阅读说明：本技术 一种栽培大豆导入一年生野生大豆染色体易位片段的方法 (Method for introducing annual wild soybean chromosome translocation fragments into cultivated soybeans ) 是由 张井勇 丁孝羊 王鹏年 张伟 张春宝 闫昊 林春晶 赵丽梅 孙寰 彭宝 李慧 于 2021-09-28 设计创作，主要内容包括：本发明提供一种栽培大豆导入一年生野生大豆染色体易位片段的方法,利用遗传改良手段,通过杂交、回交和测交等一系列育种方法,把一年生野生大豆染色体易位片段导入栽培大豆；采用非转基因及非基因编辑手段直接利用野生豆中普遍存在的染色体易位变异；方法简单、可靠、可重复,任何一个大豆育种工作者均可按照该方法进行实际操作,创制栽培大豆染色体易位等位系。(The invention provides a method for introducing chromosome translocation fragments of annual wild soybean into cultivated soybean, which utilizes a genetic improvement means to introduce the chromosome translocation fragments of the annual wild soybean into the cultivated soybean through a series of breeding methods such as hybridization, backcross, test cross and the like; directly utilizing the ubiquitous chromosome translocation variation in the wild beans by adopting a non-transgenic and non-gene editing means; the method is simple, reliable and repeatable, and any soybean breeding worker can carry out actual operation according to the method to create the soybean chromosome translocation allelic line for cultivation.)

1. A method for introducing chromosome translocation fragments of annual wild soybeans into cultivated soybeans is characterized by comprising the following steps:

and (3) hybridization combination preparation: using chromosome translocation-free cultivated soybean and chromosome translocation annual wild soybean as parent, and performing sexual hybridization to obtain F₁Seed generation, planting F₁A generation plant, which is a chromosome translocation heterozygote;

F₁identification of surrogate chromosomal translocation heterozygotes: post-hybridization F₁Generation plant, determining semi-wild single plant by morphological identification, and identification by using I₂Identifying chromosome translocation heterozygotes by a KI pollen staining method, and removing pseudo heterozygotes;

backcross transformation: with F₁Backcross is carried out by taking a generation chromosome translocation heterozygote plant as a female parent and taking a parent cultivated soybean as a male parent to obtain BC₁F₁Seed, and then BC₁F₁Backcross is carried out by taking a generation chromosome translocation heterozygote plant as a female parent and taking a parent cultivated soybean as a male parent to obtain BC₂F₁Seeds, continue selecting the hybrid plants obtained after hybridization and continuing backcrossing until backcrossed to BC₆Carrying out generation and finishing backcross;

preliminary identification of segregation population pollen fertile homozygous genotype: planting BC_nF₁(n is a natural number greater than or equal to 6) plants are selfed to obtain BC_nF₂Seed generation, BC_nF₂Planting seed generation to obtain BC_nF₂Segregating population plants for chromosomal translocation using₂-KI pollen staining to screen pollen fertile individuals, which individuals comprise two genotypes, namely homozygous translocation of the chromosome and homozygous non-translocation of the chromosome;

carrying out test cross to identify whether the fertile single plants in the segregation population have chromosome translocation conditions: at each BC_nF₂Using the pollen generation fertile single plant as a female parent and using any chromosome translocation-free soybean resource as a male parent to carry out hybridization to obtain a plurality of sets of test cross F₁Seed, test BC_nF₂Using pollen as substitute for fertile plant, and reusing₂Identification of each test cross F by KI pollen staining₁The pollen fertility rate of the single plant is that the pollen semi-fertile corresponding female parent single plant is the chromosome homozygous translocation single plant, the single plant is the cultivated soybean chromosome homozygous translocation strain, and the chromosome translocation fragment of the annual wild soybean introduced by the chromosome non-translocation cultivated soybean is completed.

2. The method for introducing chromosomal translocation fragments of annual wild soybean into soybean according to claim 1, wherein said method comprises the steps of:

step 1) obtaining F₁When the seeds are generated, the chromosome translocation-free cultivated soybean can be used as a female parent or a male parent; annual wild soybeans can also be used as female parent or male parent.

3. The method for introducing chromosomal translocation fragments of annual wild soybean into soybean according to claim 1, wherein said method comprises the steps of:

the morphological identification method in the step 2) is that F is identified₁Plant, its production method and useThe shape of the soybean is half wild type between that of cultivated soybean and wild soybean.

4. The method for introducing chromosomal translocation fragments of annual wild soybean into soybean according to claim 1, wherein said method comprises the steps of:

step 2) said I₂the-KI pollen staining method for identifying chromosome translocation heterozygote utilizes 1.2% I₂KI solution staining F₁Observing the plant pollen by using a 40-100-fold microscope, judging the dyed pollen as fertile pollen and the non-dyed pollen as sterile pollen, calculating the pollen fertility rate by using the ratio of the fertile pollen to the total number of the pollen, and selecting a single plant with the pollen fertility rate of 30.0-60.0% as a chromosome translocation heterozygote.

5. The method for introducing chromosomal translocation fragments of annual wild soybean into soybean according to claim 1, wherein said method comprises the steps of:

step 3) with F according to claim 4₁Backcross to obtain BC with the heterozygote of translocation of chromosome as female parent and cultivated soybean as male parent₁F₁Seed and BC₁F₁And (5) plant growing.

6. The method for introducing chromosomal translocation fragments of annual wild soybean into soybean according to claim 1, wherein said method comprises the steps of:

step 3) determining the BC of claim 5 by the pollen fertility rate determining method of claim 4₁F₁Obtaining a chromosome translocation heterozygote, and backcrossing by taking a single chromosome translocation heterozygote plant as a female parent and a cultivated soybean as a male parent to obtain BC₂F₁Seeds and individual plants; continuously selecting chromosome translocation heterozygote single plant obtained after backcross by the method, and carrying out continuous backcross for more than 6 generations by taking the single plant as a female parent and a cultivated soybean as a male parent to obtain BC_nF₁Seeds and individuals, where n is a natural number greater than or equal to 6.

7. The method for introducing chromosomal translocation fragments of annual wild soybean into soybean according to claim 1, wherein said method comprises the steps of:

BC described in step 4)_nF₂Identification of pollen fertile homozygous genotype of plant segregating population, BC determination by pollen staining method according to claim 4_nF₂The individual plant pollen fertility rate is determined to be a pollen fertile homozygous genotype when the fertility rate of a certain individual plant pollen is 95.0-100%, namely the pollen fertile individual plant, and is determined to be a semi-fertile genotype (individual plant) when the fertility rate of a certain individual plant pollen is 30.0-60.0%.

8. The method for introducing chromosomal translocation fragments of annual wild soybean into soybean according to claim 1, wherein said method comprises the steps of:

the screening method of the chromosome homozygous translocation single plant in the step 5) is to use the pollen fertile single plant of the claim 7 as a parent (a female parent or a male parent) to be hybridized with any one chromosome non-translocation cultivated soybean or chromosome non-translocation wild soybean to obtain a test cross F₁Seed generations and individuals, and the pollen staining method according to claim 4 is used to screen for inbred F₁Single plant with 30.0-60.0% of plant pollen fertility rate and parent BC corresponding to the single plant_nF₂And (3) breeding the single plant, namely the chromosome homozygous translocation cultivated soybean, to obtain the chromosome translocation cultivated soybean strain.

The technical field is as follows:

the invention provides a method for introducing chromosome translocation fragments of annual wild soybeans into cultivated soybeans, and relates to genetic transformation between any chromosome translocation-free cultivated soybeans and annual chromosome translocation wild soybeans.

Background art:

soybean is a major oil crop in the world and is also a major source of vegetable protein; however, the soybean is slower in the breeding aspect and lower in yield per unit, and the yield per unit is urgently required to be improved through breeding and cultivation ways; one of the factors restricting the breeding process of the cultivated soybean is that the cultivated soybean variety has low genetic diversity level and narrow genetic basis, which causes the slow breeding of excellent varieties, and the genealogical analysis research of some domestic scholars shows that the genetic background of the cultivated soybean variety bred at present comes from a few backbone parents to different degrees; compared with the cultivated soybean, the wild soybean resource has wide distribution in China and abundant genetic diversity, and the wild soybean resource has abundant excellent genes. Because the wild soybean has chromosome structure variation, particularly the chromosome translocation frequency is over 70 percent, the direct utilization of the useful gene of the chromosome translocation segment is difficult, and the conventional breeding needs to utilize the excellent gene of the wild soybean through hybridization, selfing or a series of backcrossing, and finally through a series of complex breeding programs such as phenotypic identification, and the like, so that the utilization purpose can be achieved, and the efficiency is low.

The invention aims to solve the problems that how to efficiently utilize beneficial genes of annual wild soybeans and how to directionally introduce genes of annual wild soybean chromosome translocation segments into cultivated soybeans in a non-transgenic and non-gene editing mode so as to widen the genetic basis of the cultivated soybeans.

The invention content is as follows:

the invention provides a method for introducing an annual wild soybean chromosome translocation fragment into a cultivated soybean, and relates to genetic transformation between any chromosome translocation-free cultivated soybean and an annual wild soybean with translocation of chromosomes.

The invention relates to a method for introducing annual wild soybean chromosome translocation fragments into cultivated soybeans, which comprises the following steps:

1) and (3) hybridization combination preparation: using chromosome translocation-free cultivated soybean and chromosome translocation annual wild soybean as parent, and performing sexual hybridization to obtain F₁Seed generation, planting F₁A generation plant, which is a chromosome translocation heterozygote;

2）F₁identification of surrogate chromosomal translocation heterozygotes: post-hybridization F₁Generation plant, determining semi-wild single plant by morphological identification, and identification by using I₂Identifying chromosome translocation heterozygotes by a KI pollen staining method, and removing pseudo heterozygotes;

3) go back toAnd (3) cross breeding: with F₁Backcross is carried out by taking a generation chromosome translocation heterozygote plant as a female parent and taking a parent cultivated soybean as a male parent to obtain BC₁F₁Seed, and then BC₁F₁Backcross is carried out by taking a generation chromosome translocation heterozygote plant as a female parent and taking a parent cultivated soybean as a male parent to obtain BC₂F₁Seeds, continue selecting the hybrid plants obtained after hybridization and continuing backcrossing until backcrossed to BC₆Carrying out generation and finishing backcross;

4) preliminary identification of segregation population pollen fertile homozygous genotype: planting BC₆F₁Inbreeding of the plant generations to obtain BC₆F₂Seed generation, BC₆F₂Planting seed generation to obtain BC₆F₂Segregating population plants for chromosomal translocation using₂-KI pollen staining screening pollen fertile individuals, which individuals comprise two genotypes, homozygous for a translocation and homozygous for a translocation;

5) carrying out test cross to identify whether the fertile single plants in the segregation population have chromosome translocation conditions: at each BC₆F₂Using the pollen generation fertile single plant as a female parent and using any chromosome translocation-free soybean resource as a male parent to carry out hybridization to obtain a plurality of sets of test cross F₁Seed, test BC₆F₂Using pollen as substitute for fertile plant, and reusing₂Identification of each test cross F by KI pollen staining₁The pollen fertility rate of the single plant is that the pollen semi-fertile corresponding female parent single plant is the chromosome homozygous translocation single plant which is the cultivated soybean chromosome homozygous translocation line, thus finishing the chromosome translocation of the annual wild soybean introduced by the chromosome non-translocation cultivated soybean.

In the method, F is obtained in step 1₁When the seeds are produced, the chromosome translocation-free cultivated soybean can be used as a female parent or a male parent, and similarly, the annual wild soybean can also be used as the female parent or the male parent.

The morphological identification method described in step 2, identification F₁The plant is semi-wild; said I₂-KI pollen staining method for identifying chromosomal translocation heterozygotes using 1.2% I₂KI solution staining F₁And (3) plant pollen, namely judging the dyed pollen as fertile pollen and the non-dyed pollen as sterile pollen, calculating the pollen fertility rate by using the ratio of the fertile pollen to the total number of the pollen, and selecting a single plant with the pollen fertility rate of 30.0-60.0% as a chromosome translocation heterozygote.

As described in step 3 and F₁Backcross is carried out by taking chromosome translocation heterozygote as female parent and parent cultivated soybean as male parent to obtain BC₁F₁The seeds are sown as BC₁F₁Plant growing; screening BC by using the fertile pollen judging method in the step 2₁F₁Obtaining a chromosome translocation heterozygote, backcrossing by taking a single heterozygote plant as a female parent and taking a parent cultivated soybean as a male parent to obtain BC₂F₁Sowing the seed as single plant, continuously selecting chromosome translocation heterozygote single plant obtained after backcross by the method, and continuously backcrossing for more than 6 generations by taking the single plant as female parent and taking parent cultivated soybean as male parent to obtain BC_nF₁(n is a natural number of 6 or more, the same applies hereinafter) seeds and individuals.

Cultivation method described in step 4, BC_nF₂And (3) identifying the pollen fertile homozygous genotype of the plant segregating population, namely judging the fertility rate of the pollen of the single plant by using the pollen staining method in the step (3), determining the single plant as a fertile genotype (single plant) when the fertility rate of the pollen of the single plant is 95.0-100%, and determining the single plant as a semi-fertile genotype (single plant) when the fertility rate of the pollen of the single plant is 30.0-60.0%.

The screening method of the chromosome homozygous translocation single plant in the step 5 refers to using BC_nF₂The fertile single plant is used as parent and hybridized with any chromosome non-translocation cultivated soybean or wild soybean to obtain test cross F₁Seed generation and sowing are F₁The single plant is subjected to judgment of test cross F by using the pollen staining method in the step 3₁Selecting individual plants with pollen fertility rate of 30.0-60.0%, and testing cross parent (BC) corresponding to the individual plants_nF₂Fertile single plant), namely, the soybean is cultivated with translocation of chromosome homozygous, the breeding program of the annual wild soybean for introducing the translocation segment of the chromosome is completed, and the soybean is bredThe single strain of the type can obtain a soybean line with translocation homozygous for chromosome, and the line and the original parent soybean without translocation are allelic lines.

The invention has the positive effects that:

introducing annual wild soybean chromosome translocation fragments into cultivated soybeans by a series of breeding methods such as hybridization, backcross, test cross and the like by using a genetic improvement means; directly utilizing the ubiquitous chromosome translocation variation in the wild beans by adopting a non-transgenic and non-gene editing means; the method is simple, reliable and repeatable, and any soybean breeding worker can carry out actual operation according to the method to create the soybean chromosome translocation allelic line for cultivation.

Description of the drawings:

FIG. 1a shows the plant morphology of cultivated soybean 82B;

FIG. 1b shows wild soybean OB plant morphology;

FIG. 1c shows (82 B.times.OB) F₁Plant morphology, expressed as semi-wild morphology;

FIG. 2a shows 1.2% I of pollen of 82B cultivated soybean₂Under the condition of KI staining, observing by a microscope at 100 times, wherein the chromosome is homozygous and has no translocation, the pollen is fully fertile, and the black color is fertile stained pollen;

FIG. 2b shows wild soybean OB pollen 1.2% I₂Under the condition of KI staining, observing by a microscope at 100 times, wherein the chromosome is homozygous and translocated, the pollen is fully fertile, and the black color is fertile stained pollen;

FIG. 2c is F₁Plant pollen 1.2% I₂The KI staining condition is observed by a microscope at a 100-fold ratio, the chromosome is a translocation heterozygote, the fertility rate of the plant pollen is about 43.01 percent, the black is fertile stained pollen, and the non-stained or light brown is abortion pollen);

FIG. 3 shows the introduction of annual wild soybean OB chromosome translocation fragments into cultivated soybean 82B.

The specific implementation mode is as follows:

the present invention is described by the following examples, but not limited thereto in any way, and any modification or change which can be easily made by a person having ordinary skill in the art without departing from the technical solution of the present invention will fall within the scope of claims of the present invention.

Example 1:

1) and (3) hybridization combination preparation:

sexual hybridization is carried out on the chromosome translocation-free cultivated soybean 82B (1 a) serving as a female parent and the chromosome translocation annual wild soybean OB (1B) serving as a male parent to obtain F₁Seed generation;

2）F₁identification of surrogate chromosomal translocation heterozygotes:

planting F₁Obtaining F after seed generation₁Plant generation in F₁And at the 4-6 leaf stage of the plant, identifying that the plant is a true hybrid with a semi-wild shape (shown in figure 1 c) for the first time. Further characterization of F₁Pollen abortion rate of plants in F₁At the initial flowering stage of the plants, 2 unopened buds are taken from each single plant to identify the pollen abortion rate, and the identification method adopts 1.2 percent I₂KI staining method, using 100 times microscope to observe, judging stained pollen as fertile pollen and non-stained pollen as sterile pollen, calculating pollen fertility rate by using ratio of fertile pollen to total pollen number, expressing result by average value, the pollen staining rate is about 43.01% (FIG. 2 c), and determining F₁Individuals are chromosomal translocation heterozygotes.

3) Backcross transformation:

with F₁Backcross is carried out by taking plants with the pollen generation staining rate of 30.0-60.0% as female parents and taking parent cultivated soybean 82B as male parents to obtain BC₁F₁Seed, planting BC₁F₁Seed harvesting of BC₁F₁Plant, reuse of I₂Identification of BC by KI staining method₁F₁Generating plants, determining plants with the pollen dyeing rate of 30.0-60.0% as chromosome translocation heterozygotes, and backcrossing to obtain BC by taking the plants as female parents and taking parent cultivated soybean 82B as male parents₂F₁Seeds, continue selecting the hybrid plants obtained after hybridization and continuing backcrossing until backcrossed to BC₆And finishing backcrossing.

4) Preliminary identification of segregation population pollen fertile homozygous genotype:

planting BC₆F₁Inbreeding of the plant generations to obtain BC₆F₂Seed generation, BC₆F₂Planting seed generation to obtain BC₆F₂A plant of a transgenic segregating population of chromosomal translocations, the population comprising 3 genotypes: chromosome homozygous no translocation (NN), chromosome homozygous translocation (TT), and chromosome heterozygous translocation (NT). Using 1.2% I₂Screening genotypes for the first time by a KI pollen staining method, wherein a plant with a pollen staining rate of 30.0-60.0% is a chromosome translocation heterozygote, and a plant with a pollen staining rate of 95.0-100% (figure 2 a) is a chromosome homozygous translocation-free (NN) or a chromosome homozygous translocation (TT), namely a pollen fertile single plant.

5) Carrying out test cross to identify whether the fertile single plants in the segregation population have chromosome translocation conditions:

at each BC₆F₂Taking a single plant with the pollen dyeing rate of 95.0-100% as a female parent and taking a chromosome translocation-free soybean resource JLR2 as a male parent to perform hybridization to obtain 10 sets of test cross F₁And (4) seeds.

And (3) identifying the test cross result and obtaining the translocation strain of the cultivated soybean chromosome: planting 10 test crosses F₁Seed acquisition test cross F₁Plant, utilizing I₂Identification of test cross F by KI pollen staining method₁And (3) the pollen staining rate of the single plant is 30.0-60.0% of the pollen staining rate, the single plant of the female parent is homozygous translocation single plant of the chromosome, 4 single plants are obtained in total, the single plant is the cultivated soybean chromosome translocation strain, and the introduction of the annual wild soybean chromosome translocation fragment into the cultivated soybean is completed.

Carrying out chromosome translocation import molecular level identification, taking parents and cultivated soybean leaves with translocation materials in full-bloom stage, extracting DNA, carrying out high-throughput genome sequencing, filtering and correcting original sequencing data, and carrying out mapping by using williams82 as a reference genome to obtain SNP data. Obtaining all difference SNP information of the parent-to-parent differences by using a custom PERL program; comparing all SNP sites of the cultivated soybean with translocation materials with the difference SNP between parents to obtain the translocation fragments of the annual wild soybean introduced into the cultivated soybean (figure 3).

Example 2:

1) and (3) hybridization combination preparation:

sexual hybridization is carried out by taking annual wild soybean OB (figure 1B) with translocation of chromosome as female parent and soybean 82B (figure 1 a) without translocation of chromosome as male parent to obtain F₁Seed generation;

2）F₁identification of surrogate chromosomal translocation heterozygotes:

planting F₁Obtaining F after seed generation₁Plant generation in F₁And at the 5-leaf stage of the plant, the plant is identified as a true hybrid with a semi-wild shape for the first time.

At F₁At the initial flowering stage of the plants, taking 2 unopened buds from each single plant to identify the fertility rate of the pollen, wherein the identification method adopts 1.2 percent I₂And (4) a KI dyeing method, namely judging the dyed pollen as fertile pollen and the non-dyed pollen as sterile pollen by observing through a 100-time microscope, calculating the fertility rate of the pollen by utilizing the ratio of the fertile pollen to the total number of the pollen, and expressing the result by using an average value. Determining the pollen staining rate to be 30.0-60.0% as a chromosome translocation heterozygote.

4) Backcross transformation:

with F₁Backcross is carried out by taking plants with the pollen generation staining rate of 30.0-60.0% as female parents and taking parent cultivated soybean 82B as male parents to obtain BC₁F₁Seed, planting BC₁F₁Seed harvesting of BC₁F₁Plant, reuse of I₂Identification of BC by KI staining method₁F₁Generating plants, determining plants with the pollen dyeing rate of 30.0-60.0% as chromosome translocation heterozygotes, and backcrossing to obtain BC by taking the plants as female parents and taking parent cultivated soybean 82B as male parents₂F₂Seeds, continue selecting the hybrid plants obtained after hybridization and continuing backcrossing until backcrossed to BC₆And finishing backcrossing.

5) Preliminary identification of segregation population pollen fertile homozygous genotype:

planting BC₆F₁Inbreeding of the plant generations to obtain BC₆F₂Seed generation, BC₆F₂Planting seed generation to obtain BC₆F₂A plant of a transgenic segregating population of chromosomal translocations, the population comprising 3 genotypes: chromosome homozygous no translocation (NN), chromosome homozygous translocation (NN) ((TT), chromosomal heterozygous translocation (NT). Using 1% of I₂Screening genotypes for the first time by a KI pollen staining method, wherein plants with the pollen staining rate of 30.0-60.0% are chromosome translocation heterozygotes, and plants with the pollen staining rate of 95.0-100% are chromosome homozygous translocation-free (NN) or chromosome homozygous translocation (TT), and the fertile single plants are obtained.

Cross-testing to identify chromosomal translocations in segregating populations, as per BC₆F₂Taking a single plant with the pollen dyeing rate of 95-100% as a female parent and taking a chromosome translocation-free soybean resource JLR4 as a male parent to perform hybridization to obtain 12 sets of test cross F₁Seed, planting test cross F₁Seed acquisition test cross F₁Plant, utilizing I₂Identification of test cross F by KI pollen staining method₁And (3) obtaining 3 individuals corresponding to the female parent individual, namely the chromosome homozygous translocation individual, wherein the pollen staining rate of the individual is 30.0-60.0%, and the 3 individuals are bred soybean chromosome translocation lines 82B (TT), so that the introduction of annual wild soybean chromosome translocation fragments into the cultivated soybeans is completed.