阅读说明：本技术 一种快速选育空间诱变矮杆和低镉积累水稻的方法 (Method for rapidly breeding space mutation short-stalk and low-cadmium accumulation rice ) 是由 卢卫红 曾德永 于 2021-07-27 设计创作，主要内容包括：一种快速选育空间诱变矮杆和低镉积累水稻的方法,属于水稻育种技术领域。所述方法为：对水稻种子进行空间诱变处理得到SP0代种子；种植收获主穗种子得到SP1代,种植SP1代种子得到SP2代,在其中选择具有目标性状1的突变株连续严格自交到SP5代；根据目标性状2设计查找目标基因OsHMA3；根据目标性状2基因序列设计用于qRT-PCR检测的引物；将具有目标性状2的水稻突变株在三叶期移栽种植；严格自交到SP7代,获得稳定遗传且满足目标性状的单株。本发明利用返回式卫星搭载水稻种植进行空间诱变,使得种子通过空间辐射和微重力下过产生高频、有益的突变,这减少了耗费在挑选不符合目标性状变异株上的时间,也避免了转基因技术带来的不安全因数。(A method for rapidly breeding space mutation short-stalk and low-cadmium accumulation rice belongs to the technical field of rice breeding. The method comprises the following steps: carrying out space mutagenesis treatment on rice seeds to obtain SP0 generation seeds; planting and harvesting main ear seeds to obtain SP1 generation, planting SP1 generation seeds to obtain SP2 generation, and selecting mutant strains with the target property 1 to be continuously and strictly selfed to SP5 generation; designing and searching a target gene OsHMA3 according to the target character 2; designing a primer for qRT-PCR detection according to the gene sequence of the target character 2; transplanting and planting the rice mutant strain with the target character 2 in a three-leaf stage; and strictly selfing to SP7 generation to obtain a single plant which is stably inherited and meets the target traits. According to the invention, the recoverable satellite carried rice is used for spatial mutagenesis, so that high-frequency beneficial mutation is generated under the conditions of spatial radiation and microgravity of seeds, the time consumed for selecting variant plants which do not accord with target characters is reduced, and unsafe factors caused by a transgenic technology are avoided.)

1. A method for rapidly breeding space mutation dwarf rice and low cadmium accumulation rice is characterized by comprising the following steps:

(1) carrying out space mutagenesis treatment on rice seeds to obtain SP0 generation seeds;

(2) the recovered SP0 generation seeds are planted, main ear seeds are harvested to obtain SP1 generation, SP1 generation seeds are planted to obtain SP2 generation, and mutant strains with the target character 1 are selected from the SP2 generation and are continuously and strictly selfed to the SP5 generation;

(3) searching a target Gene in a Gene Bank database according to the design of the target trait 2, wherein the target Gene is OsHMA3, and the Gene symbol is as follows: LOC 4342783;

(4) designing a primer for qRT-PCR detection according to the gene sequence of the target character 2, wherein the forward primer is as follows: GCAAGTCAAGCCACCCAATG, the reverse primer is: ACGCCCTTGATCATCTCACC, respectively;

(5) transplanting the rice mutant strain with the target character 2 to a field with a cadmium pollution background for planting in the three-leaf period;

(6) and (5) further strictly selfing until SP7 generation, and obtaining a single plant which is stably inherited and meets the target traits.

2. The method for rapidly breeding the space-mutagenized dwarf rice and the rice with low cadmium accumulation as claimed in claim 1, which is characterized in that: in the step (1), the rice seed is Dongnong 416.

3. The method for rapidly breeding the space-mutagenized dwarf rice and the rice with low cadmium accumulation as claimed in claim 1, which is characterized in that: in the step (1), the space mutagenesis treatment is a space environment, and a satellite carried by rice flies in an orbit for 10-20 days.

4. The method for rapidly breeding the space-mutagenized dwarf rice and the rice with low cadmium accumulation as claimed in claim 1, which is characterized in that: in the step (2), the target character 1 is dwarf, and phenotype identification is carried out on all SP2 generation plants to determine a mutant strain with the target character 1.

5. The method for rapidly breeding the space-mutagenized dwarf rice and the rice with low cadmium accumulation as claimed in claim 1, which is characterized in that: in the step (3), the target character 2 is low cadmium content.

6. The method for rapidly breeding the space-mutagenized dwarf rice and the rice with low cadmium accumulation as claimed in claim 1, which is characterized in that: in the step (5), mRNA of SP5 generation plants with the target character 1 is extracted and qRT-PCR detection is carried out.

7. The method for rapidly breeding the space-mutagenized dwarf rice and the rice with low cadmium accumulation as claimed in claim 1, which is characterized in that: the mutant strain with the target gene OsHMA3 overexpressed is a mutant strain with the target character 2.

8. The method for rapidly breeding the space-mutagenized dwarf rice and the rice with low cadmium accumulation as claimed in claim 1, which is characterized in that: in the step (6), the identification is carried out to evaluate the cadmium content, the plant height and the thousand seed weight of the SP7 generation plants.

Technical Field

The invention belongs to the technical field of rice breeding, and particularly relates to a method for quickly breeding space mutation short-stalk and low-cadmium-accumulation rice.

Background

The earliest aerospace embarkation and space mutagenesis experiment in China is when the 9 th recoverable scientific experiment satellite is launched at 8 months and 5 days in 1987. Scientists in China screen crop seeds such as rice, wheat, tomatoes, green peppers and other biological materials for the first time, and the beneficial attempt of carrying out space flight and space mutation experiments of the space flight is carried out by means of the ninth recoverable satellite, so that the scientists in China become the beginning of the breeding business of space flight engineering in China. The variation frequency of the rice in nature is 20 ten-thousandth, the variation frequency of the rice after space-borne space mutagenesis treatment reaches about a few percent, and the variation amplitude is obvious. The variation level of the rice character generated by space mutagenesis is more than ten times of that of the rice character generated by ground physical radiation treatment, and the large character variation range generated by space mutagenesis is difficult to obtain in nature. However, spatial mutation breeding has a certain randomness and a relatively long breeding cycle, and therefore, attention is paid to how to quickly and accurately find a breeding method for a target trait.

Cadmium is one of the most toxic heavy metals and is also widely present in the environment. The long-term exposure to cadmium can cause various diseases such as renal dysfunction, osteoporosis and cancer. In the past three decades, the degree of soil pollution caused by rapid industrialization has attracted serious attention, and cadmium pollution is one of the major environmental problems threatening food safety and agricultural sustainability in China. Compared with other crops, rice tends to accumulate more Cd, and the main source of Cd intake by people taking rice as staple food is provided. Thus, the health risks associated with eating cadmium-contaminated rice are of particular concern among asian populations that rely on rice as a staple food. It is apparent that it is important to control the absorption and accumulation of cadmium in rice to reduce the potential health risks of rice-based dietary populations. It is a feasible method to develop new rice varieties with low cadmium accumulation character to reduce cadmium entering human body. At present, the safety of the new rice variety with low cadmium accumulation directionally bred by gene editing and other transgenic technologies is controversial, and the safe breeding method for breeding the new rice variety with low cadmium accumulation is very important.

Disclosure of Invention

The invention aims to solve the problems of low breeding speed and unsafe breeding method at present and provides a method for quickly breeding space mutation short-stalk and low-cadmium accumulation rice.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for rapidly breeding space mutation dwarf and low cadmium accumulation rice comprises the following steps:

(1) carrying out space mutagenesis treatment on rice seeds to obtain SP0 generation seeds;

(2) the recovered SP0 generation seeds are planted, main ear seeds are harvested to obtain SP1 generation, SP1 generation seeds are planted to obtain SP2 generation, and mutant strains with the target character 1 are selected from the SP2 generation and are continuously and strictly selfed to the SP5 generation;

(3) searching a target gene in a GeneBank database according to the design of the target trait 2, wherein the target gene is OsHMA3, and the gene symbol is as follows: LOC 4342783; in the step, the gene (low cadmium content) with the target shape 2 is found by carrying out gene detection on the SP5 generation rice obtained in the step (2);

(4) designing a primer for qRT-PCR detection according to the gene sequence of the target character 2, wherein the forward primer is as follows: GCAAGTCAAGCCACCCAATG, the reverse primer is: ACGCCCTTGATCATCTCACC, respectively;

(5) transplanting the rice mutant strain with the target character 2 to a field with a cadmium pollution background for planting in the three-leaf period;

(6) and (5) further strictly selfing until SP7 generation, and obtaining a single plant which is stably inherited and meets the target traits.

Further, in the step (1), the rice seed is Dongnong 416.

Further, in the step (1), the space mutagenesis treatment is a space environment, and the satellite carried by the rice is a satellite in practical No. ten return time, and flies for 10-20 days in orbit.

Further, in the step (2), the target trait 1 is dwarf, and all SP2 generation plants are subjected to phenotypic identification to determine a mutant strain with the target trait.

Further, in the step (3), the target property 2 is low cadmium content.

Further, in the step (5), mRNA of SP5 generation plants with the target character 1 is extracted and qRT-PCR detection is carried out.

Furthermore, the mutant strain in which the target gene OsHMA3 is overexpressed is a mutant strain having the target trait 2.

Further, in step (6), the identification is carried out to evaluate the cadmium content, plant height and thousand kernel weight of the SP7 generation plants.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, the recoverable satellite carried rice planting is utilized to carry out space mutagenesis, so that the seeds generate high-frequency beneficial mutation through space radiation and microgravity. Plants with short stalk (lodging resistance) variation characters are selected through ground planting, mutants with target characters are quickly selected by a molecular biology method, time consumed for selecting the mutant plants which do not meet the target characters is reduced, the mutants with the target characters are accurately selected, and unsafe factors caused by a transgenic technology are avoided.

Detailed Description

The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Example 1:

step 1, the rice seeds are Dongnong 416. Dongnong 416 rice seeds were placed in A, B, C three bio-irradiation boxes practicing the Ten Return satellite. The ten-wheeled return satellite is practical to fly in the low earth orbit for 12.5 days. The total design of the satellite is in orbit for 12.5 days, the flight time is related to the mutagenesis effect, the longer the time is, the greater the effects of radiation and microgravity are, and the more obvious the mutation effect is.

And 2, after the practical No. ten recoverable satellite returns to the ground, recovering to obtain SP0 generation seeds, sowing the SP0 generation seeds, separately harvesting main spike seeds to obtain SP1 generation, sowing the SP1 generation seeds according to spikes to obtain SP2 generation, and selecting dwarf mutant plants in SP2 generation. The dwarf mutant strains are 10 strains in total, and are numbered as B-1, B-2, B-3, B-4, B-5, B-6, B-7, B-8, B-9 and B-10. All the variants were selfed strictly to SP5 generations, and 6 variants with stable inheritance were selected from B-1, B-2, B-4, B-6, B-8 and B-9.

And 3, searching a key gene OsHMA3 for controlling the cadmium accumulation of the rice in GeneBank, and designing a qRT-PCR primer according to the mRNA sequence of the key gene OsHMA 3. The forward primer is: GCAAGTCAAGCCACCCAATG, the reverse primer is: ACGCCCTTGATCATCTCACC are provided.

And 4, extracting mRNA of the SP5 generation stably inherited dwarf mutant rice, detecting the quality of the mRNA by using gel electrophoresis and a spectrophotometer, and then carrying out reverse transcription on the mRNA into cDNA. And (3) carrying out qRT-PCR detection by taking the cDNA as a template. And (3) positioning the OsHMA3 gene overexpression plant to a plant with the target character 3, namely the low-cadmium mutant. The numbers are B-1, B-6 and B-8.

And 5, transplanting 3 rice mutant strains with the target character 2 into a field with a cadmium pollution background in a three-leaf period for planting, planting the rice mutant strains to SP7 generations, and comprehensively selecting excellent mutants with low cadmium accumulation by integrating various agronomic character indexes and cadmium accumulation conditions.

The cadmium content of the soil is 1.27-1.59mg.kg^-1The cadmium content of the control group DN416(CK), the target character plants B-1, B-6 and B-8 is shown in the following table:

numbering	Plant height (cm)	Thousand Kernel weight (g)	Cadmium content
				DN416(CK)	88.7	26.8	0.452mg.kg-1
B-1	80.5	25.9	0.265mg.kg-1
				B-6	81.6	26.5	0.394mg.kg-1
B-8	83.9	25.3	0.388mg.kg-1

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention.