CN118058185A - Method for creating fertility tetraploid hybrid between cultivated rice and Australian wild rice genome - Google Patents

Abstract

The invention relates to a method for creating a fertility tetraploid hybrid between a cultivated rice genome and an Australian wild rice genome, which uses CX35-2X or YZ32-2X as a female parent material and Australian wild rice EE as a male parent material for hybridization to obtain first-generation hybrid seedlings; the first generation hybrid seedling is doubled by colchicine to obtain the seed of the hybrid tetraploid. The invention obtains the distant hybrid containing the complete genome of the wild rice through whole genome transfer and polyploidization, namely distant hybridization, and then obtains the hybrid heteropolyploid through chromosome doubling, thereby fully utilizing the excellent gene of the wild rice and the distant hybrid vigor of the polyploid.

Description

Method for creating fertility tetraploid hybrid between cultivated rice and Australian wild rice genome

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for creating a fertility tetraploid hybrid between genomes of Oryza sativa (Oryza sativa) and Oryza sativa (Oryza australiensis).

Background

Rice has abundant species resources, and in addition to Oryza sativa (Oryza sativa) and Oryza sativa (o.glaberrima), there are more than 20 wild rice species, which belong to the 10 large genome. The genetic diversity of wild rice is extremely rich, a plurality of excellent genes which are not possessed by cultivated rice or are disappeared are preserved, and the wild rice is a gene treasury for carrying out genetic improvement on the cultivated rice. The use of wild rice has been shown to strongly promote the development and progress of rice breeding. However, when wild rice resources are used, because of the distant relationship between cultivated rice and wild rice, there are problems such as difficulty in hybridization, hybrid die young, sterility of hybrid, etc., and it is difficult to efficiently use excellent genes of wild rice by hybridization and backcrossing at diploid level. Polyploid rice breeding is divided into three types of interspecific, interspecific and intergeneric polyploid heterosis utilization, and at present, interspecific and interspecific heterosis utilization has achieved a certain effect, but the defect is that the breeding time is long, the heterosis does not exist, and interspecific heterosis utilization cannot be broken through. Distant hybridization polyploid breeding makes advantageous gene combinations, utilizes the advantages of distant hybridization to enrich rice seed resources and also has multiple heterosis, but also has the problem that the fruiting rate is difficult to overcome.

The Australian wild rice is an EE genome, is originally distributed in Australian tropical zone, has excellent properties of brown planthopper resistance, black tail leafhopper resistance, drought resistance and high temperature resistance, but has serious technical obstacle on the utilization of excellent genes of the EE genome because the main cultivar AA genome is far from related relationship, and serious reproductive obstacle exists in hybridization to cause complete sterility. Although research reports that the distant hybrid F1 of the Australian wild rice and the AA genome cultivated rice with the EE genome is carried out before, and then backcrossing with the cultivated rice is continued, and an inter-hybrid BC1F1 plant (Song Lishuang, a light-transmitting, 2021) is obtained by means of embryo rescue technology, repeated backcrossing continuously dilutes the genetic background of the EE genome of the wild rice, and a certain limitation exists in the aspect of wild rice gene utilization.

Disclosure of Invention

Based on the above reasons, the invention aims to provide a method for creating and primarily identifying a fertility tetraploid hybrid between the genome of cultivated rice and Australian wild rice, overcomes the difficulty of distant hybridization and creates a new germplasm by utilizing the double advantages of distant hybridization and polyploidy, and belongs to the cultivation category of new rice varieties. Specifically, in order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:

the invention relates to a method for creating a fertility tetraploid hybrid between a cultivated rice genome and an Australian wild rice genome, which uses CX35-2X or YZ32-2X as a female parent material and Australian wild rice EE as a male parent material for hybridization to obtain first-generation hybrid seedlings; the first generation hybrid seedling is doubled by colchicine to obtain the seed of the hybrid tetraploid.

In a preferred embodiment of the invention, the wild rice EE in australia is masked to allow bud differentiation of the wild rice prior to crossing, covered in the evening in summer, uncovered in the morning and controlled to a light duration of no more than 9 hours to allow simultaneous heading and flowering of the male and female parent lines of the wild rice EE in the early stages of the february to september.

In a preferred embodiment of the invention, the anthers of wild rice EE which have just been flowering are pollinated onto the stigma of a female parent line which has been emasculated, whereupon a hybridization bag is placed.

In a preferred embodiment of the invention, the pollination hybridization is followed by a continuous 8-12 day spray addition of exogenous hormones in the following ratios: GA 50mg/ml+NAA 10mg/ml+2, 4-D2 mg/ml.

The invention is characterized in that embryo rescue and rooting of embryos after hybridization bagging are carried out for 12-15 days, after the embryos grow for 12-15 days, the peeled embryos are cultured by an improved culture medium to be called embryo rescue, the peeled embryos are placed on the improved culture medium horizontally by forceps after being sterilized on an ultra-clean workbench, the embryos are placed on the light for 15 hours and are cultured in the dark at 28 ℃ for 9 hours, the young embryos can grow into seedlings after 15 days, the improved embryo rescue culture medium is ：N6+6-BA0.5～2mg/L+NAA0.2mg/L+KT0.2～0.5mg/L+2,4-D0.1mg/L+GA0.5mg/L+Sucrose5.5％+asparagine0.5g/L+glutamine(Gln)0.5g/L. improved embryo rescue culture medium which increases nitrogen content required by rice growth, and the grown seedlings are transferred to a rooting culture medium on the ultra-clean workbench, so that the seedlings can grow into seedlings which can be transplanted to a field;

In a preferred embodiment of the invention, the method is characterized in that young spikes of true diploid hybrid F1 plants are taken for induction culture, placed in a dark condition, cultured at about 28 ℃ for about 30 days, gradually light yellow callus is grown out of the young spikes, then colchicine doubling is carried out on the callus, the callus is selected into a differentiation medium, placed in 15 hours of illumination, 9 hours of darkness and cultured at 28 ℃, after more than 30 days, the callus gradually shows green spots until bud seedlings are differentiated, then the callus is transferred into a rooting medium for emergence of seedlings, transplanted into a field for planting after seedling hardening, and seeds of hybrid tetraploids are harvested after mature plants are grown.

The invention has at least the following beneficial effects:

In order to solve the problem of utilizing the excellent genetic background of EE genome wild rice, the invention develops a technology of ' distant hybridization, embryo rescue and in-vitro doubling ' combined to efficiently create inter-genome heteropolyploid rice '. The technology is different from a gene introgression method of continuous backcrossing after hybridization on a diploid level, and utilizes whole genome transfer and polyploidization, namely, distant hybrid containing complete genome of wild rice is obtained through distant hybridization, and then hybrid heteropolyploidy is obtained through chromosome doubling, so that excellent genes of the wild rice and the distant hybrid vigor of the polyploidy are fully utilized. Aiming at the reproductive isolation barrier of cultivated rice and wild rice, the technology of hormone treatment, repeated pollination, hybrid embryo rescue, colchicine chromosome doubling and the like are comprehensively adopted, so that the heteropolyploid rice is efficiently obtained, part of AAEE tetraploid rice which can be matured is obtained at present, a good original technical system is created for the utilization of the excellent gene of EE wild rice, a method for breeding the polyploid rice variety among genomes is innovated, and precious resources are provided for the polyploid rice breeding theory.

Drawings

FIG. 1. Australian wild rice EE shade treatment;

FIG. 2. Australian wild rice EE flowering pollen-powder;

FIG. 3 is a diagram showing the growth morphology of young embryos after 12-15 days of bagging in wild hybridization pollination, wherein the right side diagram is an enlarged picture of the left side diagram;

FIG. 4. Embryo rescue of CX35-2X hybridized with EE young embryo, wherein the young embryo is taken as a legend, the left embryo is used for rescue and emergence, and the right embryo is used for rooting and seedling formation;

FIG. 5. Young embryo F1 generation plant young ear double differentiation into seedling hybridized with EE CX35-2X, young ear induction callus, double differentiation, rooting in colchicine sequentially from left to right;

FIG. 6.CX35/E stem junction site outer wall green and inner wall purple characteristics;

FIG. 7 shows the identification of the authenticity of F1 generation hybrid seedlings, QT5 is the female parent CX35-2X, the marking rate is 98.60%, F13-1 is the child generation of CX35-2X and EE, and the heterozygosity of 12 groups of chromosomes is realized;

Figure 8 morphological feature comparison of tetraploid seed with seed of parent: EE is a male parent seed, 9311-2X is a female parent seed, and tetraploid seeds after the sub-generation doubling are: YZ32/E+ -4X and CX35/E+ -4X, whereas tetraploid seeds were not obtained as a control group.

Detailed Description

In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.

Example 1:

a. Two typical indica-japonica lines (commercial indica-rice lines 9311-2X and Nippon-2X respectively) and two high-setting lines (CX 35-2X and YZ32-2X respectively) bred by a laboratory team for years are selected as female parent materials, and hybridization is carried out by taking Australian wild rice EE as male parent materials. Among them, the maternal materials CX35-2X and YZ32-2X have been reported in (Gan,L.,Huang,B.,Song,Z.et al.Unique Glutelin Expression Patterns and Seed Endosperm Structure Facilitate Glutelin Accumulation in Polyploid Rice Seed.Rice 14,61(2021).https://doi.org/10.1186/s12284-021-00500-0) literature, australian wild rice EE is reported in (Tan Yujuan, xu Yankang. Transformation study of insect resistance of Australian wild rice [ J ]. Proc. Southwest university of agriculture, 1998,020 (5): 557-562; song Lishuang, african biography, zhou Chuting, zhou Yong, & Liang Guohua (2021) & gt A method for rapid development of molecular markers specific to Australian wild rice. CN113308563A.) literature, all of which are biological materials known in the prior art. The applicant hereby ensures that CX35-2X, YZ-2X and Australian wild rice EE seeds can be issued to the public for 20 years from the date of filing.

B. Observing and determining that four female parent strains meet EE energy flowering period of male parent strain so as to pollinate

Australian wild rice EE is a short-day flowering rice line, and unlike four conventional long-day flowering adapted female parent rice lines, in the summer of Wuhan (long-day) the wild rice is subjected to a shading treatment for at least 28 days before the female parent flowering season, so that the wild rice can flower bud differentiation, red cloth is covered first and then black cloth is covered to shade illumination (as shown in figure 1), 17:30 of the evening in summer and 8:30 of the morning. Enabling the wild rice EE male parent and four conventional female parent lines to simultaneously heading and flowering during october through september (as in fig. 2);

Cutting half of the glume of female parent ear of cultivated rice, which is about to bloom, by scissors in summer evening of the previous day, picking anthers, sleeving a hybridization bag, hybridizing to begin to take anthers of wild rice EE which just bloom at two and half afternoon, at the moment, the pollen activity is strongest, pollinating the pistil column heads of four female parent strains which are already emasculated, and sleeving the hybridization bag (as shown in figure 3);

c. spraying exogenous hormone about 10 days after pollination hybridization

Because of the cross incompatibility between distant hybridized genomes, the hybridized progeny F1 can form young embryos but cannot form normal endosperm, so that exogenous spraying of hormone is required to maintain normal growth of the young embryos. The mixture ratio of the exogenous spray hormone is as follows: GA 50mg/ml+NAA 10mg/ml+2, 4-D2 mg/ml, continuously spraying exogenous hormone for about 10 days to enable the growth of the young embryo to reach the longest elongation degree (as in the embryo rescue culture medium of FIG. 4), and then carrying out in vitro culture by using the culture medium;

d. Embryo rescue and rooting seedling of embryo 12-15 days after hybridization bagging

Culturing the peeled young embryo for 12-15 days, culturing the young embryo with an improved culture medium for embryo saving, sterilizing the peeled young embryo on an ultra-clean workbench, placing the young embryo on the improved culture medium by forceps, placing the young embryo on the improved culture medium for 15 hours for illumination, culturing the young embryo at the temperature of 28 ℃ for 9 hours in darkness, and culturing the young embryo at the temperature of 28 ℃ for about 15 days, wherein the improved embryo saving culture medium is ：N6+6-BA0.5～2mg/L+NAA0.2mg/L+KT0.2～0.5mg/L+2,4-D0.1mg/L+GA0.5mg/L+Sucrose5.5％+asparagine0.5g/L+glutamine(Gln)0.5g/L, improved embryo saving culture medium, so that the nitrogen content required by rice growth is increased, the dosage and the proportion of hormone are adjusted to enable the culture medium to be more suitable for the growth of the young embryo, and the grown young embryo is transferred to a rooting culture medium on the ultra-clean workbench, so that the young embryo can grow into a seedling which can be transplanted to a field;

e. Determining the authenticity of hybrid

The rice is self-pollinated, so that on one hand, the female parent material is not thoroughly emasculated, and on the other hand, the hybrid authenticity of the hybrid seedlings needs to be identified because of the pollination condition in the emasculation period caused by the scattering of a small amount of active pollen when anthers are removed. The authenticity of the hybrid seedlings can be identified by the two modes of appearance morphological characteristics and molecular markers of the gene chip of the plants of the F1 generation of distant hybrid seedlings transplanted to the field,

Mode one: the appearance and the shape are observed, and the appearance and the shape are observed,

The simplest and most intuitive method for identifying the authenticity of hybrid seedlings in a field has two aspects: distant hybridization of the A genome and the E genome, because the gene distance is far, mature plants of the F1 generation of distant hybridization seedlings transplanted to the field are non-fertile or extremely low in fertility, and high sterility is a basis for preliminary identification; on the other hand, in the vegetative growth stage of the FI-generation hybrid plants, at each node of the stalk, the outer wall of the stalk is dark green, the inner wall of the stalk is purple, the purple is changed from node to node from dark, and the outer wall of the stalk of the conventional rice is green to form an appearance contrast, so that whether the F1 generation is a successfully hybridized plant is preliminarily judged (as shown in fig. 6).

Mode two: the molecular marker of the gene chip,

The leaves of four female parent lines and each of four F1 hybrid lines are respectively taken as gene chip molecular markers for identification, GSR40K is a newly designed high-density rice gene chip, and comprises polymorphic markers of rich Chinese rice resources, functional gene markers and haplotype markers of important agronomic characters, and markers for positioning QTL sites; GSR40K rice high-density whole genome SNP chip is based on the manufacturing technology of Illumina chip, contains 44263 sites, and the SNP sites are derived from the resequencing result of 4726 cultivars of all over the world, as shown in figure 7.

Screening 32887 high-quality sites according to genotyping results of the test sample and meeting the following criteria, wherein 32607 of the high-quality sites contain position coordinates, and 280 of the high-quality sites are transgenes or other probes;

1.GenTrain Score(The SNP cluster quality)>0.6；

2. the parental genotypes are homozygous (too many parental heterozygotes indicate poor quality markers, typically allowing less than 5% heterozygous or less);

3. the number of deletion genotypes was as small as possible (< 20%, except Indel markers);

4. the parting accuracy is high.

F. obtaining tetraploid seeds after doubling

The young spike of the true hybrid plant is taken for induction culture (as shown in figure 5), placed in a dark condition and cultured at about 28 ℃ for about 30 days, light yellow callus is gradually grown from the young spike, then colchicine doubling culture is carried out on the callus for about 48 hours, then the callus is selected into a differentiation culture medium and is placed under the condition of 2000 Lx-3000 Lx illumination for 15 hours, 9 hours are dark, the callus is gradually developed to be green until the bud is differentiated after more than 30 days, then the callus is transferred into a rooting culture medium for seedling emergence, and the rooting culture medium formula is 1/2MS+Sucrose2% + agar 7.5%o+0.2%permillage C+6-BA0.5 mg/L+NAA0.3mg/L+aspargine 0.5g/L+ glutamine (Gln) g/L. Transplanting the hybrid tetraploid into a field for planting after hardening seedlings, harvesting the seeds of the hybrid tetraploid (shown in figure 8) after growing into mature plants, doubling the first generation F1 of the hybrid field, and obtaining the tetraploid seeds of two strains YZ32/E+ -4X and CX35/E+ -4X.

Summary and discussion: because the gene distance between the Australian wild rice EE and the conventional rice AA is far, and the characters of hybridization incompatibility and filial generation sterility exist, the invention not only overcomes the situation of hybridization incompatibility and distant hybridization sterility or extremely low fruiting rate after multiple, but also utilizes the hybridization and doubling of the Australian wild rice with the conventional typical indica rice 9311-2X and the typical japonica rice Nippon-2X, and the seed description of 9311/E+ -4X and Nippon/E+ -4X is not obtained: compared with typical reference strain with common seed setting rate, the strain with high seed setting rate in the laboratory has great advantage in seed setting after distant hybridization and multiple. Tetraploid hybrid YZ32/E+ -4X and CX35/E+ -4X have been planted in the field for two seasons, the plants are stable and firm and the maturing rate is 15.92% and 21.14%, respectively, whereas conventional typical indica rice and japonica rice are used as a control group, and tetraploid hybrid cannot be obtained; the distant hybridization polyploidy breeding makes the gene combination favorable, utilizes the advantages of the distant hybridization seeds to enrich rice seed resources, and also preliminarily solves the problem of extremely low distant hybridization seed setting rate.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (6)

1. A method for creating a fertility tetraploid hybrid between a cultivated rice genome and an Australian wild rice genome comprises the steps of carrying out hybridization by taking CX35-2X or YZ32-2X as a female parent material and Australian wild rice EE as a male parent material to obtain a first-generation hybrid seedling; the first generation hybrid seedling is doubled by colchicine to obtain the seed of the hybrid tetraploid.

2. The method of claim 1, wherein prior to hybridization, wild rice EE in australia is masked to allow flower bud differentiation in wild rice, covered in the evening of summer, uncovered in the morning, and controlled to a light duration of no more than 9 hours so that both male and female parent lines of wild rice EE can be simultaneously heading and flowering between the bottom of august and the early september.

3. The method of claim 2, wherein the newly flowering anthers of wild rice EE are pollinated to the stigma of the female parent line that has been emasculated, followed by bagging.

4. A method according to claim 3, wherein the pollination hybridization is followed by a continuous 8-12 day addition of exogenous hormones in the following ratio: GA 50mg/ml+NAA 10mg/ml+2, 4-D2 mg/ml.

5. The method of claim 1, wherein the embryo of the embryo is saved and rooted for seedling 12-15 days after hybridization bagging, the peeled embryo is cultivated by using an improved culture medium for embryo saving after the embryo grows for 12-15 days, the peeled embryo is placed on an improved culture medium by forceps after being disinfected on an ultra-clean workbench, the embryo is placed under 15 hours of illumination and 9 hours of darkness and cultivated at 28 ℃ for about 15 days, so that the embryo can grow into a seedling, and the improved embryo saving culture medium is that ：N6+6-BA0.5～2mg/L+NAA0.2mg/L+KT0.2～0.5mg/L+2,4-D0.1mg/L+GA0.5mg/L+Sucrose5.5％+asparagine0.5g/L+glutamine(Gln)0.5g/L.

6. The method of claim 1, wherein young ears of true diploid hybrid F1 plants are taken for induction culture, placed in a dark condition, cultured at about 28 ℃ for about 30 days, gradually visible young ears grow light yellow callus, then colchicine doubling is carried out on the callus, the callus is selected into a differentiation medium, placed in 15 hours of illumination, 9 hours of darkness, cultured at 28 ℃, after more than 30 days, the callus gradually shows green spots until bud seedlings are differentiated, then the callus is transferred into a rooting medium for seedling emergence, transplanted into a field for planting after seedling hardening, and seeds of the hybrid tetraploids are harvested after mature plants are grown.