
    • 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
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Botany (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Husbandry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Mycology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention discloses a haploid induction line genetic purification method based on reproductive isolation traits. The invention applies the reproductive isolation character gene to the genetic purification work of an induction line to form a haploid induction line material which only receives self pollen and has a biological isolation function. The haploid inducer material with the biological isolation function is applied to commercialized DH breeding, is beneficial to avoiding the influence of external pollen on the induction performance of the inducer, solves the problem of genetic purity preservation in the breeding process of the inducer, provides a feasible scheme for solving the purity preservation work of key inducer materials in the haploid breeding process, can greatly reduce the breeding workload of the inducer and can ensure the purity of the inducer materials; provides a new idea for breeding of the popcorn and partial sweet waxy corn materials and promotes the popularization and application of the haploid breeding technology.

Description

Haploid induction line genetic purification method based on reproductive isolation traits
Technical Field
The invention belongs to the field of plant genetic breeding, and particularly relates to a haploid induction line genetic purification method based on reproductive isolation traits.
Background
Maize is a typical heterosis and cross-pollination crop, and the core of breeding is to obtain an excellent inbred line. The traditional method adopts hybridization and multi-generation backcross to obtain pure lines, the period is long, and the consumed manpower, financial resources and material resources are large. The haploid breeding technology can obtain a pure line within 1-2 years through the processes of induction, identification, doubling and the like, greatly shortens the breeding period, improves the breeding efficiency, and becomes one of the mainstream technologies of corn breeding. As one of the key materials in the technical process of haploid breeding, the work of purity preservation of an induction line is important. The key inducible gene zmpla1 on maize qhir1 was the first key site found to affect haploid inductivity (Liu et al, 2017; Kelliher et al, 2017; Gilles et al, 2017); another key inducible gene, zmdmp, on qhir8 was shown to achieve a haploid inducer doubling effect based on the presence of zmpla1 (Zhong et al, 2019). The reproductive isolation trait is present in a variety of plants, and this trait is genetically controlled and exhibits cross-incompatibility between different materials. On the corn, the burst corn has the characteristic that the Ga gene for controlling reproductive isolation has one-way hybridization incompatibility, the material with the gene can be used as a male parent to pollinate with other common materials to bear fruits, and the reverse cross is generally weak.
The development and application of the haploid breeding technology can greatly improve the breeding efficiency, and the technical application of the haploid breeding technology depends on the induction of a male parent and a female parent to produce the haploid by an induction line. The haploid inductivity of the induction line depends on the homozygosity of the induction gene, and although the purity preservation in the propagation process can be realized through strict selfing, the genetic purity preservation process in the propagation process can be simplified through the aggregation of the reproductive isolation character and the induction character, the inductivity of the induction line is maintained, and a new means is provided for the efficient utilization of the induction line.
Disclosure of Invention
The invention aims to solve the technical problem of how to keep pure the haploid induction line of the corn or how to carry out pure breeding on the haploid induction line of the corn.
In order to solve the technical problems, the invention firstly provides a method for the pure reproduction of a plant haploid induction line. The method comprises the following steps:
a1, hybridizing an induction line material carrying haploid induction related genes as a female parent and a plant carrying reproductive isolation genes as a male parent to obtain a hybrid of an F1 generation;
a2 planting said F 1 Generation hybridTo obtain the F 1 Generation of hybrid plants, said F 1 And (3) selfing the generation hybrid plants or backcrossing by taking the induction line material as a recurrent parent, detecting the genotype of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by selfing or backcrossing, and finally obtaining the selfed or backcrossed plants carrying the homozygous genotypes of the haploid induction related gene and the reproductive isolation gene, wherein the plants are plant haploid induction lines obtained by pure breeding.
In the method described above, the plant may be any one of:
1) a dicotyledonous plant;
2) (ii) a monocotyledonous plant which is,
3) a plant of the order of the gramineae,
4) a plant belonging to the family of the Gramineae,
5) a plant of the genus Zea, which plant is selected from the group consisting of Zea mays,
6) corn.
The dicot may include, but is not limited to, oilseed rape, sunflower, peanut, tomato, and the like. The monocot may include, but is not limited to, wheat, rice, and the like.
In the methods described above, the haploid induction related genes can be the maize zmpla1 and zmdmp genes. The reproductive isolation gene can be a maize Ga1-S gene. The haploid induction related gene can also be other haploid induction genes, such as a CENH3 gene, an ig gene or a PLD3 gene. The reproductive isolation gene (cross-incompatible gene) may also be other reproductive isolation genes such as SPRI1, AtLURE1, UI3a, and the like.
In the method described above, the plant haploid inducer line can be the maize haploid inducer line CAU 5. The plant haploid inducer can also be other corn haploid inducer materials. The plant haploid induction line can also be used as induction line material of other plant haploids.
In the methods described above, the plant carrying the reproductive isolation gene can be maize material 401D. The plant material carrying the reproductive isolation gene can also be other maize materials containing the reproductive isolation gene. The plant material carrying the reproductive isolation gene can also be other plant materials carrying the reproductive isolation gene. The reproductive isolation gene can also be other reproductive isolation genes, such as SPRI1, AtLURE1, UI3a, and the like.
In the method described above, the method for detecting the genotypes of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by selfing or backcrossing in A2 can comprise performing PCR amplification detection on a plant to be detected by using primers. The plant containing the segment of the reproductive isolation gene in the PCR amplification product can be a plant carrying the reproductive isolation gene A1.
The primers described above may be the primer pairs described in P1 and/or P2:
p1, and DNA molecules shown in sequence 1 and sequence 2 in the sequence table;
p2, and DNA molecules shown in sequence 3 and sequence 4 in the sequence table.
In the method, the primer can be a primer pair of P1, and the segment of the reproductive isolation gene can be a DNA molecule shown as a sequence 5 in a sequence table. The primer can also be a primer pair of P2, and the segment of the reproductive isolation gene can be a DNA molecule shown as a sequence 6 in a sequence table.
Any of the following uses of the above described method also fall within the scope of the invention:
d1, application of the method in plant induction line purification breeding;
d2, use of the method described above in a plant haploid production process;
d3, and the application of the method in the popularization of plant haploid breeding technology.
The application of the said reproductive isolation gene in the above-mentioned method in the construction of plant purity-maintaining haploid inducer line also belongs to the protection scope of the present invention.
The application of the said reproductive isolation gene in the above-mentioned method in plant induction line purification breeding also belongs to the protection scope of the present invention. The invention aims to provide a method for carrying out purity preservation work on a special corn haploid induction line by utilizing a reproductive isolation gene.
Experiments prove that: the invention utilizes a high-frequency haploid induction line CAU5 bred by Chinese agricultural breeding as a female parent, uses a cracked corn material 401D with unidirectional reproductive isolation performance as a male parent, and introduces reproductive isolation gametophyte genes in the cracked corn into the haploid induction line by a backcross method on the basis of molecular marker assisted selection to construct a haploid induction line pCAU5 which is incompatible with unidirectional hybridization. Through field verification, the haploid induction line has good fruit when selfing or pollinating other materials (including dent type, hard grain type and partial cracked corn) as a male parent, but has no fruit when pollinating the materials by common corn (without reproductive isolation gene). In addition, ZD958 test shows that the induction rate of the induction system can reach 10%.
The invention applies the reproductive isolation character to the genetic purification work of an induction line to form an induction line material which only receives self pollen and has a biological isolation function. The material is applied to commercialized DH breeding, is beneficial to avoiding the influence of foreign pollen on the induction performance of an induction line, and solves the problem of genetic purity preservation in the breeding process of the induction line.
The method provided by the invention provides a feasible scheme for solving the problem of purity preservation of key inducing line materials in the haploid breeding process, can effectively reduce the probability of field impurity bearing in breeding work of key materials such as inducing lines and the like, can reduce the breeding work task of the inducing lines, and improves the corn breeding efficiency.
Drawings
FIG. 1 is agarose electrophoresis picture of key gene locus for molecular marker detection. A is ZmPLA1 genotype identification, B is ZmDMP genotype identification, and C is reproductive isolation gene identification.
FIG. 2 is agarose electrophoresis picture of key gene locus of new target induction system containing reproductive isolation gene. A is ZmPLA1 genotype identification, B is ZmDMP genotype identification, and C is reproductive isolation gene identification.
FIG. 3 is a diagram of the plant and ear of the new target induction line pCAU 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The haploid inducer CAU5 used in the following examples was provided by chenshaojiang task group, the chinese agricultural university, the average induction rate of which was about 10%, and which had the R1-nj color marker, disclosed in the document "chenshaojiang, liliang, lihaochuan corn haploid breeding technique [ M ]. china agricultural university press 2009", which was publicly available from chinese agricultural university to repeat the present invention.
The popcorn maize line 401D was maintained by chenshaojiang topic group of chinese agricultural university (related documents: muluke, gumingxin, chenshaojiang. "selective hybridization affinity phenomenon of maize cross-incompatibility gene Ga1-s observation". 2012 national maize genetics and breeding institute and new variety display spectacular treatises and abstract collections.ed. {4}, 2012, 47. the public can obtain from the applicant, only used for experiments of repeating the present invention, and can not be used for other purposes), the existence of the reproductive isolation gene locus was ensured according to molecular marker detection, and the field verification shows that the one-way hybridization incompatibility.
Tris saturated phenol, chloroform, alcohol, isopropanol and agarose in the following examples are all products of Beijing GJIPROTENG Biotechnology Co.
The Mix reagent for PCR amplification in the following examples is product of Beijing Edelay Biotechnology Ltd.
Example 1 construction of haploid inducer lines with unidirectional reproductive isolation Properties
Breeding process of haploid inducer line with unidirectional reproductive isolation performance
To isolate the trait for unidirectional reproductionPolymerizing with haploid induced character, introducing the reproductive isolation character gene into haploid induced line, and finally making the corn haploid induced line obtain pure reproduction, firstly using CAU5 (molecular marker detection its key induced site gene ZmPLA1 and ZmDMP are recessive homozygous) as female parent and explosion corn line 401D (molecular marker detection its reproductive isolation gene, namely one-way hybridization incompatibility gene Ga1-S dominant homozygous) as male parent to make hybridization so as to obtain F whose key induced site and hybridization incompatibility gene site are both heterozygous 1 Generation hybrid seeds; planting F 1 Generation of hybrid seed to obtain F 1 Generation hybrid maize plants, using CAU5 as recurrent parent (female parent), and F 1 Backcrossing the generation hybrid corn (male parent) for 3 generations, detecting the genotype of the reproductive isolation gene locus in each generation, selecting the corn with the Ga1-S genotype homozygous or heterozygous for backcrossing, observing and recording the field phenotype and the seed character to obtain BC 3 F 1 Generation; selection of BC homozygous or heterozygous for the Ga1-S genotype and recessive homozygous for the ZmPLA1 and ZmDMP genes 3 F 1 The single plant is inbred for 2 generations, each generation carries out molecular marker detection on the gene types of Ga1-S, ZmPLA1 and ZmDMP genes, and finally, the plant in which the gene type of Ga1-S locus is dominant homozygous and the gene types of ZmPLA1 and ZmDMP genes are recessive homozygous is selected as a one-way cross incompatible haploid induction line which is named as pCAU 5.
Wherein, the method for detecting the genotypes of the key induction site genes ZmPLA1 and ZmDMP and the reproductive isolation site gene Ga1-S by molecular markers is shown in the fourth step.
Design of molecular marker primer
Detection primers for ZmPLA1 on QTL site qhir1 and ZmDMP induction site on qhir8 of CAU5 induction line are detected by using self-developed primer sequences of Chenshaojiang topic groups of China university of agriculture respectively:
PLA1-F：5'-ACGTGGAGACAGGGAGGTAC-3'；
PLA1-R：5'-GCTTCTGGGGTTGATGGCAG-3'。
pla1-F：5'-ACGTGGAGACAGGGAGCGAG-3'；
pla1-R：5'-GCTTCTGGGGTTGATGGCAG-3'。
chr9-76s1F：5'-CAAAACCACTTCAATCCAGGT-3'；
chr9-76s1R：5'-GTACGTCAGGAGTCCGGAGA-3'。
the one-way hybridization incompatibility gene Ga1-S (Zm00001d048936) sequence was downloaded through NCBI website. Autonomously designed primers CG17-F/R are used for carrying out PCR amplification and bidirectional sequencing on DNA of parent materials CAU5 and 401D to obtain gene sequences of the two, and then Primer pairs CG19-F/R and CG13-F/R are designed by using a Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /) tool on an NCBI website according to a sequencing result, and are used for detecting Ga1-S genes, wherein the specific sequences are as follows:
CG17-F：5'-ACACGACCATTAGCGAGTCC-3'；
CG17-R：5'-ATCCCGAGAAAGGGTATTGCC-3'。
CG 19-F: 5'-ACACGACCATTAGCGAGTCC-3' (sequence 1 in the sequence table);
CG 19-R: 5'-ACCTTTCGACTGTCTGGACG-3' (SEQ ID NO: 2 in the sequence Listing).
CG 13-F: 5'-ACACGACCATTAGCGAGTCC-3' (SEQ ID NO: 3 in the sequence Listing);
CG 13-R: 5'-CGGCGTATACAACGTCGTAACC-3' (sequence 4 in the sequence table), wherein the CGGC of the 4 basic groups at the CG13-R5' end is supplemented according to the content requirement of the CG of the primer sequence).
Third, DNA extraction
Maize material was sampled from individual leaves at the five-leaf stage and strictly numbered, cryopreserved, and genomic DNA extracted as described by Murray and Thompson (1980).
Fourth, genotype identification
And (3) performing PCR amplification by using the extracted DNA as a template and adopting each F/R primer pair in the second step.
PCR amplification reaction System: DNA 1. mu.L, F/R primers 0.5. mu.L each, Mix 5. mu.L, ddH 2 O3. mu.L, 10. mu.L of paraffin oil was added.
The PCR amplification reaction conditions are shown in Table 1.
TABLE 1 PCR amplification reaction conditions for different primer pairs
Figure BDA0003168767180000051
Figure BDA0003168767180000061
Agarose gel electrophoresis: preparing 2% agarose gel, detecting the PCR amplification product by electrophoresis, and carrying out sequencing verification at 160V for 12 minutes.
PLA 1-F/R: wild type ZmPLA1/ZmPLA1 (i.e. dominant homozygous genotype) and heterozygous genotype ZmPLA1/ZmPLA1 materials have bands (155bp), and recessive homozygous genotype ZmPLA1/ZmPLA1 materials have no bands. See specifically a1 in fig. 1 and a in fig. 2;
pla 1-F/R: bands (141bp) exist in recessive homozygous genotype Zmpla1/Zmpla1 and heterozygous genotype ZmPLA1/Zmpla1 materials, and no band exists in wild type, i.e. dominant homozygous genotype ZmPLA1/ZmPLA1 materials. See specifically a2 in fig. 1 and a in fig. 2;
chr9-76s 1F/R: the wild type, i.e. dominant homozygous genotype ZmDMP/ZmDMP material exists in the upper band (491bp), the heterozygous genotype ZmDMP/ZmDMP material shows upper and lower double bands (491bp and 333bp), and the recessive homozygous genotype is ZmDMP/ZmDMP material only has the lower band (333 bp). See in particular B in fig. 1 and B in fig. 2.
CG 17-F/R: the size of the dominant homozygous genotype Ga1-S/Ga1-S is about 965bp band, and the size of the recessive homozygous genotype Ga1/Ga1 material is about 860bp band. See in particular C3 in fig. 1.
CG 19-F/R: the dominant homozygous genotype Ga1-S/Ga1-S and heterozygous genotype Ga1-S/Ga1 materials have a 747bp band (sequence 5 in a sequence table), and the genotype Ga1/Ga1 materials have no band. See in particular C1 in fig. 1 and C in fig. 2.
CG 13-F/R: recessive homozygous genotype Ga1/Ga1 and heterozygous genotype Ga1-S/Ga1 materials have 741bp bands (sequence 6 in a sequence table), and dominant homozygous genotype Ga1-S/Ga1-S materials have no bands. See in particular C2 in fig. 1 and C in fig. 2.
Example 2 evaluation of the Properties of the one-way incompatible haploid inducer pCAU5
One-way incompatibility verification
In 5 months of 2020, the target inducible line pCAU5 obtained in example 1 and various conventional materials (including commercial hybrid ZD958, ND678 and JK968 and conventional inbred lines C229, Chen04_065 and Chen04_125 bred by Chenshaojiang task group, ZD958 is purchased from Beijing Ded agriculture Co., Ltd., ND678, C229, Chen04_065 and Chen04_125 are provided by Chenshaojiang task group of China agricultural university, JK968 is purchased from Beijing Shuxin agriculture GmbH, and the conventional materials do not contain Ga1-S gene and R1-nj color marker gene) are planted in different plots at Shanzhuang test station of China agricultural university.
A land parcel I: three repeat regions were set, 100 pCAU5 plants and 50 commercial hybrids ZD958, ND678 and JK968 plants were planted in each repeat region, and staggered planting ensured that the flowering phases met. The material carrying the Ga1-S gene is not contained around the plot. Strictly castrating pCAU5 before pollen scattering and bagging the female ear; pollen of three hybrids ZD958, ND678 and JK968 (as male parents) is mixed in equal proportion in the flowering period, then the pCAU5 material (as female parents) is pollinated, the fruiting condition of pCAU5 is observed 25 days after pollination, and the fruiting of pCAU5 is 0;
and (2) land parcel II: three repeat regions were provided, each repeat region was seeded with 100 pCAU5, 50 each of the commercial hybrids ZD958, ND678 and JK968, where the hybrids were distributed around pCAU5 and staggered planting ensured that the flowering phases met. The material carrying the Ga1-S gene is not contained around the plot. Strictly castrating pCAU5 before pollen scattering and not bagging female ears, and carrying out open pollination; pCAU5 set was observed about 25 days after pollen dispersal for the hybrids, and pCAU5 set was 0;
and (3) land parcel III: three repetitive areas are provided, 50 plants of 100 pCAU5, conventional inbred lines C229, Chen04-065 and Chen04-125 are planted in each repetitive area, wherein the inbred lines and the pCAU5 are planted at intervals, and staggered planting ensures that the flowering phases meet. No other inducible materials were included around the plot. All materials were open pollinated. Observing the colors of a conventional inbred line and a pCAU5 grain aleurone layer after fruit ears are harvested, wherein pCAU5 is fruited normally, the grain aleurone layer is provided with R1-nj color markers (the R1-nj color marker gene is dominant marker, and the heterozygous genotype or dominant homozygous genotype grain aleurone layer is provided with color), and the aleurone layer is purple; the seeds of the conventional inbred line not only contain the seeds with the color marks of R1-nj, but also have the color marks of R1-nj, namely normal yellow seeds.
And (4) land parcel four: three replicate regions were set up, 100 pCAU5 plants were planted per replicate region, the pCAU5 was strictly emasculated and the ears were bagged before pollen dispersal, and all materials were strictly selfed. After the ears are harvested, the pCAU5 fructification condition and the color of the aleurone layer of the seeds are observed, all single plants are normally fructified, the aleurone layer of the seeds is marked with the color of R1-nj, and the aleurone layer is purple.
The results show that the haploid induction line pCAU5 carries reproductive isolation genes, is an induction line material with a biological isolation function, can eliminate the influence of exogenous pollen when mixed with conventional materials for open pollination, and realizes the purity preservation of the induction line.
Second, induction rate test
In 5 months of 2020, three repeat regions were set up at Shanzhuang test station of Chinese agricultural university, 100 pCAU5 and 500 ZD958 were planted in each repeat region, and staggered planting ensured that the florescence met. Before pollen scattering, carrying out genotype detection on pCAU5, selecting a material with two key induction sites of zmpla1 and zmdmp genotype as recessive homozygous genotype and reproductive isolation gene Ga1-S genotype as dominant homozygous genotype (the corresponding genotype combination of the three genes of the material is zmpla1/zmpla1+ zmdmp/zmdmp + Ga1-S/Ga1-S) to carry out induction rate test on ZD958 pollination, and pollinating 5 ZD958 strains by each pCAU5, wherein the total number of 500 ZD958 strains are obtained. And (2) identifying the haploid grain by utilizing an R1-nj color marker after the fruit cluster is harvested (when ZD958 is pollinated by utilizing pCAU5, the obtained diploid embryo and aleurone layer have purple color markers, while the haploid aleurone layer has color but the haploid embryo has no color, and the haploid grain can be identified from the diploid grain according to the color difference on the embryo). Fruit ears with less than 100 single ear fruits and containing a plurality of seeds without R1-nj color markers are discarded, and the average haploid inductivity of pCAU5 to ZD958 can reach 10 +/-1.5% by statistics. Wherein, the single haploid inductivity calculation formula is as follows: the induction rate is haploid number/total fructification number.
Third, survey of agronomic characters
In 5 months of 2020, three repeat regions were established at the Shanzhuang test station at the university of agriculture in China, and 100 strains each of pCAU5, CAU5 and 401D were grown in each repeat region. Agronomic traits such as plant height, ear position, flowering phase, pollen dispersing duration, tassel branch, tassel main branch length and the like of pCAU5, CAU5 and 401D were investigated (FIG. 3), and the data are as follows:
TABLE 2 comparison of agronomic traits for novel inducible lines containing the reproductive isolation genes pCAU5, CAU5, 401D (mean. + -. standard deviation)
Figure BDA0003168767180000081
The result shows that the haploid inducer pCAU5 with reproductive isolation performance obtained by the method also has excellent agronomic characters such as short growth period, low tassel position, high tassel branch number and the like. Therefore, the induction system material has a biological isolation function, has excellent agronomic characters and has a wide application prospect in production.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> university of agriculture in China
<120> haploid induction line genetic purification method based on reproductive isolation traits
<130> GNCSQ211621
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acacgaccat tagcgagtcc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acctttcgac tgtctggacg 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acacgaccat tagcgagtcc 20
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cggcgtatac aacgtcgtaa cc 22
<210> 5
<211> 747
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acacgaccat tagcgagtcc atcgccaaca tccctgagga caacgccaaa cgctacctcc 60
ttatcctcaa acctggtgtt gtgttccgcg agaagctgtt actcggtaga agcaagcctt 120
tcatcaccat aatgtccgag gaccccatga accctgctgt tatcgtctgg aatgacactg 180
ccaccaccat gggcaaggac ggcaagcccc ttggtgtgga tggaagcagt accatggcca 240
tagagtccga ctattttgtc gcctacaacg ttgtcttcaa gaacgacgcg ccgctaccaa 300
agctagggga aaagaaaggt gaggcaccag cactgcgagt gatgggaaca aaggcaacct 360
tctacaattg caccatcgaa ggcggccagg gtgctctgta cgaccagacg ggtctgcact 420
acttcaaggc ttgtgccatc aagggaacca tcgacttcat cttcggatct gccaagtcat 480
tttatgagga atgcaaaatc gtttcggtgt tgaaggaggc attggtattg ccattggcac 540
caccggagca ggaccgctct agaaatccca tcgaaatcgc cccaggcaag agcgggttgg 600
cattcaagac ttgcacaatc gagggggaag gagaaaaaat ttacttgggt agggtgggca 660
cgcctgtgat atactcctac actaatatag gtaaggagat tgtaggcata atatctgatg 720
gtcgggacgt ccagacagtc gaaaggt 747
<210> 6
<211> 741
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acacgaccat tagcgagtcc attgccaaca tccctgacga caacaccaaa tgctacgtcc 60
ttaccctcaa acctggtgtt gtgttccgcg agaagctgtt actcggtaga agcaagccat 120
tcctcaccat aatatccgag gaccccatga acccagctat tatcgtctag aatgacactg 180
ccaccaccat gggcaaggac ggcaagcccc ttggtgtgga tggaagcagc accatggcga 240
tagagtccaa ctattttgtc gcctacaaca ttgtcttcaa gaacgacgcg ccgctaccaa 300
agctagggga aaagaaaggt gaggcaccag cactgcgagt gatgggaaca aaggcaacct 360
tctacaattg caccatccaa tgcggccagg gtgctctaaa cgaccagacg ggtctgcact 420
acttcaaggc atgtgccatc aagggaacca tcgacttcat cttcggatct gccaagtcat 480
tttatgagga atgcaaaatt gttttggtgt tgaaggaggc attgacattg ccaatcgtgc 540
caccggagca ggaccggtct agaaatccca tcaaaattgc cccaggcaag agcgggttgg 600
cattcaagac ttgcacaatc gagggggaag aagaaaaaat ttacttgagt agggtgggca 660
cgcctgtgat ctactcctac accgatatag gtaaggagat tgtaggcata atatctaatg 720
gttacgacgt tgtatacgcc g 741

Claims (4)

1. The method for the purity preservation and propagation of the plant haploid induction line is characterized by comprising the following steps: the method comprises the following steps:
a1, hybridizing an induction line material carrying haploid induction related genes as a female parent and a plant carrying reproductive isolation genes as a male parent to obtain a hybrid of an F1 generation; the plant haploid inducer is a corn haploid inducer CAU 5; the haploid induction related gene is cornzmpla1Andzmdmpa gene; the plant carrying the reproductive isolation gene is a corn material 401D; the reproductive isolation gene is cornGa1-SA gene; the above-mentionedGa1-SThe genotype of the gene is dominant homozygous;
a2 planting said F 1 Generation of hybrid to obtain the F 1 Generation hybrid plants, using the inducing line material as recurrent parent and F 1 Backcrossing the generation of hybrid plants, detecting the genotype of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by the backcrossing, selecting the genotype of the reproductive isolation gene as homozygous or heterozygous corn for backcrossing, and backcrossing for 3 generations to obtain BC 3 F 1 Plant, selecting BC that is homozygous or heterozygous for the genotype of the reproductive isolation gene and recessively homozygous for the haploid-induction-associated gene 3 F 1 And (3) selfing the single plant for 2 generations, and finally selecting a plant carrying the haploid induction related gene as recessive homozygous and the genotype of the reproductive isolation gene as dominant homozygous genotype, wherein the plant is a plant haploid induction line obtained by pure breeding.
2. The method of claim 1, wherein: the method for detecting the genotypes of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by backcrossing in A2 comprises the step of carrying out PCR amplification detection on a plant to be detected by using primers, wherein the plant containing the reproductive isolation gene segment in a PCR amplification product is a plant carrying the reproductive isolation gene A1;
the primer is the primer pair of P1 and/or P2:
p1, and DNA molecules shown in sequence 1 and sequence 2 in the sequence table;
p2, and DNA molecules shown in sequence 3 and sequence 4 in the sequence table.
3. The method of claim 2, wherein: the primer is the primer pair P1, and the segment of the reproductive isolation gene is a DNA molecule shown as a sequence 5 in a sequence table; and/or the primer is the primer pair P2, and the segment of the reproductive isolation gene is a DNA molecule shown as a sequence 6 in a sequence table.
4. Use of the method of any one of claims 1-3 for any one of the following:
d1, use of the method of any one of claims 1 to 3 for the maintenance of purity in a plant induction line;
d2, use of the method of any one of claims 1-3 in a plant haploid production process;
d3, the method of any one of claims 1-3, in the popularization of plant haploid breeding technology.
CN202110812561.1A 2021-07-19 2021-07-19 Haploid induction line genetic purification method based on reproductive isolation traits Active CN113557955B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110812561.1A CN113557955B (en) 2021-07-19 2021-07-19 Haploid induction line genetic purification method based on reproductive isolation traits

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110812561.1A CN113557955B (en) 2021-07-19 2021-07-19 Haploid induction line genetic purification method based on reproductive isolation traits

Publications (2)

Publication Number Publication Date
CN113557955A CN113557955A (en) 2021-10-29
CN113557955B true CN113557955B (en) 2022-09-16

Family

ID=78165509

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110812561.1A Active CN113557955B (en) 2021-07-19 2021-07-19 Haploid induction line genetic purification method based on reproductive isolation traits

Country Status (1)

Country Link
CN (1) CN113557955B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817036B (en) * 2021-10-21 2022-06-14 中国农业科学院生物技术研究所 DMP protein and coding gene and application thereof
CN117016378A (en) * 2023-08-24 2023-11-10 北京大学现代农业研究院 Creation method of corn haploid induction line without subspecies restriction

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104342450B (en) * 2013-07-24 2017-08-25 中国农业大学 The method for cultivating corn haploid induction line of the corn haploid-induction higher than corn haploid induction line CAU5
CN104846104B (en) * 2015-05-27 2017-07-21 中国农业大学 The selection and its primer special of a kind of corn haploid induction line
CA3090472A1 (en) * 2018-02-08 2019-08-15 China Agricultural University Maize parthenogenetic haploid-inducing gene zmpla1e and application thereof
CN111996209B (en) * 2019-05-27 2022-05-10 中国农业大学 Parthenogenesis haploid inducing gene DMP and application thereof
CN112889663A (en) * 2019-12-04 2021-06-04 中国科学院遗传与发育生物学研究所 Method for polymerizing multiple corn one-way cross incompatible genes
CN112005878B (en) * 2020-08-24 2022-06-21 中国农业大学 Method for rapidly breeding corn haploid induction line and application thereof

Also Published As

Publication number Publication date
CN113557955A (en) 2021-10-29

Similar Documents

Publication Publication Date Title
CN111165350B (en) Breeding method for efficiently identifying induction line of corn haploid immature embryo
US10415101B2 (en) Methods for producing canola plants with clubroot resistance and compositions thereof
CN112005878B (en) Method for rapidly breeding corn haploid induction line and application thereof
US11337388B2 (en) Maize plants with improved disease resistance
CN113557955B (en) Haploid induction line genetic purification method based on reproductive isolation traits
CA2982495A1 (en) Methods and compositions for producing brachytic corn plants
CN109997683B (en) Rice double haploid breeding method based on haploid induction line
WO2018036540A1 (en) Method for cultivating perennial rice using asexual propagation characteristic of oryza longistaminata
EP2190282A2 (en) Methods for incorporating multiple genes in a crop plant
CN106069719B (en) The method that rape inheritance stability group is created in the scale of rape dihaploid induction system
US20210307275A1 (en) Resistance to xanthomonas campestris pv. campestris (xcc) in cauliflower
Zhang et al. RAPD markers linked to microspore embryogenic ability in Brassica crops
CN110463599A (en) A kind of Direct-seeding Rice selection
CN107586874B (en) Primer pair for identifying yellow anther character of capsicum and application thereof
CN109402295B (en) Method for accelerating backcross breeding process of brassica napus by using orange flower color molecular markers
CN110468229B (en) Coseparation molecular marker Hxjy-1 of rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t)
CN109006456B (en) Breeding method of pimento nuclear male sterile dual-purpose line
CN115786567B (en) Semi-dominant corn dwarf related molecular marker and application thereof
CN116769955A (en) Method for quickly transferring transgenic maize inbred line and application
CN111034612A (en) Breeding method of scab and clavispora leaf spot resistant dense thorn type cucumber hybrid
CN110358862B (en) Molecular marker Hxjy-14 closely linked with rice broad-spectrum high-resistance bacterial blight gene Xa45(t)
CN106636425B (en) Molecular marker of rice purple leaf gene P L1 and identification method and application thereof
CN111512959A (en) Hybrid rape breeding method capable of early identifying seed purity
CN111808981B (en) Method for improving corn haploid ear fertility restoration and special primer thereof
CN112251525B (en) Molecular marker for gene assisted breeding related to rape dominant genic male sterility and application thereof

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