阅读说明：本技术 用核三系油菜f2代分离选育同质临保及纯合不育系方法 (Method for separating and breeding homogeneous temporary protection and homozygous sterile line by using nuclear three-line rape F2 generation ) 是由 侯剑 侯燕 侯国佐 于 2019-12-11 设计创作，主要内容包括：本发明公开了一种用核三系油菜F2代种子分离选择同质临保及纯合不育系的方法,该方法是在没有核三系亲本(即临保系和纯合不育系)育种材料的基础上,直接选择其丰产稳产性好、抗逆性强、品质优等综合经济性状好的杂交品种(组合)套袋自交,根据F2代、F3代育性表现不同分别采取可育与不育株同袋杂交、可育株自交、初花后在调查的基础上取相应株行材料的植株分别作父母本在蚊帐内制种、所产生的相应种子再对其进行育性鉴定等方法,在3-4代内直接快速分离选择出同质临保及纯合不育系的方法。(The invention discloses a method for separating and selecting homogeneous temporary protection and homozygous sterile lines by using seeds of a nuclear three-line rape F2 generation, which is characterized in that hybrid varieties (combinations) with good high yield and stable yield, strong stress resistance, excellent quality and other comprehensive economic properties are directly selected for bagging selfing on the basis of breeding materials without nuclear three-line parents (namely temporary protection lines and homozygous sterile lines), fertile plants of fertile and sterile plants are respectively adopted to be subjected to bagging hybridization, fertile plant selfing and initial flowering on the basis of investigation according to the difference of fertility performances of the F2 generation and the F3 generation, plants of corresponding plant row materials are respectively taken as parents to produce seeds in a mosquito net, and the generated corresponding seeds are subjected to fertility identification and the like, so that the homogeneous temporary protection and homozygous lines are directly and rapidly separated and selected in the 3-4 generation.)

1. The method for separating and selecting the homogeneous temporary protection and homozygous sterile line by using the nuclear three-line rape F2 generation is characterized by comprising the following steps:

s1, selecting 5-8 introduced nuclear three-line hybrid varieties or combinations through tests, respectively planting, bagging and selfing 8-10 plants in each variety or combination selected plant in a flowering period, harvesting F2 selfed seeds in different plants after maturation, and performing seed test and quality analysis to reserve seeds for later use;

s2, selecting 1-2 plant rows of F2 excellent single plants for each variety or combination; investigating the sterile plant rate of each plant line before and after the initial flowering phase, selecting plant line groups with the sterile plant rate of 18% -25%, selecting a certain number of sterile plants and fertile plants which are adjacent to each other in the group at the flowering phase, bundling the sterile plants and the fertile plants together, respectively listing and numbering the sterile plants and the fertile plants, sleeving two plant inflorescences in a paper bag by using a paper bag to keep a certain degree of openness in the paper bag so that the fertile plants and the sterile plants can pollinate, shaking the bag and lifting the bag to help pollination in the flowering phase, and enabling the pollen of the fertile plants of the sterile plants to be hybridized and fructified; respectively harvesting seeds of the fertile inbred plant and the sterile hybrid plant for later use after the seeds are mature;

s31, respectively taking 4-5g of the seeds of the selfed plant and the hybrid plant to plant corresponding plant rows, and reserving the rest seeds for later use;

s32, respectively investigating the sterile plant rate of the plant line of the inbred plant seeds and the plant line of the hybrid plant seeds in the initial flowering period;

s33, in the investigation of the plant row sterile plant rate of hybrid seed planting, the test cross male parent single plant corresponding to the plant row with full sterile plant is represented as a temporary maintainer line, so that the plant row of the seeds with corresponding numbers is found in the plant row of the self-bred seeds, the plant row single plant is represented as full fertile, and the plant is selected, bagged, matured, harvested and stored;

s34, in the investigation of the plant row sterile plant rate of hybrid seed planting, showing 45% -55% sterile plant rows, selecting 5-6 pairs of adjacent sterile plants and fertile plants to be sleeved together in a paper bag, shaking the bag in the flowering phase to carry out artificial auxiliary pollination to ensure that the sterile plants fruit in the paper bag; respectively harvesting and storing the mature fertile plants and the sterile plants in the bags;

s35, selecting a plant row of self-bred seeds corresponding to the plant row complete sterility of the hybrid seeds in S33, and taking seedlings with soil as male parents at the early flowering stage; the sterile plants in the hybrid seed plant rows show 45-55 percent of plant row plants, the sterile plants which are carefully earthed to take seedlings in the initial flowering period of each plant row are used as female parents, and the plants are transplanted with soil carefully under the condition of isolation of a mosquito net, and are pollinated manually in an auxiliary manner; after the seeds are matured, the seeds are respectively harvested and stored in a mixed manner by taking the row of the female parent plants as a unit;

s41, separating the female parent plant rows and mixed seeds for seed production in the mosquito net in the last year, sampling 2-3g of each plant row, planting the plant rows, and investigating the sterile plant rate in the flowering phase; sleeving sterile plants and fertile plants in the plant rows with 45% -55% sterile plant rows of the last-year hybrid seed plant rows together with sterile plant seeds in a bag, respectively planting 4-5 single plants in the plant rows, investigating the sterile plant rate in the flowering phase, and correspondingly comparing the sterile plant rate with the sterile plant rate of the plant rows of corresponding uneven seedlings sowed in a mosquito net, wherein when the plant rows corresponding to the uneven seedlings sowed in the mosquito net are completely sterile, the sterile plant rate of the line is 45% -55%, the material of the line is a homozygous sterile line; bagging sterile plants and fertile plants in the row continuously, and simultaneously selecting comprehensive economic characters of the plants; respectively collecting seeds of the sterile and fertile plants after maturation, carrying out examination and analysis, and reserving seeds for later use;

s42, planting the single plant selfing seed row corresponding to the full sterility in the last year hybrid row, namely, 4-5 plant rows of bagged selfing seeds in the temporary maintainer line, wherein the sterile plant rate is fully fertile in the florescence survey, selecting 8-10 plants in a plant bag, and simultaneously continuously selecting the comprehensive economic characters of the plants for the plant rows;

s43, taking the sterile plant with soil in the homozygous sterile line which is qualified in the S41 as a female parent, taking the plant with soil in the temporary maintainer line which is qualified in the S42 as a male parent, still isolating and producing seeds in the mosquito net, and harvesting and storing the seeds.

Technical Field

The invention relates to a rapid breeding method for directly separating and selecting homogeneous temporary protection and homozygous sterile line by using seeds of a nuclear three-line hybrid rape variety (combination) F2 generation, belonging to the technical field of rape variety breeding.

Background

Rape is the main oil crop in China. Rape heterosis utilization is at a leading level in the world. Since the first hybrid rape Qin you No. two in the eighties of the last century, hundreds of hybrid rapes have been developed and approved by the nation and various provinces for production, and particularly, since the nineties of the last century, the breeding of high-quality rapes and the heterosis breeding have been successfully combined, and a large number of varieties with high yield, high quality and high oil content are developed and popularized in China.

In rape heterosis breeding, the application in production is mainly realized by establishing and breeding a rape male sterile line. At present, the sterile line applied in a larger area mainly comprises cytoplasmic male sterility and nuclear male sterility, and the nuclear male sterility is divided into recessive nuclear male sterility and dominant nuclear male sterility, wherein the dominant nuclear male sterility is few in recovery source and few in bred varieties, so that the recessive nuclear male sterility is a basic material for breeding the most varieties except for the cytoplasmic male sterility.

The recessive cell nucleus male sterile material has the advantages of wide recovery source, complete and stable sterility and no environmental influence. The material is divided into a recessive cell nucleus male sterile two line and a recessive cell nucleus male sterile three line, wherein the sterile line contains 50 percent of fertile plants, and the fertile plants are pulled out before blooming in seed production, and the recessive cell nucleus male sterile three line can realize 100 percent of sterility. Therefore, the latter has the advantages of stable sterility (the cytoplasmic sterile material mainly applied at present shows trace pollen for low temperature sensitivity), thorough sterility and wide recovery source compared with the cytoplasmic male sterile material, overcomes the defect that 50 percent of fertile plants of recessive genic male sterile two-line material need to be pulled out in seed production, and is an ideal material for the current rape heterosis breeding.

The rape recessive genic male sterility three line is the same as the double recessive male sterility two lines, and the sterility is also influenced by two pairs of recessive genes (ms)₁ms₁ms₂ms₂) But the difference is that the former has a pair of episomal (RfRf) control, and when the episomal gene is recessive, RfRf, the sterility gene ms can be inhibited₁ms₁ms₂ms₂Expression of fertility, rendering it fertile. The genotype is rfrfrffms₁ms₁ms₂ms₂It is called Lin Bao series. When it is combined with sterile gene RfRfms₁ms₁ms₂ms₂When crossing, only one sterile genotype Rfrfms is generated₁ms₁ms₂ms₂Thus, it is shown as being all-sterile, the prepro-genotype RfRf ms₁ms₁ms₂ms₂Called homozygous sterile line, post-genotype Rfrfms₁ms₁ms₂ms₂Is called a hybrid sterile line. Therefore, the transformation of new homozygous sterile line and temporary maintainer line in recessive genic male sterility three-line breeding to prepare total sterile line and prepare and breed new hybrid becomes the main task of breeding.

The purpose of transformation of the new homozygous sterile line and the temporary maintainer line is to further improve the original homozygous sterile line and the temporary maintainer line by using excellent economic characters (including comprehensive excellent economic characters such as high yield, stress resistance, excellent quality and the like). The transformation method generally adopts stable homozygous sterile line as female parent, selects some excellent target characters as male parent to make hybridization, then F₂The separation is carried out to test cross, and the method has large test cross workload. Because the homozygous sterile line is used for hybridizing with the general excellent parent variety, the parent does not necessarily have a pair of homozygous upper genes of RfRf, and the genotype of the transferred material is assumed to be recessive, so that the genotype of the transferred material is the rffrfMs₁Ms₁Ms₂Ms₂Of which F₂The ratio of the separation of generation fertile and sterile is 61: 3, among the 3 sterile strains, the homozygous sterile type only accounts for 1/3, the flowering phase can not be identified, and the sterile strains are maintained by fertile strains, while among the 61 fertile strains, 1/2 fertile strains which can maintain the homozygous sterile genotype are only 4 strains in the next generation, the probability is only 4/61, so F₂The probability of obtaining homozygous sterile line by pair test crossing of generation separation is only 1/3 × 4/61 ═ 4/183; if about 4 homozygous sterile selective materials appear in the later generation of a transfer material, the F2 generation needs to be simultaneously test-crossed for more than 180 pairwise test-crossed combinations, and if two pairwise test-crossed combinations can be test-crossed on each sterile plant, about 90 sterile plant populations are needed, and the sterile plant populations can be obtained by planting more than 1800F 2 plants; simultaneously more than 180 combinations of the next year of paired test crossing need to be planted with plant rows, and the flowering period of the combined plant rows needs to be about 12 plant rows with about 1/2 sterile plant ratePaired test crossing, and paired test crossing the sterile plants with temporary maintainer line to identify the sterility gene homozygosity (RfRfms)₁ms₂ms₂Or Rfrfms₁ms₁ms₂ms₂) And each sterile plant row tested and crossed with the temporary maintainer line needs to be tested and crossed in pairs by more than 5 plants to ensure that the guarantee degree of fertility identification total sterility reaches more than 91 percent. Thus F₂The method for breeding the homozygous sterile line by generation separation test cross not only has large test cross workload, but also has large population quantity to be planted at F2 and F3, and if more materials are transferred in one year, the method can hardly be operated.

In the same way, the workload of breeding temporary maintainer lines is also great, and the occurrence probability is lower. The temporary maintainer line plant in the group fertile plant is only 1/61, and the homozygous sterile type in the sterile plant is only 1/3, so that 183 sterile plants are needed for paired test cross to breed a temporary maintainer line, and the sterile plant rate of the group is only 3/64,183 sterile plants to plant F2 plant groups of 3904 plants. Such a large population needs to be planted by selecting only one temporary maintainer line, and the test crossing of such many combinations is obviously not completed.

The breeding technology is established on the basis of the nuclear three-line homozygous sterile line, and breeding workers without the basis of the nuclear three-line breeding have no condition for completing the breeding of the method.

Disclosure of Invention

The invention aims to provide a method for selecting a homozygous temporary protection and homozygous sterile line from seeds of a Trigenic rape F2 generation so as to solve the problems in the prior art.

The core technology of the invention is as follows:

1. under the condition of no material basis of a nuclear three-line parent (and a homozygous sterile line and a temporary maintainer line), plant-row groups with the sterile plant rate of 18-25% are directly selected from a nuclear three-line variety (material) F2 segregation generation as segregation selection materials so as to shorten the breeding time and reduce the workload;

2. the adjacent sterile plants and fertile plants are bagged together to complete sterile plant hybridization and fertile plant selfing simultaneously to replace fertile plant bagging selfing and manual paired bud stripping hybridization of the sterile plants and the fertile plants, so that the workload of manual hybridization can be greatly reduced.

3. The soil-bearing seedling of sterile plants with the sterile plant rate of about 1/2 and the soil-bearing seedling of plants with the temporary protection effect in the plant line of the selfed seeds are selected in advance one year in the early flowering stage, and the plants are moved into the mosquito net for isolation seed production, so that the breeding time is shortened, the workload is saved, and the stability of purity selection of the homozygous sterile line is improved.

The invention is realized by the following steps:

the invention relates to a rapid breeding method for directly separating and selecting homogeneous temporary protection and homozygous sterile line by using seeds of a nuclear three-line hybrid rape variety (combination) F2 generation, which is realized by the following steps:

step 1: through tests, 5-8 hybrid varieties (combinations) with high yield, stable yield, strong stress resistance, high quality and the like and good comprehensive economic characters are selected from the introduced nuclear three-line hybrid varieties (combinations) and are respectively planted, each variety (combination) in the flowering period is selected and bagged for 8-10 plants to be inbred, and after the plants are matured, F2 inbred seeds are harvested in a splitting mode, and the test seeds and the quality analysis are carried out, and the seeds are reserved for later use.

Step 2: 1-2 excellent single plants of F2 are selected for each variety (combination) to plant rows, the row density of the plants is about ten thousand plants/mu, the row group of the plants reaches 600-700 plants, and the medium and high management level. The method comprises the steps of investigating sterile plant rate for each plant line before and after the initial flowering phase, selecting plant line groups with the sterile plant rate of 18% -25%, bundling left and right adjacent sterile plants and fertile plants together in the group at the flowering phase, respectively listing and numbering the sterile plants and the fertile plants, sleeving two inflorescences in a paper bag with a larger point and a special pollen isolation paper bag (such as a sulfuric acid paper bag or a non-woven fabric paper bag, hereinafter referred to as a paper bag) to keep a certain degree of vacancy in the paper bag so that the fertile plants and the sterile plants can pollinate, shaking the bag in the flowering phase to help pollination, enabling the fertile plants of the sterile plants to be hybridized and fruited, and enabling the seed quantity of each paired hybridized and selfed seed to reach more than 10 g. After the seeds are matured, the seeds of the fertile inbred plant and the sterile hybrid plant are respectively harvested and stored for standby.

And step 3: in the third planting year, the working procedure is complex, and the following sequences are respectively shown:

(1) and (4) respectively taking 4-5g of the seeds of the selfed plant and the hybrid plant to plant corresponding plant rows, and reserving the rest seeds for later use. The planting density of the plant rows can reach 2.5-3 ten thousand plants per mu, and the plant row group has about 300 plants. Moderate management levels, prevent lodging so as not to affect later work and results.

(2) The sterile plant rate of the plant line of the inbred plant seeds and the plant line of the hybrid plant seeds is firstly investigated respectively in the initial flowering period.

(3) In the investigation of the plant row sterile plant rate of hybrid seed planting, the test cross male parent single plant corresponding to the full sterile plant row is a temporary maintainer line, so that the plant row of the seeds with corresponding numbers can be found in the plant row of the selfing seeds, the plant row single plant is fully fertile, and the plant is selected and bagged for about 20 plants, and the plants are harvested and stored after being mature.

(4) In the investigation of the plant row sterile plant rate of hybrid seed planting, about 1/2 (45-55 percent) of sterile plant rows (homozygous sterile lines are possible to breed) are shown, 5-6 pairs of adjacent sterile plants and fertile plants are selected to be sleeved together in a paper bag, and the sterile plants are grown in the paper bag by shaking bags in the flowering phase for artificial auxiliary pollination. After maturation, the fertile plants and the sterile plants in the bags are respectively harvested and stored.

(5) In step 3(3), selfing seed plant row corresponding to the full sterility of hybrid seed plant row can be maintained, and fine soil is taken as male parent in the initial flowering period; sterile plants in the hybrid seed plant rows show about 1/2 (45-55%) plant row plants, sterile plants about 20 carefully earthed seedlings in the early flowering period of each plant row are taken as female parents and are transplanted to a female parent row 1 area under the condition of mosquito net isolation, and after 1 row of male parents are planted in every 4 rows, the male parents are watered, earthed and guaranteed to survive, strictly isolated and artificially pollinated in an auxiliary manner. After the seeds are matured, the seeds are respectively harvested and stored in a mixed manner by taking the row of the female parent plant as a unit.

And 4, step 4: the following are distinguished:

(1) mixing seeds of female parent plant rows separated for seed production in the mosquito net in the last year, sampling 2-3g of each plant row, planting plant rows with the row length of 3 m, the row spacing of 0.4 m, 1 row area, sowing seeds in different rows, applying no or little fertilizer, and investigating the sterile plant rate in the flowering phase; sterile plants in sterile plant rows with the plant row expression of about 1/2 of the last year hybrid seeds and sterile plant seeds in fertile plants are bagged together, 4-5 single plants are respectively planted in the plant rows with the density of 12000 plants/mu and 80-100 plant row groups, the sterile plant rate is investigated in flowering phase and is correspondingly compared with the sterile plant rate of the plant rows of corresponding non-uniform seedlings sowed in a mosquito net, when the plant rows corresponding to the non-uniform seedlings sowed in the mosquito net are completely sterile, the sterile plant rate of the row is about 50 percent (45 percent to 55 percent), and the material of the row is a homozygous sterile line. Bagging sterile plants and fertile plants together, and simultaneously selecting high yield, stable yield, stress resistance, quality and other comprehensive economic characters of the plants in the row.

(2) And (3) bagging 4-5 plant lines of selfing seeds in the plant line (temporary maintainer line) of the single plant selfing seeds corresponding to the full sterility in the last year of the planting of the hybrid plant line, wherein the sterile plant rate is fully fertile in the florescence survey, selecting 8-10 plants in a bag, and continuously selecting the comprehensive economic characters such as high yield and stable yield, stress resistance, quality and the like of the plants on the plant line.

(3) Taking the sterile plant with soil in the homozygous sterile line which is qualified in the step 4(1) as a female parent, taking the plant with soil in the temporary maintainer line which is qualified in the step 4(2) as a female parent, still isolating and producing seeds in the mosquito net, harvesting and storing, and identifying the fertility of the newly selected homozygous sterile line and the temporary maintainer line in the next year, and carrying out combined test cross or seed production by using the generated full sterile line.

Compared with the prior art, the method can directly select hybrid varieties (combinations) with good high yield and stable yield, strong stress resistance, excellent quality and other comprehensive economic characters on the basis of breeding materials without nuclear three-line parents (namely a temporary maintainer line and a homozygous sterile line), and directly and quickly separate and select homogeneous temporary maintainer and homozygous sterile lines in 2-3 generations by selfing separation, test crossing and hybridization on the basis of F2 generation plants, respectively adopting fertile plants and sterile plants for same-bag hybridization, fertile plant selfing, taking plants of corresponding plant row materials on the basis of investigation after initial flowering as parent plants to produce seeds in a mosquito net and carrying out fertility identification on the generated corresponding seeds and the like according to different fertility performances of F3 plant rows.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments.

The technical scheme and the technical principle of the invention are as follows:

(1) the invention has the technical characteristics that:

① the hybrid variety (combination) of the three lines of nucleus is directly used as the selection material, and there is no material basis of the three lines of nucleus parent (and homozygous sterile line, temporary maintainer line).

② the workload is relatively small, the adjacent sterile plants and fertile plants are bagged together to complete sterile plant hybridization and fertile plant selfing at the same time to replace fertile plant bagging selfing and the manual paired bud stripping hybridization of the sterile plants and the fertile plants, thus greatly reducing the workload of manual hybridization

③, the time is fast, the hybrid seeds F2 generation seeds are used as the starting point, the plant-row population with the sterile plant rate of 18-25% is selected as the separation selection material, and the stable homozygous sterile line and temporary maintainer line can be ensured to be respectively generated by 2-3 generations.

(2) The main key technology and technical principle are as follows:

the hybrid of three nuclear lines (combination) is prepared by using the complete sterile line of three nuclear lines as female parent and high-quality restoring line as male parent. The genotype of the female parent nuclear three-line total sterile line is Rfrfms₁ms₁ms₂ms₂The gamete generated by hybridization of it as female parent has only two Rfms₁ms₂And rfms₁ms₂The paternal restorer line may have several genotypes, but since the restorer line is a stable variety material, the same gene is either dominant or recessive at the same time, so that conventional high quality restorer lines have only four genotypes ① RfRfMs₁Ms₁Ms₂Ms₂②rfrfMs₁Ms₁Ms₂Ms₂③RfRfMs₁Ms₁ms₂ms₂(or ms)₁ms₁Ms₂Ms₂)③rfrfMs₁Ms₁ms₂ms₂(or ms)₁ms₁Ms₂Ms₂) It has only four basic gametes, heterozygosis with female parentThe complete sterile line is crossed to generate 6 genotypes of F1 seeds, and after the plants planted with the 6 genotypes of F1 seeds are fruited, F2 generation seeds are generated and then planted, and then a plurality of genotypes are separated. The genotypes of the conventional high-quality restorer lines in the sequences are sequences, and the probability that the F2 generation seeds have several available genotypes to occupy the total population is respectively as follows: the sterile plants respectively account for: 5.47%, 2.34%, 21.88%, 9.38% (table 1) wherein homozygous sterile plants account for 3.91%, 0.78%, 15.63%, 3.13%, respectively, and temporary plants account for: 0.83%, 4.0%, 8.0%, 3.4%, homozygous fertile plants account for: 16.53%, 3.2%, 40.0%, 6.9%, various genes

TABLE 1 genotype of possible segregating plants of the nuclear three-line hybrid (Material) F1 generation and several segregation performances of the F2 plants

The hybrid combination separation ratio of the restorer line configuration is very different, and ③ th hybrid combination has RfRf Ms₁Ms₁ms₂ms₂(ms₁ms₁Ms₂Ms₂) The hybrid (combination) in which the restorer of the genotype is used as the male parent is the most preferable. Thereby having RfRfMs₁Ms₁ms₂ms₂(ms₁ms₁Ms₂Ms₂) Genotype is an example: the planting of 600 left and right groups F2 generation plant groups can generate 131 sterile plants, which can completely meet the requirement of 100 combinations for hybridization, the 100 pairwise combinations can ensure that 5.7 strains and 28 strains of line temporary protection and homozygous sterile lines can respectively occur in the F3 generation after fertile plants in the groups are hybridized with homozygous sterile types in the sterile plants; the sterile plants of F2 group of other several restoring lines need to be respectively planted into 2349, 5598 and 1396 plants of F2 group to achieve the number of the sterile plants, 100 paired test cross combinations are enough, the occurrence probability of temporary maintainer lines is only 0.59 plant line, 1.3 plant lines and 1.15 plant lines respectively, and the occurrence probability of homozygous sterile lines is only 11.81 plant lines, 1.33 plant lines and 2.3 plant lines respectively. It can be seen that the sterile plant rate of other recovery line hybrid F2 populations is low,meanwhile, the temporary maintainer line and the homozygous sterile line which appear in the progeny of the same number of hybrid combinations are lower.

Therefore, in the breeding design, several combinations are planted, and the variety (combination) with the sterile plant rate of about 20 percent (18 to 25 percent) is selected as a selection group, so that the workload can be greatly reduced, and the breeding process can be accelerated

In the plant rows planted by F2 generation hybrid seeds, the plant rows with the sterile plant rate of about 1/2 are the basis for breeding the homozygous sterile line. In the step two population pairwise test crossing, the filial generations planted by a plurality of hybrid seeds of homozygous A multiplied by homozygous B homozygous A multiplied by heterozygous B heterozygous A multiplied by homozygous B all show about 1/2 sterility, the total occurrence probability reaches 57%, but only after homozygous A multiplied by homozygous B

TABLE 2 plant-row ratio of the sterile plants 1/2 appearing in the plant-row population planted with the hybrid seeds and the respective genotype expressions

Generations still appeared as homozygous a and homozygous B populations (table 1), accounting for about 28% of the total population, while the progeny in the other few populations were homozygous sterile and a mixture of heterozygous and sterile. Therefore, the true homozygous sterile line can be selected only after the plant rows with about 1/2 sterile plant rate are identified by hybridization with the temporary maintainer line.

The advantages of the technology of the invention and the original transformation technology are compared:

compared with the prior breeding technology of the common temporary maintainer line and the homozygous sterile line, the invention has more advantages that:

1. advantages in Material selection

First, the present invention provides for the selection of a hybrid variety (material) of the nuclear trilineage, rather than the nuclear trilineage parent. This is of particular interest to breeders who do not have a nuclear trilineage parent. Secondly, the invention selects the population with sterile plant rate of about 20 percent as the basic selection material in the second step, the F1 generation hybrid is two genotypes of RfRf Ms1Ms1Ms2Ms2 and RfrfMs1Ms1Ms2Ms2, the hybrid is generated by the hybridization of the complete sterile line (hybrid and sterile line) for the hybrid seed production and the restorer line with RfRfMs1Ms 2Ms2 (gametophyte type: RfMs1Ms2) (table 3), the F2 generation population sterile plant rate and the temporary plant rate are respectively 21.88 percent and 8 percent, the intra-population test cross 100 combination only needs to plant the 457 plant population; in other technologies, the sterile plant rate and the temporary plant rate of the F2 generation group are respectively 2.34-25% and 0-8%, the average rate is respectively 11.72% and 3.20%, and 400-4274 plants need to be planted in 100 combinations of in-group test crossing.

TABLE 3 probability of adjacent and homozygous sterile lines in F2 and F3 generations after cross-breeding of different genic three-line sterile lines and different conventional varieties

The plant rows of the temporary maintainer line and the homozygous sterile line of the F2 generation plant rows of the invention can respectively reach 5.71 percent and 28.57 percent through pairing test cross F3 generation plant rows in the colony, other technologies are respectively only 0 to 2.56 percent and 1.04 to 50.0 percent, the average is respectively 0.88 percent and 13.6 percent, wherein, the occurrence probability of the homozygous sterile line of the two technologies is as high as 25 percent and 50 percent, but because the episomal genes of the male parent and the female parent are all dominant, no temporary maintainer line plant occurs, and the breeding of the temporary maintainer line can not be completed.

Therefore, the advantageous genotypes of the temporary maintainer line and the homozygous sterile line of the offspring are higher than those of other technologies, and the later-stage selection is facilitated; the F2 population planted by 100 combinations of paired test cross is smaller than others, taking 600 plants planted by each material as an example, the occupied area is 0.05 mu, and the occupied area is small, so that a plurality of hybrid varieties from different sources can be generated in F2 generations, and the method is favorable for the inspection and selection of the sterile plant rate reaching the standard in the flowering phase.

2. Advantage in breeding time

Table 4 lists the time required for the invention to complete the selection of the temporary maintainer line and the homozygous sterile line together with other technologies, and shows that the invention only requires 4 sowing years, wherein the workload of the first year is very small. For example, in the regional test or multi-point test of the quality of the nuclear three-line, the plant selection and bagging in the flowering period are only needed. Other techniques took 6-7 years of seeding, respectively (table 4). More difficult, in the F2 segregating population test cross, because the occurrence probability of the temporary plant is low, the artificial pair test cross quantity is insufficient, and the temporary line can not be found in the result, the result is not obtained in the selection for many years. This is why some breeders have failed to blindly test cross selection of a provisional and homozygous sterile line in hybrid varieties (combinations).

TABLE 4 time period required for the present invention and conventional transformation techniques

3. Advantages in breeding efforts

Only by using the sterile plants and adjacent fertile plants to be bagged together to replace artificial hybridization in paired test crossing as an example, table 5 lists 100 paired test crossing combinations which are also made for comparison, and shows that 100 hybridization combinations are made in F2 generation of other technologies, test crossing and hybridization with temporary maintainer lines are performed in groups of about F3 generation 1/2 sterile plants to identify the purity of the groups, 285 personal hybridization combinations are respectively made, 635 hybridization combinations are totally achieved, 7 hybridization technologies are completed according to each skilled worker day, 90 more than 90 manual workloads are performed in total, bagging, bag drawing and the like are respectively performed on the male parent plants and the female parent plants, and the total number of labor is more than 110; the invention adopts the seed production in the mosquito net, reduces the corresponding paired test cross, reduces the number of the paired test cross combination to 385, does not need to carry out artificial hybridization, has less bags covering and drawing than the artificial hybridization, increases the work load of bag shaking, needs 7-8 workers to complete the work, and in addition, needs about 5 workers to complete the seed production in the mosquito net, and the invention needs about 13 workers to complete 100 paired test cross, and needs about 100 workers to complete the same number of artificial paired test cross, and nearly ten times less.

In the aspect of selfing and hybrid seeds harvested by paired test crossing, the whole inflorescence of the parents is bagged, so that the time for the parents to seed is relatively long, and the seeds are more than those obtained by artificial crossing.

In addition, the labor amount can be reduced in the planting period and the land area.

TABLE 5 comparison of workload for pairwise crossing of sterile and fertile plants (to make 100 pairwise crossing sets) between the present invention and conventional transformation techniques

4. The invention compares the quantity and quality stability of temporary maintainer line and homozygous sterile line cultivated by other techniques

In the aspect of the quantity of possibly bred temporary maintainer lines and homozygous sterile lines, the invention can breed 5-6 temporary maintainer lines and 28 homozygous sterile lines in 3-4 generations by taking 100 combinations of paired test cross as an example, and the temporary maintainer lines and the homozygous sterile lines bred by other technologies in 6-7 generations are respectively 0-5.71 and 1-50, and the average number is 1.49 and 15.47. Although the homozygous sterile line which appears by two techniques (the genes on the transferring male parent and the transferring female parent are not dominant at the same time) is higher, the temporary maintainer line does not appear.

In the aspect of pure sterile line purity identification of sterile plant rows of about 1/2, other technologies only hybridize 5 plants (so that the workload is large) to the sterile plant rows of about 1/2 and the temporary maintainer line in the F3 generation, and then purity identification is carried out, while 20 sterile plants sampled in mosquito net seed production are 20 plants, so the fertility stability of the latter is much higher.

TABLE 6 comparison of the quality stability and the quantity of temporary maintainer line and homozygous sterile line of the present invention with those of other techniques

Implementation example:

the invention is used to breed homogeneous homozygous sterile line C112A and temporary maintainer line L105B, homozygous sterile line C113A and temporary maintainer line L107B. The process is as follows:

in 2014 and 2015, respectively selecting combined bagging selfings with different sources and good comprehensive properties from a nuclear three-line variety comparison test in spring, respectively planting 5-6 plant rows in autumn, respectively planting 5-6 plant rows in the flower period in 2015 in spring with sterile plant rate of 2.01-19.59%, and 5 plant rows in the flower period in 2016 in spring with sterile plant rate of 1.9-20.61%, wherein one plant row has sterile plant rate of about 20% in two years (Table 7), respectively selecting sterile plants and adjacent fertile plants in the group to be bagged 105 and 111, and performing bag drawing and shaking for artificial auxiliary pollination when the weather in the flower period is good, and respectively reserving sterile and fertile single plant seeds in mature offspring for later use.

Expression of sterile plant rates in F2 populations of several varieties (combinations) planted in spring of tables 72015 and 2016, respectively

Respectively sampling 5g of sterile and fertile plant seeds remained in the last season in autumn of 2015 and 2016 to plant corresponding plant rows, wherein the density is 2-2.5 ten thousand plants/mu, the plant rows are left with about 300 seedlings, the lodging is prevented by the medium fertilization management level, the sterile plant rate of the plant rows planted by sterile single plants is investigated in the flowering phase, and the results are listed in table 8: wherein, the F3 generation hybrid seeds of 2016 are planted in 105 rows, and the investigation result shows that 6 rows represent complete sterility, accounting for 5.71% of the total row, 55 rows with about 1/2 sterility accounting for 52.8% of the total row; the F3 plant row hybrid seeds of 2017 are planted in 107 plant rows, and the investigation result shows that 5 plant rows represent complete sterility, accounting for 4.68% of the total plant rows, and 57 plant rows with about 1/2 sterility account for 53.27% of the total plant rows. For the plants which show complete sterility, the fertile plant rows which are bagged together with the fertile plants can be found out from the plant rows which are planted by selfing of the fertile plants, the plant rows can be preliminarily determined as temporary maintainer lines, and each row is bagged with 15-20 plants for selfing and seed reserving; for 1/2 male sterile plant rows, firstly selecting 5 male sterile plants and about 5 female sterile plants from each plant row, bagging the male sterile plants and adjacent fertile plants together for test crossing in pairs, respectively reserving seeds of the male sterile plants and the fertile plants after maturation, and taking 20 male sterile plants with soil from each plant row and transplanting the seedlings with soil from the temporary maintainer line preliminarily determined in the selfing plant rows into an isolation mosquito net for seed production, wherein 55 female parent plant rows and 57 female parent plant rows are respectively produced in two years. Due to soil-carrying transplantation, the plant-row female parent population is easy to survive in the mosquito net in spring, about 20 plants are arranged, the mixed seed amount of the mature female parent plant rows is more than 100g, and the requirement of next generation fertility identification can be met.

Tables 82016 and 2017Q 026133 and combination F2 paired test cross seed Performance in F3 plant line, respectively

In 2016 and 2017, 55 rows and 57 rows of mixed seeds of the female parent plant rows for the previous-year mosquito net seed production are respectively planted, 3-5g of seeds are taken from each plant row, uneven seedlings are sowed in a row, no fertilizer or little fertilizer is applied, the sterile plant rate of the plant rows is investigated in the flowering phase, the number of plant lines expressing total sterility is respectively 27 plants and 29 plants, and the plant lines respectively account for 50.94% and 51.9% of the total plant rows; simultaneously planting preliminarily determined temporary maintainer line rows, planting 4-5 selfing single plants in each row, selecting growth vigor, uniformity and comprehensive oil excellent characters, and performing indoor seed test analysis and reserving seeds for later use after maturity; seeds of sterile plants in the last year sterile and fertile plant pairwise test cross in about 1/2 sterile plant rows are planted, according to the investigation result of the sterile plant rate, 27 and 29 plant rows corresponding to the above full sterility and providing the last year seed production are preliminarily determined to be homozygous sterile lines, in the plant rows, 4-5 selfing single plants are planted in each plant row, the growth vigor, the uniformity and the comprehensive oil excellent characters are selected in the same way, and the seeds are tested indoors for analysis and reserved for use after the plants are matured.

In order to further identify the fertility stability of the preliminarily selected temporary maintainer line and the homozygous sterile line, on the basis of observation and selection, 11-12 excellent homozygous sterile lines and excellent temporary maintainer lines are respectively selected in the plant rows preliminarily determined as the temporary maintainer line and the homozygous sterile line in the F4 group, seedlings are picked with soil and transplanted into a mosquito net for seed production, seed production seeds are respectively planted and identified, the sterile plant rate of the produced seeds reaches 100 percent, and the pure sterile line and the temporary maintainer line which are correspondingly provided for seed production are stable breeding materials.

TABLE 9 expression of F4 and F5 seeds of F3 and F4 generation seed production female parent plant rows in mosquito net in fertility identification

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.