CN117016378A - Creation method of corn haploid induction line without subspecies restriction - Google Patents
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- 235000005822 corn Nutrition 0.000 title claims abstract description 69
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Abstract
The invention relates to the technical field of crop breeding, in particular to a method for creating a corn haploid induction system without subspecies restriction, which comprises the steps of selecting a haploid induction system containing Ga1-S genes, haploid induction genes (zmpla 1 and zmdmp genes) and color marker genes as male parents, and selecting a haploid induction system containing haploid induction genes (zmpla 1 genes) and color marker genes as female parents for hybridization to obtain a corn haploid induction system without subspecies restriction. The creation method also comprises an evaluation system for the obtained haploid induction line: the environmental adaptability of the corn haploid induction line is evaluated by observing the agronomic characters of the corn haploid induction line under different environmental conditions; haploid induction is performed by crossing with a target corn material, and seed setting and induction rate are counted to evaluate the applicability of the material in haploid breeding. The creation method provided by the invention can create a haploid induction line without subspecies limitation, which has wide adaptability and strong applicability.
Description
Technical Field
The invention relates to the technical field of crop breeding, in particular to a method for creating a corn haploid induction line without subspecies limitation.
Background
The pollination induction of a target maize breeding material by a haploid induction line to induce haploids is the most effective way for obtaining haploid breeding intermediate materials from maize.
The existing haploid induction lines are mostly selfing lines containing haploid induction genes (zmpla 1 and or zmdmp) and color marker genes R1-Navajo (R1-nj). When the inbred line material is used as a pollination parent, the defect of weak growth and limited pollen quantity exists in most cases.
Chen Shaojiang et al, patent No. ZL201110332389.6 and issued publication No. CN103081797B, describe a method for inducing haploid of corn, wherein the obtained offspring is marked as induced line hybrid by utilizing different haploid induced line inbred lines, the agronomic character of the induced line hybrid is greatly improved relative to the inbred line, and especially, plant growth vigor, powder scattering amount, powder scattering duration, adaptability, resistance and the like are greatly improved, thereby providing favorable conditions for improving haploid induction efficiency and providing possibility for large-scale application of haploid induction.
In the corn haploid breeding practice, the growth vigor, lodging resistance, tassel branch number, loose powder amount and environmental adaptability of an induction system have important influence on the application of the haploid induction system. The growth vigor and lodging resistance determine the difficulty of cultivation management, the number of branches of tassel and the quantity of loose powder have direct influence on haploid induction efficiency, and the adaptability of different cultivation modes such as open field and greenhouse facility cultivation, different cultivation seasons such as spring, summer and autumn, different planting areas such as northwest corn planting areas and Huang-Huai-Hai corn planting areas directly determine the application range. Although hybrids generally have advantages over their parent inbred lines in terms of vigour, lodging resistance, tassel branching number, pollen-spreading capacity, etc., specific analysis and evaluation of agronomic performance and adaptability to different environments for a particular hybrid combination is also required.
At present, after a corn haploid induction line pollinates a target corn breeding material, haploid seeds are screened mainly by using an embryo-endosperm color observation method, and the problem of low accuracy in haploid screening and haploid induction rate calculation is caused by the fact that false positive haploid seeds exist in the screened haploid seeds. The high oil haploid induction line is used for measuring the difference of the oil content of the seed embryo of the induced haploid seed and the false positive haploid seed, and the false positive haploid seed can be identified, but the method needs special equipment, and a general laboratory cannot apply the method due to the lack of expensive equipment. The identification of the chromosome through the root tip chromosome is the most direct and effective method, but no simple and efficient method for observing the corn root tip chromosome is reported at present.
The pop corn can prevent other subspecies of corn pollen in corn seeds from pollinating the pop corn due to the Ga1-S gene, but pollen can pollinate other subspecies of corn, and the phenomenon is called unidirectional hybridization incompatibility. The existing corn haploid induction system is mainly formed by breeding corn subspecies other than the pop corn subspecies in corn seeds, such as dent corn, sweet corn, waxy corn and the like, and cannot be used as the haploid induction system of the pop corn because the haploid induction system does not contain Ga1-S genes. There is no haploid inducer line that is suitable for popping corn that has been publicly published.
Aiming at the problems, the invention aims to provide a method for creating a corn haploid induction line without subspecies limitation, which promotes the large-scale application of the corn haploid induction line in breeding practice.
Disclosure of Invention
The invention aims to provide a creation method of a corn haploid induction system without subspecies limitation aiming at the problems, by using the method, a series of corn haploid induction systems which are good in adaptability and high in haploid induction rate and can break through subspecies limitation can be created, and an evaluation system capable of accurately and comprehensively evaluating the applicability and the adaptability of the obtained corn haploid induction system without subspecies limitation is provided.
In order to achieve the aim, the invention discloses a method for creating a corn haploid induction line without subspecies limitation, which comprises the following steps: selecting a haploid induction line containing Ga1-S genes, haploid induction genes and color marker genes as a male parent, selecting a haploid induction line which is different from the genetic background of the male parent and contains the haploid induction genes and the color marker genes as a female parent, and hybridizing to obtain the maize haploid induction line hybrid without subspecies restriction.
Preferably, the haploid inducer gene comprises a zmpla1 gene.
Preferably, the haploid inducer gene of the male parent further comprises a zmdmp gene.
Preferably, the creation method further comprises an evaluation system of the obtained maize haploid inducer line without subspecies limitation, the evaluation system comprising an applicability evaluation: the haploid induction line to be evaluated is respectively mixed with different subspecies of corn, and the subspecies of corn comprise dent corn, sweet corn, waxy corn and pop corn, and the haploid induction line is evaluated through seed setting statistics and induction rate tests.
Preferably, the method for testing the inductivity comprises the following steps: randomly selecting a plurality of hybrid seeds obtained by hybridizing a haploid induction line to be tested with a certain subspecies of corn, screening haploid seeds by an embryo-endosperm color observation method, and then identifying false positive haploid seeds by adopting a root tip chromosome counting method on the screened haploid seeds, wherein the number of the true haploid seeds accounts for the percentage of the number of the selected hybrid seeds, namely the haploid induction rate. The root tip chromosome counting method eliminates false positive haploid seeds, so that the haploid induction rate can be more accurately identified by adopting the embryo-endosperm color observation and the root tip chromosome counting two-step method.
Preferably, the specific operation flow of the root tip chromosome counting method is as follows: placing haploid seeds in a two-layer filter paper culture dish for germination acceleration at 25 ℃, placing the culture dish at 4 ℃ when the root length reaches 2-3cm, keeping the filter paper moist, keeping the root tips close to the filter paper, culturing for 18-24 hours, cutting off the root tips from the position of 0.5-1cm above the root crowns, placing the root tips in a 10ml penicillin bottle, fixing the root tips in 10ml Carnot fixing solution, tabletting the root tips after fixing the root tips for 12 hours at room temperature, and observing the chromosome number by using an Olympus CX43 phase contrast microscope.
Preferably, the evaluation system further comprises an adaptability evaluation under different environmental conditions: observing the agronomic characters of the corn haploid induction line hybrid to evaluate the environmental suitability thereof; the observed agronomic traits include: growing vigor, lodging resistance, branch number of tassel and powder scattering amount. Different environmental conditions include different cultivation modes, different planting times and different planting areas; different cultivation modes comprise open field cultivation and greenhouse cultivation, different planting time comprises spring and autumn, and different planting areas comprise a Huang-Huai-Hai corn planting area (Shandong Fang) and a northwest corn planting area (Xinjiang Changji).
Experiments prove that: the method of the invention is used to prepare a hybridization combination to obtain a corn haploid induction line F by taking a common corn haploid induction line CHOI4 polymerized with R1-nj and zmpla1 genes as a female parent and taking a corn haploid induction line S241-12 polymerized with Ga1-S, R1-nj, zmpla1 and zmdmp genes as a male parent 1 Population by comparison with F 1 The population carries out adaptability evaluation based on agronomic characters and breeding adaptability evaluation based on a two-step method of embryo-endosperm color observation-root tip chromosome counting, and the excellent corn haploid induction line 22Co00020 is obtained through confirmation, and the corn haploid induction line has the advantages of good agronomic characters, strong environmental adaptability and high haploid induction rate, can overcome unidirectional hybridization incompatibility among subspecies, and can completely meet the practical requirement of large-scale breeding.
The invention has the beneficial effects that:
1. the invention provides a method for creating a corn haploid induction system without subspecies limitation, which can be used for creating a haploid induction system without subspecies limitation (applicable to all corn types including burst corn), and has wide adaptability and strong applicability.
2. The invention also provides an agronomic character adaptability evaluation system of the corn haploid induction system, which can screen the induction system with stable growth vigor and powder scattering amount (less affected by the environment) by observing the agronomic character of the induction system under different environmental conditions so as to evaluate the adaptability of the induction system, thereby simplifying the field cultivation and screening management of the induction system and solving the problems of less powder scattering amount and larger affected by the environment of the haploid induction system in the prior art.
3. The invention also provides an evaluation method of haploid induction line breeding applicability, which comprises the steps of preparing hybridization combination of a haploid induction line to be tested and various subspecies of corn materials, testing the haploid induction rate, screening the haploid induction line with high induction rate aiming at a specific subspecies, and solving the problem of low induction rate in the prior art.
4. The invention also provides a method for identifying haploid induction rate by using embryo-endosperm color observation-root tip chromosome counting two-step method, wherein false positive haploid seeds are removed by using the root tip chromosome counting method, and the two-step method identification data is more accurate than the existing embryo-endosperm color observation method.
Drawings
FIG. 1 is a roadmap of the method disclosed in the present invention for creating a maize haploid inducer line without subspecies restriction;
FIG. 2 is an engineered roadmap for maize haploid inducer line 22Co 00020;
FIG. 3 is a photograph of field performance of CHOI4, S241-12 and its hybrid 22Co00020 plants;
FIG. 4 is a photograph of the branching manifestations of the tassel of CHOI4, S241-12 and their hybrids 22Co 00020;
FIG. 5 is a photograph showing 22Co00020 haploid and diploid hybrid seeds after haploid induction on a high-speed tester lamp panel;
FIG. 6 is an enlarged view of the haploid and diploid hybrid seed of FIG. 5;
FIG. 7 is a photograph of chromosome number differences of haploid and diploid hybrid seeds after haploid induction of 22Co 00020;
FIG. 8 is a table of pollination seed set and haploid induction of the hybrid 22Co00020 of CHOI4 and S241-12 on different female parent varieties (lines).
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The invention discloses a method for creating a corn haploid induction line without subspecies limitation. As shown in fig. 1, the creation method includes the steps of: selecting a haploid induction line containing Ga1-S genes, haploid induction genes and color marker genes as a male parent; and selecting a haploid induction line which is different from a male parent genetic background and contains a haploid induction gene and a color marker gene as a female parent, and hybridizing to obtain a corn haploid induction line without subspecies restriction. In particular, haploid inducer genes include, but are not limited to, zmpla1 genes and/or zmdmp genes, and color marker genes include, but are not limited to, R1-nj genes.
Male parent (S241-12): after crossing with the maize haploid induction line CAU5 bred by Chinese agricultural crops as female parent and the burst maize hybrid WC12-1 as male parent, carrying out continuous selfing for 7 generations, selecting single plants with Ga1-S, zmpla1 and zmdmp genes existing simultaneously and excellent agronomic characters through molecular marker assistance from the second generation to the seventh generation, and carrying out single plant screening containing R1-nj genes through the colors of seed embryo and endosperm top end to breed the maize haploid induction line S241-12 with polymerized Ga1-S, R1-nj, zmpla1 and zmdmp genes. Through experiments, S241-12 can be used for haploid induction of burst corn and common corn haploid induction, but the loose powder amount is unstable, the loose powder amount is different under different environmental conditions, and the uncertainty factor of haploid induction by serving as a male parent is large.
Female parent (CHOI 4): CHOI4 is a haploid induced inbred line obtained by the national corn improvement center Chen Shaojiang team of China university of agriculture through Stock 6 series induction line crossing with high oleic corn, successive selection of offspring. CHOI4 is a corn haploid induced inbred line which is widely used at present because of the aggregation of R1-nj and zmpla1 genes and the control of high seed oil content genes. Although CHOI4 continuously selects agronomic characters in the breeding process, the defects of weak stems and easy lodging still exist in the breeding application practice.
As shown in fig. 2, hybridization combinations were formulated: the hybridization combination is prepared by taking a haploid induction line inbred line CHOI4 as a female parent and a haploid induction line inbred line S241-12 as a male parent. Only 50% of pollen of the hybrid contains Ga1-S genes, namely only 50% of pollen is effective in pollination with burst corn, but the amount of the corn pollen is very large, the cross incompatibility is reduced to very low, the induction efficiency is still very high, and the high-low evaluation of the induction efficiency depends on the test effect on different varieties.
When the hybrid combination is prepared, the artificial supplementary pollination mode is adopted.
In order to solve the problem of small amount of the pollen of S241-12, the number of the plants of S241-12 is as much as possible.
To adjust flowering phase, S241-12 was planted in three batches, the first batch was sown contemporaneously with CHOI4, the second batch was sown 7 days later than CHOI4, and the third batch was sown 7 days later than the second batch.
And (3) carrying out emasculation treatment on the CHOI4 before powder scattering, and manually pulling out the tassel before the tassel is pulled out and the powder is not scattered. And (3) before the female tassel filaments are not spitted, sleeving the female tassel of CHOI4 by using a first hybridization bag with the length of 11 x 18 cm, sleeving the male tassel of the male parent S241-12 in the full bloom stage by using a second hybridization bag with the length of 17 x 30 cm in the afternoon of the day before pollination when the filaments grow to 3-4 cm length, and fixing the opening of the second hybridization bag by using a clip.
And 6-7 hours in the morning of pollination, removing the first hybridization bag on the CHOI4 female spike, and cutting off the upper part of the CHOI4 female spike filament by scissors, and keeping the length of 1-2 cm. Dew is dried after 10 points, and pollination is carried out after the sun comes out. And (3) beating a second hybridization bag of the S241-12 sets by hand one day before pollination, taking down the second hybridization bag after collecting bright yellow fresh corn pollen in the second hybridization bag, pouring the collected pollen on the filaments of the CHOI4, sleeving the first hybridization bag back on the female spike of the CHOI4, and writing the pollination date on the first hybridization bag to finish hybridization.
After pollination for about 50-60 days, the seed milk line disappears, and the hybrid seeds can be harvested after hardening, thus obtaining the maize haploid induction line hybrid F 1 Designated 22Co00020.
The problem of flowering phase incompatibility can be solved by adopting staggered sowing, the problem of insufficient pollen quantity can be compensated by increasing the number of male parent plants, and the hybridization success rate can be improved by emasculation bagging, timely artificial pollination, timely harvesting and the like.
Adaptability (growth vigor, lodging resistance, tassel branch number and loose powder amount) evaluation of maize haploid inducer hybrid 22Co 00020:
the harvested hybrid seeds (maize haploid induction line hybrid 22Co 00020) are selected from a test greenhouse of an ecological economy development area of the Shandong Weifang urban isthmus mountain of the modern agricultural institute of Beijing university and a mountain open field test base, and the adaptability (growth vigor, lodging resistance, tassel branch number and powder scattering amount) of the Xinjiang Changji open field test base is evaluated. The evaluation method was performed by direct observation.
Through a greenhouse planting test in a mountain-east Weifang ecological economy development area of a modern agricultural institute of Shandong university in autumn 2022 and a spring-Beijing university in 2023, an open-field planting test in a mountain-east Weifang ecological economy development area of a modern agricultural institute of Shandong university in 2023 and an open-field planting test in Xinjiang Changji in 2023, as shown in fig. 3 and 4, the observation shows that a corn haploid induction line 22Co00020 is vigorous in growth vigor, compared with female parent CHOI4, the lodging resistance is obviously enhanced, the number of branches of tassel is obviously increased, and the pollen quantity is obviously increased compared with that of male parent S241-12.
Suitability evaluation of maize haploid inducer line 22Co 00020:
5 maize varieties (lines) including GH jingke 968 (dent maize), jingke sweet 608 (sweet maize), agro-jade 368 (waxy maize), pop maize inbred lines 1 and 2 were selected for suitability evaluation.
The hybrid corn haploid induction line 22Co00020 of CHOI4 and S241-12 is used as a male parent, five varieties (lines) of GH Beijing 968 (dent corn), beijing 608 (sweet corn), fangke Yu 368 (waxy corn) and pop corn inbred lines 1 and 2 are used as female parents for pollination, and 7-13 spikes are pollinated for each variety (line).
And (5) respectively mixing and collecting the varieties (lines) after maturation, and respectively counting seed setting numbers. 100 grains are randomly selected respectively, and haploid seeds are screened by embryo-endosperm color observation method, and the colors of the embryo and the top of endosperm are identified by using a high-speed tester lamp panel or a lamp box of a seed tester, wherein the color of the embryo is white, and the purple-black seeds at the top of endosperm are haploid seeds. And then, identifying false positive haploid seeds (the chromosome number of the false positive haploid seeds is twice that of the true haploid seeds) by adopting a root tip chromosome counting method, wherein the percentage of the true haploid seeds accounting for 100 seeds is the haploid induction rate. Repeating the steps for 3 times, taking the average, and obtaining the average haploid induction rate of the hybrid maize haploid induction line 22Co00020 of the CHOI4 and the S241-12 on each variety (line).
The root tip chromosome counting method comprises the following specific operation procedures: accelerating germination of haploid seeds at 25 ℃ in a two-layer filter paper culture dish, when the length of roots reaches 2-3cm, placing the culture dish at 4 ℃, keeping filter paper moist, keeping root tips close to the filter paper, culturing for 18-24 hours, cutting off root tips from the position of 0.5-1cm above root crowns, placing the root tips in a 10ml penicillin bottle, fixing the root tips in 10ml Carnot fixing solution (95% alcohol: glacial acetic acid=3:1), tabletting the root tips after fixing the root tips at room temperature for 12 hours, and observing the chromosome number by using an Olympus CX43 phase contrast microscope.
As shown in FIG. 8, seed count statistics indicate that CHOI4 has good affinity with the S241-12 hybrid maize haploid inducer line 22Co00020 for 5 maize varieties (lines) without unidirectional cross incompatibility. The haploid induction rate shows that the average haploid induction rate of the hybrid corn haploid induction line 22Co00020 of the CHOI4 and S241-12 is between 8% and 18.3%.
In conclusion, compared with the hybrid maize haploid induction line 22Co00020 of the CHOI4 and the S241-12, the lodging resistance of the female parent CHOI4 is obviously enhanced, the number of branches of tassel is obviously increased, the pollen quantity is obviously increased compared with that of the male parent S241-12, the unidirectional hybridization incompatibility among subspecies can be overcome, the hybrid maize haploid induction line can be used as a maize haploid induction line of different types, and the haploid induction rate is between 8% and 18.3%, and can completely meet the breeding practice requirement, so that the hybrid maize haploid induction line 22Co00020 of the CHOI4 and the S241-12 is an excellent maize haploid induction line with good agronomic characters and high haploid induction rate and without restriction among subspecies. Therefore, the creation method disclosed by the invention can create a haploid induction system with no subspecies limitation, wide agronomic character adaptability and strong breeding applicability.
The corn haploid induction system 22Co00020 without subspecies limitation can be comprehensively and accurately evaluated by using the evaluation system provided by the invention. In addition, the evaluation system can also be used for screening the induction system with stable growth vigor and powder scattering amount (less affected by environment) in variety cultivation, so that the field cultivation and screening management of the induction system are simplified, and the problems of less powder scattering amount and greater affected by environment of the haploid induction system in the prior art are solved; and screening haploid induction lines with high induction rate aiming at specific subspecies, and solving the problem of low induction rate in the prior art.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and substitutions can be made by those skilled in the art without departing from the technical principles of the present invention, and these modifications and substitutions should also be considered as being within the scope of the present invention.
Claims (7)
1. A method for creating a maize haploid inducer without subspecies limitation, which is characterized by comprising the following steps: and selecting a haploid induction line containing Ga1-S genes, haploid induction genes and color marker genes as male parents, selecting a haploid induction line containing haploid induction genes and color marker genes which are different from the genetic background of the male parents as female parents, and hybridizing to obtain the maize haploid induction line without subspecies restriction.
2. The method of creating a maize haploid inducer line without subspecies restriction of claim 1, characterized in that the haploid inducer gene comprises a zmpla1 gene.
3. The method for creating a maize haploid inducer line without subspecies restriction according to claim 2, characterized in that the haploid inducer gene of the male parent further comprises zmdmp gene.
4. A method of creating a maize haploid inducer line without subspecies limitation as claimed in any one of claims 1-3, further comprising an evaluation system of the obtained maize haploid inducer line without subspecies limitation comprising an applicability evaluation: the haploid induction lines to be evaluated are respectively hybridized and combined with different subspecies of corns, and the subspecies of corns comprise dent corns, sweet corns, waxy corns and pop corns, and are evaluated through seed setting statistics and inductivity tests.
5. The method of creating a maize haploid inducer line without subspecies restriction of claim 4 characterized in that the method of inducer testing comprises: randomly selecting a plurality of hybrid seeds obtained by hybridizing a haploid induction line to be tested with a certain subspecies of corn, screening haploid seeds by an embryo-endosperm color observation method, and then identifying false positive haploid seeds by adopting a root tip chromosome counting method on the screened haploid seeds, wherein the number of the true haploid seeds accounts for the percentage of the number of the selected hybrid seeds, namely the haploid induction rate.
6. The method for creating a corn haploid inducer line without subspecies restriction of claim 5, wherein the root tip chromosome enumeration method comprises the following specific operation procedures: placing haploid seeds in a two-layer filter paper culture dish for germination acceleration at 25 ℃, placing the culture dish at 4 ℃ when the root length reaches 2-3cm, keeping the filter paper moist, keeping the root tips close to the filter paper, culturing for 18-24 hours, cutting off the root tips from the position of 0.5-1cm above the root crowns, placing the root tips in a 10ml penicillin bottle, fixing the root tips in 10ml Carnot fixing solution, tabletting the root tips after fixing the root tips for 12 hours at room temperature, and observing the chromosome number by using an Olympus CX43 phase contrast microscope.
7. The method of creating a maize haploid inducer line without subspecies restriction of claim 4 wherein the evaluation system further comprises an adaptive evaluation under different environmental conditions: observing agronomic characters of a corn haploid induction line; the observed agronomic traits include: growing vigor, lodging resistance, branch number of tassel and powder scattering amount; different environmental conditions include different cultivation modes, different planting times and different planting areas; different cultivation modes comprise open field cultivation and greenhouse cultivation, different planting time comprises spring and autumn, and different planting areas comprise a Huang-Huai-Hai corn planting area and a northwest corn planting area.
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| CN113317197A (en) * | 2021-08-03 | 2021-08-31 | 中国农业科学院生物技术研究所 | Rapid chromogenic parthenogenesis induction line and application thereof in identification of corn haploid |
| CN113557955A (en) * | 2021-07-19 | 2021-10-29 | 中国农业大学 | Haploid induction line genetic purification method based on reproductive isolation traits |
| CN115053803A (en) * | 2022-06-29 | 2022-09-16 | 北京市农林科学院 | Induction line breeding method with enhanced color marker and high-frequency haploid induction rate and application thereof |
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| CN113557955A (en) * | 2021-07-19 | 2021-10-29 | 中国农业大学 | Haploid induction line genetic purification method based on reproductive isolation traits |
| CN113317197A (en) * | 2021-08-03 | 2021-08-31 | 中国农业科学院生物技术研究所 | Rapid chromogenic parthenogenesis induction line and application thereof in identification of corn haploid |
| CN115053803A (en) * | 2022-06-29 | 2022-09-16 | 北京市农林科学院 | Induction line breeding method with enhanced color marker and high-frequency haploid induction rate and application thereof |
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