CN115104525A - Breeding method of maize haploid breeding combined with conventional breeding and selection and its application - Google Patents

Abstract

The invention discloses a breeding method combining corn haploid breeding with conventional breeding and selection and its application. The present invention provides a method for obtaining a corn DH line, comprising the following steps: combining a corn haploid breeding method with a conventional breeding method, and performing breeding by combination of induction and selection; Contemporary selection for excellent traits is carried out at the same time. The method of the invention can greatly improve the production efficiency of excellent DH lines, reduce the frequency of DH production that does not meet the breeding target, and reduce ineffective labor and costs, thereby improving the breeding efficiency.

Description

Breeding method combining maize haploid breeding with conventional breeding and selection and its application

technical field

The invention relates to the field of biotechnology, in particular to a breeding method combining maize haploid breeding with conventional breeding and selection and application thereof.

Background technique

The preparation of pure lines has always been a key link in crop breeding. Conventional breeding methods mainly adopt self-crossing or backcrossing for material background purification, which generally requires more than 8 generations, while the use of doubled haploid technology (DH technology) only It takes 2 generations to obtain a pure line (Gerald et al., 2013). According to CIMMTY estimates, about 70% to 80% of the parents of new corn varieties produced by major seed companies in North America and Europe currently contain one or more DH (doubled haploid) lines. 600 to 750 million mu (CIMMTY, 2015). Many foreign seed companies have realized the large-scale application of haploid breeding. Domestic haploid breeding started late. At present, domestic corn breeding is mostly a combination of conventional breeding and haploid breeding. Conventional maize breeding mostly adopts the method of bicyclic breeding, that is, generation-by-generation selfing or backcrossing, which has rich genetic variation, controllable target traits, and is easier to aggregate multiple excellent genes. Each generation is artificially selected and inherited. The response is obvious. But the conventional breeding cycle is long, the workload is heavy, and the work is cumbersome. Compared with conventional breeding, haploid breeding has the following advantages (1) greatly shortens the breeding cycle, and only requires two generations to obtain a pure line with stable traits; (2) saves years of continuous self-cultivation in the purification process of inbred lines. (3) Homozygous genetic background can be combined with molecular marker technology to screen DH lines to improve the accuracy and efficiency of selection (Rober and Gordillo, 2009). et al., 2005; Geiger and Gordillo., 2009); (4) the DH line is completely homozygous, and the hybrids produced by using the DH line are more likely to meet the requirements of variety, stability and consistency (Geiger and Gordillo, 2009). But at the same time, haploid breeding also has the disadvantages of low genetic exchange frequency and uncontrollable target traits. In order to maintain the excellent characters, the breeding process of pure lines is accelerated. At present, the combination of conventional breeding and haploid breeding is often adopted. In order to reduce the production rate of ineffective DH, the screened F ₂ or BC ₁ and above generations are generally used as the basic material for DH production. However, the process of combining conventional breeding and haploid breeding currently adopted is two relatively independent stages of breeding, and it has not been able to truly realize the simultaneous development of haploid induction and excellent trait selection. Therefore, how to select individual plants of the basic population of DH production, select the best for induction, select the best haploid and double the harvested ear, and truly achieve the combination of inducement and selection, is one of the important means to improve the production efficiency of excellent DH lines. In order to make the breeding goal clearer, it is of great significance to create a method combining conventional breeding with haploid breeding and selection, which is of great significance to accelerate the breeding process of elite pure lines and reduce ineffective operations, thereby promoting the development of maize breeding technology.

SUMMARY OF THE INVENTION

In order to solve the problems of long conventional breeding cycle and poor controllability of haploid breeding, and to achieve the goal of combining haploid induction and target trait screening, the present invention provides a feasible solution, which can greatly improve the production efficiency of the target DH line, This reduces the cost of breeding and speeds up the breeding process.

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

The method for obtaining a maize DH line claimed in the present invention may include the following steps: combining the maize haploid breeding method with the conventional breeding method, and breeding through the combination of induction and selection; the combination of induction and selection refers to haploid induction of contemporary At the same time, the selection of excellent traits was carried out.

Further, the generation generation of the combination of inducement and selection can be the F ₂ generation or the BC ₁ generation or the F ₂ generation or more generations or the BC ₁ generation or more generations with segregation of the target trait.

Further, the target of the combination of inducement and selection may be a haploid-induced hybrid contemporary plant or fruit ear.

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

Further, the method may include the following steps:

(A1) Select maize inbred lines with differences in target traits, assemble hybrids, and then self-cross or backcross, and separate the obtained F ₂ generation or BC ₁ generation or F ₂ generation or more generation or BC with the target trait. ₁ or more generations as the basic group;

(A2) carrying out hybrid induction between the maize haploid inductive line and the basic population, then according to the breeding requirements, select the contemporary plant traits and/or ear traits of the hybrid induction, retain the selected ear, and then carry out haploid selection. Identification and doubling to produce the DH line.

In the step (A2), before the haploid induction is performed, the following steps may be further included: selecting the plant traits for the individual plants in the basic population obtained in the step (A1), removing the individual plants that do not meet the breeding target, and leaving the remaining individual plants Strictly emasculated, and the ears are strictly bagged. After the filaments emerge, select the best for induction. In order to ensure the acquisition of DH lines containing the target traits, generally retain as many excellent individual plants as possible for haploid induction.

In step (A2), the selected ear can be based on one or more of the identified plant traits and/or ear traits, and the hybrid-induced contemporary ear is arranged in an order from superior to inferior, and the top 20 % of the ear.

In step (A1), the target trait may be one or more of the plant traits and/or ear traits.

In step (A2), the basic population is sown, and the maize haploid induction line is staggered to ensure that the flowering period meets.

In the above, the plant traits include but are not limited to: powdering stage, silking stage, plant height, ear position, leaf angle, leaf length, leaf width, lodging resistance, disease resistance, drought resistance, salt and alkali resistance Wait. The ear traits include, but are not limited to: ear length, ear diameter and/or ear row number.

In some cases of the present invention, in step (A1), the maize inbred lines to be assembled are Jing 1110 and Jing J2418; in step (A2), the maize haploid inducible line is a maize haploid inducible line CAU6.

In the method, the selected ear of the hybrid induction generation can be harvested in a specific period in combination with the later-stage haploid doubling method. If immature embryos are doubled, the ear can be harvested 15-20 days after pollination. Haploid grains or sprouts are doubled, and the ears are harvested after maturity.

If required, the following step may be included after step (A2): identifying and comparing the target traits of the obtained DH lines to verify the effectiveness of hybrid induction of contemporary selection. The specific operation can be as follows: screen the plant traits and/or ear traits at the time of induction hybridization, sort the induced hybrid ear by single trait or multiple traits or composite traits, and select 20% of the front and rear respectively, which are used for haploid identification and DH production. Comparing the classification index characters of the DH lines obtained by the above classification, according to the significance of the difference between the two, it can prove the effectiveness of the contemporary selection of induced hybridization.

The above-mentioned plant traits and/or ear traits are screened before haploid induction or at the time of hybridization induction. Depending on the breeding goal, the selection method of focusing on plant traits or ear traits or both can be selected. .

In the second aspect, the present invention claims the application of the method described in the first aspect above in corn breeding.

Experiments have proved that: the present invention combines haploid breeding with conventional breeding methods, and achieves the goal of simultaneous induction and screening. By selecting excellent individual plants in the basic population of DH production, combined with key plant traits, excellent individual plants are selected for induction. , In the harvest period of the ear, the hybrid induced ear is screened again, and the important ear traits are screened, and the haploid is selected for doubling to produce the DH line. The method can greatly improve the production efficiency of excellent DH lines, reduce the frequency of DH production that does not meet the breeding target, and reduce ineffective labor and costs, thereby improving the breeding efficiency.

Description of drawings

Figure 1 shows the comparison of DH obtained from the screening of contemporary plant height and ear position traits induced by hybridization.

Figure 2 is a comparison of DH obtained by screening for the traits of contemporary leaf angle, leaf length and leaf width induced by hybridization.

Figure 3 shows the difference in ear length and ear diameter induced by hybridization of F ₂ population.

Figure 4 shows the distribution of ear length, ear diameter and ear row number of DH obtained by induction after hybrid contemporary selection.

Detailed ways

The technical scheme of the present invention is as follows:

1. Material preparation

Firstly, according to the breeding goal, the basic material for producing the inductive line DH is assembled, and there are obvious differences in at least one key plant trait or ear trait between the parents of the basic material. In order to obtain the segregating population of the target trait, the F ₁ obtained from the above-mentioned parental combination is self-crossed or backcrossed, so as to obtain the F ₂ or BC ₁ or other high-generation segregating population with rich variation of the target trait.

2. Screening of plant traits in trait segregated populations

The segregated population was sown, and the haploid inducer line was sown at a staggered period to ensure that the flowering period met. Before using the induction line to induce the basic material, screen and identify the single plant of the basic population according to the breeding goal. The identification indicators can include key plant traits such as loose powder silking stage, plant height, ear position, leaf angle, and male and female coordination. , Eliminate the single plant that does not meet the breeding goal, the remaining single plant is strictly emasculated, the female ears are strictly bagged, and the optimal induction is selected after the filaments are all out. Haploid induction. After the pollination is completed, before the hybrid induction ear is harvested, the plant disease resistance (such as rust, spot disease, etc.), lodging resistance (such as stem strength, stem breaking force, aerial root development, etc.), stress resistance, etc. can be further improved. (such as drought resistance, salinity resistance, disease and pest resistance, etc.) and other key traits are screened, and then the best ones are harvested to induce hybrid ears, and the obtained ears are classified and sorted according to the breeding goals. Identification and doubling to obtain DH lines that meet the breeding goals.

3. Screening of ear traits of trait segregated populations

In the same way, according to the breeding target to induce the basal population, before the haploid identification and doubling, the contemporary ear traits of the induced hybrids should be identified and screened. The specific screening indicators may mainly include important ear traits such as ear length, ear diameter and number of ear rows. , according to the breeding goal, 20% of the induced hybrid ear before and after were retained, and then the haploid identification and doubling were carried out to produce the DH line.

4. Validation of the effectiveness of contemporary selection in induced hybridization

Screening of plant traits or ear traits in the generation of induced hybridization, sorting of single trait or multiple traits or compound traits to induce hybrid ear, select 20% before and after respectively, for haploid identification and DH production. Comparing the classification index characters of the DH lines obtained by the above classification, according to the significance of the difference between the two, it can prove the effectiveness of the contemporary selection of induced hybridization. Therefore, the method of combination of induction and selection can be applied to subsequent breeding work.

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The high-frequency haploid induction line CAU6 in the following examples, its average induction rate is 12%, has R1-nj mark, in document "Zhong Yu, Liu Chenxu, et al.Mutation of ZmDMP enhances haploid induction in maize.[ It was disclosed in J].Nature plants, 2019, 5(6).”, and the public can obtain it from China Agricultural University with consent. This material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

In the following examples, the corn (Zea mays L.) inbred line Jing 1110 (announcement No. 20211000927) and Jing J2418 (announcement No. 20201000757) are well-known materials, and both are obtained by It is self-selected and bred by the Maize Research Center of Beijing Academy of Agriculture and Forestry, and the public can obtain it from Beijing Academy of Agriculture and Forestry to repeat the experiment of this application and cannot be used for other purposes.

In the following implementation cases, the haploid doubling used is colchicine, which is a product of Beijing Boyouhang Biotechnology Co., Ltd.

Embodiment 1. The combination method of induction and selection based on plant traits

1. Material selection and preparation

In the spring of 2020, the inbred lines Jing 1110 and Jing J2418 were planted in Beijing, and the F ₁ was combined. In the winter of 2020, the F ₁ was sown/crossed in Hainan to obtain F ₂ seeds, and F ₂ was sown in the second season of Hainan to obtain the F ₂ population (ie, the basic population). , and at the same time sow the maize haploid induction line CAU6 (carrying R1-nj color mark, the induction rate is about 12%) in order to meet the flowering period of the basal population.

2. Individual plant screening and haploid induction

For the F ₂ segregated population, the plants with obvious unfavorable traits, such as weak seedlings, lodging, and disease susceptibility, were eliminated, and the pollen-scattering stage and silking stage of each plant were investigated, and the female ears were strictly bagged before silking to prevent foreign pollen pollution. After that, the pollen of the inductive line CAU6 was taken and pollinated with the individual plants of the basic population to complete the haploid induction.

3. Identification and screening of contemporary plant traits induced by hybridization

After haploid induction and pollination, plant traits such as plant height, ear position, leaf-leaf angle, leaf length and leaf width were measured about 10 days after pollination. The traits of the target plant were investigated and sorted, and 20% of the fruit ears before and after the sorting were selected according to a single trait, of which the first 20% were marked as group A, and the last 20% were marked as group B. Combined with the later haploid doubling method, harvest and select at a specific period. For fruit ears, if the young embryos are doubled, the ears are harvested 15-20 days after pollination. If the haploid grains or sprouts are doubled, the ears are harvested after maturity. Ears were selected for haploid identification and doubling to produce DH lines.

4. Verification of the combined effect of hybrid induction of contemporary plant traits

The characteristics of plant height, ear position, leaf angle, leaf length and leaf width of the contemporary induced hybridization were investigated. According to the single character, 20% of the front and back were taken and marked as A and B groups. The two types of DH were compared, and four plants were selected for each DH for character collection. The results showed that in terms of plant height and ear position (Fig. 1, Table 1), the variation range of DH plant height and ear position produced by group A was 188.00- 259.00cm and 45.67-128.33cm; the variation ranges of DH plant height and ear position obtained in group B were 145.25-217.50cm and 28.00-98.33cm, respectively; it can be seen from the distribution of DH plant height and ear position produced by the two groups in Figure 1 The plant height and ear position of DH in group A were generally higher than those in group B. Overall, the average plant height and ear position of DH produced by group A were 211.08 cm and 86.56 cm, respectively, which were significantly higher than those of 183.26 cm and 60.83 cm in group B. cm (P<0.05), the average plant height and ear position of DH obtained in group A were 27.82 cm and 25.73 cm higher than those obtained in group B, respectively. In terms of leaf traits (Figure 2, Table 2), leaf angle, leaf length and leaf width, after classification and production of DH, there were significant differences between the two types of DH selected traits (P<0.05). According to the leaf angle, leaf length and leaf width, the average difference between the two produced DH reached 4.07°, 6.90cm and 0.82cm, respectively. And the distribution of DH in group A was significantly higher than that in group B in leaf angle, leaf length and leaf width. This shows that the screening of the basic population for plant traits in the induction generation can more effectively produce DH lines that meet the breeding goals. It also proves that the hybrid induction generation combines haploid breeding with conventional breeding to achieve the combination of induction and detection. effectiveness.

Table 1. DH performance obtained from the screening of contemporary plant height and ear position traits induced by hybridization

Note: Within the same item, different letters represent significant differences at the 0.05 level, and the same letters represent insignificant differences at the 0.05 level.

Table 2. DH performance obtained from the screening of contemporary leaf angle, leaf length and leaf width traits induced by hybridization

Note: Within the same item, different letters represent significant differences at the 0.05 level, and the same letters represent insignificant differences at the 0.05 level.

Embodiment 2, the combination method of induction and selection based on ear character

1. Material preparation and haploid induction

Material preparation and haploid induction are the same as in Example 1, without selecting plant traits, taking pollen from the inducer line and individual plants of the basic population for pollination, and performing hybrid induction on all plants in the basic population to complete the haploid induction work.

2. Identification and classification of contemporary ear traits induced by hybridization

Combined with the late haploid doubling method, the hybrid induced ear is harvested in a specific period. If the immature embryo is used for doubling, the ear is selected 15-20 days after pollination. If the haploid grain or sprout is doubled, the selection is Harvest the ears when ripe. After the ear is harvested, before identifying the young haploid embryos or grains, statistical classification is performed for key ear traits such as ear length, ear diameter, and number of ear rows, and 20% of the ear before and after the sorting are selected for a single trait, of which the top 20% Labeled as group A and the bottom 20% as group B for haploid identification and doubling to produce DH lines.

3. Verification of the combination effect of hybrid induction of contemporary ear traits

According to the performance of the hybrid induced ear (Fig. 3), the target traits were sorted for the long, short, thick and thin F ₂ individual plants, the haploids were identified respectively, and the haploids were doubled. Thus, the DH line was produced. The obtained DH is planted in the field, and _four ears of each DH line are taken to measure the target characters, and then the performance of the obtained DH is compared. DH obtained by classification in several aspects found that the distribution peaks of DH obtained in group A were significantly higher than those in group B, and the distributions of the two were significantly different (Figure 4). The variation ranges of DH obtained in group A on these three traits were 110.23-162.61 cm, 38.50-50.03 cm and 13-19 cm, respectively, while the variation range of DH obtained in group B on these three traits was 87.18-151.35 cm, respectively. 36.37-46.10cm and 10-17, comparing the final average value, it is found that there are significant differences in the target traits (P<0.05), among which the difference in the average ear length of the obtained DH is 14.27mm, the difference in ear thickness is 2.77mm, and the difference in the number of ear rows 2.22 was reached (Table 3). This shows that the selection of key traits of hybrid induction of contemporary ear can effectively improve the performance of DH lines in terms of target traits, and also proves the effectiveness of the combination of induction and selection in the selection of ear traits.

Table 3. DH performance obtained from the screening of different ear traits of contemporary hybrid induction

Note: Within the same item, different letters represent significant differences at the 0.05 level, and the same letters represent insignificant differences at the 0.05 level.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

Claims (10)

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;

(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.

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.

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