Crossbreeding method for self-compatible line of strong-winter brassica campestris L.

Abstract

The invention provides a crossbreeding method of a self-compatible line of strong-winter brassica campestris L., and belongs to the technical field of breeding. The crossbreeding method of the self-compatible line of the strong-winter brassica rapa comprises the following steps: hybridizing self-compatible brassica rapa with the strong-winter brassica rapa to obtain an F1 generation, and carrying out backcrossing for multiple times by taking the strong-winter brassica rapa as a recurrent parent until a BC1F4 generation is obtained; single plants with the affinity index larger than or equal to 6 and the relative affinity index larger than or equal to 60% are selected from the BC4F1-generation population for isolated propagation, bagging selfing is continuously conducted for three generations, and a BC4F4 generation is obtained; and selecting rape plants with the self-compatible plant rate of 100%, the affinity index of more than or equal to 6 and the relative affinity index of more than or equal to 60% in the BC4F4 generation as the strong winter brassica campestris self-compatible line. The breeding method provided by the invention provides germplasm and technology for breeding the pure line variety of the strong winter brassica campestris.

Description

[0001] This application is a divisional application. The application number of the original application is: 202311006867.3, the application date is 2023.08.11, and the name of the invention is: A method for breeding a self-compatible line of winter rapeseed with strong winter properties. Technical Field

[0002] The invention relates to the technical field of breeding, and in particular to a hybrid breeding method of a self-compatible line of winter rapeseed of strong winter property. Background Art

[0003] The winter rapeseed of the strong winter type has the characteristics of cold resistance and early maturity, and plays an important role in agricultural production in the cold and arid areas of the north. However, the main varieties in production are cross-pollination varieties with self-incompatibility, that is, comprehensive varieties with high heterogeneity. The self-incompatibility characteristics easily lead to mixed varieties, poor variety stability, and short service life.

[0004] Therefore, breeding self-compatible varieties is an effective way to improve the quality of northern strong winter-resistant cabbage-type winter rapeseed and to use hybrid vigor to increase yield and economic benefits. It is also the key to solving the problem of self-depression in offspring and homozygous parents during quality breeding and utilization of hybrid vigor. Summary of the invention

[0005] The purpose of the present invention is to provide a method for breeding a self-compatible line of winter rapeseed of the Chinese cabbage type with strong winter properties, so as to transform the self-incompatibility of the northern winter rapeseed of the Chinese cabbage type into a self-compatible line, thereby providing a basis for cultivating pure line varieties, using pure line parents to solve the bottleneck restricting the breeding of three-line hybrids of the Chinese cabbage type winter rapeseed, and greatly improving the yield of the Chinese cabbage type winter rapeseed.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for breeding a self-compatible line of winter rapeseed of Chinese cabbage type with strong winter properties, comprising the following steps:

[0008] (1) Using the self-compatible winter rapeseed variety Ganza 1 of cabbage as the female parent and the self-incompatible winter rapeseed variety Longyou 7 of Chinese cabbage as the male parent, hybridization was performed to obtain the F1 generation;

[0009] (2) Using the F1 generation plants as the female parent and the self-incompatible variety Longyou 7 of the strong winter-resistant Chinese rapeseed as the recurrent parent, backcrossing was performed to obtain the BC1F1 generation;

[0010] (3) Selecting self-compatible BC1F1 plants as the female parent, emasculating the inflorescence of the female parent branches, and using the self-incompatible variety Longyou 7 of the strong winter-resistant Chinese cabbage type winter rapeseed as the recurrent parent for backcrossing to obtain the BC2F1 generation;

[0011] (4) Repeat step (3) and continue backcrossing until the BC4F1 generation is obtained;

[0012] (5) Selecting individual plants with affinity index ≥6 and relative affinity index ≥60% from the BC4F1 generation plant population, bagging and self-pollinating for three consecutive generations to obtain the BC4F4 generation;

[0013] (6) Select plants with a self-compatible plant rate of 100%, a compatibility index ≥ 6, a relative compatibility index ≥ 60% and uniform traits in the BC4F4 generation for isolation and reproduction to obtain a self-compatible line of winter rapeseed with strong winter properties.

[0014] Preferably, the self-compatible BC1F1 generation plants in step (3) are self-compatible BC1F1 generation plants with an affinity index ≥ 6 and a relative affinity index ≥ 60%.

[0015] Preferably, the self-compatible BC1F1 generation plants also have the characteristic of not bolting when sown in spring.

[0016] Preferably, the temperature for spring sowing is 10-25°C, and the growing period is 70-90 days.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention breeds a self-pollination high-compatibility line of winter rapeseed with strong winter characteristics for the first time, and the line is named as LRWCOM-12. LRWCOM-12 has stable self-pollination compatibility, a self-pollination compatibility plant rate of 100%, a self-pollination compatibility index of ≥6, and a relative compatibility index of ≥60%; excellent agronomic traits, a plant height of 130 cm, a branch position of 40 cm, 7 branches at one time, 200 siliques per plant, ≥14 siliques, 5 cm long, 0.5 cm long beak, 3.7 g thousand-grain weight, and 12 g per plant yield; petals are spread, anthers are normally developed, and pollen activity is 87%; and cold resistance is good.

[0019] The present invention successfully breeds self-compatible strong winter-type Brassica rapa winter oilseed rape by hybridizing self-incompatible Brassica rapa with self-compatible Brassica oleracea, thereby solving the problems of fast degeneration and poor variety stability of northern winter-type Brassica rapa winter oilseed rape varieties, providing germplasm and technology for breeding pure-line varieties of strong winter-type Brassica rapa winter oilseed rape, and at the same time solving the contradiction between the need for parental homozygosity and self-depression in utilizing hybrid vigor of Brassica rapa winter oilseed rape, thereby making it possible for northern strong Brassica rapa winter oilseed rape to utilize hybrid vigor to increase yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 The figure shows the comparison of the characteristics of the inflorescence tips of the self-compatible line LRWCOM-12 bred in Example 1 and the self-incompatible Brassica rapa Longyou 7, wherein the left figure shows LRWCOM-12 and the right figure shows Longyou 7;

[0022] Figure 2 The self-compatible line LRWCOM-12 bred in Example 1 showed silique fruiting performance in the field by self-pollination and open pollination in cellophane bags;

[0023] Figure 3 The left figure shows the angular fruit state of the self-incompatible line Longyou 7 in open pollination and bagged self-pollination, and the right figure shows the angular fruit state of the self-compatible line LRWCOM-12 in open pollination and bagged self-pollination;

[0024] Figure 4 for Figure 3 Comparison of the angle of the self-incompatible line Longyou 7 and the self-compatible line LRWCOM-12 after bagging and self-pollination without the bagging cellophane bag and open pollination;

[0025] Figure 5 Comparison of open-pollinated siliques (left) and selfed siliques (right) of the self-compatible line LRWCOM-12;

[0026] Figure 6 The results are a comparative study of the stigma proteomics of the self-incompatible line Longyou 7 and the self-compatible line LRWCOM-12 of the strong winter-type Brassica rapa. DETAILED DESCRIPTION

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] From autumn 2009 to spring 2010, hybridization was carried out in the Shangchuan winter rapeseed experimental field in Lanzhou, using the self-compatible winter cabbage-type rapeseed Ganza No. 1 as the female parent and the strongly winter cabbage-type winter rapeseed self-incompatible variety Longyou No. 7 (original code name Mxw-1) as the male parent to obtain F1 generation seeds.

[0030] The F1 representative is now a radish pod with about 4 pods, high self-incompatibility, and the self-pollinated offspring show abnormal traits such as male sterility, with large variation and difficulty in stability.

[0031] In the autumn of 2010, the F1 seeds obtained by hybridizing Ganza No. 1 and Longyou No. 7 were sown with Longyou No. 7 seeds in Shangchuan, Lanzhou in autumn. After they turned green and bloomed in the spring of 2011, backcrossing was carried out with the F1 plants as the female parent and Longyou No. 7 as the recurrent parent (male parent) to obtain BC1F1 seeds.

[0032] In the autumn of 2011, BC1F1 seeds and Longyou No. 7 seeds were sown in Shangchuan, Lanzhou. In the spring of 2012, BC1F1 plants in the flowering period were bagged and self-pollinated. Each single plant was bagged with 3 inflorescences, and each inflorescence was bagged and self-pollinated with 10-12 flower buds. After 10 days, the self-pollination fruitfulness was examined; BC1F1 plants with high self-pollination affinity (affinity index ≥6, relative affinity index ≥60%) were selected as female parents, and backcrossed with the recurrent parent (Longyou No. 7) to obtain BC2F2 generation seeds;

[0033] Self-compatibility index = total number of siliques in bagged self-pollination / total number of pollinated flowers;

[0034] Relative self-compatibility index = average number of siliques in bagged self-pollinated siliques / average number of siliques in open-pollinated siliques × 100%.

[0035] From autumn 2012 to spring 2014, according to the working steps from autumn 2011 to spring 2012, self-compatible plants with strong self-compatibility (self-compatibility index ≥ 6, relative affinity index ≥ 60%) and cabbage-type plant shape were selected, and backcrossing with the recurrent parent was continued until BC4F1 generation seeds were obtained.

[0036] In the autumn of 2014, BC4F1 seeds were sown; after flowering in the spring of the following year, the BC4F1 plant population was bagged and self-pollinated during the flowering period to detect changes in self-pollination compatibility; individual plants with an affinity index ≥6 and a relative affinity index ≥60% were selected, and the selected self-pollination-compatible individual plants were tested at maturity, and the self-pollination-compatibility index was calculated, and the individual plants with an affinity index ≥6 and a relative affinity index ≥60% were selected to obtain BC4F2 seeds;

[0037] Since the characteristics and traits of the BC4F2 generation, such as self-compatibility and temperature sensitivity, were separated, BC4F2 seeds were sown individually in the spring of 2016 at the Shangchuan base in Lanzhou (the main purpose of spring sowing is to eliminate plants with weak temperature sensitivity, because semi-winter plants with weak temperature sensitivity can generally undergo vernalization, bolt, bloom, set horns and mature under spring sowing conditions or under higher temperature growth conditions, while strong winter plants can remain in a vegetative growth state, only growing leaves without bolting). After 70-90 days of growth after germination, the bolting plants were removed and eliminated, and the creeping plants that did not bolt were allowed to continue growing. When the temperature dropped to around -10°C in winter, the plants were covered with mulch to prevent them from being damaged by vegetative growth. The long-term growth and large vegetative body will cause freezing and death; after flowering in the spring of 2017, the BC4F2 generation plants were bagged and self-pollinated during the flowering period to detect changes in self-pollination affinity; individual plants with affinity index ≥6 and relative affinity index ≥60% were selected, and the selected self-pollination-compatible individual plants (the average number of siliques in bagged self-pollination is greater than 1, which is a self-pollination-compatible plant) were tested and the self-pollination affinity index was calculated during maturity, and the self-pollination-compatible plant rate was counted, and individual plants with affinity index ≥6 and relative affinity index ≥60% were selected and threshed to obtain BC4F3 seeds; BC4F3 seeds were sown in the autumn of 2017, and in the spring of 2018, the flowering period continued to be bagged and self-pollinated, and individual plants with affinity index ≥6 and relative affinity index ≥60% were selected. BC4F4 generation seeds were obtained in June 2018.

[0038] The rate of self-compatible plants = the number of self-compatible plants in the population / the total number of plants in the population × 100%.

[0039] In the autumn of 2018, 35 communities were divided at the Lanzhou Shangchuan base, each with an area of ​​20m 2 . The BC4F4 generation seeds were sown by artificial furrowing and spot sowing, and 500 plants were sown in each plot. In the spring of 2019, bagging and self-pollination were carried out during the flowering period. 100 plants were bagged in each plot, and 40 flower buds were bagged for each plant (4 inflorescences, 10 flower buds in each inflorescence). At the same time, a nylon isolation mesh cover (150 mesh) was set on the outside of each plot. After the plants matured, the self-compatible plant rate (%), self-compatible index and relative self-compatible index were calculated; after the plants matured, the self-compatible plant rate (%) was calculated, and the self-compatible index and relative self-compatible index were calculated.

[0040] The lines with a self-compatible plant rate of 100%, a self-compatible index ≥ 6, and a relative self-compatible index > 60% were selected as self-compatible lines and named LRWCOM-12. After 2019, the self-compatible line LRWCOM-12 was sown in autumn, and the morphological characteristics and stigma proteomics of LRWCOM-12 at various stages were recorded and analyzed. The results are as follows Figure 1-6 shown.

[0041] Figure 1This is a comparison of the characteristics of the inflorescence tops of the self-compatible line LRWCOM-12 and the Chinese rapeseed rape Longyou 7. The left picture shows LRWCOM-12, and the right picture shows Longyou 7. It can be seen that the top of the inflorescence of LRWCOM-12 is an unopened bud, and the blooming flowers are below the buds, while the unopened buds of Longyou 7 are covered by the blooming flowers;

[0042] Figure 2 This is the performance of the self-compatible line LRWCOM-12 in the field after self-pollination in glassine bags and open pollination. It can be seen that the siliques of the bagged self-pollination developed normally, and the length of the siliques was similar to that of the open pollination siliques.

[0043] Figure 3 The figures show the seed angles of the self-incompatible line Longyou 7 and the self-compatible line LRWCOM-12 after open pollination and bagged selfing. The left figure shows the seed angles of the self-incompatible line Longyou 7 after open pollination and bagged selfing, and the right figure shows the seed angles of the self-compatible line LRWCOM-12 after open pollination and bagged selfing. By comparison, it was found that the self-incompatible line rarely set seeds, while the number of seeds set by the self-compatible line was close to that of open pollination, indicating that the high affinity of the self-compatible line LRWCOM-12 is real.

[0044] Figure 4 for Figure 3 The siliques of the self-incompatible line Longyou 7 and the self-compatible line LRWCOM-12 after bagging and self-pollination without the cellophane bags were compared with those of open pollination. The number of siliques of the self-incompatible line bagged and self-pollinated was very small, which was very different from the number of siliques of open pollination. However, the number of siliques of the self-compatible line bagged and self-pollinated was close to that of open pollination, indicating that the self-compatible line LRWCOM-12 has high compatibility.

[0045] Figure 5 This is a comparison between the open-pollinated siliques (left) and self-pollinated siliques (right) of the self-compatible line LRWCOM-12. It was found that the number of siliques produced by bagged self-pollinated lines was close to that produced by open pollination.

[0046] Figure 6 The results are a comparative study of the stigma proteomics of the self-incompatible line Longyou 7 and the self-compatible line LRWCOM-12 of the strong winter-type Brassica rapa.

[0047] The self-compatible line LRWCOM-12 was sown in the autumn of 2020, with a total of 12 groups. The self-compatibility index, outcrossing compatibility index (i.e., the number of pods per silique under open pollination conditions) and relative compatibility index were calculated in June 2021. The results are shown in Table 1.

[0048] Table 1 LRWCOM-12 self-compatibility index in the 2021 harvest season (mid-June)

[0049] serial number Self-compatibility index Outcrossing compatibility index Relative affinity index % LRWCOM-12-1 8.56 11.22 76 LRWCOM-12-2 15.33 16.22 95 LRWCOM-12-3 7.37 12.10 61 LRWCOM-12-4 6.78 9.22 74 LRWCOM-12-5 7.12 8.59 83 LRWCOM-12-6 6.47 9.4 69 LRWCOM-12-7 8.06 13.06 62 LRWCOM-12-8 9.08 15.24 60 LRWCOM-12-9 7.37 12.10 61 LRWCOM-12-10 9.47 12.80 74 LRWCOM-12-11 6.57 7.93 83 LRWCOM-12-12 7.80 10.27 76 average 8.33 11.51 73

[0050] As shown in Table 1, the self-pollination compatibility index of individual plants in the LRWCOM-12 population was greater than 6.47, and the relative compatibility index was above 60%.

[0051] In the autumn of 2021, the self-compatible line LRWCOM-12 was continued to be sown, with a total of 11 groups, and one group of Longyou No. 7 was sown at the same time as a control. Each group was treated with bagging self-pollination and open pollination, and the number of seeds, affinity index and relative affinity index were counted. The results are shown in Table 2.

[0052] Table 2 Results of LRWCOM-12 affinity index determination in the 2022 harvest season (mid-June)

[0053]

[0054]

[0055] As shown in Table 2, the 60% index of self-pollination compatibility of individual plants in the LRWCOM-12 population was greater than 6.47, and the relative compatibility index was above 60%.

[0056] From the above examples, it can be seen that the present invention provides a method for breeding a self-compatible line of winter rapeseed with strong winter properties, and successfully breeds a self-compatible high-compatibility line of winter rapeseed with strong winter properties, LRWCOM-12. LRWCOM-12 has stable self-compatibility, a self-compatibility plant rate of 100%, a self-compatibility index of ≥6, and a relative affinity index of ≥60%.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for breeding a self-compatible line of winter rapeseed of strong winter-resistant Chinese cabbage type, characterized in that: The following steps are involved: (1) Using the self-compatible winter rapeseed variety Ganza 1 of cabbage type as the female parent and the self-incompatible winter rapeseed variety Longyou 7 of Chinese cabbage type as the male parent, hybridization was performed to obtain the F1 generation; (2) Using the F1 generation plants as the female parent and the self-incompatible variety Longyou 7 of the strong winter-resistant Chinese rapeseed as the recurrent parent, backcrossing was performed to obtain the BC1F1 generation; (3) Selecting self-compatible BC1F1 plants as the female parent, emasculating the inflorescence of the female parent branches, and using the self-incompatible variety Longyou 7 of the strong winter-resistant Chinese cabbage type winter rapeseed as the recurrent parent for backcrossing to obtain the BC2F1 generation; (4) Repeat step (3) and continue backcrossing until the BC4F1 generation is obtained; (5) Selecting individual plants with affinity index ≥6 and relative affinity index ≥60% from the BC4F1 generation plant population, bagging and self-pollinating for three consecutive generations to obtain the BC4F4 generation; (6) selecting plants with a self-compatible plant rate of 100%, an affinity index ≥ 6, a relative affinity index ≥ 60% and uniform traits in the BC4F4 generation for isolation and propagation to obtain a self-compatible line of winter rapeseed with strong winter properties; The self-compatible BC1F1 generation plants in step (3) are BC1F1 generation plants with a self-compatibility index ≥6 and a relative affinity index ≥60%; The self-compatible BC1F1 generation plants also have the characteristic of not bolting after spring sowing; The temperature for spring sowing is 10-25°C, and the growing period is 70-90 days.

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