CN115104525A

CN115104525A - Breeding method combining haploid breeding with conventional breeding and induced selection of corn and application of breeding method

Info

Publication number: CN115104525A
Application number: CN202210743465.0A
Authority: CN
Inventors: 陈琛; 王元东; 赵久然; 付修义; 陈传永; 吴珊珊; 张华生; 张春原; 张亮; 郭成恩; 王志浩; 彭研科; 孙玉和
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2022-09-27

Abstract

The invention discloses a breeding method combining haploid breeding and conventional breeding selection and application thereof. The invention provides a method for obtaining a corn DH line, which comprises the following steps: combining a corn haploid breeding method with a conventional breeding method, and breeding by combination of induced selection; the selective binding refers to excellent character selection at the same time of haploid induction generation. The method can greatly improve the production efficiency of the excellent DH line, reduce the production frequency of DH which does not meet the breeding target, and reduce ineffective labor and cost, thereby improving the breeding efficiency.

Description

Breeding method combining haploid breeding and conventional breeding selection of corn and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a breeding method combining corn haploid breeding and conventional breeding selection and application thereof.

Background

The preparation of pure lines is always a key link of crop breeding, the conventional breeding mode mainly adopts a self-crossing or backcross mode to purify the material background, generally more than 8 generations are needed, and only 2 generations are needed to obtain the pure lines by using a haploid breeding technology (double connected technology, DH technology) (Gerald et al, 2013). According to CIMMTY estimates, about 70% to 80% of the maize new variety parents currently produced by North American and European seed companies contain one or more DH (double hatched) lines, and DH-based maize hybrids cover about 6.0 to 7.5 million acres (CIMMTY, 2015) of the global area. Many foreign industry companies have realized the large-scale application of haploid breeding, the domestic haploid breeding starts late, and at present, the domestic maize breeding is mostly in a mode of combining conventional breeding and haploid breeding. The conventional breeding of the corn mostly adopts a two-cycle line breeding mode, namely generation-by-generation selfing or backcrossing, the corn has rich genetic variation, controllable target characters, easy polymerization of a plurality of excellent genes, manual selection of each generation and obvious genetic response. But the conventional breeding cycle is long, the workload is large and the work is complicated. Compared with conventional breeding, haploid breeding has the following advantages that (1) the breeding period is greatly shortened, and pure lines with stable characters can be obtained only by two generations; (2) saves the purification process of the inbred line for many yearsThe large manpower, land and capital costs incurred by continuous selfing and selection (Geiger and Gordillo, 2009); (3) the homozygous genetic background can be combined with molecular marker technology to screen DH lines, so as to improve the accuracy and efficiency of selection (Rober et al, 2005; Geiger and Gordillo, 2009); (4) DH line is completely homozygous, and hybrid species matched by using DH line can meet the requirements of variety on diversity, stability and consistency more easily (Geiger and Gordillo, 2009). But haploid breeding also has the defects of low genetic exchange frequency and uncontrollable target characters. In order to maintain excellent characters and accelerate the breeding process of the pure line. At present, a mode of combining conventional breeding with haploid breeding is mostly adopted. To reduce the production rate of ineffective DH, the F after screening is generally used ₂ Or BC ₁ And the superior groups of the above generations as the basic materials for DH production. However, the conventional breeding and haploid breeding combined process adopted at present is two relatively independent stages of breeding, and haploid induction and excellent character selection cannot be truly realized at the same time. Therefore, how to select and preferentially induce the single plants of the DH production basic population, preferentially screen haploid and double the induced clusters, really achieve the combination of induction and selection, and is one of the important means for improving the production efficiency of excellent DH lines. In order to make the breeding target more clear, the establishment of a method combining conventional breeding and haploid breeding selection has important significance for accelerating the breeding process of an excellent pure line and reducing invalid operations, thereby promoting the development of a corn breeding technology.

Disclosure of Invention

In order to solve the problems of long conventional breeding period and poor haploid breeding controllability and realize the goal of combining haploid induction and target character screening, the invention provides a feasible scheme, which can greatly improve the production efficiency of a target DH line, further reduce the breeding cost and accelerate the breeding process.

In a first aspect, the invention claims a method of obtaining a maize DH line.

The method for obtaining the corn DH line claimed by the invention can comprise the following steps: combining a corn haploid breeding method with a conventional breeding method, and breeding by combination of induced selection; the induced combination refers to haploid induction of the current generation and excellent character selection.

Further, the generation of the mutagenic binding may be F isolated with the desired trait ₂ Generation or BC ₁ Is substituted or F ₂ Generation or BC ₁ Generation or more.

Further, the target of the induced binding can be a haploid induction cross current plant or fruit cluster.

Further, the mode of action of the induced combination can be selection for plant traits and/or ear traits.

Still further, the method may comprise the steps of:

(A1) selecting maize inbred lines with difference in target characters, combining hybrid seeds, then carrying out inbred or backcross, and obtaining F with target character separation ₂ Generation or BC ₁ Is substituted or F ₂ Generation or BC ₁ Generation or more as a basic group;

(A2) and performing hybrid induction on the corn haploid induction line and the basic population, selecting the plant character and/or the ear character of the hybrid induction current generation according to breeding requirements, reserving the selected ears, and then performing haploid identification and doubling respectively to produce a DH line.

Before haploid induction is carried out in step (a2), the method may further comprise the following steps: and (D) selecting the plant characters of the single plants in the basic population obtained in the step (A1), removing the single plants which do not accord with the breeding target, strictly emasculating the rest single plants, and strictly bagging the female ears. And (3) performing preferential induction after filaments are completely grown, and generally reserving as many excellent single plants as possible for haploid induction in order to ensure that a DH line containing target characters is obtained.

In step (a2), the selected ears may be ears ranked in order from superior to inferior, the top 20% of the ears ranked in order based on the identified one or more of the plant traits and/or ear traits.

In step (a1), the trait of interest may be one or more of the plant trait and/or the ear trait.

In step (a2), the basal population is sown while the maize haploid inducer lines are sown in time-staggered intervals to ensure that the flowering time meet.

In the above, the plant traits include, but are not limited to: the pollen scattering period, the silking period, the plant height, the ear position, the leaf angle, the leaf length, the leaf width, the lodging resistance, the disease resistance, the drought resistance, the salt and alkali resistance and the like. The ear traits include, but are not limited to: ear length, ear thickness and/or ear row number.

In some embodiments of the present invention, in the step (a1), the corn inbred lines to be combined are jing 1110 and jing J2418; in step (a2), the corn haploid inducer line is corn haploid inducer line CAU 6.

In the method, a later haploid doubling method can be combined, the selected fruit cluster of the current generation is harvested in a specific period through hybridization induction, if young embryo doubling is adopted, the fruit cluster is harvested 15-20 days after pollination, and if haploid grain or bud doubling is adopted, the fruit cluster is harvested after maturation.

If necessary, the following steps can be further included after the step (a 2): and identifying and comparing the target characters of the obtained DH lines, and verifying the effectiveness of the hybrid induction contemporary selection. The specific operation can be as follows: screening plant characters and/or cluster characters in the current induced hybridization generation, sequencing induced hybrid clusters with single characters or multiple characters or composite characters, respectively selecting front and back 20%, and using for haploid identification and DH production. The classification index characters of the DH lines obtained by classification are compared, and the effectiveness of induced hybridization contemporary selection can be proved according to the difference significance of the characters.

The screening period of the plant characters and/or the ear characters is carried out before haploid induction or in the current generation of hybrid induction, and according to different breeding targets, a mode of screening the plant characters or the ear characters or both the plant characters and the ear characters can be selected.

In a second aspect, the invention claims the use of the method of the first aspect as described hereinbefore in maize breeding.

Experiments prove that: the invention combines the haploid breeding with the conventional breeding method, realizes the aim of synchronous induction and screening, selects the excellent single plant of the DH production basic population, combines the key plant character, selects the excellent single plant for induction, screens the cross-induced ears again in the ear harvesting period, screens according to the important ear character, selects the haploid preferentially to double, and produces the DH line. The method can greatly improve the production efficiency of the excellent DH line, reduce the production frequency of DH which does not meet the breeding target, and reduce ineffective labor and cost, thereby improving the breeding efficiency.

Drawings

FIG. 1 shows the comparison of DH obtained by screening the plant height and spike position character of the cross-induced current generation.

FIG. 2 is a comparison of DH obtained by screening the characters of hybrid induction of the leaf angle, leaf length and leaf width in the current generation.

FIG. 3 is F ₂ Group hybridization induces the ear length and ear thickness difference of the current fruit.

FIG. 4 shows the distribution of the length of the ears, the thickness of the ears, and the number of rows of ears of DH obtained by respective induction after the contemporary selection of hybridization.

Detailed Description

The technical scheme of the invention is as follows:

1. material preparation

Firstly, according to the breeding goal, the basic material for producing induction system DH is assembled, and at least one key plant character or fruit cluster character of the basic material parent has obvious difference. Combining the above parents to obtain F for obtaining target character segregation population ₁ Selfing or backcrossing to obtain F with rich variation of target characters ₂ Or BC ₁ Or other high-generation segregating populations.

2. Trait segregation population plant trait screening

The segregating population is sown, and the haploid inducing line is sown in a staggered period to ensure that the flowering phases meet. Before the induction system is used for inducing a base material, individual plants of a basic group are screened and identified according to breeding targets, identification indexes can comprise key plant characters such as a loose powder spinning period, a plant height, a spike position, a leaf angle, male and female harmony and the like, the individual plants which do not accord with the breeding targets are eliminated, the remaining individual plants are strictly emasculated, the female spikes are strictly bagged, and after filaments are produced, the optimal induction is carried out, and in order to ensure that a DH line containing the target characters is obtained, excellent individual plants as many as possible are generally reserved for carrying out haploid induction. After pollination is completed, before harvesting the hybrid induction ears, further screening key characters such as plant disease resistance (such as rust disease, leaf spot disease and the like), lodging resistance (such as stalk strength, stalk breaking force, air root development condition and the like), stress resistance (such as drought resistance, salt and alkali resistance, disease and insect resistance and the like), then preferentially harvesting the induction hybrid ears, classifying and sequencing the obtained ears according to breeding targets, respectively selecting front and back 20% of ears, and identifying and doubling the haploids of the ears, thereby obtaining a DH line meeting the breeding targets.

3. Character segregation population ear character screening

In the same way, according to a breeding target induction basic population, before haploid identification and doubling, identification and screening are carried out on induced hybrid current-generation fruit cluster characters, specific screening indexes mainly comprise important ear part characters such as ear length, ear thickness and ear row number, 20% of induced hybrid fruit clusters before and after are respectively reserved according to the breeding target, and then haploid identification and doubling are carried out to produce a DH line.

4. Validation of induced hybrid contemporary selection

Screening plant characters or cluster characters in the current induced hybridization generation, sequencing induced hybrid clusters with single characters or multiple characters or composite characters, and respectively selecting front and back 20% for haploid identification and DH production. The classification index characters of the DH lines obtained by classification are compared, and the effectiveness of induced hybridization contemporary selection can be proved according to the difference significance of the characters. Thereby applying the method of combination of selection and selection to the subsequent breeding work.

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 high-frequency haploid inducer line CAU6 in the following examples, which has an average induction rate of 12% and has an R1-nj marker, is disclosed in the document "Zhong Yu, Liu Chenxu, et al.mutation of ZmDMP enghanes haploided induction in mail [ J ] Nature plants,2019,5 (6)", and is available from the university of agriculture under public consent, and is used only for repeating the experiments related to the present invention, and is not used for other purposes.

In the following examples, maize (Zea mays L.) inbred line jing 1110 (new plant species application No. 20211000927) and jing J2418 (new plant species application No. 20201000757) are all known materials, and were bred by the maize research center of the agroforestry academy of agriculture and forestry, beijing, and publicly available from the agroforestry academy of agriculture and forestry, beijing, to repeat the application experiments, and not used for other purposes.

In the following embodiment, colchicine, a product of Biotech limited of Boyou navigation, Beijing, is used for the haploid doubling.

Example 1 plant trait-based selection and binding method

First, material selection and preparation

Planting inbred lines Jing 1110 and Jing J2418 in Beijing in 2020 spring, and preparing a composition F ₁ Sowing/hybridization in the southern winter 2020 ₁ Obtaining F ₂ Seeds are sown in Hainan two seasons F ₂ Obtaining F ₂ The population (i.e. the basal population) and simultaneously the corn haploid inducer line CAU6 (carrying the R1-nj color marker with an induction rate of about 12%) are sown in stages so as to meet the flowering stage of the basal population.

Second, screening single plant and haploid induction

For F ₂ Separating the colony, eliminating weak seedling, lodging and susceptible plants with obvious adverse characters, investigating individual plant pollen scattering period and spinning period, and strictly bagging before spinning for female ear to preventWhen the external pollen is polluted and the filaments are completely grown, pollen of an induction line CAU6 is taken and pollinated with the single plant of the basic group, and the haploid induction work is completed.

Third, identification and screening of cross-induced current plant characters

After haploid induced pollination is completed, plant characters such as plant height, ear position leaf angle, leaf length, leaf width and the like of a single plant are measured about 10 days after pollination. Inspecting and sequencing the target plant characters, selecting 20% of ears before and after sequencing according to a single character, wherein the first 20% is marked as an A group, the last 20% is marked as a B group, harvesting the selected ears in a specific period by combining a later haploid doubling method, harvesting the ears 15-20 days after pollination if young embryo doubling is adopted, and harvesting the ears after maturation if haploid seeds or buds are doubled. Ears were selected for haploid identification and doubling, thereby producing DH lines.

Fourth, the combination effect verification of the character selection of the current plant induced by hybridization

Investigating the characteristics of the plant height, the ear position, the leaf-leaf included angle of the ear position, the leaf length and the leaf width of the induced hybridization current-generation single plant, respectively taking front and back 20% according to the single characteristic, marking the front and back 20% as groups A and B, respectively comparing two types of DH aiming at the target characteristic, selecting four plants for each DH to carry out characteristic collection, and finding out that the plant height and the ear position are shown in the figure 1 and the table 1, and the variation ranges of the plant height and the ear position of the DH produced by the group A are 188.00-259.00cm and 45.67-128.33cm respectively; the variation ranges of the height and the spike position of the DH produced by the group B are 145.25-217.50cm and 28.00-98.33cm respectively; as can be seen from the distribution of the plant height and the ear position of the DH produced in the two groups shown in FIG. 1, the plant height and the ear position of the DH in the group A are generally higher than those in the group B, and the average plant height and the ear position of the DH produced in the group A are 211.08cm and 86.56cm respectively, which are significantly higher than 183.26cm and 60.83cm (P <0.05) of the group B, and the average plant height and the ear position of the DH produced in the group A are 27.82cm and 25.73cm respectively higher than those of the DH produced in the group B. In the aspect of leaf traits (figure 2, table 2), leaf angle, leaf length and leaf width, after DH is produced by classification, the two types of DH have significant difference in selectivity (P < 0.05). Average differences of DH obtained by classified production according to leaf angle, leaf length and leaf width also reach 4.07 degrees, 6.90cm and 0.82cm respectively. And the distribution of the DH obtained by the group A in three properties of leaf angle, leaf length and leaf width is obviously higher than that of the DH obtained by the group B. Therefore, the basic population is screened aiming at the plant characters in the current induction generation, the DH line meeting the breeding target can be produced more effectively, and the effectiveness of combination induction is realized by combining the haploid breeding with the conventional breeding through the current cross induction generation.

TABLE 1 DH expression screening of the hybrid induced current plant height and panicle position traits

Note: within the same item, different letter representations differ significantly at the 0.05 level and the same letter representations differ not significantly at the 0.05 level.

TABLE 2 hybrid-induced current-generation leaf angle, leaf length, leaf width traits screening resulting DH expression

Example 2 ear trait-based selection and binding method

First, material preparation and haploid induction

The material preparation and haploid induction are the same as in example 1, the plant characters are not selected, the pollen of the induction line is taken to pollinate the single plants of the basic group, and all the plants of the basic group are subjected to cross induction to complete the haploid induction work.

Second, identification and classification of characters of current-generation ears induced by hybridization

And (3) combining a later haploid doubling method, harvesting the hybrid induction fruit cluster in a specific period, if young embryos are adopted for doubling, harvesting the fruit cluster 15-20 days after pollination, and if haploid grains or buds are adopted for doubling, harvesting the fruit cluster after maturation. After harvesting, before identifying haploid young embryos or seeds, performing statistical classification on key ear properties such as ear length, ear thickness and ear row number, selecting 20% of ears before and after sorting according to single properties, wherein the first 20% is marked as a group A, and the second 20% is marked as a group B, and using the groups for haploid identification and doubling to produce a DH system.

Third, the combination effect verification of hybrid induction current ear character selection

Ear performance induced by crosses (FIG. 3), for long, short, coarse and fine ears F ₂ And (4) sequencing the target characters of the single plants, respectively identifying haploids, and doubling the haploids so as to produce a DH line. The obtained DH is planted in the field, four ears of each DH line are taken for target character measurement, and then the obtained DH performances are compared, the result shows that F is treated ₂ After population hybridization induction of the current fruit cluster is screened, the DH obtained by classification in the aspects of ear length, ear thickness and ear row number is compared, and the property value of the DH obtained from the group A on the distribution peak value of the three properties is obviously higher than that of the DH obtained from the group B, and the two properties have obviously different distribution conditions (figure 4). The DH obtained from the group A has the variation ranges of 110.23-162.61cm,38.50-50.03cm and 13-19 on the three traits respectively, while the DH obtained from the group B has the variation ranges of 87.18-151.35cm, 36.37-46.10cm and 10-17 on the three traits respectively, and the comparison of the final average values shows that the DH has obvious difference on the target traits (P is the average value of P)<0.05), wherein the average ear length of the obtained DH differed by 14.27mm, the ear thickness differed by 2.77mm, and the ear row number differed by 2.22 (Table 3). Therefore, the selection of key traits of the hybrid-induced current ears can effectively improve the expression of the DH line on target traits, and the effectiveness of the combination of the selection and the ear trait selection is proved.

TABLE 3 DH expression screening of different ear traits in the current generation induced by hybridization

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.

Claims

1. A method of obtaining a maize DH line, comprising the steps of: combining a corn haploid breeding method with a conventional breeding method, and breeding by combination of induced selection; the induced combination refers to haploid induction of the current generation and excellent character selection.

2. The method of claim 1, wherein: the generation of the induced combination is F with target character separation ₂ Generation or BC ₁ Is substituted or F ₂ Generation or BC ₁ Generation or more.

3. The method according to claim 1 or 2, characterized in that: the inducing and combining action object is to induce the current generation plant or fruit cluster of the cross by the haploid.

4. A method according to any one of claims 1-3, characterized in that: the mode of action of the combination of the induction and the selection is to select the plant character and/or the ear character.

5. A method for obtaining a maize DH line, comprising the steps of:

(A1) selecting maize inbred lines with difference in target characters, combining hybrid seeds, then carrying out inbred or backcross, and separating the obtained maize inbred lines with the target charactersF of (A) ₂ Generation or BC ₁ Is substituted or F ₂ Generation above or BC ₁ Generation or more as a basic group;

6. The method of claim 5, wherein: in the step (a2), before haploid induction is performed, the method further comprises the following steps: and (D) selecting the plant characters of the single plants in the basic population obtained in the step (A1), and removing the single plants which do not meet the breeding target.

7. The method of claim 5, wherein: in step (a2), the selected ears are the ears of the current generation of cross induction, which are ranked in order of superior to inferior according to the identified one or more plant traits and/or ear traits, and the first 20% of the ears are ranked.

8. The method according to any one of claims 4-7, wherein: the plant traits comprise: a powder scattering period, a spinning period, plant height, spike position, leaf angle, leaf length, leaf width, lodging resistance, disease resistance, drought resistance and/or salt and alkali resistance;

and/or

The ear traits comprise: ear length, ear thickness and/or ear row number.

9. The method according to any one of claims 4-8, wherein: in the step (A1), the corn inbred lines to be combined are Jing 1110 and Jing J2418; and/or

In step (a2), the corn haploid inducer is corn haploid inducer CAU 6.

10. Use of the method of any one of claims 1 to 9 in maize breeding.