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

A method for creating fertile tetraploid hybrids between the genomes of cultivated rice and Australian wild rice

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a method for creating a fertile tetraploid hybrid between the genomes of cultivated rice (Oryza sativa) and Australian wild rice (Oryza australiensis).

Background technique

The genus Oryza has rich species resources. In addition to Asian cultivated rice (Oryza sativa) and African cultivated rice (O. glaberrima), there are more than 20 wild rice species belonging to 10 major gene groups. Wild rice has extremely rich genetic diversity and preserves many excellent genes that cultivated rice does not have or has disappeared. It is a gene bank for genetic improvement of cultivated rice. History has proved that the use of wild rice has effectively promoted the development and progress of rice breeding. However, when utilizing wild rice resources, due to the distant relationship between cultivated rice and wild rice, there are difficulties such as hybridization difficulties, hybrid death, and hybrid sterility. It is difficult to efficiently utilize the excellent genes of wild rice by using diploid hybridization and backcrossing. Polyploid rice breeding is divided into three types: inter-subspecies, inter-species, and inter-genome polyploid hybrid advantage utilization. At present, the utilization of inter-subspecies and inter-species hybrid advantages has achieved certain results, but the disadvantages are that the breeding time is long, there is no hybrid advantage, and it has not been able to break through the utilization of inter-subspecies hybrid advantages. Distant hybridization polyploidy breeding allows for favorable gene combinations, utilizing the advantages of distant hybrids to enrich rice seed resources, and also has the hybrid advantages of polyploidization, but there is still a problem that is difficult to overcome with low fruit set rates.

Australian wild rice is an EE genome, originally distributed in tropical Australia, and has excellent characteristics of resistance to brown planthoppers, black-tailed leafhoppers, drought resistance and high temperature resistance. However, due to the distant relationship between the main cultivated variety AA genome and it, there are serious reproductive barriers in hybridization, resulting in complete infertility. Therefore, there are serious technical barriers to the use of excellent genes in the EE genome. Although previous studies have reported that the EE genome Australian wild rice and the AA genome cultivated rice distant hybrid F1, and then continued to backcross with cultivated rice and obtained interspecific hybrid BC1F1 plants with the help of embryo rescue technology (Song Lishuang, Yi Chuandeng, 2021), repeated backcrossing continuously diluted the genetic background of the wild rice EE genome, and there are still certain limitations in the use of wild rice genes.

Summary of the invention

Based on the above reasons, the purpose of the present invention is to propose a method for creating and preliminarily identifying fertile tetraploid hybrids between the genomes of cultivated rice and Australian wild rice, overcoming the difficulties of distant hybridization and utilizing the dual advantages of distant hybridization and polyploidy to create new germplasm, which belongs to the category of breeding new rice varieties. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

On one hand, the present invention relates to a method for creating a fertile tetraploid hybrid between the genomes of cultivated rice and Australian wild rice, which comprises hybridizing CX35-2X or YZ32-2X as the maternal material and Australian wild rice EE as the paternal material to obtain first-generation hybrid seedlings; the first-generation hybrid seedlings are doubled with colchicine to obtain hybrid tetraploid seeds.

In a preferred embodiment of the present invention, it is characterized in that before hybridization, the Australian wild rice EE is covered with cloth so that the wild rice can differentiate flower buds, the cloth is covered in the summer evening and uncovered in the morning, and the light duration is controlled not to exceed 9 hours, so that the male and female lines of the wild rice EE can simultaneously head and bloom from the end of August to the beginning of September.

In a preferred embodiment of the present invention, it is characterized in that the anthers of wild rice EE that have just bloomed are taken and pollinated on the stigma of the emasculated female parent line, followed by a hybridization bag.

In a preferred embodiment of the present invention, it is characterized in that after pollination and hybridization, spraying and adding of exogenous hormones are carried out for 8-12 consecutive days, and the ratio of the exogenous spraying hormones is: GA 50mg/ml+NAA 10mg/ml+2,4-D 2mg/ml.

In a preferred embodiment of the present invention, it is characterized in that the embryos are rescued and rooted into seedlings 12-15 days after hybridization bagging: after the young embryos grow for 12-15 days, the stripped young embryos are cultured with a modified culture medium, which is called embryo rescue. After the stripped young embryos are disinfected on a clean bench, they are placed flat on the modified culture medium with tweezers and cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28°C. It takes about 15 days for the young embryos to grow into seedlings. The modified 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. The improved embryo rescue medium increases the nitrogen content required for rice growth. The grown seedlings are transferred to the rooting medium on the clean bench so that the seedlings can grow into mature seedlings that can be transplanted to the field.

In a preferred embodiment of the present invention, it is characterized in that the young spikelets of the true diploid hybrid F1 generation plants are taken for induction culture, placed in dark conditions, and cultured at about 28°C for about 30 days, and gradually pale yellow callus can be seen growing from the young spikelets, and then the callus is doubled with colchicine, and then it is picked into a differentiation medium and cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28°C. After more than 30 days, the callus gradually shows green spots until it differentiates into seedlings, and then it is transferred to a rooting medium for germination, and after hardening the seedlings, it is transplanted to the field for planting, and after growing into mature plants, the hybrid tetraploid seeds are harvested.

The present invention has at least the following beneficial effects:

In order to solve the problem of utilizing the excellent genetic background of wild rice in the EE genome, the present invention has developed a technology of "combining distant hybridization, embryo rescue and in vitro doubling to efficiently create inter-genomic allopolyploid rice". This technology is different from the gene introgression method of continuous backcrossing after hybridization at the diploid level. Instead, it uses the whole genome transfer and polyploidization, that is, obtaining distant hybrids containing the complete genome of wild rice through distant hybridization, and then obtaining hybrid allopolyploids through chromosome doubling, so as to make full use of the excellent genes of wild rice and the advantages of polyploid distant hybrids. In view of the reproductive isolation barrier between cultivated rice and wild rice, hormone treatment and repeated pollination, hybrid embryo rescue and colchicine chromosome doubling are comprehensively adopted to efficiently obtain allopolyploid rice. At present, some AAEE tetraploid rice that can bear fruit has been obtained, which has created a good original technical system for the utilization of excellent genes of EE wild rice, innovated the method of selecting polyploid rice varieties between genomes, and provided valuable resources for the theory of polyploid rice breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. EE shading treatment of Australian wild rice;

Figure 2. Australian wild rice EE blooms and sheds pollen;

Figure 3. Growth morphology of young embryos after 12-15 days of pollination bagging in wild hybridization, where the right picture is an enlarged picture of the left picture;

Figure 4. Embryo rescue of immature embryos of the hybrid of CX35-2X and EE, wherein the immature embryos are used as illustrations, the left image shows embryo rescued seedlings, and the right image shows rooted seedlings;

Figure 5. The young spikelets of the F1 generation plants of the hybrid of CX35-2X and EE doubled and differentiated into seedlings. From left to right, the young spikelets were callus induced, the callus doubled in colchicine, differentiated, and rooted;

Figure 6. Characteristics of green outer wall and purple inner wall at the node of CX35/E stem;

Figure 7. Identification of the authenticity of the F1 hybrid seedlings, QT5 is the female parent CX35-2X, the marker rate is 98.60%, F13-1 is the offspring of CX35-2X and EE, the heterozygous status of 12 sets of chromosomes;

Figure 8. Comparison of morphological characteristics between tetraploid seeds and parental seeds: EE is the paternal seed, 9311-2X is the maternal seed, and the tetraploid seeds after doubling in the second generation are: YZ32/E+-4X and CX35/E+-4X, while no tetraploid seeds were obtained from the control group.

Detailed ways

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

Embodiment 1:

a. Select two typical stable and conventional indica and japonica lines (the commercially available indica lines 9311-2X and Nipponbare-2X) and two high-fruiting lines (CX35-2X and YZ32-2X) bred by the laboratory team over many years as the female parent materials, and use Australian wild rice EE as the male parent material for hybridization. Among them, the maternal materials CX35-2X and YZ32-2X have been reported in the literature (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), and Australian wild rice EE has been reported in the literature (Tan Yujuan, Xu Yankang. Research on the breeding of insect resistance in Australian wild rice [J]. Journal of Southwest Agricultural University, 1998, 020 (5): 557-562; Song Lishuang, Yi Chuandeng, Zhou Chuting, Zhou Yong, & Liang Guohua. (2021). A method for rapid development of Australian wild rice-specific molecular markers. CN113308563A.), both of which are biological materials known in the prior art. The applicant hereby guarantees that it will be able to distribute CX35-2X, YZ32-2X and Australian wild rice EE seeds to the public within 20 years from the date of application.

b. Observe and confirm that the four female lines and the male line EE can meet at the same flowering period to carry out pollination

Australian wild rice EE is a short-day flowering rice strain, which is different from the four conventional long-day flowering female rice strains. In the Wuhan summer (long-day), before the female plant flowering season, the wild rice must be covered with cloth for at least 28 days to allow the wild rice to differentiate flower buds. First, cover with red cloth and then with black cloth to block light (as shown in Figure 1). Cover the cloth at around 17:30 in the evening of summer and remove it at around 8:30 in the morning. This allows the wild rice EE male plant and the four conventional female plant strains to simultaneously produce heading and flowering from August to September (as shown in Figure 2);

On the summer evening of the previous day, cut the husk of the cultivated rice female parent ear that was heading and about to bloom in half with scissors, pick out the anthers, and put them in the hybridization bag. At 2:30 pm on the day of hybridization, start to take the anthers of the wild rice EE that has just bloomed. At this time, the pollen activity is the strongest. Pollinate the stigma of the pistil of the four female parent lines that have been emasculated, and then put them in the hybridization bag (as shown in Figure 3);

c. After pollination and hybridization, spray and add exogenous hormones for about 10 consecutive days.

Due to the hybridization incompatibility between genomes of distant hybridization, the hybrid offspring F1 can form immature embryos but cannot form normal endosperm, so exogenous spraying of hormones is required to enable the immature embryos to maintain normal growth. The exogenous spraying hormone ratio is: GA 50mg/ml+NAA 10mg/ml+2,4-D 2mg/ml. After spraying the exogenous hormones for about 10 days continuously, the growth of the immature embryos reaches the longest elongation (as shown in the immature embryo morphology in the embryo rescue medium in Figure 4), and then the medium is used for in vitro culture;

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

After the young embryos grow for 12-15 days, the peeled young embryos are cultured with a modified culture medium, which is called embryo rescue. After the peeled young embryos are generally disinfected on a clean bench, they are placed flat on the modified culture medium with tweezers and cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28°C. After about 15 days, the young embryos can grow into seedlings. The modified 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. The modified embryo rescue culture medium increases the nitrogen content required for rice growth, adjusts the dosage and ratio of hormones to make the culture medium more suitable for the growth of young embryos, and the grown seedlings are transferred to the rooting culture medium on the clean bench so that the seedlings can grow into mature seedlings that can be transplanted to the field.

e. Determine the authenticity of the hybrid

Rice is self-pollinating. On the one hand, due to the incomplete emasculation of the maternal material, and on the other hand, due to the scattering of a small amount of active pollen during the removal of the anthers, pollination occurs during the emasculation period. Therefore, it is necessary to identify the authenticity of the hybrid seedlings. The authenticity of the hybrid seedlings of the F1 generation of distant hybrid seedlings transplanted to the field can be identified by both appearance morphological characteristics and gene chip molecular markers.

Method 1: Observation of appearance and morphology,

There are two simplest and most intuitive methods for identifying the authenticity of hybrid seedlings in the field: distant hybridization of the A genome and the E genome. Due to the long genetic distance, the mature plants of the F1 generation of distant hybrid seedlings transplanted to the field are infertile or have extremely low fertility. High sterility is a basis for preliminary identification; on the other hand, in the vegetative growth stage of the F1 generation hybrid plants, at each node of the stem, the outer wall of the stem is green and the inner wall is purple, and the purple changes from dark to light from node to node, which forms an appearance contrast with the green inner and outer walls of the stem of conventional rice, and a preliminary judgment is made as to whether the F1 generation is a successfully hybridized plant (as shown in Figure 6).

Method 2: Gene chip molecular markers,

Leaves from the four maternal lines and each plant of the four F1 hybrid lines were taken for gene chip molecular marker identification. GSR40K is a newly designed high-density rice gene chip, which contains polymorphic markers of rich Chinese rice resources, functional gene markers and haplotype markers of important agronomic traits, and markers for locating QTL loci. The GSR40K rice high-density whole-genome SNP chip is a SNP chip made based on Illumina chip manufacturing technology, containing 44,263 loci. The SNP loci are derived from the resequencing results of 4,726 cultivated rice varieties from all over the world, as shown in Figure 7.

A total of 32,887 high-quality loci were screened based on the genotyping results of the test samples and meeting the following criteria, of which 32,607 contained position coordinates and 280 were transgenic or other probes;

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

2. The parental genotype is homozygous (too many parents are heterozygous, indicating that the marker quality is poor, and usually less than 5% heterozygous or less is allowed);

3. The number of missing genotypes should be as small as possible (<20%, except for Indel markers);

4. The accuracy of classification is high.

f. Obtain tetraploid seeds after doubling

Take the young spikelets of real hybrid plants for induction culture (as shown in Figure 5), place them in dark conditions, and culture them at about 28°C for about 30 days. Gradually, light yellow callus can be seen growing from the young spikelets. Then, the callus is double-cultured with colchicine for about 48 hours, and then it is picked into the differentiation medium and placed under light conditions of 2000Lx~3000Lx for 15 hours, dark for 9 hours, and cultured at 28°C. After more than 30 days, the callus gradually shows green spots until it differentiates into seedlings. It is then transferred to the rooting medium for germination. The rooting medium formula is 1/2MS+Sucrose2%+agar7.5‰+0.2‰C+6-BA0.5mg/L+NAA0.3mg/L+asparagine0.5g/L+glutamine(Gln)0.5g/L. After hardening, the seedlings are transplanted to the field for planting. After they grow into mature plants, the tetraploid seeds of the hybrid are harvested (as shown in Figure 8). After doubling the first generation F1 of the wild hybrid, tetraploid seeds of the two lines YZ32/E+-4X and CX35/E+-4X are obtained.

Summary and discussion: Due to the long genetic distance between Australian wild rice EE and conventional rice AA, there are traits of hybrid incompatibility and offspring sterility. The present invention not only overcomes hybrid incompatibility, but also overcomes the sterility of distant hybridization or extremely low fruit setting rate after polyploidization. By hybridizing and doubling Australian wild rice with conventional typical indica rice 9311-2X and typical japonica rice Nipponbare-2X, no seeds of 9311/E+-4X and Nipponbare/E+-4X were obtained. Description: Compared with typical reference strains with average fruit setting rate, the strains with high fruit setting rate in this laboratory have great advantages in fruit setting after distant hybridization polyploidization. The tetraploid hybrid varieties YZ32/E+-4X and CX35/E+-4X have been planted in the field for two seasons. The plants have stable fruit set and the fruit set rates are 15.92% and 21.14%, respectively. However, the conventional typical indica rice and japonica rice used as the control group failed to produce tetraploid hybrid varieties. Distant hybridization polyploidy breeding has enabled favorable gene combinations, utilized the advantages of distant hybrids to enrich rice seed resources, and the hybrid advantages of polyploidization have preliminarily solved the problem of extremely low fruit set rates in distant hybridization.

The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims (6)

1. A method for creating a fertile tetraploid hybrid between the genomes of cultivated rice and Australian wild rice, wherein CX35-2X or YZ32-2X is used as the maternal material and Australian wild rice EE is used as the paternal material for hybridization to obtain first-generation hybrid seedlings; the first-generation hybrid seedlings are doubled with colchicine to obtain hybrid tetraploid seeds. 2. The method according to claim 1 is characterized in that before hybridization, the Australian wild rice EE is covered with cloth so that the wild rice can differentiate flower buds, the cloth is covered in the evening in summer and uncovered in the morning, and the light duration is controlled to be no more than 9 hours, so that the male and female lines of the wild rice EE can simultaneously head and bloom from the end of August to the beginning of September. 3. The method according to claim 2, characterized in that the anthers of wild rice EE that have just bloomed are taken and pollinated on the stigma of the female parent line that has been emasculated, and then a hybridization bag is put on. 4. The method according to claim 3 is characterized in that after pollination and hybridization, spraying and adding of exogenous hormones are carried out for 8-12 consecutive days, and the ratio of exogenous spraying hormones is: GA 50mg/ml+NAA 10mg/ml+2,4-D 2mg/ml. 5. method according to claim 1 is characterized in that, embryo rescue and rooting of embryos in 12-15 days after hybridization bagging: after young embryos grow for 12-15 days, the young embryos of peeling off are cultivated with improved culture medium and are called embryo rescue, the young embryos of peeling off are placed flat on improved culture medium with tweezers after disinfection on the clean bench, placed in 15 hours of illumination, 9 hours of darkness, and cultivated under 28 ℃ condition, so that young embryos can grow seedlings in about 15 days, and 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. 6. The method according to claim 1 is characterized in that the spikelets of the true diploid hybrid F1 generation plants are taken for induction culture, placed in dark conditions, and cultured at about 28°C for about 30 days, and gradually pale yellow callus can be seen growing from the spikelets, then the callus is doubled with colchicine, and then it is picked into a differentiation medium and cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28°C. After more than 30 days, the callus gradually shows green spots and blocks until it differentiates into seedlings, and then it is transferred to a rooting medium for germination, and after hardening, it is transplanted to the field for planting, and after it grows into a mature plant, the hybrid tetraploid seeds are harvested.