Method for carrying out backcross breeding by using plant male sterile material and application
阅读说明：本技术 一种利用植物雄性不育材料进行回交育种的方法及应用 (Method for carrying out backcross breeding by using plant male sterile material and application ) 是由 龙起樟 黄永兰 万建林 王会民 芦明 唐秀英 朱雪晶 于 2020-07-02 设计创作，主要内容包括：为了在多系品种的培育中提高不同品系培育所需回交工作的效率,本发明提供了一种利用植物雄性不育材料进行回交育种的方法及应用,向多系品种背景亲本导入核隐性雄性不育基因并用以辅助回交选育的技术方案,本发明提供的技术方案可大幅度减轻田间人工杂交的工作量,进而使得扩大回交群体规模变得较为容易,从而提高回交育种的工作效率,缩短育种年限。本发明的提供的方法有助于推动多系品种的培育。(In order to improve the efficiency of backcross work required by breeding different strains in the breeding of multiple varieties, the invention provides a method for carrying out backcross breeding by utilizing plant male sterile materials and application thereof, and a technical scheme that nuclear recessive male sterile genes are introduced into background parents of the multiple varieties to assist backcross breeding is provided. The method provided by the invention is beneficial to promoting the cultivation of multiple varieties.)
1. A method for carrying out backcross breeding by using plant male sterile materials comprises the following steps:
1) modifying recurrent parents into a male sterile line of which the sterile character is controlled by a single-nucleus recessive male sterile gene locus;
2) hybridizing the recurrent parent sterile line obtained by transforming in the step 1) as a female parent and a donor parent containing a certain known beneficial target gene as a male parent to obtain a hybrid F1；
3) Propagation of hybrid F1Hybridizing with the sterile strain line as male parent and recurrent parent as female parent to obtain BC1F1Seed growing;
4) propagation of BC1F1And (3) selecting a fertile or sterile individual plant which has the agronomic characters which are visible by naked eyes and tend to recurrent parent and contains the beneficial genes of the donor parent to continue backcrossing:
when the selected individual plant is fertile, the selected individual plant is used as male parent and the recurrent parent sterile plant line is used as female parent to carry out hybridization to obtain BC2F1Seed growing;
when the selected single plant is the sterile plant, the single plant is taken as the female parent and the original recurrent parent or the recurrent parent sterile gene heterozygous fertile plant line is taken as the male parent to carry out hybridization to obtain BC2F1Seed growing;
5) propagation of BC2F1The group is continuously backcrossed for more than 1 time according to the backcross method in the step 4) to obtain BCnF1A population;
6) BC obtained in the last backcross of step 5)nF1Selecting fertile plants which contain target genes and have obvious difference between agronomic characters visible to naked eyes and recurrent parents from the population for selfing, harvesting seeds of the fertile plants for propagation to obtain BCnF2Separating the colony, and selecting fertile plants which are homozygous for the advantageous genes of the donor parents and do not contain sterile genes from the colony to complete backcross breeding.
2. The method of claim 1, wherein the plant is a diploid plant.
3. The method for breeding by backcrossing with plant male sterile material as claimed in claim 1, wherein the method for modifying in step 1) comprises: gene editing technology, mutation breeding technology and backcross breeding technology.
4. The method of claim 1, wherein the genetic locus of the nuclear recessive male sterility gene controlling the male sterility has the following characteristics in step 1): sterile plants and normal fertile plants generated by the control method are easy to identify in the field; does not cause the change of agronomic traits except male sterility.
5. The method of claim 1, wherein the nuclear recessive male sterile gene in step 1) is on a different chromosome from the advantageous target gene in step 2).
6. A method of backcross breeding using plant male sterile material as claimed in claim 1, wherein the recurrent parent sterile lines are propagated by harvesting seeds of individual plants whose sterile genes are heterozygous or asexually by using vegetative organs of homozygous sterile plants.
7. The method of claim 1, wherein the TDR gene is used for modifying the male sterility of recurrent parent when the plant is rice.
8. The method of claim 7, wherein the male sterility trait is obtained by knocking out the normally functioning TDR gene in the recurrent parent through CRISPR \ Cas9 system-mediated gene knock-out technology.
9. Use of the method of any one of claims 1-8 for backcross breeding using plant male sterile material in multi-line variety breeding.
10. The use of claim 9, wherein said multiple cultivars are plant cultivars from a mixture of two or more near isogenic lines or two or more lines with no apparent difference in agronomic traits visible to the naked eye.
Technical Field
The invention belongs to the technical field of plant breeding, and relates to a method for carrying out backcross breeding by using a plant male sterile material and application thereof in multi-variety cultivation.
Background
With the identification and cloning of a large number of plant favorable trait control genes (or genetic loci), the application of breeding to these genes (or genetic loci) has become a major task in current breeding. The breeding of known genes (or genetic loci) is mainly carried out by a molecular marker assisted selection method, and the current mainstream method is to aggregate a few advantageous genes into a variety by molecular marker assisted selection, and as the number of the advantageous genes increases, the aggregation of the advantageous genes into a variety becomes very difficult, and if different genes on the same chromosome are aggregated into a variety, the aggregation becomes more difficult. The breeding of multiple varieties can avoid the difficulties and achieve the effect similar to the aggregation of multiple beneficial genes.
The multi-line variety is a variety formed by mixing different lines in a certain proportion. The different strains forming the multi-line variety can be near isogenic lines, or can be strains which have no obvious difference in agronomic traits and have larger genetic difference than that of the near isogenic lines. The core step of multi-line variety breeding is the breeding of different lines which are composed of the multi-line variety, and the multiple lines which have almost identical genetic background and agronomic characters but different specific excellent characters respectively are generally bred by a backcross method. The breeding of different strains of multiple varieties is realized by molecular marker assisted backcross breeding of multiple genes, so that for crops which are not easy to castrate, a great deal of labor is consumed for castration during hybridization, and the situation is more serious when more hybrids need to be harvested, so that the backcross importing work of multiple genes becomes very difficult, the breeding period is longer, and the breeding of multiple varieties is limited. Therefore, a method for improving the working efficiency is needed in practice to accelerate the breeding process of multiple varieties.
Plant male sterility is often used in production to avoid selfing, for example for heterosis utilization and recurrent selection. The male sterility which is naturally generated in plants or generated by mutagenesis is generally divided into two types of cytoplasmic male sterility and nuclear male sterility, and compared with the cytoplasmic male sterility, the nuclear male sterility is more common, and in the nuclear male sterility, recessive male sterility is the most common, so far, a plurality of recessive male sterility genes are identified in different crops. Because of its great ease of creation, researchers in the field of genetic breeding are actively exploring the use of recessive male-sterile genes, but nuclear recessive sterility is really less applied to production than cytoplasmic male sterility, which has been widely applied to hybrid production. Up to now, nuclear male sterility has been applied only to recurrent selection and spt (seed production) technology as a new generation hybrid production technology, and has not been widely popularized and applied.
Disclosure of Invention
The invention aims to overcome the defects of large workload and low efficiency of the conventional multi-variety cultivation of plants, and provides a method for backcross breeding by using a plant male sterile material and application thereof.
In order to achieve the technical purpose, the invention is realized by the following technical scheme:
in a first aspect, the present invention provides a method for backcross breeding using plant male sterile material, comprising the steps of:
1) modifying recurrent parents into a male sterile line of which the sterile character is controlled by a single-nucleus recessive male sterile gene locus;
2) hybridizing the recurrent parent sterile line obtained by transforming in the step 1) as a female parent and a donor parent containing a certain known beneficial target gene as a male parent to obtain a hybrid F1；
3) Propagation of hybrid F1Hybridizing with the sterile strain line as male parent and recurrent parent as female parent to obtain BC1F1Seed growing;
4) propagation of BC1F1Population, selection of macroscopic agronomic traits tending toAnd (3) continuing backcrossing the fertile or sterile single plants which are backcrossed to the parents and contain the beneficial genes of the donor parents:
when the selected individual plant is a fertile individual plant, the fertile individual plant is used as a male parent and the recurrent parent sterile plant line is used as a female parent to carry out hybridization to obtain BC2F1Seed growing;
when the selected individual plant is sterile, hybridization is carried out by taking the selected individual plant as a female parent and taking the original recurrent parent or the recurrent parent sterile gene heterozygous fertile plant line as a male parent to obtain BC2F1Seed growing;
5) propagation of BC2F1The group is continuously backcrossed for more than 1 time according to the backcross method in the step 4) to obtain BCnF1A population;
6) BC obtained in the last backcross of step 5)nF1Selecting fertile plants which contain target genes and have obvious difference between agronomic characters visible to naked eyes and recurrent parents from the population for selfing, harvesting seeds of the fertile plants for propagation to obtain BCnF2Separating the colony, and selecting fertile plants which are homozygous for the advantageous genes of the donor parents and do not contain sterile genes from the colony to complete backcross breeding.
Preferably, the plant is a diploid plant.
Generally, a good parent is selected as a background parent and is determined as a recurrent parent for backcross improvement; preferably, in step 1), the method for retrofitting comprises: gene editing techniques, (traditional) mutagenic breeding techniques and backcross breeding techniques, more preferably, selective gene editing techniques.
Preferably, in step 1), the nuclear recessive male sterility gene locus for controlling male sterility has the following characteristics: sterile plants and normal fertile plants generated by the control method are easy to identify in the field; does not cause the change of agronomic traits except male sterility.
Preferably, the nuclear recessive male sterility gene described in step 1) is on a different chromosome from the advantageous gene of interest described in step 2). More preferably, when the number of advantageous genes of interest introduced into the recurrent parent is large and this requirement cannot be met with one male-sterility gene, a second male-sterility gene, which is located on a different chromosome from the first male-sterility gene, is introduced.
Preferably, the recurrent parent sterile lines are propagated by harvesting seeds of the individual plants whose sterile genes are heterozygous or asexually by using vegetative organs of homozygous sterile plants. For example, rice can be asexually propagated using rice stakes of homozygous sterile plants.
Preferably, when the plant is rice, the TDR gene (the MSU rice genome database is LOC _ Os02g 02820; the RAP rice genome database is Os02g0120500) is applied to the male sterility transformation of the recurrent parent.
More preferably, the TDR gene with normal function in the recurrent parent is knocked out by a CRISPR \ Cas9 system-mediated gene knockout technology so as to obtain the male sterility character, and the method refers to the Chinese invention patent 202010401496.9.
In a second aspect, the invention provides an application of the method for backcross breeding by using the plant male sterile material, namely the method is applied to the cultivation of multiple varieties.
Preferably, the multi-line variety refers to a plant variety formed by mixing more than two near isogenic lines or more than two lines with no obvious difference in agronomic traits visible to the naked eye.
More preferably, part or all of the lines of the multi-line variety are bred by the backcross breeding method using the plant male sterile material.
Advantageous effects
Under the situation that a calling multi-line variety is produced and the breeding process is slow, the invention provides a method for carrying out backcross breeding by using a male sterile material, and the method is applied to breeding of different lines of the multi-line variety, so that the workload of artificial hybridization in the backcross process can be greatly reduced, an implementer of the invention can simultaneously carry out backcross introduction work of more favorable genes on one hand, and can enlarge the hybridization scale to increase the yield of the hybrid on the other hand, thereby increasing the scale of a progeny segregation population, improving the probability of occurrence of a single plant with agronomic characters close to recurrent parents, shortening the backcross breeding process, further improving the breeding efficiency of the whole multi-line variety and finally accelerating the process of popularization of the multi-line variety to the market, which is the first beneficial effect of the invention.
The second beneficial effect of the invention is that after all strains of multiple varieties are obtained by the method provided by the invention, the male sterile line used in the backcross breeding process can be conveniently hybridized with different strains of multiple varieties to synthesize an initial population for recurrent selective breeding, and then the operation of recurrent selective breeding is further carried out to breed multiple varieties or separate pure lines from the multiple varieties which meet certain requirements.
Drawings
FIG. 1: the invention discloses a process schematic diagram for carrying out backcross breeding by using plant recessive male sterile materials. In the figure, A/a represents an allele for controlling male sterility, A is a wild type, a is a mutant type, and a plant shows male sterility when the mutant gene is pure; B/B denotes an allele which controls an agronomic trait, B is a favorable allele (capitalization does not necessarily mean dominant), and B is an allele without the corresponding favorable function. The genetic symbols in all upper or lower case indicate that the two alleles of the locus are homozygous, and the upper and lower case are present in combination, indicate heterozygosity. The content within the dashed box in the figure represents the 3 backcrossing patterns. The male parent and the female parent respectively represent the female parent and the male parent when in hybridization, the deletion line in the genotype symbol represents that the genotype plant should be abandoned, and the x and
FIG. 2: the rice male sterile gene and the low cadmium gene sequence utilized by the backcross breeding of the invention are compared with the wild type sequence in the conventional variety. The upper part shows a male sterility gene TDR, and the lower part shows a low cadmium gene Nramp 5; the figure shows the position of the base in the open reading frame of the TDR gene sequence of the Nipponbare reference genome; WT, wild type; MUT, mutant; underlined letters indicate newly inserted bases; short horizontal lines indicate missing bases; compared with a wild type sequence, the TDR mutant is deleted for 9 bases and simultaneously inserted with a new base A; the Nramp5 mutant was 28 bases deleted compared to the wild type.
FIG. 3: high-resolution melting curve typing effect of the low-cadmium donor parent OsNramp5 gene mutant and the wild type used in backcross breeding.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
The invention provides a method for carrying out backcross breeding by using a plant male sterile material, wherein the backcross process is shown as figure 1 and mainly comprises the following steps:
1) transforming recurrent parents into a male sterile line (aabb) of which the sterile character is controlled by a single-nucleus recessive male sterile gene locus (A/a);
2) hybridizing the recurrent parent sterile strain obtained by transformation in the step 1) serving as a female parent (AABB) and a donor parent containing a certain known favorable target gene (B) serving as a male parent (AABB) to obtain a hybrid F1(AaBb)；
3) Propagation of hybrid F1Hybridizing the male parent strain AaBb male parent strain and the recurrent parent sterile strain as female parent (AaBb male parent) to obtain BC1F1Species (AaBb );
4) propagation of BC1F1And (3) selecting a fertile or sterile individual (AaBb or aaBb) which has the agronomic characters which are visible to the naked eye and tend to recurrent parent and contains the beneficial genes of the donor parent to continue backcrossing:
when the selected individual plant is fertile, the fertile individual plant is used as a male parent (AaBb male parent) and the recurrent parent sterile plant is used as a female parent (aabbb female parent) to carry out hybridization to obtain BC2F1Seeds (as shown in backcross mode 1 of FIG. 1);
when the selected individual is a sterile individual, the method comprisesWhich is used as female parent (aaBb) and an original recurrent parent or a recurrent parent sterile gene heterozygous fertile line as male parent (AAbb or Abbb) to carry out hybridization to obtain BC2F1Seeds (as shown in backcross pattern 2 or 3 of FIG. 1);
5) propagation of BC2F1The group is continuously backcrossed for more than 1 time according to the backcross method in the step 4) to obtain BCnF1A population;
6) BC obtained in the last backcross of step 5)nF1Selecting a fertile plant (AaBb) which contains a target gene and has no obvious difference between the agronomic characters visible to the naked eye and the recurrent parent in the population for selfing, harvesting the seeds of the fertile plant for propagation to obtain BCnF2Separating the population, and selecting fertile strains (AABB) which are homozygous for the donor parent and do not contain the sterile genes from the population to finish backcross breeding.
The experimental procedures in the following examples, in which specific conditions are not specified, are generally performed under conventional conditions, such as those described in book "molecular cloning, laboratory Manual (3 rd edition) (J. SammBruk, D.W. Lassel, Huang Peyer et al, J. Paecio, science publishers, 2008), or under conditions recommended by manufacturers of manufacturing reagents or equipment.
The agronomic traits (agronomictraits) refer to the related traits of the crop such as growth period, plant height, leaf area, fruit weight and the like, which can represent the characteristics of the crop variety. The "no obvious difference in agronomic traits visible to the naked eye" of different crops is determined according to the existing determination standard in the field, and is not limited herein.
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