CN111543310A - Method for breeding by using rice indica subspecies cross/doubled tetraploid recombinant inbred line - Google Patents
Method for breeding by using rice indica subspecies cross/doubled tetraploid recombinant inbred line Download PDFInfo
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Abstract
The invention discloses a method for breeding by using a tetraploid recombinant inbred line for interspecific hybridization/doubling of indica-japonica subspecies of rice. The method comprises the following steps: hybridizing indica rice and japonica rice; doubling to tetraploid; selfing the tetraploid, and selecting a tetraploid recombinant selfing line with target characters; obtaining the diploid with the target character by a chromosome set ploidy reduction means, namely the rice breeding material with the target character. The invention overcomes sterility of indica-japonica F1 hybrid, utilizes the high tolerance capability of tetraploid to genome instability, takes the tetraploid as a platform capable of rapidly inducing a large amount of genome variation, rapidly obtains a tetraploid recombination inbred line with genome containing genome variation such as partial homologous chromosome fragment exchange and the like and various phenotypes in the inbred progeny of the tetraploid, and ensures that the diploid restoring line induced by a chromosome set ploidy reduction means can mostly inherit the phenotype and resistance characteristics of the parental tetraploid. The invention has important significance for rice breeding.
Description
Technical Field
The invention relates to the field of biotechnology and agriculture, in particular to a method for breeding by using a tetraploid recombinant inbred line for interspecific hybridization/doubling of indica-japonica subspecies of rice.
Background
Rice is one of important staple food crops, supports more than half of the population of the world, and the yield and the quality of the rice are important for maintaining the global food safety. In order to obtain a rice variety with high yield, strong stress resistance, high rice quality and good taste, breeders culture new rice varieties by various rice breeding means and expand rice germplasm resources. The existing rice breeding methods comprise crossbreeding, mutation breeding, space breeding, transgenic breeding and the like, each of which has great success, but also has certain limitations, and the methods are listed as follows:
and (3) cross breeding: the method is characterized in that two rice varieties are specified to be hybridized, and individuals which have excellent properties and meet the screening expectation are selected from filial generations of the hybrids by utilizing heterosis to cultivate the rice varieties. The limitations include: a, the selection of hybrid parents has limitation, two proper rice varieties (usually, the varieties with relatively close relationship of broad relatives or relatives are required to be selected) are required to be selected as the parents for hybridization, so as to avoid sterility of F1 generation hybrid seeds; b, the rice hybrid vigor is not shown in all hybrid combinations (many rice varieties show hybrid debility after hybridization), so the success rate of obtaining the rice variety with the super-parent characteristic by the hybrid breeding is not 100 percent; and c, the breeding time is long, and in the case of diploid rice, the F1 hybrid needs at least 7 generations of selfing to obtain the hybrid progeny with non-separated characters and homozygous loci.
Mutation breeding: the method is characterized in that rice seeds are treated by chemical reagents (such as EMS) and physical rays (gamma rays and the like) to induce genome mutation and phenotypic variation, and excellent rice varieties are screened from the mutants. The limitations include: a, the beneficial variation induced by chemical reagents or physical rays is less, and a large amount of rice seeds are required to be treated; b, the direction and the property of mutagenesis cannot be controlled, and the success rate is low; and c, the quantitative trait is improved less effectively, and genome single-base mutation (point mutation) is usually caused, so that the quantitative trait is influenced less.
Space breeding: the method is a breeding method for breeding new rice varieties by breeding or selecting and breeding rice seeds or asexual breeding strains in a space environment 200-400 km away from the earth by using a recoverable satellite or spacecraft, inducing plant seeds or materials to generate heritable variation by using microgravity, cosmic rays, high vacuum, a weak magnetic field, an taiyang ion and the like of the outer space. The limitations include the need for return satellites or space vehicles for airborne mutagenesis, which is a real rare opportunity.
Transgenic/gene editing breeding: refers to a breeding method which improves the existing rice variety by means of transgene/gene editing and leads the rice variety to have correspondingly better performance. The limitations include: a, because the rice gene editing technology relates to the callus induction and regeneration process, the rice gene editing technology is only suitable for a part of rice varieties which can successfully induce the callus and regenerate; b, aiming at a target trait, the genetic basis of the trait needs to be mastered (for example, genes for controlling the trait need to be known), and the trait improvement with unclear genetic basis is difficult; c, the control of the transgenic/gene edited crops is strict in China, and the transgenic/gene edited crops have a certain distance from practical production application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel rice breeding method.
In a first aspect, the present invention claims a method for obtaining rice breeding material with a desired trait.
The method for obtaining the rice breeding material with the target characters, which is claimed by the invention, can comprise the following steps:
(A1) hybridizing indica rice and japonica rice to obtain F1 generation hybrid (current generation sterile, diploid);
(A2) doubling the F1 generation hybrid obtained in (A1) into tetraploids;
(A3) subjecting the tetraploid obtained in (A2) to continuous multi-generation selfing, and selecting a tetraploid recombinant inbred line with the target character from the obtained tetraploid recombinant inbred lines;
(A4) carrying out chromosome group doubling on the tetraploid recombinant inbred line with the target character obtained in the step (A3) to obtain a diploid with the target character; the diploid is rice breeding material with the target character.
In step (a1), the indica rice may be any indica rice variety; the japonica rice can be any japonica rice variety. In a particular embodiment of the invention, the indica is indica cultivar 93-11; the japonica rice is specifically japonica rice variety Nipponbare.
In the step (a1), the hybridization between indica rice and japonica rice may be performed in both positive and negative directions, or in one direction, who is the male parent and who is the female parent.
In a specific embodiment of the invention, indica rice and japonica rice are hybridized into indica rice variety 93-11 and japonica rice variety Nipponbare, which are hybridized in the positive and negative directions to obtain hybrid seeds of F1 generation in the positive and negative directions.
In step (a2), doubling the F1 generation into tetraploids may be achieved by any prior art means. In a particular embodiment of the invention, the doubling of the F1 generation into tetraploids is in particular performed with colchicine.
In the step (a3), the tetraploid recombinant inbred line may be a tetraploid recombinant inbred line inbred for more than 8 generations. In a specific embodiment of the present invention, the tetraploid recombinant inbred line is specifically a tetraploid recombinant inbred line inbred for 15 generations.
Further, in a specific embodiment of the present invention, the tetraploid recombinant inbred line inbred for 15 generations is obtained as follows: the 1 generation selfing, the 2 generation selfing, the 9 generation selfing and more than 9 generations selfing are single-grain methods, and the rest generations are equal-grain passage methods (namely, each strain selects the same seed grain number for downward passage so as to ensure that the progeny material is a random population).
In the step (a4), a diploid having the desired trait may be obtained by anther culture of the tetraploid recombinant inbred line having the desired trait; the diploid with the target character can also be obtained by hybridizing the tetraploid recombination inbred line with the target character with a rice haploid induction line.
The invention also claims rice breeding materials with target characters obtained by the method, including complete plants, seeds, callus tissues and the like.
In a second aspect, the invention claims a method for obtaining a tetraploid rice recombinant inbred line with a trait of interest.
The method for obtaining the rice tetraploid recombinant inbred line with the desired trait claimed in the present invention may comprise the steps (a1) - (A3) of the method described in the first aspect.
The invention also claims a tetraploid rice recombination inbred line with target characters obtained by the method, which comprises a complete plant, seeds, callus tissues and the like.
In a third aspect, the invention claims any of the following applications:
(B1) use of a method as hereinbefore described in the first aspect in rice breeding.
(B2) Use of a rice breeding material having a desired trait prepared by the method of the first aspect in rice breeding.
(B3) Use of a method as hereinbefore described in the second aspect in rice breeding;
(B4) the method of the second aspect is used for preparing the rice tetraploid recombination inbred line with the target character in rice breeding.
In a fourth aspect, the invention claims a method of breeding rice.
The rice breeding method as claimed in the present invention may comprise the steps (a1) to (a4) in the method described in the first aspect, and the following step (a 5):
(A5) crossing the diploid having the desired trait obtained in (A4) as one of the parents with a rice variety having excellent other existing traits but not having the desired trait to obtain a new rice variety having the desired trait together with the genetic background of the existing rice variety.
The invention also claims a new rice variety which is obtained by the method and has the genetic background of the existing rice variety and the target character, and the new rice variety comprises a complete plant, seeds, callus and the like.
In each of the above aspects, the trait of interest may be any trait. In a particular embodiment of the invention, the trait of interest is embodied in salt tolerance, cold resistance or high utilization of nitrogen (nitrate nitrogen) as evidenced by chlorate sensitivity.
Experiments prove that the method for breeding by using the rice indica-japonica hybrid and the partial heterotetraploid (segmented allotetraploid) recombinant inbred line obtained by the whole genome doubling provided by the invention, the method can overcome sterility of indica-japonica hybrid F1 generation and induce a large amount of genome variation (mainly part of homologous recombination exchange between chromosomes, Homoeologous exchange, HE, also including other types of genetic and epigenetic variation) and phenotypic (including stress resistance) diversity characteristics rapidly and randomly, after 15 generations of continuous selfing (experience shows that the internal surface of the basic line is stable after 8-10 generations), selecting a tetraploid recombination selfing line with excellent target characters, and carrying out anther culture to recover diploid rice with the same genome composition (genomic composition), thereby achieving the purposes of expanding rice germplasm resources and applying to breeding. The obvious heterosis among indica rice varieties is utilized to overcome F1 hybrid sterility, so that HE distribution in the recombinant inbred line after tetraploid inbred for multiple generations is more uniform and wide, corresponding genome variation is more, and phenotypic diversity is more prominent compared with that in the diploid-level recombinant inbred line. The invention has important significance for rice breeding.
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FIG. 1 shows PCR identification of N9, 9N hybrids. N9 represents a diploid F1 hybrid obtained by crossing japonica rice Nipponbare (N) as a female parent and indica rice 93-11(9) as a male parent; 9N represents a diploid F1 hybrid obtained by crossing indica 93-11(9) as a female parent and japonica rice Nipponbare (N) as a male parent. NPB represents japonica rice Nipponbare; mix represents a mock hybrid sample in which DNA of Nipponbare and indica 93-11 was mixed at a ratio of 1:1 (molar ratio). The upper panel shows the detection result of the primer pair mPL-4, and the lower panel shows the detection result of the primer pair mPL-5.
FIG. 2 is a diagram showing the result of cytologically identifying the number of chromosomes in the doubled rice material, wherein the number of chromosomes in the tetraploid rice is 48.
FIG. 3 is a pattern diagram of the passage flow of 512 rice indica-japonica internode segment heterotetraploid recombinant inbred lines (S15 generation). The material set is subjected to single-grain passage of 6 successive generations at the time of S9 generation to finally obtain S15 independent recombinant inbred lines. The different colors represent the genomic differences between different individuals (individuals of early generations) or different lines (tetraploid recombination inbred lines) caused by the multiple recombination of partially homologous chromosomes during meiosis. The dashed arrows indicate that there was selfing passage during the actual passage but not drawn in the pattern.
FIG. 4 is a schematic diagram showing the genome composition of 32S 6 generation rice tetraploid populations. Each row represents a single plant tested, and each column represents a chromosome of rice. Different colors represent different ratios of Nipponica (NPB) to 93-11 derived homeologous fragments in a particular genomic segment, e.g., green for NPB:93-11 ═ 0:4 and red for NPV:93-11 ═ 1: 3.
FIG. 5 is a table showing the phenotype of partially recombined inbred lines (S15) and diploid parents Nipponbare (NPB) 93-11 of rice between indica and japonica subspecies. A, the whole plant type; b, sword leaf angle; c, the length and width of the sword leaf; d, seed length and width; and E, setting percentage. In the diagram E, the two rows of seeds below the spike are full solid grains and abortive flat grains from top to bottom respectively.
FIG. 6 shows phenotypic characteristics of the set of indica-japonica subspecies tetraploid recombinant inbred lines (S15) displayed by Boxplot, wherein four phenotypes are respectively plant height, effective tiller number, stalk diameter and tiller angle from top to bottom, and L1-L10 are 10 tetraploid recombinant inbred lines (S15) randomly selected.
FIG. 7 is a graph showing phenotype of rice Nipponbare (NPB), 93-11 and 4 tetraploid recombinant inbred lines (S15) under normal culture conditions and after salt stress using the first salt treatment method. A, normal culture conditions; b, salt treatment conditions of 150mM NaCl. The time points of the photographs were the same for both culture conditions, and from top to bottom, they were representative of the time points before salt treatment, after treatment and after recovery, where two lines, S15-18-4X (line 18) and S15-31-4X (line 31), were salt-resistant lines, two lines, S15-6-4X (line 6) and S15-23-4X (line 23), were salt-sensitive lines, and bar was 5 cm.
FIG. 8 is a bar graph showing the average survival rates of the rice plants Nipponbare (NPB), 93-11 and the corresponding 4 tetraploid recombinant inbred lines (S15) of FIG. 7 under normal culture conditions and after salt stress using the first salt treatment method.
FIG. 9 is a graph showing germination of rice under normal culture conditions and after salt stress using the second salt treatment method, Nipponbare (NPB) 93-11 and tetraploid recombinant inbred lines corresponding to the 4S 15 generations in FIG. 7. The left half of the graph is normal culture conditions; the right half shows 200mM NaCl salt treatment conditions. The time points for the photographs were the same for both culture conditions, from left to right, before salt treatment (just the seeds broke the chest) and 10 days after treatment, where both lines S15-18-4X (line 18) and S15-31-4X (line 31) were salt-resistant lines, both lines S15-6-4X (line 6) and S15-23-4X (line 23) were salt-sensitive lines, DAG, day (S) after germination, bar 1 cm.
FIG. 10 is a graph showing phenotype of rice Nipponbare (NPB), 93-11 and 4 tetraploid recombinant inbred lines (S15) under normal culture conditions and after cold stress at cold treatment intensity at 4 ℃. A, normal culture conditions; b, cold treatment at 4 ℃. The time points of the photographs were the same for both culture conditions, and from top to bottom, they were phenograms of the time points before cold treatment, after treatment and after recovery, where two lines, S15-3-4X (line 3) and S15-14-4X (line 14), were cold resistant lines, two lines, S15-27-4X (line 27) and S15-38-4X (line 38), were cold sensitive lines, and bar was 5 cm.
FIG. 11 is a bar graph showing the average survival rates of rice Nipponbare (NPB), 93-11 and the tetraploid recombinant inbred lines corresponding to the 4S 15 generations in FIG. 10 under normal culture conditions and after cold stress using the 4 ℃ cold treatment method.
FIG. 12 shows potassium chlorate (KClO) at 2mM3) Under treatment, rice Nipponbare (NPB), 93-11 and 4 tetraploid recombinant inbred lines (S15) were phenotypically shown under normal culture conditions and after potassium chlorate stress. A, normal culture conditions; b, 2mM potassium chlorate (KClO)3) And (4) processing conditions. The same time points were taken for both culture conditions, from top to bottom, and are respectively the time point phenotype photographs before cold treatment, after treatment and after recovery, wherein two lines, S15-7-4X (line 7) and S15-15-4X (line 15), were chlorate sensitive lines, i.e., lines with high nitrogen utilization, two lines, S15-23-4X (line 23) and S15-34-4X (line 34), were chlorate resistant lines, i.e., lines with low nitrogen utilization, and bar was 5 cm.
FIG. 13 is a bar graph showing the use of 2mM potassium chlorate (KClO)3) The average survival rates of the rice Nipponbare (NPB), 93-11 and the tetraploid recombinant inbred lines corresponding to the 4S 15 generations in FIG. 12 under normal culture conditions and after chlorate stress.
FIG. 14 is a schematic flow chart of the anther culture technique used in the present invention and the tetraploid recombination inbred line 18 line (S15-18-4X) and the corresponding diploid regeneration line (S15-18-2X) obtained by anther culture thereof. A is a flow schematic diagram of an anther culture technology; b is a phenotype chart of the 18 th line (S15-18-4X) of the tetraploid recombination inbred line and a corresponding diploid regeneration line (A-18-2X) obtained by anther culture of the tetraploid recombination inbred line.
FIG. 15 is a schematic diagram of the identification of rice karyotypes using the two-color oligonucleotide probe fluorescence in situ hybridization (Oligo-FISH) technique. A, a distribution pattern diagram of a double-color Oligo-FISH probe on 12 rice chromosomes; b, applying the bicolor Oligo-FISH technology to the chromosome layout of diploid rice; c, identifying a tetraploid recombination inbred line (S15-18-4X) of the 18 th line of rice as an example graph of euploid by using a two-color Oligo-FISH technology; d, an exemplary diagram for identifying a rice regeneration plant cultivated by the anther of the tetraploid recombination inbred line 18 (S15-18-4X) as diploid euploid (A-18-2X) by using the double color Oligo-FISH technique.
FIG. 16 is a diagram showing phenotypes of the S15 generation tetraploid recombinant inbred line (S15-18-4X) and its diploid regeneration line (A-18-2X) obtained by anther culture under normal culture conditions and after salt stress. A, normal culture conditions; b, 150mM NaCl salt treatment conditions. The time points were the same for both cultures, and from top to bottom are the time point phenotype photographs before salt treatment, After treatment and After recovery, respectively, DAP, day(s) After Planting in seed trays, bar 5 cm.
FIG. 17 is a diagram showing the phenotype of tetraploid recombinant inbred lines (S15-3-4X, S15-14-4X) of generation S15 and diploid regenerated lines (A-3-2X, A-14-2X) thereof obtained by anther culture under normal culture conditions and after cold stress. A, normal culture conditions; b, cold treatment at 4 ℃. The time points of the photographs were the same for both culture conditions, and from top to bottom, the photographs were taken of the phenotype before cold treatment, After treatment and After recovery, DAP, day(s) After Planting in seed trays and bar 5 cm.
FIG. 18 is a diagram showing phenotype of tetraploid recombinant inbred lines (S15-7-4X, S15-15-4X) of generation S15 and diploid regenerated lines (A-7-2X, A-15-2X) thereof obtained by anther culture under normal culture conditions and after chlorate stress. Left panel, normal culture conditions; right panel, 2mM potassium chlorate (KClO)3) And (4) processing conditions. The time points of the photographs were the same for both culture conditions, and from top to bottom, the photographs were taken of the phenotype before, after and after chlorate treatment, DAP, day(s) after planting in seed trays, and bar was 5 cm.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Breeding Using tetraploid recombinant inbred line crossed/doubled between indica and japonica subspecies of Rice
In this embodiment, a set of rice indica-japonica subspecies allotetraploids are successfully created by carrying out forward and reverse hybridization on indica-rice representative variety 93-11 and typical japonica rice nipples, carrying out whole genome doubling by processing forward and reverse F1 hybrid sprouts with colchicine, 512 independent rice indica-japonica subspecies interspecies segment allotetraploid recombination inbred lines (hereinafter referred to as rice tetraploid recombination inbred lines) of S15 generations are obtained by continuous 15 generations of inbred breeding, 38 rice tetraploid recombination inbred lines with different target traits (such as salt resistance, cold resistance or high nitrogen utilization rate) are randomly selected from the inbred lines for anther culture, fertile diploids are obtained, and whether the diploids have stable target traits or not are verified.
First, hybridization process, obtaining of positive and negative cross F1 hybrid
The parent (japonica rice: Nipponbare; indica rice: 93-11) material is cultivated in a staggered period in a greenhouse of the rice research institute of agricultural academy of Jilin province, and the sowing difference period of the parent and the female parent is reasonably determined (the flowering period is arranged to meet the spike beginning and the full bloom of the parent and the female parent at the same period). Before blooming, castration is carried out on female parent material (before anther powder is scattered in the early morning), and pollination and hybridization processes are carried out by respectively taking parents as male and female parents during blooming of male parents (different flowering time of different materials) at about ten am in midsummer.
The molecular means PCR technology is used to identify hybrid and eliminate pseudo hybrid. The two rice varieties Nipponbare and 93-11 have difference in genome sequence, and we utilize the insertion polymorphism (which means that there is insertion of mPing in Nipponbare and there is no insertion of mPing in 93-11 at a specific position in the genome, or vice versa) of transposition element mPing between the two varieties, wherein there are roughly 53 mPing insertion sites in wild type Nipponbare genome and roughly 10 mPing insertion sites in wild type 93-11 genome, 10 genomic sites with difference in insertion of Nipponbare and 93-11 are selected, and primers (10 pairs of primers are named mPL-1-mPL-10, and the specific primer sequence is shown in Table 1) are designed on mPing and its insertion side wing sequence respectively (Wang H, Chai Y, Chu X, et al Bmc plant biology,2009,9(1): 63-63). FIG. 1 shows the PCR results of two pairs of primers (mPL-4 and mPL-5) among which, in the hybrids, there are two bands of Nipponbare and 93-11, respectively, which are true hybrids, and otherwise, pseudo hybrids.
Table 110 Pair mPin insertion site primer sequence information
Double colchicine
1. Preparing a colchicine solution: dissolving colchicine solid with appropriate amount of anhydrous ethanol (generally dissolving 1g colchicine solid with 2ml of anhydrous ethanol) in a fume hood, filtering and sterilizing the colchicine solution with a filter membrane with pore diameter of 0.38 μm in a super clean bench, and preparing colchicine liquid (RO water + colchicine) with concentration of 0.2% (namely 2g/L) with sterilized RO water (deionized water).
2. Performing aseptic germination on the cross-bred F1 hybrid obtained in the first step: peeling off residual seed coats, pouring the seeds into a sterilized conical flask, cleaning the surfaces of the seeds for 30s by using 75% alcohol, cleaning twice by using sterilized RO water, soaking for 10-12mins by using sodium hypochlorite disinfectant, cleaning for 3-4 times by using the sterilized RO water, soaking for germination by using the sterilized RO water, adding a proper amount of colchicine treatment solution prepared in the step 1 after the hybrid seeds break the chest and germinate (just send out <0.5cm), soaking for three days, and replacing the treatment solution in a superclean bench every day.
3. 1/2MS seed culture medium (without sucrose) is prepared, and the seeds after being washed for several times are planted in the culture medium for subsequent observation.
4. After the rice seedlings in the culture medium grow to the top cover, uncovering the rice seedlings and hardening the rice seedlings for 2 to 3 days, and then transferring the rice seedlings to soil for culture.
5. And when the doubled plants grow to the mature stage, observing the seed phenotype, carrying out dark culture on the seeds until the seeds root, and taking the root tips of the plants for common cytological identification when the root length is 1-1.5 cm.
The karyotype of the rice material after the doubling is identified by cytology is shown in figure 2, and the picture is shown as a schematic diagram of 48 rice chromosomes in a tetraploid rice chromosome map.
The step is verified to successfully obtain tetraploids of S0 generations in two directions (NN99 refers to tetraploid materials obtained by doubling the whole genome of an F1 hybrid obtained by hybridization of Nippon nitrile as a female parent and 93-11 as a male parent, 99NN refers to tetraploid materials obtained by doubling the whole genome of an F1 hybrid obtained by hybridization of Nippon nitrile as a male parent and 93-11 as a female parent).
Third, selfing passage process
And (3) continuously selfing the tetraploids (NN99 and 99NN) of the S0 generations in two directions obtained in the step two according to the mode shown in the figure 3, respectively breeding and breeding offspring (the prepared seeds are raised for seedling and are sent to field for transplanting after being cultivated for about 30-40 days) in Tianjin and Hainan every year, controlling the offspring to be selfed, and continuously selfing for 15 generations to obtain 512 tetraploid recombinant selfing lines.
1. In this step, the inventors of the present invention performed genome-wide re-sequencing (whole genome re-sequencing) on 32 direct ancestral individuals (S6 in fig. 3) of the tetraploid recombinant inbred line, and found that there were a large number of differences in relative copy number of the homoeological fragments among the 32 tested samples, indicating that there were a large number of Homoeological Exchanges (HEs) occurring, and suggesting that there would be a large difference in chromosome arrangement (rewiring) among the tetraploid recombinant inbred lines.
The inventor conducts whole genome re-sequencing relative to rice genome coverage of 10 multiplied by 10 on individual plants of direct ancestors of S6 generations of 32 rice tetraploid recombination inbred lines. The re-sequencing data of each sample are respectively aligned to a modified Nipponbare genome reference sequence MSU.7.0 by using BWA software (all Nipponbare and 93-11 genome are replaced by base N), SNP information of each sample is counted by using a GATK process, and the recombination points of part of homologous chromosomes of each tested rice tetraploid single strain and the relative copy number of part of homologous fragments of the whole genome range are finally determined by a series of Perl and R scripts (Zhang Z, Fu T, Liu Z, et al. extension exchange gene expression and alternative hybridization genes. thermal and Applied Genetics,2019,132(8): 2295: 8.). Based on the tetraploid characteristics of the material, the relative copy number of the homologous fragments/genes in tetraploid for any one genomic locus or gene includes 93-11(9) 0:4, 1:3, 2:2, 3:1 and 4:0 (Sun Y, Wu Y, Yang C, et al. Segmentative allelic genetic sensitive expression reuse and phenotypic diversity of the genomic expression level in 2017,26(20): 5451) 5466). The re-sequencing data analysis results show that for a given genomic locus or gene, the relative copy number of the five partially homologous fragments/genes are indeed present in the 32S 6 generation rice tetraploids, and the proportion of homozygous segments (N:9 ═ 0:4 and 4:0) is about 60% of the average proportion in the population tested. When all sites/genes in the genome are considered together, there will be 5n possibilities (n is the number of sites or genes) for relative copy number combinations of partially homologous fragments/genes at multiple sites in each tetraploid, resulting in large differences in relative copy number combinations of partially homologous fragments/genes between different tetraploid individuals. FIG. 4 is a schematic genomic organization of 32S 6 generation rice tetraploid populations showing in detail the relative copy number combinatorial variation of the dramatic partially homologous fragments/genes among each of the tested S6 generation tetraploid individuals. The results strongly suggest that huge differences of chromosome arrangement (rewiring) exist among tetraploid recombinant inbred lines (S15) obtained by continuous 9-generation selfing of the tetraploid individuals of the S6 generations, and the differences can also be used as genomics basis for characteristics of huge phenotypic diversity existing among the tetraploid rice recombinant inbred lines, stress resistance differences under various stress conditions and the like
2. In this step, 512 rice tetraploid recombinant inbred lines (S15) were found to exhibit phenotypic diversity between lines at the population level.
The inventor of the invention has carried out the measurement of up to 21 rice agronomic traits at population level (selecting 30-40 individuals per line) on the 512 rice tetraploid recombination inbred lines (S15) at the same planting place (Tianjin Diwu area) for two consecutive years (2018, 2019), and the results of the phenotype survey show that the individual phenotypes in the rice tetraploid recombination inbred lines are highly consistent, but huge phenotypic diversity exists among the lines (figure 5 and figure 6).
The 21 rice agronomic traits measured included: flowering period, plant height, sword leaf length, sword leaf width, sword leaf angle, maximum tillering angle, stem diameter, effective tillering number, spike length, primary branch number, secondary branch number, per spike glume number, per spike grain number, per spike glume density, seed setting rate, thousand seed weight, single plant yield, seed length, seed width, seed length-width ratio and single plant aboveground biomass.
3. In this step, some of the rice tetraploid recombinant inbred lines (S15) were found to have strong stress resistance.
(1) Strong salt tolerance
The inventors of the present invention performed two different salt stress treatments on 38 rice tetraploid recombinant inbred lines (S15) randomly selected (also used for anther culture in the next step) and diploid parental nipponica and 93-11 population thereof, and each salt stress treatment was performed in three independent experiments.
The first method of salt stress is to transfer the material to be tested to the three-leaf one-heart stage, culture in the rice nutrient solution containing 150mM NaCl for 7 days, and then transfer to the normal rice nutrient solution for 7 days. The results of three independent salt stress experiments show that each experimental line grows well under the contrast condition, under the salt treatment intensity, the diploid rice parents Nipponbare 93-11 and most of the rice tetraploid recombinant inbred lines die completely, and 5 recombinant inbred lines (numbered as line 3, line 12, line 18, line 21 and line 31) show extremely strong salt tolerance and can be normally inbred and fructified after the salt stress is recovered; also during the stress period, the inventors found 12 rice four-fold systems that were extremely sensitive to salt stress (the stress phenotype appeared early and died rapidly), and found that these salt stress sensitive systems were more salt tolerant than their diploid parents 93-11 but less salt tolerant than Nipponbare by the shortened salt treatment days (FIGS. 7 and 8). It is presumed that at least 60 salt-resistant lines and 160 salt-sensitive lines could be selected from 512 rice tetraploid recombinant inbred lines.
Another method for salt stress is to soak seeds of each experimental line to break the chest, immediately transfer the seeds after breaking the chest to a rice nutrient solution containing 200mM NaCl salt solution for high-intensity salt stress treatment, count the germination rate of each experimental line after 10 days, then reduce the salt stress intensity to 60mM NaCl, and observe the seedling formation condition of each line after 4 weeks. The results of three independent experiments of the salt treatment method all show that all experimental lines can normally bud and grow under the control condition, and under the salt stress, diploid parents Nipponbare and 93-11 and most of rice tetraploid recombinant inbred lines can not normally bud, only 5 lines (namely the numbers in the table 2: line 3, line 12, line 18, line 21 and line 31) can normally bud and grow into seedlings, and the seedlings are consistent with the resistance lines screened by the first salt treatment method, and the time and the growth speed required by the germination are obviously reduced compared with those of the control condition (figure 9); resistant lines capable of budding and seedling formation were consistently cultured under low salt stress (in rice nutrient solution containing 60mM salt solution) and found to be still selfable. Through the experimental results, the invention discovers that the salt resistance systems and the salt sensitivity systems screened by two different salt treatment methods are consistent. From the above experimental results, the present inventors found that the salt-resistant line and the sensitive line screened by the two different salt treatment methods are identical, so that in the following experiments, the present inventors will use only the salt treatment method which is easy to observe, easy to operate and stable in result, i.e., the first treatment method (treatment in the three-leaf one-heart period of rice) described above.
(2) Resistance to cold
The inventors of the present invention performed three independent cold stress treatment experiments on the above randomly selected (also used for anther culture in the next step) 38 rice tetraploid recombinant inbred lines (S15) and their diploid parental japanese sunny and 93-11 populations. When the experimental materials grow to a three-leaf one-heart stage in normal rice nutrient solution, transferring the materials to a4 ℃ incubator for three-day cold stress treatment, and then transferring the materials to a 25 ℃ phytotron for recovery for 7 days. The results of three independent cold stress experiments show that each experimental line grows well under the contrast condition, and under the stress of cold treatment at 4 ℃, the diploid rice parents 93-11 and most of the tetraploid recombinant inbred lines of the rice die completely, and 6 recombinant inbred lines (namely the serial numbers in the table 2 are: line 3, line 8, line 14, line 18, line 26 and line 33) have the same cold resistance as the parent Nipponbare and can be normally selfed and fructified after the cold stress is recovered; meanwhile, in the stress process, 10 rice tetraploid recombination inbred lines which are extremely sensitive to cold stress (the stress phenotype appears very early and the death is rapid) are discovered by the invention. Figures 10 and 11 show the phenotype after stress and survival rate, respectively, for the two resistant lines, line 3 and line 14, and the two sensitive lines, line 27 and line 38. It is presumed that at least 80 cold stress resistant lines and 130 cold sensitive lines can be selected from 512 rice tetraploid recombinant inbred lines.
(3) Enhanced nitrogen availability (Low Nitrogen tolerance stress)
The inventors of the present invention performed three independent chlorate stress experiments on the above randomly selected (also used for anther culture in the next step) 38 rice tetraploid recombinant inbred lines (S15) and their diploid parental japanese nitrile and 93-11 populations. Chlorate (potassium chlorate, KClO is used as medicine)3) Is a toxic analog of nitrate, which can be absorbed by nitrate transporters and then converted into hypochlorite, which has a toxic effect on plants, under the action of nitrate reductase. When the experimental materials grow to one leaf and one heart in the normal rice nutrient solution, transferring the experimental materials into a nitrogen-free nutrient solution with the concentration of 2mM potassium chlorate for culturing for 4 days, and then transferring the experimental materials into the normal rice nutrient solution for recovering for 7 days. The results of three independent chlorate stress experiments all show that each experiment line grows well under the control condition, the diploid rice parents 93-11 and most of the rice recombinant inbred lines die under the potassium chlorate treatment, and only 5 lines (namely, the serial numbers of the lines 7, 15, 25, 32 and 38 in the table 2) show strongerChlorate sensitivity, indicating a higher utilization of nitrate nitrogen, while during stress 9 were also found to show resistance to chlorate, indicating a lower utilization of nitrate nitrogen (figures 12 and 13). It is presumed that at least 60 lines with high nitrogen utilization and 120 lines with low nitrogen utilization can be selected from 512 rice tetraploid recombinant inbred lines.
Fourthly, anther culture process
(1) Drawing the anther: sowing diploid parent Nipponbare, 93-11 and 38 rice tetraploid recombination inbred lines (numbered as line 1-line 38) randomly selected from 512 tetraploid recombination inbred lines (S15) in the three steps in a test field, and at the time of 7-8 months, the booting stage of the rice is generally 8: 00-10: 00 in the morning or 16: 00-18: 00 in the afternoon of sunny days, the cells are in a vigorous division stage. Taking young spikes when the distance between the sword leaf and the next leaf is about 10cm, wherein the anther is about 1/3 of glume, and observing the anther in a microscope at the time of the single-core side approaching period; the edge-near period of the single nucleus is the optimal period for culturing the rice anther. The induction rate of the callus is affected by the fact that the anthers are too old or too tender. The retrieved young ears are wrapped by preservative films and then put in a refrigerator at 4 ℃ for low-temperature pretreatment, the low-temperature pretreatment is the most conventional and effective means for improving the anther culture efficiency, and the induction rate of the rice callus can be improved by several times to dozens of times. The low-temperature treatment has the action mechanisms of delaying the degeneration of the pollen, maintaining the physiological environment for the development of the pollen, improving the level of endogenous auxin, reducing the content of ethylene, starting the development of an androgenesis and the like. After being stored for 7-14 days, the sample is taken out and is subjected to the next experiment in an ultra-clean workbench. Each strain was taken down (individual plants were not distinguished): 20-30 young ears.
(2) Anther culture: the following items were prepared in an ultraclean bench: alcohol burner, 75% ethanol, sodium hypochlorite (atenmin), sterilized deionized water, scissors, tweezers, culture dish, sterilized filter paper, sterilized beaker, waste liquor jar, rice anther culture callus induction medium (solid).
The formula of the solid culture medium (1L) for inducing the callus of the rice anther culture is as follows, wherein, the macro-elements, the trace elements, the ferric salts, the organic medicines and the hormones are adopted; and the r is other necessary steps or medicines:
the anther culture specific operation process is as follows:
in a clean bench, the bottom of a rice glume (anthers are carefully prevented from being cut, the aim is to cut anther filaments so that the anthers can be easily shaken out) is cut off by scissors, the top of the glume is clamped by tweezers, the anthers are shaken down to the center of a callus induction culture medium for rice flower culture, approximately 500 anthers are respectively packed in each bottle, strains and dates are marked after sealing, and the callus is induced under the condition of dark culture at 25 ℃. After about 25 days, the callus is generated, at this time, the callus is transferred to a new callus induction culture medium for rice anther culture, subcultured for 1-2 times, transferred to a rice regeneration culture medium, and placed in a continuous illumination incubator at 32 ℃ under 12000lx illumination intensity for induction and differentiation. The green seedlings are differentiated from the calluses about 20 to 25 days, the calluses are continuously cultured in a light incubator until the regenerated seedlings grow to the height of a top cover, the cover of a culture bottle is opened, the seedlings are hardened for 1 to 2 days, then the seedlings are placed in a rice nutrient solution to be cultured in a climatic chamber (the constant temperature is 25 ℃, the illumination is 12000 lx), and the nutrient solution is replaced once a day (the pictures of all steps of anther culture are shown in figure 14 in detail). And (3) taking root tips after about 5-7 days of culture, identifying the ploidy of the material by a cytological method, and identifying the karyotype of the diploid restorer rice by a two-color Oligo-FISH technology.
(3) The method for identifying the tetraploid karyotype of the rice by using the two-color Oligo-FISH technology comprises the following steps: the oligonucleotide probe fluorescence in situ hybridization (Oligo-FISH) is developed on the basis of Fluorescence In Situ Hybridization (FISH), and is mainly characterized in that a probe for completing the in situ hybridization process is derived from oligonucleotides (Oligos) with the length of 45nt, which are designed autonomously by a user and are synthesized in parallel by DNA synthesis companies on a large scale independently from oligonucleotides. The probes in the two-color Oligo-FISH technique used in the present invention were designed based on the rice genome reference information, and the two pools of oligonucleotides (Oligos) were provided by professor Gong Shing cloud, university of Yangzhou (Liu X, Sun S, Wu Y, et al, Dual-color Oligo-FISH can novel chromosomal variations and evolution in Oryza species, plant journal 2020,101(1):112-121.), with different location and number distributions on different chromosomes. By identifying the number and the position distribution of the two probes on the chromosome, the technology can accurately distinguish 12 chromosomes of rice. The invention identifies the karyotype of the diploid restoration line obtained by culturing the tetraploid recombinant inbred line anther by the two-color Oligo-FISH technology (as shown in figure 15).
Fifth, anther culture experiment results
The anther culture uses a rice anther culture callus induction culture medium, 25 rice tetraploid recombination inbred lines (numbered as line 1-line 38) randomly selected from 512 tetraploid recombination inbred lines (S15) in the step three successfully obtain diploid restoring lines with the same genome mosaic composition with the rice tetraploid recombination inbred lines, and the specific callus induction condition and the differentiation seedling emergence condition are shown in Table 2.
Table 2 statistics table of anther culture callus induction and differentiation emergence.
Note: v represents success; x represents failure.
Sixthly, identifying the stress resistance of the diploid rice line obtained by anther culture
1. Some tetraploid recombinant inbred lines (S15) have strong salt tolerance, and diploid rice lines obtained by anther culture inherit the strong salt tolerance.
And (4) performing salt stress identification on the diploid rice line which is obtained by anther culture of the tetraploid recombinant inbred line (S15) with strong salt tolerance and has the same genome composition as the tetraploid recombinant inbred line. (the salt treatment method is completely consistent with the first salt treatment method described in the third step) experiments prove that the diploid rice lines with the same genome composition obtained by anther culture of the salt stress resistance lines can inherit the strong salt resistance property of the corresponding parent tetraploid recombination inbred lines. Using the S15-18 quadruple system (S15-18-4X, i.e., line 18 in Table 2) and its corresponding anther culture doubler system (A-18-2X) as examples, the salt stress display diagram is shown in FIG. 16.
2. Some tetraploid recombinant inbred lines (S15) have strong cold resistance, and diploid rice lines obtained by anther culture thereof also inherit the strong cold resistance.
And (4) carrying out cold resistance identification with the same strength as that of the tetraploid recombinant inbred line on the diploid rice line with the same genome composition obtained by anther culture of the tetraploid recombinant inbred line (S15) with strong cold resistance identified by the step three. (the cold treatment method is completely identical to the cold treatment method described in step three). Experiments prove that the diploid rice line which is obtained by anther culture of the salt stress resistance lines and has the same genome composition with the salt stress resistance lines can inherit the strong cold resistance of the corresponding parent tetraploid recombination inbred line. Using two quadruple systems S15-3 and S15-14 (S15-3-4X and S15-14-4X, i.e., lines 3 and 14 in Table 2) and their corresponding anther culture doubler systems (A-3-2X and A-14-2X) as examples, the cold stress is shown in FIG. 17.
3. Some tetraploid recombinant inbred lines have higher nitrogen utilization (chlorate stress), and diploid rice lines obtained by anther culture inherit the high nitrogen utilization characteristics.
And (3) carrying out high utilization rate (chlorate stress) identification on the diploid rice line which is obtained by anther culture and has the same genome composition with the tetraploid recombinant inbred line (S15) which is identified to have high nitrogen utilization rate (chlorate stress) in the third step (the chlorate treatment method is completely consistent with the chlorate treatment method described in the third step). Experiments prove that the diploid rice line which is obtained by anther culture of the lines with high nitrogen utilization rate and has the same genome composition with the diploid rice line can inherit the high-efficiency nitrogen utilization characteristic of the corresponding parent tetraploid recombination inbred line. Using two quadruple systems S15-7 and S15-15 (S15-7-4X and S15-15-4X, i.e. lines 7 and 15 in Table 2) and their corresponding anther culture doubler systems (A-7-2X and A-15-2X) as examples, the chlorate stress is shown in FIG. 18.
The above results show that the diploid restorer line induced by the tetraploid recombinant inbred lines (S15) through anther culture and having the same genomic composition (genomic composition) can mostly inherit the phenotype and resistance characteristics of the parent tetraploid. The obtained diploid restorer line with the desired traits can be used as a rice breeding material. The hybrid rice is hybridized with the existing rice variety with other excellent properties without target properties, so that a new rice variety with the genetic background of the existing rice variety and the target properties can be obtained.
Claims (10)
1. A method for obtaining a rice breeding material having a desired trait, comprising the steps of:
(A1) hybridizing indica rice and japonica rice to obtain F1 hybrid seeds;
(A2) doubling the F1 generation hybrid obtained in (A1) into tetraploids;
(A3) subjecting the tetraploid obtained in (A2) to continuous multi-generation selfing, and selecting a tetraploid recombinant inbred line with the target character from the obtained tetraploid recombinant inbred lines;
(A4) carrying out chromosome group doubling on the tetraploid recombinant inbred line with the target character obtained in the step (A3) to obtain a diploid with the target character; the diploid is rice breeding material with the target character.
2. The method of claim 1, wherein: in the step (A1), the indica rice is indica rice variety 93-11; and/or the japonica rice is japonica rice variety Nipponbare; and/or
In the step (a1), the hybridization between indica rice and japonica rice is reversible hybridization or unidirectional hybridization.
3. The method according to claim 1 or 2, characterized in that: in step (a2), doubling the F1 generation to tetraploid is doubled with colchicine.
4. A method according to any one of claims 1-3, characterized in that: in the step (a3), the tetraploid recombinant inbred line is a tetraploid recombinant inbred line inbred for more than 8 generations;
further, the tetraploid recombinant inbred line is a tetraploid recombinant inbred line inbred for 15 generations.
5. The method of claim 4, wherein: in the step (a3), the tetraploid recombinant inbred line inbred for 15 generations is obtained as follows: the inbred 1 generation, the inbred 2 generation, the inbred 9 generation and more than 9 generations are single-grain methods, and the rest generations are equal-grain passage methods.
6. The method according to any one of claims 1-5, wherein: in the step (a4), obtaining a diploid having the desired trait by anther culture of the tetraploid recombinant inbred line having the desired trait; or
In the step (a4), the diploid with the target trait is obtained by crossing the tetraploid recombinant inbred line with the rice haploid inducer.
7. A method for obtaining a rice tetraploid recombinant inbred line with a trait of interest comprising the steps (a1) - (A3) of the method of any one of claims 1-6.
8. Any of the following applications:
(B1) use of the method of any one of claims 1 to 6 in rice breeding;
(B2) use of a rice breeding material having a desired trait produced by the method according to any one of claims 1 to 6 in rice breeding;
(B3) use of the method of claim 7 in rice breeding;
(B4) the use of the rice tetraploid recombinant inbred line with the desired trait prepared by the method of claim 7 in rice breeding.
9. A rice breeding method comprising the steps (A1) to (A4) of the method of any one of claims 1 to 6, and the step (A5) of:
(A5) and (c) hybridizing the diploid having the desired trait obtained in (a4) as one of parents with an existing rice variety not having the desired trait to obtain a new rice variety having the desired trait together with the genetic background of the existing rice variety.
10. The method or use according to any one of claims 1-9, wherein: the target characters are salt resistance, cold resistance and high nitrogen utilization rate.
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Application publication date: 20200818 |