Construction method and application of corn high-frequency haploid induction line breeding population
By constructing a high-frequency haploid induction system breeding group of corn, using methods such as double-line hybridization and semi-compatriot selection, the problems of long breeding cycle and low induction rate of corn haploid induction system were solved, and the breeding efficiency was significantly improved and stability of induction rate.
Patent Information
- Application Number
- CN202411736848.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-02-14
AI Technical Summary
The selection and breeding of corn haploid induction systems has problems such as long cycle, large workload, difficult to fix the induction rate, and low induction rate, which is difficult to effectively improve the induction efficiency of haploid.
By constructing a high-frequency haploid induction system breeding population of corn, selecting haploid induction systems with higher induction rates as the population to construct parents, performing double-line hybridization, balancing two generations, selecting selected single plants for backcrossing and seed mixing. Screening and improvement generation by generation to form a high-frequency haploid induction breeding group.
The breeding efficiency of corn high-frequency haploid induction systems has been significantly improved, the breeding cycle has been shortened, the stability and high frequency of induction rates have been improved, and the problem of difficulty in improving induction rates has been solved.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for constructing a high-frequency haploid induction line breeding population of corn in the field of biotechnology and its application. Background Art
[0002] Corn is an important food crop. Technological innovation and germplasm creation related to corn breeding are important means to achieve scientific and technological self-reliance in the seed industry. The essence of corn breeding lies in the utilization of hybrid vigor, and the creation of pure line parents is the key link in the utilization of hybrid vigor. Conventional breeding methods mainly use self-pollination or backcrossing to purify the material background, which generally requires more than 8 generations, while the use of haploid breeding technology only takes 2 generations to obtain pure lines. The induction of haploids is the primary link in haploid breeding technology, which can directly affect the efficiency of haploid breeding.
[0003] Studies have shown that the haploid induction trait of maize is a quantitative trait controlled by micro-effect polygenes. Currently, there are 8 genes or QTLs reported to control the haploid induction trait of maize. How to use so many micro-effect genes to aggregate and breed high-frequency induction lines is the bottleneck for achieving high-frequency induction of haploids and an important means to improve the efficiency of haploid breeding. The breeding of high-frequency haploid induction lines of maize has the problems of long cycle, large workload, difficulty in fixing the induction rate, and low induction rate. The problem of improving the induction efficiency of haploids needs to be solved urgently. Summary of the invention
[0004] A technical problem to be solved by the present invention is how to breed high-frequency haploid induction lines of corn.
[0005] In order to solve the above problems, the present invention first provides a method for constructing a high-frequency haploid induction line breeding population of corn, comprising the following steps:
[0006] A1. Select several or more haploid induction lines with high induction rates as parents for population construction;
[0007] A2, the population is constructed by double-row hybridization of parents and mixed harvesting seeds to obtain the initial selection population C0; the initial selection population C0 is balanced for two generations to obtain the selection population C2;
[0008] A3, selecting individual plants with normal plant growth, harmonious male and female sexes, and large pollen quantity from the selected line population C2, numbering them, taking the individual plants with induction rates in the top 30% among the numbered individual plants as the selected individual plants of the selected line population C2, mixing the pollen of the entire population of the selected line population C2, backcrossing the selected individual plants of the selected line population C2, and collecting all the backcross seeds to obtain the population C3;
[0009] A4. Thereafter, in each generation, the plants with the top 30% induction rates in the contemporary population are selected as the contemporary selected plants, and the pollen of the contemporary selected plants are mixed and backcrossed to the contemporary selected plants, and the seeds of the contemporary selected plants are mixed in equal amounts to obtain a higher generation population; the next generation of the population C3 is population C4, and so on; the population C4 or the higher generation population is the constructed corn high-frequency haploid induction line breeding population.
[0010] In the above construction method, the number is 3 or more, for example 3, 4, 5, etc.; preferably 4.
[0011] In the above construction method, the haploid induction line with a higher induction rate described in A1 has an induction rate of more than 10%.
[0012] In the above construction method, the population construction parents can all be haploid induced lines with R1-nj markers, so that haploid grains can be identified according to the R1-nj markers during the breeding process. Specifically, both the heterozygous diploid embryo and endosperm appear purple-red, while the haploid grains only have colored endosperm and colorless embryo.
[0013] In the above construction method, the population construction parents may be composed of CAU5, CHOI4, CHOI5 and Jingkeyou109.
[0014] In the above construction method, A2 is described as two balanced generations, each generation group is planted with more than 2,000 plants, isolated and open pollinated, and the seeds are mixed after removing the single plants that fall over, break, or have serious diseases, degenerate male ears, or have poor grain color markings.
[0015] In the above construction method, the single plants with the induction rate in the top 30% are selected, and the selection is made after the induction rate is detected by planting test varieties when the planting group is matched.
[0016] The present invention also provides a method for breeding a corn high-frequency haploid induction line, comprising the step of breeding a target corn high-frequency haploid induction line from the corn high-frequency haploid induction line breeding population constructed by the above method.
[0017] In the above breeding method, the breeding is to induce the corn high-frequency haploid induction line breeding population to obtain haploid embryos and then double them to obtain multiple induction line DH lines, detect the induction rate of the induction line DH line, and combine it with agronomic traits to obtain the target corn high-frequency haploid induction line.
[0018] In the above breeding method, the induction rate of the corn high-frequency haploid induction line is above 20%.
[0019] The present invention also provides a method for constructing the corn high-frequency haploid induction line breeding population and application of the constructed corn high-frequency haploid induction line breeding population in corn breeding.
[0020] The present invention also provides a breeding method for the corn high-frequency haploid induction line and application of the bred corn high-frequency haploid induction line in corn breeding.
[0021] The present invention firstly performs two rounds of genetic balance by free pollination, removes single plants with obvious unfavorable traits, then sows and selects excellent single plants for haploid induction rate determination, takes pollen of the entire population and pollinates the selected single plants at the same time, performs half-sib selection on the selected single plants in combination with the induction rate, obtains an improved population, then sows and selects excellent single plants for haploid induction rate determination, takes pollen of the selected single plants and pollinates the selected single plants at the same time, performs half-sib selection on the selected single plants in combination with the induction rate, mixes excellent single plants in equal amounts after screening to obtain an improved population, and plants the remaining seeds in ear rows to obtain a low-generation high-frequency haploid induction system.
[0022] The present invention evaluates haploid induction lines from different sources, screens excellent haploid induction lines, and establishes a basic population for haploid induction line breeding; then breaks the conventional method of two-ring system for induction line breeding, combines haploid induction line breeding with population improvement and half-sib selection, and realizes rapid aggregation of multiple genes controlling induction traits of haploid; and uses haploid breeding technology to feed back for haploid induction line breeding, and quickly fixes haploid induction traits by creating an induction line DH system, thereby breeding high-frequency haploid induction lines of corn. This provides a feasible solution to solve the problem of long breeding cycle of haploid induction lines of corn and difficulty in improving induction rate, which can accelerate the aggregation improvement of induction rate of haploid induction lines and significantly improve the efficiency of high-frequency haploid induction line breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The figure is a comparison of haploid induction rates of different induced lines in Example 1 of the present invention. Different letters in the figure represent significant differences at the 0.05 level, and the same letters represent insignificant differences at the 0.05 level. DETAILED DESCRIPTION
[0024] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0025] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
[0026] The high-frequency haploid induction lines CAU5 and CHOI3 in the following examples have an average induction rate of 10% and have R1-nj markers, which have been disclosed in the document "Jiao Yanyan. Evaluation of maize haploid induction lines and contemporary genetic effect research. [D]. China Agricultural University, 2017."
[0027] The high-frequency haploid inducer lines CHOI4 and CHOI5 in the following examples are disclosed in the document “Liu Chenxu, et al. Development of high-oil maize haploid inducer with a novel phenotyping strategy”.
[0028] In the following embodiments, the maize (Zea mays L.) haploid induced lines Jingkeyou 109 (plant variety right application number 20184298.9), Jingkeyou 118 (plant variety right application number 20184301.4), Jingkeyou 129 (plant variety right application number 20184303.2), Jingkeyou 138 (plant variety right application number 20184299.8), Jingkeyou 158 (plant variety right application number 20184300.5) and Jingyou 004 (plant variety right application number 20241000135), and the maize hybrid NK815 (plant variety right authorization number CNA20171510.8) are known materials, all of which are varieties bred by the Maize Research Institute of Beijing Academy of Agricultural and Forestry Sciences. The public can obtain them from the applicant, Beijing Academy of Agricultural and Forestry Sciences, for use in repeating the relevant experiments of the present invention.
[0029] Example 1 Construction of breeding population for high-frequency haploid induction lines of maize
[0030] 1. Screening and identification of parents for population construction
[0031] In the spring of 2020, Beijing planted maize haploid induction lines CAU5, CHOI3, CHOI4, CHOI5, Jingkeyou 109, Jingkeyou 118, Jingkeyou 129, Jingkeyou 138 and Jingkeyou 158 as male parents, all of which carried the R1-nj color marker, and planted maize variety NK815 as female parent. To ensure that the flowering period of the parents meets, the female parent is sown in one phase, and the male parent is sown in three phases, that is, one phase is sown 3 days earlier than the female parent, one phase is sown at the same time, and one phase is sown 3 days later. The parents are strictly weeded, the female parent is strictly emasculated before tasseling, and the female ears are bagged. The above-mentioned induced lines (CAU5, CHOI3, CHOI4, CHOI5, Jingkeyou 109, Jingkeyou 118, Jingkeyou 129, Jingkeyou 138 and Jingkeyou 158) were used as male parents and hybridized with NK815 respectively. Each induced line was pollinated with more than 5 ears. The ears with less than 50 grains were removed at harvest time. Haploid grains were identified based on the R1-nj marker. Both the embryo and endosperm of the heterozygous diploid appeared purple-red, while the haploid grains only had the endosperm colored and the embryo colorless, so haploids were identified, and the haploid induction rate was statistically analyzed.
[0032] Results Figure 1 There are obvious differences in the induction rates of different induction lines, among which CHOI5 and Jingkeyou 109 have induction rates of 14.49% and 15.56%, respectively, which are significantly higher than other induction lines except CAU5 and CHOI4. In addition, the induction rates of CAU5 and CHOI4 are also high, and CAU5 has better lodging resistance and large pollen volume. In addition to carrying the R1-nj marker, CHOI4 is also a high-oil induction line.
[0033] The purple stems can be used to identify haploid grains and seedlings. Therefore, CAU5, CHOI4, CHOI5 and Jingkeyou 109 were finally selected as parents for the new induced line breeding population.
[0034] 2. Construction of selected population and genetic balance
[0035] In the winter of 2020 in Hainan, while screening parents for the construction of the above-mentioned new induced line breeding population, double-row hybridization was carried out with the induced lines CAU5, CHOI3, CHOI4, CHOI5, Jingkeyou 109, Jingkeyou 118, Jingkeyou 129, Jingkeyou 138 and Jingkeyou 158 in preparation for subsequent population construction.
[0036] Based on the above evaluation results, CAU5, CHOI4, CHOI5 and Jingkeyou 109 were selected as parents for population construction. Six hybrid F1s were obtained by diaristic hybridization of selected induced lines in Beijing planting in the spring of 2021. The details are as follows:
[0037] 1) Using CAU5 as the female parent and CHOI4 as the male parent to cross, obtain the F1 of CAU5 / CHOI4 combination;
[0038] 2) Using CAU5 as the female parent and CHOI5 as the male parent to crossbreed to obtain the F1 of the CAU5 / CHOI5 combination;
[0039] 3) Using CHOI5 as the female parent and Jingkeyou 109 as the male parent to obtain the F1 of CHOI5 / CAU5 combination;
[0040] 4) Using CAU5 as the female parent and Jingkeyou 109 as the male parent to cross, obtain the F1 of CAU5 / Jingkeyou 109 combination;
[0041] 5) Using CHOI4 as the female parent and CHOI5 as the male parent to crossbreed to obtain the F1 of the CHOI4 / CHOI5 combination;
[0042] 6) Using CHOI4 as the female parent and Jingkeyou 109 as the male parent to obtain the F1 of the combination of CHOI4 / Jingkeyou 109;
[0043] Pollinate more than 5 ears for each combination, harvest all the ears obtained from the above 6 double-row hybridizations, thresh and mix them to obtain C0 seeds of the initial population of the selected line.
[0044] In the early batch of Hainan in winter 2021, more than 2,000 selected initial groups C0 were planted for isolated open pollination. At harvest, the individual plants with lodging, breaking, serious disease, degenerated male ears, or poor grain color markings were removed, and the other ears were threshed and mixed to obtain the genetically balanced first generation group C1; in the Hainan main season of 2021, the same method will continue to plant group C1 for isolated open pollination to break the linkage, perform genetic balance, and obtain the selected group C2.
[0045] 3. Reincarnation selection and group improvement
[0046] In the spring of 2022, 2,000 selected population C2 plants were planted in Beijing, and the test variety NK815 was planted at the same time. The female ears of the parents were bagged before they spun silk, and the female parent NK815 was emasculated. During the pollen shedding period, 500 plants with normal growth, coordinated male and female sexes, and large pollen quantity were selected and numbered. Pollen from each selected plant was hybridized with NK815. The pollen of each selected plant was hybridized with more than 5 ears to ensure that more than 3 hybrid ears were harvested for induction rate determination. At the same time, pollen from all 2,000 selected population C2 plants was mixed and pollinated with the 500 selected plants. During the harvest period, the 500 plants were harvested according to the numbered ears after mixed pollination.
[0047] The haploid induction traits of hybrid ears used for induction rate determination were investigated and counted. First, the hybrid ears with less than 50 grains or unclear color markings were eliminated. Then, haploids were selected according to color markings, and the haploid induction rate was calculated. The male plant numbers corresponding to the hybrid ears with induction rates in the top 30% were obtained. All the ears after mixed pollination of the male plants with the corresponding induction rates in the top 30% were mixed and threshed. That is, the selected plants of the selected population C2 were improved using the half-sib selection method, thereby obtaining the population C3 improved by recurrent selection.
[0048] 2000 plants of the improved population C3 after recurrent selection were planted, and excellent individual plants were selected to measure the induction rate as the test variety NK815, and the individual plants with induction rates in the top 30% were screened as the selected individual plants. However, when the selected individual plants were pollinated, the pollen used only came from the selected individual plants for mixed pollen, rather than the entire population, so as to accelerate the aggregation of the high-frequency induction line, and the individual plants with induction rates in the top 30% at the final screening were used as the mother parent. After mixed pollination, the fruit ears were threshed separately, and 50 seeds from each ear were taken and mixed in equal amounts to obtain the next round of improved population C4, which was used for the next round of population improvement; in addition, the remaining seeds of the selected individual plants were retained and planted in ear rows in the next generation to obtain ear rows of high-frequency induction lines, so as to prepare for the selection of low-generation high-frequency induction lines after the individual plant induction rate was measured.
[0049] Example 2 Genetic background purification of haploid induction line and rapid fixation of haploid induction performance
[0050] 1. Obtaining the induced DH line
[0051] In the second season of Hainan in winter 2022 and Beijing in spring 2023, hybridization was carried out with the induced line Jingyou 004 as the male parent and the above-mentioned population C4 as the female parent. 15 days after pollination, the hybrid ears were taken for embryo peeling. Since the induced line population itself carries the R1-nj marker, the haploid immature embryos cannot be identified by color marking, but there is a ploidy difference between haploid embryos and heterozygous diploid embryos, so their immature embryos are significantly different. The haploid immature embryos are significantly smaller than the heterozygous diploid immature embryos, and the embryos are wedge-shaped, thereby realizing the haploid identification of the induced line, and then combined with the haploid immature embryo tissue culture doubling, the induced line DH line is obtained. At the same time, Jingyou 004 is used as the male parent to induce the selected line initial population C0, and the induced line DH line is produced as a control.
[0052] 2. Evaluation and screening of induced DH lines
[0053] In the winter of 2023, the induced DH lines and the test variety NK815 from the two populations were planted in Hainan respectively. During the pollen shedding and silking period, the bad DH lines were eliminated, such as lodging, breaking, male spike degeneration, empty stems, poor coordination between males and females, etc. The remaining DH lines were used as the male parents for hybridization with NK815, and the haploid induction rate was evaluated. The results showed that the induction rate of the induced DH lines from the initial selection population C0 ranged from 3.20-17.86%, with an average induction rate of 10.53%; while the induction rate of the induced DH lines from population C4 ranged from 6.25-23.65%, with an average induction rate of 15.15%. This shows that the induction rate of the induced line breeding population improved by this method has been significantly improved. In addition, statistics on high-frequency induction lines showed that among the induction lines from the initial selection population C0, there were 18 induction lines with an induction rate of more than 15%, and an induction coefficient of more than 20% was 0; while among the induction lines from population C4, there were 138 induction lines with an induction rate of 15% and 15 induction coefficients of more than 20%, respectively. Combined with agronomic traits, three high-frequency induction lines (with induction rates of more than 20%) were screened and named Jingyou001, Jingyou002, and Jingyou003.
[0054] Table 1 Comparison of DH lines induced by different sources
[0055]
[0056] **Indicates significant differences in the same group at the 0.01 level.
[0057] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.
Claims
1. A method for constructing a high-frequency haploid induction line breeding population of corn, characterized in that: The steps include: A1. Select several or more haploid induction lines with high induction rates as parents for population construction; A2, the population is constructed by double-row hybridization of parents and mixed harvesting seeds to obtain the initial selection population C0; the initial selection population C0 is balanced for two generations to obtain the selection population C2; A3, selecting individual plants with normal plant growth, harmonious male and female sexes, and large pollen quantity from the selected line population C2, numbering them, taking the individual plants with induction rates in the top 30% among the numbered individual plants as the selected individual plants of the selected line population C2, mixing the pollen of the entire population of the selected line population C2, backcrossing the selected individual plants of the selected line population C2, and collecting all the backcross seeds to obtain the population C3; A4. Thereafter, in each generation, the plants with the top 30% induction rates in the contemporary population are selected as the contemporary selected plants, and the pollen of the contemporary selected plants are mixed and backcrossed to the contemporary selected plants, and the seeds of the contemporary selected plants are mixed in equal amounts to obtain a higher generation population; the next generation of the population C3 is population C4, and so on; the population C4 or the higher generation population is the constructed corn high-frequency haploid induction line breeding population.
2. The method according to claim 1, characterized in that The several are 3 or more.
3. The method according to claim 2, characterized in that The number is 4.
4. The method according to claim 1, characterized in that: A1 is a haploid induction line with a relatively high induction rate, and the induction rate is above 10%.
5. The method according to claim 4, characterized in that The population construction parents are all haploid induction lines with R1-nj markers.
6. The method according to claim 5, characterized in that The population construction parents consist of CAU5, CHOI4, CHOI5 and Jingkeyou109.
7. A method for breeding high-frequency haploid induction lines of maize, characterized in that: The method comprises the step of breeding the target corn high-frequency haploid induction line from the corn high-frequency haploid induction line breeding population described in any one of claims 1 to 6.
8. The method according to claim 7, characterized in that The breeding is to induce the corn high-frequency haploid induction line breeding population to obtain haploid embryos and then double them to obtain multiple induction line DH lines, detect the induction rate of the induction line DH lines, and combine agronomic traits to obtain the target corn high-frequency haploid induction line.
9. Use of the method according to any one of claims 1 to 6 and / or a maize high-frequency haploid induction line breeding population constructed by the method according to any one of claims 1 to 6 in maize breeding.
10. Use of the method according to claim 7 or 8 and / or the high-frequency haploid induced maize line bred by the method according to claim 7 or 8 in maize breeding.