CN115119744A - Apple non-integration system four-group sports planting method - Google Patents
Apple non-integration system four-group sports planting method Download PDFInfo
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- 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
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- 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
-
- 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
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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Abstract
The invention discloses a four-group sports method of an apple apomixis system, which comprises the steps of selecting apomixis base materials of wild natural groups and sequencing genomes; mutagenesis is carried out to create a base material mutant library, and excellent character strains are selected as mutagenesis progeny groups; collecting various source pollen; the flowering time is prolonged; collecting seeds and sowing; carrying out chromosome ploidy identification on the target character seedlings; performing homologous comparison with a wild type after re-sequencing; selecting a strain with complete replacement of the genome or ploidy increase and target characters as a filial generation population; by utilizing the apomixis characteristic, offspring with high apomixis capacity is selected from the mutagenic offspring population and the filial offspring population as an excellent stable population. Based on the facultative apomixis characteristic of the apples, the diversified allopolyploid apples are quickly created and identified by utilizing various source pollen pollination, flow cytometry and genome re-sequencing, and the method greatly enriches the breeding approaches of the apple polyploids.
Description
Technical Field
The invention belongs to the technical field of plant breeding, and particularly relates to a method for non-fusion one-line four-group physical breeding of apples.
Background
Apomixis is a special asexual reproduction in which asexual embryos and asexual seeds are directly produced without fertilization combination of sperms and ova, and progeny of the asexual reproduction does not generate a separation phenomenon, which is favorable for fixing heterosis. Facultative apomixis refers to a mode of reproduction that performs both sexual and apomixis. By utilizing the sexual reproductive capacity of the male parent, the male parent can be selected to be hybridized, and a novel allopolyploid is purposefully created; the species superiority obtained by hybridization can be fixed by utilizing the apomixis ability of the hybrid, and the hybrid is propagated through seed propagation.
Polyploidization is an important way for realizing speciation and biological diversification, and polyploidization breeding is an important means for crop breeding. In general, polyploid plants have superior traits of vigorous growth, strong stress resistance, large organs, high nutritional value, and the like, compared with diploid progenitors thereof. The allopolyploid is usually rendered sterile by male and female gametes, resulting in its reproduction by apomixis, and the allopolyploid fixes a chromosome set from parents with natural heterosis, so that the allopolyploid breeding is particularly important in the whole polyploid breeding work.
However, the problem that the polyploid apple with the multi-resistance and apomixis characteristic is difficult to create at present restricts the application of the apomixis in the polyploid breeding and asexual seed production of the apple. Particularly, in the breeding of polyploid apples, the problems of narrow genetic basis of crop varieties, long breeding period, low resource utilization rate and incapability of transmitting high apomixis characters to offspring through sexual hybridization exist.
Disclosure of Invention
The invention provides a method for producing four-group sports varieties of non-integration one-line apples, which overcomes the defects that the non-integration reproduction varieties and subspecies of the apples are difficult to hybridize, natural filial generations are few, the characters of the filial generations are separated, excellent characters are degenerated by means of vegetative propagation and the like, realizes rapid and high-purpose creation of polyploids, saves great time and space, and can rapidly obtain multi-source distant polyploidy apples.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for cultivating four groups of sports seeds of an apple non-integration system, which comprises the following steps:
(1) selecting wild natural population apomixis apples as a breeding base material, and performing genome sequencing;
(2) creating a mutant library of breeding base materials by using mutation breeding, and selecting strains with excellent target characters as variant progeny populations;
(3) collecting various source pollen, and airing for later use;
(4) prolonging the flowering time of wild type breeding base materials;
(5) carrying out artificial pollination by using the pollen in the step (3) and the breeding base material in the step (4), bagging until the flowering phase is finished, and collecting seeds after fruits are mature;
(6) the seeds in the step (5) are subjected to stratification treatment and germination and then transplanted to a field for sowing, and a plant line with target characters obviously superior to those of a wild type is selected;
(7) extracting the cell nucleus suspension of the strain in the step (6), and performing chromosome ploidy identification after dyeing;
(8) taking the genome in the step (1) as a reference genome, performing re-sequencing on the strain in the step (6), performing homology comparison with the reference genome, and selecting the strain with complete replacement of the genome or increased ploidy as a filial generation population;
(9) and (3) selecting the offspring with high apomixis ability from the variant offspring population in the step (2) and the filial generation population in the step (8) as an excellent stable population by utilizing the apomixis characteristic to obtain a new species of the apomixis apples.
Further, the breeding base material is triploid or tetraploid apomixis apple material; the triploid apomixis apple material comprises Malus hupehensis Rehd, Malus hupehensis Rehd and Malus sieboldii; the tetraploid apomictic apple material comprises Trifolium pratense and Malus micromalus.
Further, the breeding base material comprises a maternal material and a paternal material; the maternal material is a triploid or tetraploid apomixis apple material; the paternal material is tetraploid apomictic apple material.
Further, the mutation breeding in the step (2) comprises EMS mutagenesis, radiation mutagenesis, latent bud and bud mutation.
Further, the pollen includes but is not limited to apple species pollen, apple varieties include cylindrical apple, M9, shanding nut, fuji, gala, and other diploid apple materials.
Further, the method for prolonging the flowering time of the wild type breeding base material in the step (4) comprises the steps of spraying a preservative, reducing the temperature, increasing the humidity and reducing the illumination.
Further, when the flowering time of the wild type breeding basic material is prolonged by spraying the preservative, the formula of the preservative comprises the following components: 8-hydroxyquinoline potassium sulfate, silver nitrate, citric acid and sucrose.
Further, if the breeding base material is a maternal material in the step (5), pollen is adopted to carry out artificial pollination on the maternal material; if the breeding base material is a father material, the diploid apple material is subjected to artificial pollination by using pollen of the father material.
Further, the specific steps of the step (7) are as follows: and (3) shearing young and tender leaves of the strain in the step (6), cleaning the surfaces, draining, putting into a culture dish, adding dissociation liquid, quickly cutting, filtering a filter membrane with 300 meshes and 400 meshes into a centrifuge tube, incubating, adding a staining solution to obtain a stained cell nucleus suspension, and identifying the ploidy of the chromosome of the strain by using a flow cytometer and taking the Malus octandra as a diploid control and the Malus hupehensis as a triploid control.
Further, the complete chromosome set in the step (8) is replaced by a strain with a genome homology comparison result of 60% -70%; the ploidy increase is a line identified as tetraploid for chromosome ploidy.
Further, if the breeding base material is tetraploid apomictic apple material, the mutation breeding in step (2) and the resequencing in step (8) are omitted, and the strains in step (6) are all directly used as the filial generation population.
Further, the superior stable population has an apomictic reproductive capacity greater than 80%.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the method selects the apomixis base material of the wild natural population and carries out genome sequencing; rapidly creating a base material mutant library by using mutagenesis, and selecting an excellent-character strain as a mutagenic progeny population; collecting various source pollen; artificially prolonging the flowering time of apomictic apples in the flowering period; collecting seeds, and sowing the seeds after layering treatment; carrying out chromosome ploidy identification on the seedlings with the target characters by a flow cytometer; re-sequencing the genome of the seedling with the target character and performing homologous comparison with a wild type; selecting a strain with complete replacement of the genome or ploidy increase and excellent target characters as a filial generation group; by utilizing the apomixis characteristics, offspring with high apomixis capacity is selected from the mutagenic offspring population and the filial offspring population as an excellent stable population. Based on the facultative apomixis characteristic of the apples, the diversified heteropolyploid apples are quickly created and identified by utilizing various source pollen pollination and flow cytometry, and the method greatly enriches the ways of apple polyploid breeding. The method of the invention has the advantages of simple required flow, easy mastering of operation technology and wide application prospect.
Drawings
FIG. 1 is a wild population of Yimeng mountain area Pingyi sweet tea;
FIG. 2 is a characteristic diagram of the genome of wild Malus hupehensis Rehd;
FIG. 3 is a BUSCO quality assessment of wild Malus hupehensis Rehd genome;
FIG. 4 is a ploidy analysis of the wild Malus hupehensis Rehd chromosome;
FIG. 5 shows different leaves of the mutagenized progeny population of Malus hupehensis;
FIG. 6 shows the heights of seedlings of different strains of a mutagenized progeny population of Malus hupehensis Rehd;
FIG. 7 shows different fruit strains of Malus hupehensis mutant progeny population;
FIG. 8 shows the root systems of different strains of the mutagenized progeny population of Malus hupehensis Rehd;
FIG. 9 shows Malus hupehensis Rehd tea during pollination;
FIG. 10 shows Malus hupehensis Rehd leaf obtained after pollination;
FIG. 11 shows fruits and seeds of Malus hupehensis Rehd fruit line, wherein the left is Malus hupehensis Rehd, the right is wild type, and the scale is 1 cm;
FIG. 12 shows the homology between the re-sequencing results of different Malus hupehensis Chun and the wild type, wherein the hatching indicates the homologous portion and the white indicates the non-homologous portion;
FIG. 13 is a flow chart of a method for apple non-fusion 'wild natural population-mutagenic progeny population-filial progeny population-excellent stable population' one line of four groups of sports seeds.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.
The four populations are 'natural wild population-mutagenic progeny population-filial generation population-excellent stable population', wherein:
(1) wild natural population: the apple belongs to a ploidy facultative apomixis material with different chromosomes. The facultative apomixis triploid can be used as a hybrid female parent, and other excellent sexual reproduction characters such as drought resistance, column shape and the like are introduced into the facultative apomixis triploid to obtain heterosis; the facultative apomixis tetraploid can be used for hybridizing male parents and is mainly used for introducing apomixis characters into excellent diploid materials to create novel apomixis triploid and realize apomixis of sexual varieties; if necessary, a material such as a pentaploid may be optionally used.
(2) And (3) hybridizing progeny populations: parents of different traits or genes are subjected to genome recombination or superimposed offspring collection through sexual hybridization. Taking triploid Malus hupehensis (2 n: 3X: 51) as an example: two sexual embryo sacs (n ═ X ═ 17, n ═ 2X ═ 34) can be generated by crossing and recombined with different paternal gamete chromosomes to form filial generations (2n ═ X + X ═ 17+17, 2n ═ 2X + X ═ 34+17, 2n ═ X +2X ═ 17+34, 2n ═ 2X +2X ═ 34+ 34); the generated maternal clone gamete (n ═ 3X ═ 51) can also receive different paternal gamete chromosomes to form filial generations with the maternal chromosomes superimposed (2n ═ 3X + X ═ 51+17, 2n ═ 3X +2X ═ 51+ 34); aneuploidy is not considered.
(3) Mutagenizing the progeny population: collection of mutants obtained by natural variation and artificial mutagenesis. When collecting natural variation material, wild material seed and filial generation seed can be induced by artificial mutagenesis to obtain excellent character or potential excellent gene, F 2 The generation genetic stable facultative apomictic individuals can also realize the rapid creation of novel allopolyploid through hybridization.
(4) Good stable population: and (3) selecting a good strain set which has high apomictic reproduction capability and can be used for production from the filial generation population and the mutant line. According to the castration bagging condition, a large amount of fertile seeds can be generated, the high apomixis capacity of the offspring can be rapidly judged under two conditions of the character separation degree of the offspring under the natural condition, the production application value of the offspring can be determined through later grafting affinity, biotic stress, abiotic stress and other tests, and the offspring finally enters the market.
Example 1
A method for apple non-integration into a series of four groups of sports seeds (figure 13) comprises the following steps:
(1) selecting apple facultative apomictic species 'Malus hupehensis' or 'Malus micromalus' as breeding basic material, performing whole genome sequencing on the apple facultative apomictic species 'Malus hupehi' by adopting but not limited to comprehensive means such as PacBio Hifi sequencing, Oxford Nanopore Technology sequencing and Illumina second-generation sequencing platform, assembling the obtained raw data by using Canu Hifi software, correcting the assembly result by using NextPolish software and second-generation Illumina data and assembling high-quality genome, performing Contig phase splitting and hanging by using published genome sequence of apples such as Hanfu, golden crown and the like and Hi-C data by using ALLHiC software, finally obtaining genome sequence of apomictic reproduction target material hung to all chromosomes, performing GaONT cloning on the genome hung to chromosomes by using NECAT corrected sequencing data, and then performing Gap cloning by using PacBio Hifi data again to obtain final apomictic reproduction target material with chromosome level, carrying out chromosome ploidy analysis;
(2) mixing wild type seeds with moist sandy soil (the moist degree of sandy soil is that the seeds are held into a ball without dripping water), placing the mixture in a refrigerator at 4 ℃ for stratification, and obtaining seeds to be germinated after 30 days; washing out seeds with clear water, carrying out light-shielding treatment on the seeds for 16 hours by using EMS mutagenesis solutions with different concentrations, then carrying out disinfection treatment by using a 0.1M sodium thiosulfate solution, continuously laminating for 20 days at 4 ℃, and then carrying out field sowing to quickly obtain a large number of mutagenesis progeny groups; if the tetraploid malus micromalus is selected as a breeding base material, the step is not needed;
(3) collecting multiple varieties of apple pollen from multiple regions, air drying for later use, wherein the pollen of the variety later than the Malus hupehensis Chun or Malus micromalus Chun blossoms can be stored in a refrigerator at-20 deg.C after air drying, and can be used in the next year; if the basic material is selected to be the malus micromalus, collecting pollen of the malus micromalus and airing for later use;
(4) when wild Malus hupehensis Rehd or Malus hupehensis Rehd is in the bell flowering phase, selecting branches with the same flowering phase, picking off buds at other different periods, castrating the buds for experiments, spraying a preservative on the buds, bagging after spraying is finished, avoiding spraying on leaves as much as possible, spraying once every day, and continuously spraying until the stigma loses activity; wherein, the formula of the antistaling agent is as follows:
(5) carrying out artificial pollination on the Malus hupehensis or Malus micromalus by using collected diploid apple pollen, bagging until the flowering phase is finished, and collecting seeds after fruits are mature; or carrying out artificial pollination on the diploid apple by using collected pollen of the malus micromalus, bagging until the flowering phase is finished, and collecting seeds after the fruit is mature;
(6) uniformly mixing the seeds collected in the step (5) with moist sandy soil (the seeds are held into a cluster without dripping water), then placing the mixture in a refrigerator at 4 ℃ for 40 days for stratification, germinating at room temperature for 2 days, carrying out soil transplantation, sowing the mixture in a field, and selecting plant lines with changed target characters, such as large fruits, strong drought resistance, dwarf plants or semi-dwarf plants and the like;
(7) preparation of cell nucleus suspension: shearing young leaves (about 1g) of the strain in the step (6), washing the surface with distilled water, draining the filter paper, putting the filter paper into a precooled culture dish, and adding precooled dissociation liquid (Tris-MgCl) 2 ：200mmol/L Tris、4mmol/L MgCl 2 0.5% (v/v) Triton X-100, pH 7.5, 4 ℃) about 1-2mL, rapidly chopped at one time with a sharp blade, the material must be submerged in the dissociation liquid throughout the process in order to better free the nuclei; absorbing dissociation liquid in the culture dish, filtering the 300-400-mesh filter membrane into a 1.5mL centrifuge tube, and then placing the centrifuge tube in a refrigerator at 4 ℃ for incubation for 5 min; centrifuging at 4 deg.C (1000rpm for 5min), discarding supernatant, adding 100 μ L of precooled dissociation liquid, and simultaneously adding 150 μ L of precooled staining solution (comprehensive staining solution prepared from 0.1% Ethidium Bromide (EB) and 0.1% Propidium Iodide (PI)), wherein each sample contains about 250 μ L of cell nucleus suspension; dyeing for 10min in a refrigerator at 4 ℃ in a dark place, and transferring to a sample loading tube;
(8) and (3) carrying out ploidy identification on the sample chromosome: an Epics Altra flow cytometer produced by Beckman-Coulter company in America is adopted, the fluorescence intensity promoted by dyeing DNA molecules is detected, and the fluorescence intensity is analyzed by computer software connected with a measuring device, and finally the DNA content is drawn, wherein the Malus spectabilis used as a diploid control, and the Malus hupehensis is used as a triploid control; the cell nucleus content of all the detected samples is a relative value taking a contrast as a standard, each sample is repeated twice, and at least 10000 cell nuclei are collected in each detection;
(9) re-sequencing the triploid plants of the Malus hupehensis from the step (6) by taking the genome from the step (1) as a reference genome, comparing the percentage of homologous sequences with the genome, and selecting the plants with complete replacement of the chromosome set (the genome homology is between 60 and 70 percent) or ploidy increase (the tetraploid identified from the step (8)) as a filial generation group; or taking all the 'malus micromalus' triploids in the step (6) as a filial generation group;
(10) flower thinning: when the strains in the step (6) grow to the bell florescence, only 2-3 flowers are reserved in each inflorescence, and the rest flowers are removed; a total of 100 inflorescences were treated, 50 of which were emasculated and bagged as emasculation group, and the other 50 of which were not emasculated and served as control group. Marking: at the bottom of the short branch, the number of flowers emasculated from the branch or the number of flowers treated in the control are marked. Investigation of apomixis rate: after the fruit ripened in autumn, all the fruits were harvested and the number of seeds full in the fruit was investigated in the fresh state. Counting the number of the hundred-flower knots N1 (control group) and N2 (emasculation group), and calculating the apomixis capacity: N2/N1 × 100%;
(11) selecting the offspring with high apomixis reproductive capacity more than 80% from the materials of the mutagenic offspring population and the filial offspring population as an excellent stable population, and screening to obtain a new species of the apomixis apples.
Example 2
Now, taking Malus facultative apomixis triploid Malus hupehensis sweet tea as an example, the detailed introduction of the apple amalgamation-free series four-group sports seed method disclosed by the invention comprises the following steps:
(1) selecting Malus highly apomictic triploid species, Malus Lepidus tea, as breeding base material, sampling leaves of wild old tree (figure 1) in Yimeng mountain area, rapidly freezing with liquid nitrogen, sending to Beijing Baimaike Biotech limited for sequencing, and extracting genome by Baimaike;
(2) whole genome sequencing is carried out by using PacBio Hifi sequencing, Oxford Nanopore Technology sequencing and Illumina second generation sequencing platform, Canu Hifi software is used for assembling PacBio Hifi data, NextPolish software and second generation Illumina data are used for correcting the assembly result, genome sequence of 'Hanfu' and Hi-C data are used for carrying out phase splitting and hanging of Contig by using ALLHiC software, finally obtaining the genome sequence of the Pingyi sweet tea which is hung to 51 chromosomes, wherein the chromosome hanging rate is 94.33%, Gap cloning is carried out on the genome which is hung to the chromosomes by using ONT sequencing data corrected by NECAT, and then Gap cloning is carried out by using PacBio Hifi data again, thus obtaining the final chromosome level split phase split-separated Pingyi sweet tea genome total 1.88Gb (figures 2 and 3), wherein the result of ploidy analysis of the wild sweet tea chromosome ploidy is shown in figure 4;
(2) mixing wild Malus hupehensis with moist sandy soil (moist degree of sandy soil is that the mixture is held by hand to form a ball without dripping water), placing in a refrigerator at 4 deg.C, and laminating for 30 days to obtain seeds to be germinated; washing seeds with clear water, processing the seeds for 16 hours in a dark way by using 0.5 percent EMS mutagenesis solution (each 100mL mutagenesis solution contains 0.5mL EMS, 9.2mL 10M sodium dihydrogen phosphate solution, 0.8mL 1M disodium hydrogen phosphate solution, 5mL DMSO solution and 84.5mL deionized water), then sterilizing by using 0.1M sodium thiosulfate solution, continuously laminating for 20 days at 4 ℃, sowing in a field, and quickly obtaining a large number of mutagenesis progeny groups (table 1 and figures 5-8);
TABLE 1 different trait ratios after EMS mutagenesis of Malus hupehensis Rehd seeds
(3) Collecting apple pollen of various varieties from multiple regions, air drying for later use, wherein the pollen of the variety later than the blossom of the Malus hupehensis Rehd can be air dried and stored in a refrigerator at-20 deg.C for the next year;
(4) when the wild Pingyi sweet tea is in the bell flowering period, branches with the same flowering period are selected, buds in other different periods are removed, the buds for experiments are subjected to castration, a preservative is used for spraying the buds, bags are sleeved after spraying is completed, the buds are prevented from being sprayed on leaves as much as possible, and the buds are sprayed once every day and continuously sprayed until the stigma loses activity; wherein, the formula of the antistaling agent is the same as that of the embodiment 1:
(5) carrying out artificial pollination on the Malus hupehensis Rehd in the step (4) by using collected column-shaped apples, M9 and golden crown pollen (male parent) (figure 9), bagging until the flowering phase is finished, and collecting seeds after fruits are mature (figure 10);
(6) uniformly mixing the seeds obtained in the step (5) with moist sandy soil (the seeds are held into a cluster without dripping), placing the mixture in a 4-DEG refrigerator for 40 days, germinating the mixture at room temperature for 2 days, transplanting the soil, sowing the soil in a field, and selecting a plant line with enlarged fruits in the embodiment (figure 11);
(7) preparation of cell nucleus suspension: shearing young leaves (about 1g) of the strain in the step (6), cleaning the surface with distilled water, putting the leaves into a precooled culture dish after the filter paper is sucked dry, and adding precooled dissociation liquid (Tris-MgCl) 2 ：200mmol/L Tris、4mmol/L MgCl 2 0.5% (v/v) Triton X-100, pH 7.5, 4 ℃) about 1-2mL, rapidly chopped at one time with a sharp blade, the material must be submerged in the dissociation liquid throughout the process in order to better free the nuclei; absorbing the dissociation liquid in the culture dish, filtering the 300-400-mesh filter membrane into a 1.5mL centrifuge tube, and then placing the centrifuge tube in a 4-degree refrigerator for incubation for 5 min; centrifuging at 4 deg.C (1000rpm, 5min), discarding supernatant, adding 100 μ L of precooled dissociation liquid, and simultaneously adding 150 μ L of precooled staining solution (comprehensive staining solution prepared from 0.1% Ethidium Bromide (EB) and 0.1% Propidium Iodide (PI)), about 250 μ L of cell nucleus suspension per sample; dyeing for 10min in a refrigerator at 4 ℃ in a dark place, and transferring to a sample loading tube;
(8) and (3) carrying out ploidy identification on the sample chromosome: by adopting an Epics Altra flow cytometer produced by Beckman-Coulter company in America, detecting the fluorescence intensity promoted by dyed DNA molecules, analyzing by computer software connected with a measuring device, and finally drawing the DNA content, wherein the Malus spectabilis is used as a diploid control, and the Malus hupehensis is used as a triploid control; wherein the cell nucleus content of all the detected samples is a relative value taking a contrast as a standard, each sample is repeated twice, and at least 10000 cell nuclei are collected in each detection;
(9) taking the chromosome-level split Malus hupehensis Rehd genome in the step (2) as a reference genome, re-sequencing the triploid line in the step (6) (completed by Beijing Baimai Ke Biotechnology Co., Ltd.), comparing the percentage of homologous sequences with the genome (shown in figure 12), and selecting a line with complete replacement of the chromosome group (with the genome homology of 60-70%) or ploidy increase (tetraploid identified in the step (8)) as a filial generation group;
(10) flower thinning: when the strains in the step (6) grow to the bell florescence, only 2-3 flowers are left in each inflorescence, and the rest are thinned out; a total of 100 inflorescences were treated, 50 of which were emasculated and bagged as emasculation group, and the other 50 of which were not emasculated and served as control group. Marking: at the bottom of the braches, the number of flowers emasculated from the branch or the number of flowers emasculated from the control was marked. Investigation of apomixis rate: after the fruit ripened in autumn, all the fruits were harvested and the number of seeds full in the fruit was investigated in the fresh state. Counting the number of the hundred-flower knots N1 (control group) and N2 (emasculation group), and calculating the apomixis capacity: N2/N1 × 100%;
selecting the offspring with apomixis reproductive capacity more than 80% from the materials of the mutagenic offspring population and the filial offspring population as an excellent stable population, and screening to obtain a new species of the apomixis apples.
(11) And (3) filial generation of Malus hupehensis Rehd: as can be seen from the degree of apomixis in Table 2, although about 42% of the strains showed an advantage of apomixis, 83.42% of 181 apomictic bodies were on the lowest 2 grades (50-60% and 61-70%) of apomixis with respect to the degree of apomixis.
TABLE 2 regeneration and reproduction of triploid Malus hupehensis Rehd
Example 3
In this embodiment, a method for using tetraploid is described by taking malus micromalus as an example:
(1) collecting pollen of Malus micromalus, air drying, or storing in refrigerator at-20 deg.C; removing male in the flowering period of diploid Gala bell, carrying out artificial pollination on Gala by using collected Malus micromalus pollen, bagging until the flowering period is finished, and collecting seeds after fruits are mature;
(2) collecting multiple varieties of apple pollen from multiple regions, air drying for later use, wherein the variety later than flowering of Malus micromalus can be stored in a refrigerator at-20 deg.C after air drying, and used in the next year; removing the male bag when the wild Malus micromalus is in the small bell flowering phase, carrying out artificial pollination on the Malus micromalus by using collected Mala and golden crown pollen, bagging until the flowering phase is finished, and collecting seeds after the fruits are mature;
(3) uniformly mixing the seeds in the step (1) or (2) with moist sandy soil (the seeds are held by hands to form a cluster without dripping water), placing the mixture in a refrigerator with 4 ℃ for enough days for stratification, germinating at room temperature for 2 days, and transplanting soil to obtain a strain;
(4) preparation of cell nucleus suspension: shearing young leaves (about 1g) of the strain in the step (3), cleaning the surface with distilled water, draining the filter paper, putting the filter paper into a precooled culture dish, and adding precooled dissociation liquid (Tris-MgCl) 2 ：200mmol/L Tris、4mmol/L MgCl 2 0.5% (v/v) Triton X-100, pH 7.5, 4 ℃) about 1-2mL, rapidly chopped at one time with a sharp blade, the material must be submerged in the dissociation liquid throughout the process in order to better free the nuclei; absorbing the dissociation liquid in the culture dish, filtering the 300-400-mesh filter membrane into a 1.5mL centrifuge tube, and then placing the centrifuge tube in a 4-degree refrigerator for incubation for 5 min; centrifuging at 4 deg.C (1000rpm, 5min), discarding supernatant, adding 100 μ L of precooled dissociation liquid, and simultaneously adding 150 μ L of precooled staining solution (comprehensive staining solution prepared from 0.1% Ethidium Bromide (EB) and 0.1% Propidium Iodide (PI)), about 250 μ L of cell nucleus suspension per sample; dyeing in a refrigerator at 4 ℃ in a dark place for 10min, and transferring to a sample loading tube;
(5) and (3) carrying out ploidy identification on the sample chromosome: an Epics Altra flow cytometer produced by Beckman-Coulter company in America is adopted, the fluorescence intensity promoted by dyeing DNA molecules is detected, and the fluorescence intensity is analyzed by computer software connected with a measuring device, and finally the DNA content is drawn, wherein the Malus spectabilis is used as a diploid control, and the Malus hupehensis is used as a triploid control. The cell nucleus content of all the detected samples is a relative value taking a contrast as a standard, each sample is repeated twice, and at least 10000 cell nuclei are collected in each detection;
(6) flower thinning: and (5) when the strains in the step (4) grow to the bell florescence, only 2-3 flowers are reserved in each inflorescence, and the rest flowers are removed. A total of 100 inflorescences were treated, 50 of which were emasculated and bagged as emasculation group, and the other 50 of which were not emasculated and served as control group. Marking: at the bottom of the braches, the number of flowers emasculated from the branch or the number of flowers emasculated from the control was marked. Investigation of apomixis rate: after the fruit ripened in autumn, all the fruits were harvested and the number of seeds full in the fruit was investigated in the fresh state. Counting the number of the hundred-flower knots N1 (control group) and N2 (emasculation group), and calculating the apomixis capacity: N2/N1X 100%.
Selecting offspring with apomixis ability more than 80% from the triploid filial generation population materials as an excellent stable population, and screening new species of the apomixis apples.
(7) Malus micromalus filial generation: when malus micromalus is used as a female parent (table 3), 67% of apomictic bodies are classified into two stages with low apomictic reproduction, and a strain with apomictic reproduction capability more than 80% is not found, possibly because the number of obtained filial generations is small; when malus micromalus is used as a male parent (table 4), about 10% of the lines have apomictic reproduction capability of more than 80%, 60% of the lines belong to two stages with low apomictic reproduction, and meanwhile, offspring with high apomictic reproduction capability are basically triploid, which shows that the introduction of apomictic reproduction character by using facultative apomictic tetraploid is efficient.
TABLE 3 reproduction of tetraploid Malus salmoides plants (Malus salmoides as female parent)
TABLE 4 reproduction of tetraploid Malus micromalus plants (Malus micromalus as male parent)
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A method for four groups of sports seeds of an apple non-integration system is characterized by comprising the following steps:
(1) selecting wild natural population apomixis apples as breeding base materials, and performing genome sequencing;
(2) creating a mutant library of breeding base materials by using mutation breeding, and selecting strains with excellent target characters as variant progeny populations;
(3) collecting various source pollen, and airing for later use;
(4) prolonging the flowering time of wild type breeding base materials;
(5) carrying out artificial pollination by using the pollen in the step (3) and the breeding base material in the step (4), bagging until the flowering phase is finished, and collecting seeds after fruits are mature;
(6) carrying out stratification treatment and germination on the seeds in the step (5), transplanting the seeds into a field for sowing, and selecting a strain with target properties obviously superior to those of a wild type;
(7) extracting the cell nucleus suspension of the strain in the step (6), and performing chromosome ploidy identification after dyeing;
(8) taking the genome in the step (1) as a reference genome, performing re-sequencing on the strain in the step (6), performing homology comparison with the reference genome, and selecting the strain with complete replacement of the genome or increased ploidy as a filial generation population;
(9) and (3) selecting the offspring with high apomixis ability from the variant offspring population in the step (2) and the filial generation population in the step (8) as an excellent stable population by utilizing the apomixis characteristic to obtain a new species of the apomixis apples.
2. The method for breeding an apple apomixis trilinear line as recited in claim 1, wherein said breeding base material is a triploid or tetraploid apomictic apple material; the triploid apomixis apple material comprises Malus hupehensis Rehd, Malus hupehensis Rehd and Malus sieboldii; the tetraploid apomixis apple material comprises Malus trilobata and Malus schoenoprassis.
3. The method as claimed in claim 1, wherein the mutation breeding in step (2) comprises EMS mutagenesis, radiation mutagenesis, latent bud and bud mutation.
4. The method as claimed in claim 1, wherein the step (4) of prolonging the flowering time of the wild type breeding base material comprises spraying a preservative, reducing the temperature, increasing the humidity and reducing the illumination.
5. The method for breeding the apples in the three-line non-fusion manner according to claim 4, wherein when the antistaling agent is sprayed to prolong the flowering time of the wild breeding basic materials, the antistaling agent comprises the following components in percentage by weight: 8-hydroxyquinoline potassium sulfate, silver nitrate, citric acid and sucrose.
6. The method as claimed in claim 2, wherein in the step (5), if the breeding base material is a maternal material, the maternal material is artificially pollinated with pollen; if the breeding base material is a father material, the diploid apple material is subjected to artificial pollination by using pollen of the father material.
7. The method for breeding the apple of the three-line non-fusion breeding method according to claim 1, wherein the specific steps of the step (7) are as follows: and (3) shearing young and tender leaves of the strain in the step (6), cleaning the surfaces, draining, putting into a culture dish, adding dissociation liquid, quickly cutting, filtering a filter membrane with 300 meshes and 400 meshes into a centrifuge tube, incubating, adding a staining solution to obtain a stained cell nucleus suspension, and identifying the ploidy of the chromosome of the strain by using a flow cytometer and taking the Malus octandra as a diploid control and the Malus hupehensis as a triploid control.
8. The method as claimed in claim 1, wherein the complete set of genome replacement in step (8) is a line with a genome homology comparison result of 60% -70%; the ploidy increase is a line identified as tetraploid for chromosome ploidy.
9. The method of claim 2, wherein if the breeding base material is tetraploid apomictic apple material, the mutation breeding in step (2) and the resequencing in step (8) are omitted, and the lines in step (6) are directly used as the filial generation population.
10. The method of claim 1, wherein the apomictic reproductive capacity of the elite stable population is greater than 80%.
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