A method for creating a fertile tetraploid hybrid between an oryza sativa and an oryza australiensis genome

Abstract

The application relates to a method for creating a fertile tetraploid hybrid between a cultivated rice and an Oryza australiensis genome, which comprises the following steps: using CX35-2X or YZ32-2X as a female parent material, and Oryza australiensis EE as a male parent material to perform hybridization, thereby obtaining first-generation hybrid seedlings; doubling the first-generation hybrid seedlings by colchicine, thereby obtaining hybrid tetraploid seeds. The application utilizes the whole genome transfer and polyploidization, that is, a distant hybrid containing a complete wild rice genome is obtained through distant hybridization, and then a hybrid allopolyploid is obtained through chromosome doubling, so that the excellent genes of the wild rice and the distant hybridization advantage of the polyploid are fully utilized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for creating a fertile tetraploid hybrid between Oryza sativa and Oryza australiensis genomes. BACKGROUND

[0002] Rice has rich species resources. In addition to Oryza sativa and O. glaberrima, there are more than 20 wild rice species, which belong to 10 genomes. Wild rice has extremely rich genetic diversity and preserves many excellent genes that cultivated rice does not have or has lost, and is a gene treasure house for genetic improvement of cultivated rice. History has proved that the use of wild rice has greatly promoted the development and progress of rice breeding. However, in the use of wild rice resources, due to the distant genetic relationship between cultivated rice and wild rice, there are difficulties in hybridization, hybrid death, and hybrid sterility. It is difficult to efficiently use the excellent genes of wild rice by using diploid level hybridization and backcrossing. There are three types of intersubspecific, intergeneric and intergenomic polyploid hybridization advantage utilization, and at present, intersubspecific and intergeneric hybridization advantage utilization has achieved certain results, but the disadvantages are long breeding time and no hybridization advantage, and the intersubspecific hybridization advantage utilization has not been broken through. Polyploid breeding by distant hybridization makes it possible to combine favorable genes, use the advantages of distant hybridization species to enrich rice resources, and have the hybridization advantage of polyploidization, but there is still the problem of low seed setting rate.

[0003] Oryza australiensis is of EE genome, originally distributed in the tropics of Australia, and has excellent characteristics of resistance to brown planthopper, resistance to black-tailed leafhopper, drought tolerance and high temperature tolerance. However, due to the distant genetic relationship between the main cultivated variety AA genome and EE genome, hybridization has serious reproductive obstacles leading to complete sterility, so the use of EE genome excellent genes has serious technical obstacles. Although previous studies have reported that on the basis of F1 of EE genome O. australiensis and AA genome cultivated rice distant hybridization, subsequent backcrossing with cultivated rice and the use of embryo rescue technology have obtained intergeneric hybrid BC1F1 plants (Song Lishuang, Yi Chuan Deng, 2021), but repeated backcrossing continuously dilutes the genetic background of wild rice EE genome, and there are still certain limitations in the use of wild rice genes. SUMMARY

[0004] Based on the above reasons, the purpose of the present application is to propose a method for creating and preliminarily identifying a fertile tetraploid hybrid between Oryza sativa and Oryza australiensis genomes, overcoming the difficulty of distant hybridization and creating new germplasm by using the double advantages of distant hybridization and polyploidy, which belongs to the category of new rice variety breeding. Specifically, in order to achieve the purpose of the present application, the present application proposes the following technical solutions:

[0005] The present application relates to a method for creating a fertile tetraploid hybrid between Oryza sativa and Oryza australiensis, which comprises the following steps: using CX35-2X or YZ32-2X as the female parent material, and O. australiensis EE as the male parent material to perform hybridization, and obtaining the first generation hybrid seedlings; doubling the first generation hybrid seedlings by colchicine to obtain hybrid tetraploid seeds.

[0006] In a preferred embodiment of the present application, the O. australiensis EE is covered with a cloth before hybridization to allow the wild rice to differentiate flower buds, the cloth is covered in the evening and uncovered in the morning to control the light duration to less than 9 hours, so that the wild rice EE male parent and female parent lines can simultaneously bloom in late August to early September.

[0007] In a preferred embodiment of the present application, the anther of the wild rice EE is taken and pollinated to the stigma of the female parent line which has been emasculated, and then a hybridization bag is put on.

[0008] In a preferred embodiment of the present application, after the pollination and hybridization, an exogenous hormone is sprayed and added for 8-12 days, and the exogenous hormone spraying ratio is: GA 50mg / ml+NAA 10mg / ml+2,4-D 2mg / ml.

[0009] In a preferred embodiment of the present application, the embryo rescue and rooting of the embryo are performed 12-15 days after the hybridization bagging: after the embryo grows for 12-15 days, the peeled embryo is cultured in a modified medium for embryo rescue, the peeled embryo is sterilized on a clean bench, and then placed flat on the modified medium with tweezers, and cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28℃, so that the embryo can grow into seedlings in about 15 days, and the modified embryo rescue medium is: N6+6-BA 0.5-2mg / L+NAA 0.2mg / L+KT 0.2-0.5mg / L+2,4-D 0.1mg / L+GA 0.5mg / L+Sucrose 5.5%+asparagine 0.5g / L+glutamine (Gln) 0.5g / L. The modified embryo rescue medium increases the nitrogen content required for the growth of rice, and the seedlings are transferred to a rooting medium on a clean bench to grow into seedlings that can be transplanted to the field;

[0010] In a preferred embodiment of the present application, the young panicles of the real diploid hybrid F1 generation plants are taken for induction culture, placed in dark conditions, cultured at about 28°C for about 30 days, and gradually the young panicles grow into yellow callus. Then the callus is doubled by colchicine, and then it is picked into differentiation medium and placed in 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 buds are differentiated, and then the buds are transferred into rooting medium to grow into seedlings, and after hardening, the seedlings are transplanted into the field for planting, and the mature plants are harvested to obtain hybrid seeds of tetraploid.

[0011] The present application has at least the following beneficial effects:

[0012] To solve the problem of utilizing the excellent genetic background of EE genome wild rice, the present application develops a technology of "high-efficiency creation of inter-genome allopolyploid rice by combining distant hybridization, embryo rescue and in vitro doubling". This technology is different from the gene introgression method by continuous backcrossing after hybridization at the diploid level, but by whole genome transfer and polyploidization, i.e. by obtaining a distant hybrid containing the complete genome of wild rice through distant hybridization, and then obtaining a hybrid allopolyploid through chromosome doubling, so as to fully utilize the excellent genes of wild rice and the distant hybridization advantage of polyploid. In view of the reproductive isolation barrier between cultivated rice and wild rice, the technologies of hormone treatment, repeated pollination, hybrid embryo rescue, and colchicine chromosome doubling are comprehensively used to efficiently obtain allopolyploid rice. At present, some AAEE tetraploid rice with seed setting has been obtained, which creates a good original technology system for the utilization of EE wild rice excellent genes, innovates the method of inter-genome polyploid rice breeding, and provides valuable resources for polyploid rice breeding theory. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 . Oryza australiensis EE covering treatment;

[0014] Figure 2 . Oryza australiensis EE flowering and pollen shedding;

[0015] Figure 3 . Growth morphology of young embryos after hybridization pollination and bagging of cultivated rice and wild rice for 12-15 days, wherein the right side figure is an enlarged picture of the left side figure;

[0016] Figure 4 . Embryo rescue of hybrid young embryos of CX35-2X and EE, wherein the young embryo of is taken as an example, the left figure shows seedling emergence after embryo rescue, and the right figure shows rooting and seedling growth;

[0017] Figure 5 . Young panicle doubling and differentiation into seedlings of F1 generation plants of hybrid young embryos of CX35-2X and EE, from left to right, are induction of young panicle into callus, doubling of callus in colchicine, differentiation, and rooting;

[0018] Figure 6 the characteristic of green outer wall and purple inner wall of CX35 / E stalk joint position;

[0019] Figure 7 Identifying the authenticity of F1 hybrid seedlings, QT5 is the female parent CX35-2X, the marker rate is 98.60%, F13-1 is the child generation of CX35-2X and EE, the heterozygous condition of 12 groups of chromosomes;

[0020] Figure 8 Comparison of morphological characteristics of tetraploid seeds and seeds of parents: EE is the father seed, 9311-2X is the mother seed, the tetraploid seeds of the doubled child generation are YZ32 / E+-4X and CX35 / E+-4X, and the control group fails to obtain tetraploid seeds. DETAILED DESCRIPTION

[0021] In order to further understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0022] Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available products, which can be purchased through commercial channels.

[0023] Example 1:

[0024] a. Select two typical indica japonica conventional lines (9311-2X and Nipponbare-2X, which are commercially available indica rice lines, and two high seed setting lines (CX35-2X and YZ32-2X) bred by the laboratory team over the years as female materials, and cross with O. australiensis EE as male material. Among them, the female 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 O. australiensis EE is reported in the literature (Tan Yujuan, Xu Yankang. Research on the transfer of O. australiensis insect resistance [J]. Journal of Southwest Agricultural University, 1998, 020(5):557-562; Song Lishuang, Yi Chuan Deng, Zhou Chuting, Zhou Yong, & Liang Guohua. (2021). A method for rapidly developing O. australiensis specific molecular markers. CN113308563A.), which are known biological materials in the prior art. The applicant hereby guarantees that the seeds of CX35-2X, YZ32-2X and O. australiensis EE can be distributed to the public for 20 years from the date of application.

[0025] b. Observe and determine that the four female lines can meet the flowering period of the male line EE to pollinate

[0026] O. australiensis EE is a short-day flowering rice line, unlike the four conventional long-day flowering female rice lines, which need to be covered for at least 28 days before the female flowering season in Wuhan in summer (long-day) to allow the wild rice to differentiate flower buds. Cover the cloth with red cloth first and then black cloth to block the light (such as Figure 1 ), cover the cloth at around 17:30 in the summer evening, and uncover the cloth at around 8:30 in the morning. This allows the wild rice EE male parent and the four conventional female parent lines to bloom at the same time from August to September (such as Figure 2 );

[0027] The day before, the summer evening, the spike of the cultivated rice female parent that will bloom soon is cut in half with scissors, and the anthers are picked out and put into a hybridization bag. On the day of hybridization, the anthers of the wild rice EE just after flowering are taken at 2:30 pm, when the pollen activity is strongest, and pollinated onto the pistil stigma of the four female parent lines that have been emasculated, and then the hybridization bag is put on (such as Figure 3 );

[0028] c. Spraying of exogenous hormones for 10 days after pollination and hybridization

[0029] Due to the cross-incompatibility between genomes of distant hybridization, the hybrid offspring F1 can form a young embryo but cannot form a normal endosperm, so it is necessary to spray exogenous hormones to enable the young embryo to maintain normal growth. The exogenous hormone spraying ratio is: GA 50 mg / ml + NAA 10 mg / ml + 2,4-D 2 mg / ml, and the continuous spraying of exogenous hormones for about 10 days enables the growth of the young embryo to reach the maximum elongation (such as Figure 4 Embryo rescue medium for young embryo morphology), followed by in vitro culture with the medium;

[0030] d. Embryo rescue and rooting of hybrid seedlings 12-15 days after bagging

[0031] After the growth of the young embryo for 12-15 days, the peeled young embryo is cultured with the improved medium for embryo rescue. The peeled young embryo is placed on the improved culture medium with tweezers after general disinfection on the clean bench, and is cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28°C. The young embryo can grow into seedlings in about 15 days. The improved embryo rescue medium is: N6 + 6-BA 0.5-2 mg / L + NAA 0.2 mg / L + KT 0.2-0.5 mg / L + 2,4-D 0.1 mg / L + GA 0.5 mg / L + Sucrose 5.5% + asparagine 0.5 g / L + glutamine (Gln) 0.5 g / L. The improved embryo rescue medium increases the nitrogen content required for rice growth, adjusts the amount and ratio of hormones to make the culture medium more suitable for the growth of young embryos. The seedlings grown can be transferred to the rooting medium on the clean bench to grow into seedlings that can be transplanted to the field;

[0032] e. Determination of the authenticity of hybrid seeds

[0033] Rice is self-pollinated. On the one hand, there is incomplete detasseling of the female material, and on the other hand, a small amount of active pollen is scattered when the anthers are removed, so there is pollination during detasseling. Therefore, it is necessary to identify the hybrid authenticity of hybrid seedlings. The plants of the F1 generation of distant hybrid seedlings transplanted to the field can be identified for hybrid authenticity from the two ways of external morphological characteristics and gene chip molecular markers,

[0034] Method one: morphological observation,

[0035] The simplest and most intuitive method for identifying the authenticity of hybrid seedlings in the field has two aspects: A genome and E genome distant hybridization. Due to the long distance between genes, the mature plants of the F1 generation of the transplanted distant hybrid seedlings are infertile or have very low fertility. High sterility is a preliminary basis for identification. On the other hand, during the vegetative growth stage of the FI generation hybrid plants, the outer wall of the stem at each node is green, and the inner wall is purple. The purple color changes from dark to light from node to internode, which is in contrast to the green color of the inner and outer walls of the conventional rice stem, and the appearance is different. Preliminary judgment of F1 generation whether it is a successful hybrid plant (as shown in Figure 6 ).

[0036] Method two: gene chip molecular markers,

[0037] The leaves of the four female parent lines and the leaves of each F1 hybrid line were taken for gene chip molecular marker identification. GSR40K is a newly designed high-density rice gene chip, which contains a large number of polymorphic markers of Chinese rice resources, functional gene markers of important agronomic traits, haplotype markers, and markers for QTL site positioning, etc. GSR40K rice high-density whole genome SNP chip is a SNP chip based on Illumina chip manufacturing technology, containing 44263 sites. The SNP sites are derived from the resequencing results of 4726 cultivated rice varieties from all over the world, as shown in Figure 7 .

[0038] According to the genotyping results of the test samples and meeting the following standards, 32887 high-quality sites were screened, of which 32607 contained position coordinates, and 280 were transgenic or other probes;

[0039] 1. GenTrain Score (The SNP cluster quality) > 0.6;

[0040] 2. Parent genotype is homozygous (too many parent heterozygotes indicate poor marker quality, usually allow less than 5% heterozygous or less);

[0041] 3. The number of missing genotypes is as few as possible (<20%, except Indel markers);

[0042] 4. High accuracy of genotyping.

[0043] f. After doubling, four sets of seeds are obtained

[0044] Take the young panicles of the true hybrid plants for induction culture (as shown in Figure 5), placed in dark conditions, about 28℃ for about 30 days, gradually visible yellowish callus out of the young ear, then the callus is doubled culture 48h or so, and then it is picked into differentiation medium placed in light 2000Lx-3000Lx conditions for 15 hours, 9 hours dark, 28℃ conditions for culture, more than 30 days after the callus gradually showed green point fast until differentiation of seedlings, and then it is transferred into rooting medium seedling, rooting medium formula is 1 / 2MS + Sucrose 2% + agar 7.5‰ + 0.2‰C + 6-BA 0.5mg / L + NAA 0.3mg / L + asparagine 0.5g / L + glutamine (Gln) 0.5g / L. After acclimatization, transplanting to the field planting, after growing into mature plants, harvesting hybrid four-ploid seeds (such as Figure 8 )Wild hybrid F1 doubling obtained YZ32 / E+-4X and CX35 / E+-4X two lines of tetraploid seeds.

[0045] Summary and discussion: Because the gene distance between Australian wild rice EE and conventional rice AA is far, there are hybrid incompatibility, sterile traits, the present application not only overcomes the hybrid incompatibility, but also overcomes the distant hybrid sterility or the low seed setting rate after polyploidization, using Australian wild rice and conventional typical indica rice 9311-2X and typical japonica rice Nipponbare-2X hybridization and doubling, and no 9311 / E+-4X and Nipponbare / E+-4X seeds are obtained, which shows that compared with the typical reference lines with general seed setting rate, the high seed setting rate lines in the laboratory have great advantages in distant hybrid polyploidization. Tetraploid hybrids YZ32 / E+-4X and CX35 / E+-4X have been planted in the field for two seasons, and the plants are stable and the seed setting rates are 15.92% and 21.14% respectively, while the conventional typical indica rice and japonica rice as a control group failed to obtain tetraploid hybrids; distant hybrid polyploid breeding makes use of the advantages of distant hybrids to enrich rice resources, and the hybridization advantage of polyploidization preliminarily solves the problem of low seed setting rate of distant hybridization.

[0046] The above describes the preferred embodiments of the present application, but it is not intended to limit the present application. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present application.

Claims

1.A method for creating fertile tetraploid hybrids between Oryza sativa and Oryza australiensis, which comprises the following steps: using CX35-2X or YZ32-2X as the female parent material, and O. australiensis EE as the male parent material to perform hybridization, and obtaining the first generation hybrid seedlings; doubling the first generation hybrid seedlings by colchicine to obtain hybrid tetraploid seeds; before hybridization, covering the O. australiensis EE to make the wild rice perform flower bud differentiation, covering the cloth in the evening and uncovering it in the morning to control the light duration to be less than 9 hours, so that the male parent and female parent lines of the wild rice EE can both bloom in the end of August to the beginning of September; the hybridization refers to taking the anthers of the wild rice EE just after blooming to pollinate the stigma of the female parent line after emasculation, then covering the hybridization bag, and continuously spraying exogenous hormones for 8-12 days after hybridization, the exogenous hormone spraying ratio is: GA 50mg / ml+NAA10mg / ml+2, 4-D 2mg / ml, embryo rescue and rooting of the seedlings 12-15 days after hybridization: after the embryo grows for 12-15 days, the peeled embryo is cultured in the improved medium for embryo rescue, the peeled embryo is placed on the improved medium in the sterile workbench after sterilization, and is cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28℃, so that the embryo can grow into seedlings in 15 days, the improved embryo rescue 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; taking the young panicles of the real diploid hybrid F1 generation plants to perform induction culture, and culturing under dark conditions at 28℃ for 30 days, and gradually yellow callus can be seen on the young panicles, then the callus is doubled by colchicine, and is then picked into the differentiation medium and cultured under the conditions of 15 hours of light, 9 hours of darkness, and 28℃, and after 30 days or more, the callus gradually shows green spots and then differentiates into seedlings, which are then transferred into the rooting medium to grow into seedlings, and after hardening, the seedlings are transplanted into the field to grow into mature plants, and then the hybrid tetraploid seeds are harvested.

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