阅读说明：本技术 一种提高水稻性细胞基因组变异诱导效率的方法 (Method for improving rice sexual cell genome variation induction efficiency ) 是由 郭涛 陈淳 李丹丹 黄翠红 周丹华 夏澳运 王慧 陈志强 于 2019-10-18 设计创作，主要内容包括：本发明公开了一种提高水稻性细胞基因组变异诱导效率的方法,属于植物育种技术领域。该方法包括如下步骤：水稻种子萌发后,待幼苗长出时移植在营养液中生长,控制其分蘖,只保留少数主茎；待水稻幼穗发育期,选取适宜的水稻主茎幼穗,用酒精棉球消毒幼穗苞叶表面,在幼穗上部位置慢慢注入浓度为0.01％～0.1％的化学诱变剂EMS,待药液从穗顶部中心孔溢出时,停止注射并等待5-10min,再次注入同浓度EMS,操作时不断对幼穗苞叶表面消毒；EMS处理的植株继续在营养液中培养,待水稻幼穗发育结实并收获全部种子,取样萌发后进行基因组测序,根据测序数据计算突变频率。该诱导方法处理水稻性细胞可获得更高的诱导效率,同时具有更高的安全性。(The invention discloses a method for improving the efficiency of rice sexual cell genome variation induction, and belongs to the technical field of plant breeding. The method comprises the following steps: after the rice seeds germinate, transplanting the rice seeds to grow in nutrient solution when seedlings grow out, controlling tillering of the rice seeds and only keeping a few main stems; selecting proper main stem young ears of the rice at the young ear development period of the rice, disinfecting the surfaces of the bracts of the young ears by using an alcohol cotton ball, slowly injecting a chemical mutagen EMS with the concentration of 0.01-0.1% into the upper position of the young ears, stopping injecting and waiting for 5-10min when liquid medicine overflows from the center hole at the top of the ears, injecting the EMS with the same concentration again, and continuously disinfecting the surfaces of the bracts of the young ears during operation; and continuously culturing the plant treated by the EMS in the nutrient solution, obtaining all seeds after the young ear of the rice grows and fructifies, sampling and germinating, then carrying out genome sequencing, and calculating mutation frequency according to sequencing data. The induction method can obtain higher induction efficiency and higher safety when used for treating the rice sexual cells.)

1. A method for improving the efficiency of inducing the rice sexual cell genome variation is characterized by comprising the following steps:

(1) after the rice seeds germinate, transplanting the rice seeds to grow in nutrient solution when seedlings grow out, controlling tillering of the rice seeds and only keeping a few main stems;

(2) selecting proper main stem young ears of the rice at the young ear development period of the rice, disinfecting the surfaces of the bracts of the young ears by using an alcohol cotton ball, slowly injecting a chemical mutagen EMS with the concentration of 0.01-0.1% into the upper position of the young ears, stopping injecting and waiting for 5-10min when liquid medicine overflows from the center hole of the top of the ears, then repeatedly injecting the EMS with the same concentration into the same position, and continuously disinfecting the surfaces of the bracts of the young ears in the operation process;

(3) continuing culturing the plant treated by EMS in nutrient solution until young ears of rice are developed and fructified and harvesting all seeds;

(4) and randomly selecting 50 harvested seeds, germinating, taking seed embryos for sequencing, calculating mutation frequency, and determining the planting number of the offspring mutagenesis group according to the mutation frequency.

2. The method for improving the efficiency of inducing sexual cytogenome variation in rice according to claim 1, wherein the chemical mutagen EMS is prepared from 0.1M phosphate buffer solution with pH 6.98.

3. The method for improving the efficiency of inducing sexual genomics variation in rice plants in accordance with claim 1, wherein the seedlings in step (1) are transplanted in a nutrient solution to grow, and the growth degree of the seedlings is 1-2 cm.

4. The method of claim 1, wherein the method is used for improving the sexual cell genome variation of riceThe method for inducing efficiency is characterized in that the seedlings in the step (1) are transplanted to a nutrient solution for growth when growing out, and the formula of the nutrient solution is as follows: NaH₂PO₄·2H₂O0.7485g，K₂SO₄1.305g，MnCl₂·4H₂O 0.054054g，(NH₄)₆MoO₂₄·4H₂O 0.002754g，H₃BO₃0.03294g，ZnSO₄·7H₂O0.001337g，CuSO₄·5H₂O 0.001248g，FeCl₃·6H₂O0.29214g, citric acid hydrate 0.44625g, MgSO4 & 7H₂O 25.08g，Ca(NH₄).(NO₃)₃3.9g are dissolved in 30L of water and the pH of the solution is adjusted to about 4.5 by adding 4.8ml of 5M NaOH.

5. The method for improving the induction efficiency of the sexual cell genome variation of the rice according to claim 1, wherein in the step (1), when the seedling grows out, the seedling is transplanted in a nutrient solution to grow, tillering of the seedling is controlled, only a few main stems are reserved, specifically, a hole is punched in the middle of a sponge with the diameter of 2cm, a seed with the length of 1-2cm of the seedling is placed in the hole and floats on the nutrient solution, and the small tillering is removed during the growth of the rice plant, and only 1 main stem tillering is reserved.

6. The method for improving the efficiency of inducing sexual genomics variation in rice as claimed in claim 1, wherein the young ears of main stem of rice suitable in step (2) are young ears of flag leaf pillow and young ear of inverted two-leaf pillow.

7. The method for improving the efficiency of inducing sexual genomics variation in rice according to claim 1, wherein the young ear injection site in step (2) is 2cm away from the occipital lobe of the two-leaf rice at the upper part of the young ear, and the concentration of the EMS chemical mutagen is 0.05%.

8. The method for improving the induction efficiency of sexual cytogenome variation of rice as claimed in claim 1, wherein the EMS-treated plant of step (3) is cultured under the condition of 30 ℃ temperature and natural illumination, and the nutrient solution is changed once a week.

9. The method for improving the efficiency of inducing the rice sexual cell genome variation according to claim 1, wherein 50 harvested seeds are randomly selected in the step (4), and the seed embryos are taken for sequencing after germination, wherein the sequencing is whole genome sequencing, and the sequencing data volume is 40G.

10. The method of claim 1, wherein 50 harvested seeds are randomly selected in the step (4), the seed embryos are taken after germination for sequencing, the genome variation data after EMS processing is counted, and the number of the mutagenized groups planted is determined according to the variation data, and the calculation formula is as follows: the number of single plants needed to be planted in a mutagenic group is equal to the number of expected mutants X3/(the probability of single gene variation X1 multiplied by the probability of non-synonymous mutation X2); wherein, the single gene variation probability X1 is the number of SNPs in a gene region/40000, and the nonsynonymous mutation probability X2 is the number of nonsynonymous mutation SNPs/the number of all SNP mutations.

Technical Field

The invention belongs to the technical field of plant breeding, and particularly relates to a method for improving the efficiency of rice sexual cell genome variation induction.

Background

Ethyl Methane Sulfonate (EMS) is one of the alkylating agents and is the most commonly used chemical mutagen (wann et al, 2016). EMS can induce and generate a large amount of point mutations (Zhangyi, etc., 2011), avoids the problems of safety and stability (True-Qing, etc., 2013) caused by transgenosis, and has wide application in genetic research and genetic improvement at present. Its mutagenesis mechanism is the addition of alkyl groups to the nucleotide guanines of DNA, resulting in erroneous transitions during DNA replication, point mutations (i.e. G: C base pairs to a: T base pairs), or EMS agents cause these alkylated guanines to degrade automatically, resulting in gaps in the DNA strand, eventually DNA strand breaks, base translocations and even cell death.

In recent years, with the application of molecular marker technology, the EMS mutagenesis technology has a breakthrough in genetic mechanism analysis. At present, EMS mutagenesis technology has been widely applied to genetic improvement and breeding research of plants such as tomatoes (Gady et al, 2009), strawberries (Bhat et al, 2017), corn (avaiw et al, 2008), wheat (Mishraet al, 2016), and the like. For example, ZHU et al (year) screened 1 floret development-delayed mutant drm1 from the Arabidopsis mutant pool and has been applied to the study of floret development. Wangbeiing, etc. (2000) utilize EMS to treat soybean, and screen out a batch of excellent mutant lines with high protein (45% -50%), high linoleic acid (more than 60%) and low linolenic acid.

The mutant progeny generated by inducing rice plants by EMS has abundant variation among different strains and varieties, and the stability of the gene mutation is good, so that sequencing analysis is easy to carry out (Yangfeng et al, 2017). Therefore, EMS is gradually mature in technical means of research on mutagenic rice breeding (Nianqiang et al, 2016), and is also more and more widely applied. The varieties of indica rice Guang-Lu-Shi-4, Nippon and Ning-Jing 28 and the like are mutagenized by EMS (American society for plant research) (1991), Raney-Bosheng (2008), Wang-Caifen and the like (2011), and valuable mutation occurrence is obtained. Chen faithful et al (2004) mutagenize indica rice "9311" by EMS and screen large-grain mutant "M316" and long ear neck mutant "9311 ER". EMS mutagenesis treatment is carried out on Wangfeng (2011) by carrying out good indica restorer line red silk-restoring No. 10 of rice, and 24 parts of semi-dwarf mutants are screened out; EMS mutagenesis treatment is carried out on the Xiuhui 09 of the late japonica rice variety by Dungxiamei and the like (2012) to obtain 1 yellow-green leaf mutant Ygl in the whole growth period, which is expressed as yellow-green leaves and can be stably inherited, 1 pair of invisible genes are successfully positioned, the mutant characters of the yellow-green leaves are verified to be controlled by the pair of genes, and meanwhile, the mutant is detected to have single base mutation. Raytal (2000) has applied 1 mutant of susceptible bacterial disease screened from a rice mutant bank to rice disease resistance studies. Huxi et al (2013) screened 1 lateral root deletion mutant hts1 sensitive to high temperature by using a rice EMS mutant library, and studied the response of the mutant in seedling stage characters and agronomic characters to different temperature gradients by performing preliminary genetic analysis on the mutant.

The most commonly used treatment for EMS chemical mutagenesis is the seeds of the plant (Wen Niu et al, 2015; Liuwei et al, 2017; Yuxipu et al, 1994; Zhu Baoge et al, 1997), and a large number of mutant populations can be constructed in a short time. However, other biological tissues of cell membranes of embryo parts of seeds can prevent EMS from entering embryo cells, thereby reducing the mutagenesis effect; EMS also chemically interacts with water to produce ethanol and methanesulfonic acid, both compounds significantly affecting seed vigor and even leading to death of the individual. The rice seeds are typical diploid somatic cell tissues, mutant cells generated by EMS mutagenesis compete with normal cells, and the mutant cells are usually inhibited or disappeared; sometimes chimeras are easily formed, affecting the selection effect (Shenfafu et al, 1999; Yuejie et al, 1996; Xue Shaw Wang et al, 1998). In addition, since EMS is mostly recessive, dominant mutations of the population are shown only in the M2 generation and recessive mutations in the M3 generation, which requires a long time to obtain mutants. Therefore, there are limiting factors such as difficulty in increasing the mutation frequency, long cycle, and low activity of the treated seeds when the seeds are subjected to EMS mutagenesis treatment. The EMS is utilized to mutate the young ear, pollen, zygote and other sexual cells of the plant, so that chimera can be avoided, and the mutagenesis frequency is improved; because the generated mutation can be directly inherited to form seeds, the identification of the mutant can be advanced by one generation, and the screening time is saved; the mixed sequencing is carried out on the seeds generated by the sexual cell variation, so that the mutation frequency can be calculated, the planting scale of a mutagenic group is further guided, and the waste of manpower and material resources is avoided. However, there is no systematic study on the method for inducing rice sexual cell variation by using EMS, and the frequency and molecular characteristics of mutation lack critical data.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for improving the efficiency of inducing the rice sexual cell genome variation, which mainly adopts an EMS injection method to treat young ears of rice at a specific period to induce the rice sexual cell genome variation, and provides a method for calculating the frequency of mutagenized offspring.

In order to achieve the purpose, the invention adopts the following technical scheme:

(1) after the rice seeds germinate, transplanting the rice seeds to grow in nutrient solution when seedlings grow out, controlling tillering of the rice seeds and only keeping a few main stems;

(2) selecting proper main stem young ears of the rice at the young ear development period of the rice, disinfecting the surfaces of the bracts of the young ears by using an alcohol cotton ball, slowly injecting a chemical mutagen EMS with the concentration of 0.01-0.1% into the upper position of the young ears, stopping injecting and waiting for 5-10min when liquid medicine overflows from the center hole of the top of the ears, then repeatedly injecting the EMS with the same concentration into the same position, and continuously disinfecting the surfaces of the bracts of the young ears in the operation process;

(3) continuing culturing the plant treated by EMS in nutrient solution until young ears of rice are developed and fructified and harvesting all seeds;

(4) and randomly selecting 50 harvested seeds, germinating, taking seed embryos for sequencing, calculating mutation frequency, and determining the planting number of the offspring mutagenesis group according to the mutation frequency.

Further, the chemical mutagens EMS were all prepared with 0.1M phosphate buffer solution at pH 6.98.

Further, transplanting the seedlings in the step (1) to grow in a nutrient solution when the seedlings grow out, wherein the growth degree of the seedlings is 1-2 cm.

Further, in the step (1), when the seedlings grow out, the seedlings are transplanted in a nutrient solution for growth, and the formula of the nutrient solution is as follows: NaH₂PO₄·2H₂O 0.7485g，K₂SO₄1.305g，MnCl₂·4H₂O 0.054054g，(NH₄)₆MoO₂₄·4H₂O0.002754g，H₃BO₃0.03294g，ZnSO₄·7H₂O0.001337g，CuSO₄·5H₂O 0.001248g，FeCl₃·6H₂O0.29214g, citric acid hydrate 0.44625g, MgSO4 & 7H₂O 25.08g，Ca(NH₄).(NO₃)₃3.9g are dissolved in 30L of water and the pH of the solution is adjusted to about 4.5 by adding 4.8ml of 5M NaOH.

Further, transplanting the seedlings in the step (1) to grow in nutrient solution when the seedlings grow out, controlling the tillers of the seedlings, and only keeping a few main stems, specifically, punching a hole in the middle of a sponge with the diameter of 2cm, placing seeds with the length of 1-2cm of the seedlings in the hole, floating on the nutrient solution, removing the small tillers in the growth process of the rice plants, and only keeping 1 main stem tillering.

Further, the rice stem young ear suitable in the step (2) is judged to be a proper young ear to be processed if the flag leaf pillow is flush with the inverted two-leaf pillow.

Furthermore, in the step (2), a chemical mutagen EMS with the concentration of 0.01-0.1% is slowly injected into the upper part of the young ear, the injection part is the position of the upper part of the young ear 2cm away from the occipital lobe of the inverted two leaves, and the concentration of the EMS chemical mutagen is 0.05%.

Further, the plant treated by EMS in the step (3) is continuously cultured in nutrient solution, all seeds are harvested after the young ears of rice are developed and fructified, the culture condition is 30 ℃, the natural illumination condition is adopted, and the nutrient solution is changed every week.

Further, in the step (4), 50 harvested seeds are randomly selected, and after germination, a seed embryo is taken for sequencing, wherein the sequencing is whole genome sequencing, and the sequencing data volume is 40G.

Further, in the step (4), 50 harvested seeds are randomly selected, the seed embryos are taken for sequencing after germination, genome variation data after EMS processing are counted, the number of the mutagenized groups is determined according to the mutation frequency, and the calculation formula is as follows: the number of single plants needed to be planted in a mutagenic group is equal to the number of expected mutants X3/(the probability of single gene variation X1 multiplied by the probability of non-synonymous mutation X2); wherein, the single gene variation probability X1 is the number of SNPs in a gene region/40000, and the nonsynonymous mutation probability X2 is the number of nonsynonymous mutation SNPs/the number of all SNP mutations.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention uses EMS to induce the young ear cells of rice in a proper development period to generate variation, the sexual cells are highly sensitive to mutagens and easy to generate variation, and can avoid chimeras generated by seed cell variation induced by the existing EMS and improve the mutagenesis frequency; juvenile cells are more easily permeabilized by the mutagen EMS than are seed somatic cells.

(2) The mutation induced by the rice sexual cells can be directly inherited to form seeds, so that the mutant identification can be about 6 months earlier than the prior art by one generation, and the breeding time is obviously shortened.

(3) The invention provides a method for performing mixed sequencing on seeds generated by sexual cell variation, and the planting scale of a mutagenic group is accurately guided through the mutation frequency at the molecular level, so that the defect that the planting scale of the mutagenic group is difficult to scientifically estimate by the traditional method is avoided, and manpower and material resources are effectively saved.

(4) The method carries out EMS mutagenesis on the young ear cells of the rice, has high operation safety, small harm to the plant, basically normal activity of the harvested seeds and overcomes the defects of death of a large amount of seeds and sterile offspring after the seeds are soaked by the traditional EMS.

(5) Compared with the existing EMS treatment of rice seeds, the proportion of the transversing single base variation (SNP) generated by mutating rice sexual cells by the EMS young spike injection method is relatively high. Induction of transversion SNPs is relatively difficult, suggesting that higher induction efficiencies can be achieved in EMS-treated cells.

Drawings

FIG. 1 shows the maturing rate of young ears of rice in example 1 after EMS treatment.

Figure 2 is a graph of the effect of different EMS treatments on seed vigor in example 1.

FIG. 3 shows EMS after EMS processing in example 1₀The main agronomic character value of the generation plant.

FIG. 4 is a global distribution of EMS-induced variation in example 1.

FIG. 5 is a flow chart of the technique of example 1.

Detailed Description

The test material selected by the application is japonica rice Nipponbare. Planting the seeds in a greenhouse of an agricultural college of southern China university in a water culture manner in 2018 from 5 months to 7 months.