CN112913679A - Transformation method for corn recessive disease-resistant gene - Google Patents
Transformation method for corn recessive disease-resistant gene Download PDFInfo
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Abstract
The invention relates to a method for transferring recessive disease-resistant genes of corn, which adopts the idea of reverse backcross, uses recurrent parents as female parents all the time, selects pollen of partial excellent single plants in a backcross segregation generation to mix, and uses the pollen as a male parent to pollinate silks of the recurrent parents, thereby realizing the backcross transfer of the recessive disease-resistant genes of the corn. Compared with the traditional method for realizing recessive gene transfer by positive backcross, the method omits 3-generation selfing links, shortens the breeding period, improves the working efficiency, has the recurrent parent genetic background recovery rate of over 90 percent, and can provide reference for character improvement of other crops controlled by the recessive gene.
Description
Technical Field
The invention relates to a transformation method for a recessive disease-resistant gene of corn, belonging to the field of crop disease-resistant breeding.
Background
Corn is the first large food crop in the world, and the total corn production has exceeded 10 hundred million tons worldwide. In China, corn is used as the first large grain crop, the planting area in 2019 is up to 6.1 hundred million acres, the total yield is 2.6 hundred million tons, and the corn has a significant effect on grain safety in China. However, corn diseases are a key factor influencing the corn yield, especially in recent years, the straw returning for many years in production causes disease residue accumulation, sufficient pathogenic bacteria and increased disease pressure, so that important diseases such as corn stalk rot and leaf spot are easy to occur frequently, the disease becomes the most main limiting factor of corn production, and the yield loss caused each year accounts for more than 10% of the total yield. The corn belongs to high-density planted crops, and diseases of the corn mostly occur in the middle and later growth periods of the corn, and the corn has the characteristics of outbreak and difficult control, so that planting disease-resistant varieties becomes the most economic and effective measure for treating corn diseases, and meanwhile, the popularization and application of the disease-resistant varieties have positive significance for reducing the use of pesticides, protecting the environment and reducing the investment.
Because the corn variety belongs to the hybrid, the precondition for breeding the disease-resistant hybrid is to culture the disease-resistant inbred line (parent), and the culture of the disease-resistant inbred line is always one of the main breeding targets of the breeder. The disease-resistant genes of plants are divided into quality traits and quantitative traits, and the functional characteristics of the main disease-resistant genes can be divided into recessive genes (RR), dominant genes (RR) and the like. However, most of the diseases reported at present, such as corn stalk rot, rough dwarf, large leaf spot, small leaf spot, and the like, are quantitative, and the disease resistance characteristics of the host are controlled by the major genes and the micro-effective polygenes together. The location and action characteristic analysis of multiple disease-resistant genes of corn shows that main disease-resistant sites for resisting fusarium graminearum stem rot (qRgf3), rough dwarf disease (qZD-MRDD8-1) and small leaf spot (rhm) are recessive genes, if a hybrid (rr) resisting recessive gene diseases is selected, the parents (male parent and female parent) of the hybrid are required to contain the recessive disease-resistant genes (rr), and the hybrid F prepared in such a way is1The genotype (rr) of (a) can show the characteristics of disease resistance.
Backcross transformation of disease-resistant genes is one of the commonly used breeding methods, and usually an inbred line with excellent agronomic characters is selected as a recurrent parent, and germplasm containing the disease-resistant genes is used as a donor parent. As for the dominant disease resistance gene (RR), in different backcross segregating generations, a single plant (RR or Rr) containing the target dominant gene can show the characteristic of disease resistance in a resistance identification nursery, and can be selected as a target plant to be crossed with a recurrent parent (RR). However, for recessive disease-resistant genes (Rr), the single plants of backcross segregating populations are mostly heterozygous genotypes (Rr) and show the characteristic of no disease resistance, and only after selfing, disease-resistant single plants (Rr) containing homozygous recessive genes can be found to be backcrossed with recurrent parents. This presents two problems: on the one hand, if the breeding is carried out in a resistance identification garden with destructive diseases, such as rough dwarf disease, the disease-resistant plants containing RR and Rr cannot be harvested by selfing because the disease is infected; on the other hand, because of the need of selfing, the backcross transformation period of recessive disease-resistant genes is almost prolonged by 1 time compared with the backcross of dominant genes, the breeding period is too long, and the efficiency is low. Therefore, according to the characteristics of self pollination of the corn: only one pollen grain pollinates one filament and only one seed is grown, and the corn recessive disease-resistant gene transfer method is provided aiming at the backcross transfer of the corn recessive disease-resistant gene.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for transferring a corn recessive disease-resistant gene, which adopts the reverse backcross thought, uses a recurrent parent as a female parent all the time, selects part of good single-plant pollen in a backcross segregating generation to be mixed and used as a male parent to pollinate the filaments of the recurrent parent, and realizes the backcross transfer of the corn recessive disease-resistant gene.
In order to achieve the purpose, the invention adopts the technical scheme that:
a transformation method aiming at a corn recessive disease-resistant gene comprises the following steps:
(1) selecting inbred line with excellent characters as recurrent parent P1The material containing target disease-resistant gene is used as a donor parent P2(ii) a Seeding recurrent parent P1And the donor parent P2Seed to recurrent parent P1As female parent, donor parent P2As male parent, group P1×P2Obtaining F1Seeds;
(2) seeding recurrent parent P1And F1Seed according to P1×F1Or F1×P1Preparing to obtain BC1F1Generation seeds;
(3) seeding recurrent parent P1And BC1F1Generation seed, using recurrent parent as female parent, BC1F1Mixed pollen as male parent to compound P1×BC1F1Obtaining BC2F1Generation seeds;
(4) seeding recurrent parent P1And BC2F1Generation seed, using recurrent parent as female parent, BC2F1Mixed pollen as male parent to compound P1×BC2F1Obtaining BC3F1Generation seeds;
(5) sowing 300-500 BC3F1Generation seeds are selfed in the stage of loose powder and silking, and single ear is harvested, threshed and preserved to obtain BC3F2Generation seeds;
(6) sowing BC in disease identification nursery or field with serious disease all year round3F2The generation seeds are planted with 300-500 ear rows, 15-25 plants in each row are selfed in the stage of powder scattering and silk spinning, and only selfed ears on the disease-resistant single plant are harvested and stored according to ear threshing to obtain BC3F3Generation seeds;
(7) sowing BC in disease identification nursery or field with serious disease all year round3F3Generation seeds are selfed in the stage of loosening and spinning, only the ears of disease-resistant single plants are harvested and threshed according to ears for preservation, and BC is obtained3F4Generation seeds;
(8) seeding BC3F4Carrying out test crossing on generation seeds and test seeds in the period of loose powder spinning; the selected plant line which has the advantages of the same heterosis as the recurrent parent and similar agronomic characters and contains the recessive disease-resistant gene is the plant line which is successfully transferred and contains the target recessive disease-resistant gene and can be used for the combination of disease-resistant hybrid combination.
The recurrent parent in the step (1) is the inbred line Zheng 58, and the donor parent is the rough dwarf resistant germplasm D863F.
The specific method of the step (3) is as follows: 150-180 BC plants are sown1F1Generation seed, 15-25 recurrent parent P1From BC during the dusting and spinning stage1F1Selecting 30-50 excellent individual plant pollen from generation group, using 10 pollen as a group, mixing uniformly, pollinating 3-5 recurrent parent plants respectively, harvesting 3-5 pollinated ears from the recurrent parent plants, threshing, mixing uniformly to obtain BC2F1Generation seed.
The specific method of the step (4) comprises the following steps: seeding 200-300 BC2F1Generation seed, 25-35 recurrent parent P1From BC during the dusting and spinning stage2F1Selecting 50-100 excellent individual plant pollen in a generation group, taking 10 pollen as a group, uniformly mixing, pollinating 5-10 recurrent parent plants respectively, harvesting 5-10 pollinated ears from the recurrent parent plants, uniformly mixing the seeds after threshing to obtain BC3F1Generation seed.
The specific method of the step (6) is as follows: sowing BC in disease identification nursery or field with serious disease all year round3F2The generation seeds are planted with 300-500 ear rows, each ear is planted with 1 row, each row is 15-25 plants, ear rows with serious diseases are eliminated in the peak period of disease occurrence, excellent single plants in the ear rows with disease resistance of target diseases are selfed, and selfed ears of the disease-resistant single plants are only harvested and stored according to ear threshing, wherein the selfed ears are BC3F3Generation seed.
The specific method of the step (7) is as follows: sowing BC in disease identification nursery or field with serious disease all year round3F3The generation seeds are planted according to ear rows, each ear is planted for 1 row, each row is 25-35 plants, ear rows with serious diseases are eliminated in the peak period of disease occurrence, excellent single plants in the ear rows with disease resistance of the target diseases are selfed, and the selfed ears of the disease-resistant single plants are only harvested and stored according to ear threshing, wherein the seeds are BC3F4Generation seed.
The test cross breeding in the step (8) is Chang 7-2.
The invention has the beneficial effects that:
(1) at present, technologies such as gene editing, molecular marker assisted selection and the like have many successful reports and cases on crop genetic improvement, but most of the application of the technologies still stays in a laboratory, and the laboratory requiring operation has advanced equipment and technical strength and the like. However, most corn seed industry enterprises and breeders in China currently do not have the advanced instruments, equipment, molecular biology knowledge and the like. The transformation method provided by the invention does not need the advanced equipment and technical force, only needs the conventional breeding technical operation, and is convenient for the operation of workers engaged in the traditional corn breeding.
(2) For some destructive diseases, such as maize rough dwarf disease, if non-disease resistant plants containing RR and Rr genotypes are planted in a disease nursery, seeds cannot be harvested by selfing due to the rough dwarf disease. The method of the present invention, backcrossed low generation BC1～BC3The selection is not planted in a disease garden, hybrid plants containing Rr genotypes are effectively reserved, the recessive gene r is inherited continuously, and guarantee is provided for obtaining resistant plants containing the homozygous Rr genotypes.
(3) The traditional backcross transformation of recessive genes needs to be performed through the process of backcross-selfing-backcross, and before each generation of backcross, one generation of backcross is needed again, so that the disease-resistant plants of the homozygous recessive Rr genotype of heterozygous single plants containing the Rr genotype in the backcross segregation generation can be expressed, screened and identified as target plants to be hybridized with recurrent parents, and the breeding of the disease-resistant inbred lines is expressed as long period and low efficiency. According to the method, partial good single plant pollen is selected from backcross segregating generations to be mixed to serve as a male parent, the filaments of recurrent parents are pollinated, compared with the traditional method of realizing recessive gene transfer through forward backcross, the method omits 2-4 generation selfing links, shortens breeding years and improves working efficiency. In addition, reference can be provided for genetic improvement of traits controlled by recessive genes of other crops.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1 transformation of maize rough dwarf recessive disease resistance Gene (qZD-MRDD8-1)
Maize rough dwarf is a worldwide maize disease, and once a plant is attacked, the maize rough dwarf cannot be prevented and controlled, so the maize rough dwarf is called maize cancer, mainly occurs in Huang-Huai-Hai regions in China, is caused by the spread of rice black-streaked dwarf viruses carried by Laodelphax striatellus, and poses serious threat to maize production. At present, a plurality of loci are reported to control the resistance of maize rough dwarf disease, wherein the major disease-resistant locus qZD-MRDD8-1 located on the 8 th chromosome is a recessive disease-resistant locus and has an important effect on the resistance of the maize rough dwarf disease.
Selecting the core inbred line Zheng 58 of China as the recurrent parent P1Anti-rough dwarf germplasm D863F as donor parent P2Successfully transfers the recessive anti-rough dwarf locus in D863F into Zheng 58, and obtains a Zheng 58 improved strain resisting rough dwarf. The specific operation is as follows:
(1) seeding and recurrent parent P in the test field of Yuanyang county in Xinxiang city in 2012 summer1And the donor parent P2Seed to recurrent parent P1As female parent, donor parent P2As male parent, matched with Zheng 58 XD 863F, the harvested seed is F1。
(2) In 2012 winter, in Hainan three-basic-land, the inbred lines Zheng 58 and F were sown1Seeds, during the period of spinning and powder scattering, Zheng 58 XF1Or F1X Zheng 58 combination is BC1F1Generation seed.
(3) In the summer of 2013, 20 Zheng 58 and 170 BC are sown in the test land of Yuanyang county in New county1F1Generation seed, at the stage of loose powder spinning, from BC1F1Selecting 40 good individual plants of pollen in generation group, mixing with 10 plants of pollen, and blending according to Zheng 58 × BC1F1The combination mode of (1) and respectively pollinates 4 recurrent parent Zheng 58. Harvesting 4 pollination ears from recurrent parents in the maturation period, and uniformly mixing the seeds after separation to obtain BC2F1Seeds of a generation.
Frequency of recurrent parent Gene recovery according to the publication 1- (1/2)n+1Calculating (n is the number of backcrosses), BC2F1The gene recovery rate of the generation recurrent parent Zheng 58 is 87.5%.
(4) In 2013, in Hainan Sanxia, 30 Zheng 58 and 280 BC were sown2F1Generation seed, at the stage of loose powder spinning, from BC2F1Selecting pollen of 70 excellent individuals in the generation group, mixing with 10 pollen, and collecting the pollen of Zheng 58 × BC2F1The combination mode of (1) respectively pollinating 8 recurrent parent Zheng 58, harvesting 8 pollinated ears from the recurrent parent in the maturation period, and uniformly mixing the threshed seeds to obtain BC3F1Seeds of a generation. BC3F1The gene recovery rate of the generation recurrent parent Zheng 58 is 93.75%.
(5) In summer of 2014, 460 BC are sown in the test land of Yuanyang county in New county3F1Generation seeds are selfed in the stage of loose powder and silking, and the selfed ears of 381 are harvested in the later stage by combining the bearing property, ear position and lodging resistance, and the seeds are threshed and preserved in single ear, and are BC3F2Seeds of a generation.
(6)2015 spring, 5 months and 15 days, sowing 381 harvested BC in field with serious perennial rough dwarf of Dulianzhen in Kanghai city, Henan province3F2The generation of the ears, 1 row of seeds is planted in each ear, 20 seeds are planted in each row, and 381 ear rows are counted. In the seedling stage (5-leaf stage) of the current year, about 15 heads of single plant of the laodelphax striatellus exist, after the plant is sowed for 40 days, field investigation shows that the rough dwarf disease naturally occurs seriously, about 90 percent of ear rows cannot normally spin and scatter powder, the height of a disease-susceptible plant is about 45cm, and only 23 rows show better resistance. In the peak period of disease occurrence, the ear rows with serious diseases are eliminated, the excellent single plants in the ear rows with target disease resistance are selfed, only the selfed ears of the disease-resistant single plants are harvested and threshed according to ears for storage, the selfed ears of 56 plants are harvested in the mature period, the single ears are harvested, threshed and stored, and the plant is BC3F3Seeds of a generation.
(7)2016 spring, 5 months and 13 days, in Henan province, Kaifeng city, Dulianzhen, field with serious perennial rough dwarf disease, and sowing BC3F3The generation seeds are 56 rows per ear, and 1 row is planted per earIn 30 plants per row, field investigation in the current year shows that the rough dwarf disease naturally occurs seriously, and resistant plants exist in most ear rows. In the peak period of disease occurrence, the ear rows with serious diseases are eliminated, the excellent single plants in the ear rows with target disease resistance are selfed, and only the selfed ears of the disease-resistant single plants are harvested and stored according to ear threshing. 16 resistant and well-fruited fruit ears are harvested in the mature period, and the single ear is harvested, threshed and preserved and is BC3F4Seeds of a generation.
In 2015 and 2016, infected plants are eliminated through two continuous generations of planting, screening and identifying in disease nursery, and 16 disease-resistant strains are obtained.
(8) In 2016 winter, 16 BC disease-resistant strains were sown in rows of ears in the Hainan three-test field3F4The generation seeds (No. MD 161-MD 1616) are 20 plants per row, the row performance is uniform, except the selfing is carried out in the period of spinning and powder scattering, the generation seeds are respectively matched with Chang 7-2, and the heterosis and the biological characteristics are compared with the Control (CK) combination Zheng 58X Chang 7-2. The selected plant line which has the advantages of the same heterosis as the recurrent parent and similar agronomic characters and contains the recessive disease-resistant gene is the plant line which is successfully transferred and contains the target recessive disease-resistant gene and can be used for the combination of disease-resistant hybrid combination.
(9) In 2017 spring, the combination matched with Chang 7-2 and the control are planted in an Kaifeng Duliang rough dwarf disease resistance identification nursery, and the resistance of the Kaifeng Duliang rough dwarf disease resistance identification nursery is investigated; in summer, the combination and the contrast of the components are planted in summer. Through resistance identification, field character investigation and yield measurement (table 1), 2 strains which have the same heterosis and similar agronomic characters with recurrent parents and contain recessive rough dwarf resistant genes are selected and named as MD1611 and MD167 respectively to be used as rough dwarf resistant germplasm.
TABLE 1 Rough dwarf resistance, agronomic performance and yield of the tested combinations
By utilizing the steps of the method, strains with resistance to the small spot and the stem rot can be obtained. In addition, reverse backcross transformation is carried out on backbone inbred lines such as Chang 7-2, PH6WC, PH4CV and the like, so that an improved line which has the recurrent parent genetic background recovery rate of more than 90% and contains recessive rough dwarf resistant genes can be obtained and applied to breeding units.
(10) The performance test is to further clarify the difference of the obtained characteristic characteristics of MD1611 and MD167 from the recurrent parent zheng 58. In 2018, MD1611 and MD167 and the recurrent parent Zheng 58 are planted in an unsealed Duliang rough dwarf disease resistance identification garden in spring, and the resistance of the Duliang rough dwarf disease resistance identification garden is investigated; in summer, resistance identification and agronomic character investigation are carried out on other main diseases according to a corn disease resistance identification technical program (Wangxinging, a corn disease and insect pest field manual-disease and insect pest identification and resistance identification. Beijing: Chinese agricultural science and technology publisher, 2010.). Agronomic trait surveys showed that the growth period, tassel and ear characteristics and recurrent parent differences were not significant (table 2). The resistance identification result of the rough dwarf disease shows that MD1611 and MD167 show disease resistance, while the recurrent parent Zheng 58 shows high sense; the resistance of MD1611 and MD167 to curvularia leaf spot is superior to that of Zheng 58; the resistance of MD1611 to both small spot and southern rust is better than Zheng 58; the resistance of other diseases was comparable to zheng 58 (table 3).
TABLE 2 comparison of agronomic traits for improved lines and recurrent parents
TABLE 3 comparison of disease resistance of improved lines with recurrent parents
Note: HR indicates high resistance, R indicates resistance, MR indicates medium resistance, S indicates feeling, and HS indicates high feeling.
(11) The program and the number of progeny populations are consistent for the reverse backcross transformation of the maize stalk rot recessive disease resistance gene (qRgf3) and the northern leaf blight recessive disease resistance gene (rhm). However, there are two keys, namely, to find a suitable source of resistance as a donor parent, and to provide a suitable pathogenic environment or resistanceA sexual identification garden for artificial inoculation identification to ensure the BC in a planting separation generation3F2And BC3F3When the disease nursery is selected, the disease is fully developed, the disease-sensitive plants are eliminated, and the disease-resistant plants are reserved.
Claims (7)
1. A transformation method aiming at a corn recessive disease-resistant gene is characterized by comprising the following steps:
(1) selecting inbred line with excellent characters as recurrent parent P1The material containing target disease-resistant gene is used as a donor parent P2(ii) a Seeding recurrent parent P1And the donor parent P2Seed to recurrent parent P1As female parent, donor parent P2As male parent, group P1×P2Obtaining F1Seeds;
(2) seeding recurrent parent P1And F1Seed according to P1×F1Or F1×P1Preparing to obtain BC1F1Generation seeds;
(3) seeding recurrent parent P1And BC1F1Generation seed, using recurrent parent as female parent, BC1F1Mixed pollen as male parent to compound P1×BC1F1Obtaining BC2F1Generation seeds;
(4) seeding recurrent parent P1And BC2F1Generation seed, using recurrent parent as female parent, BC2F1Mixed pollen as male parent to compound P1×BC2F1Obtaining BC3F1Generation seeds;
(5) sowing 300-500 BC3F1Generation seeds are selfed in the stage of loose powder and silking, and single ear is harvested, threshed and preserved to obtain BC3F2Generation seeds;
(6) sowing BC in disease identification nursery or field with serious disease all year round3F2Planting 300-500 ear rows of 15-25 plants in each row, selfing in the stage of powder scattering and silk spinning, harvesting selfing ears only on the disease-resistant single plant, and storing by ear threshing to obtain the final productGet BC3F3Generation seeds;
(7) sowing BC in disease identification nursery or field with serious disease all year round3F3Generation seeds are selfed in the stage of loosening and spinning, only the ears of disease-resistant single plants are harvested and threshed according to ears for preservation, and BC is obtained3F4Generation seeds;
(8) seeding BC3F4Carrying out test crossing on generation seeds and test seeds in the period of loose powder spinning; the selected plant line which has the advantages of the same heterosis as the recurrent parent and similar agronomic characters and contains the recessive disease-resistant gene is the plant line which is successfully transferred and contains the target recessive disease-resistant gene and can be used for the combination of disease-resistant hybrid combination.
2. The method for transforming the maize recessive disease-resistant gene as claimed in claim 1, wherein the recurrent parent in step (1) is Zheng 58 of inbred line, and the donor parent is germplasm D863F for resisting rough dwarf disease.
3. The method for transforming the maize recessive disease-resistant gene as claimed in claim 1, wherein the specific method in the step (3) is as follows: 150-180 BC plants are sown1F1Generation seed, 15-25 recurrent parent P1From BC during the dusting and spinning stage1F1Selecting 30-50 excellent individual plant pollen from generation group, using 10 pollen as a group, mixing uniformly, pollinating 3-5 recurrent parent plants respectively, harvesting 3-5 pollinated ears from the recurrent parent plants, threshing, mixing uniformly to obtain BC2F1Generation seed.
4. The method for transforming the maize recessive disease-resistant gene as claimed in claim 1, wherein the specific method in the step (4) is as follows: seeding 200-300 BC2F1Generation seed, 25-35 recurrent parent P1From BC during the dusting and spinning stage2F1Selecting 50-100 excellent individual plants of pollen from the generation group, taking 10 plants of pollen as a group, mixing uniformly, pollinating 5-10 recurrent parent plants respectively, and pollinating the recurrent parent plantsHarvesting 5-10 pollination ears, threshing and mixing seeds uniformly to obtain BC3F1Generation seed.
5. The method for transforming the maize recessive disease-resistant gene as claimed in claim 1, wherein the specific method in the step (6) is as follows:
sowing BC in disease identification nursery or field with serious disease all year round3F2The generation seeds are planted with 300-500 ear rows, each ear is planted with 1 row, each row is 15-25 plants, ear rows with serious diseases are eliminated in the peak period of disease occurrence, excellent single plants in the ear rows with disease resistance of target diseases are selfed, and selfed ears of the disease-resistant single plants are only harvested and stored according to ear threshing, wherein the selfed ears are BC3F3Generation seed.
6. The method for transforming the maize recessive disease-resistant gene as claimed in claim 1, wherein the specific method in the step (7) is as follows:
sowing BC in disease identification nursery or field with serious disease all year round3F3The generation seeds are planted according to ear rows, each ear is planted for 1 row, each row is 25-35 plants, ear rows with serious diseases are eliminated in the peak period of disease occurrence, excellent single plants in the ear rows with disease resistance of the target diseases are selfed, and the selfed ears of the disease-resistant single plants are only harvested and stored according to ear threshing, wherein the seeds are BC3F4Generation seed.
7. The method for transforming a maize recessive disease-resistant gene as claimed in claim 1, wherein the test cross of step (8) is Chang 7-2.
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| 杨青等: "玉米粗缩病抗病基因定位和抗病育种研究进展", 《分子植物育种》 * |
| 邸垫平等: "玉米粗缩病抗性遗传研究", 《植物病理学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114303931A (en) * | 2021-12-24 | 2022-04-12 | 吉林省农业科学院 | Backcross transformation method for controlling genetic characters by plant recessive genes |
| CN116530409A (en) * | 2023-05-22 | 2023-08-04 | 河南省农业科学院植物保护研究所 | Creation method of multi-resistance corn germplasm |
| CN116732226A (en) * | 2023-06-16 | 2023-09-12 | 袁隆平农业高科技股份有限公司 | Method for rapidly transferring and breeding Ruifeng 125 transgenic maize inbred line and application |
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