CN110583480A - Method for improving rice sexual cell genome variation induction efficiency - Google Patents
Method for improving rice sexual cell genome variation induction efficiency Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
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.
Description
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.
Example 1
1. Planting of rice material
The method for planting the rice by adopting the water planting mode comprises the following specific steps:
(1) seed soaking and germination accelerating: soaking a certain amount of seeds in water for one day, wrapping with wet rag, and germinating in 30 deg.C incubator.
(2) Seedling raising and transplanting: taking a proper amount of seeds for accelerating germination, putting the seeds into a germination box, arranging two layers of filter paper in the box, then adding a proper amount of nutrient solution, putting the seeds into a 30-degree incubator for culturing, and taking out and transplanting the seeds to the nutrient solution when the seedlings of the seeds grow to 1-2 cm.
(3) Preparing a nutrient solution, wherein the formula of the nutrient solution is as follows: NaH2PO4·2H2O 0.7485g，K2SO41.305g，MnCl2·4H2O0.054054g，(NH4)6MoO24·4H2O 0.002754g，H3BO30.03294g，ZnSO4·7H2O 0.001337g，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.
(4) Punching a hole in the middle of a sponge with the diameter of 2cm, placing seeds with the length of 1-2cm of seedlings in the hole, floating on the nutrient solution, removing tillers in the growth process of rice plants, and only keeping 1 main stem tillerer. During the growth process, the nutrient solution is replaced once a week.
(5) The culture condition is natural illumination and the temperature is 30 ℃.
EMS mutagenesis treatment
(1) When the rice plant grows until the flag leaf pillow is equal to the inverted two-leaf pillow, pollen mother cells in the anther of the young rice spike are in the meiotic stage, are sensitive to a mutagen and are a proper period for EMS mutagenesis treatment.
(2) The concentrations of the chemical mutagen EMS treatment were 0.01%, 0.025%, 0.05%, and 0.1%, respectively, and the four treatments (No. M0.01M 0.025M 0.05M 0.1) were prepared in 0.1M phosphate buffer at pH 6.98, and treated with phosphate buffer to prepare a CK control group.
(3) Method for treating EMS-mutagenized cells (injection method): carrying out mutagenesis treatment at 9-11 am every day, firstly selecting young ears at a proper period, wiping the surfaces of bracts of the young ears by using a 70% alcohol cotton ball, probing the upper positions of the young ears, slowly injecting liquid medicine, and when the liquid medicine overflows from the center holes at the tops of the ears, indicating that the liquid medicine is full of the young ears. During the operation, alcohol and sodium thiosulfate are continuously used for disinfection to prevent pollution. After the liquid medicine seeps out of the young ear, EMS with the same concentration is injected again, and the CK control strain is injected with phosphate buffer solution in the same way. Finally, hanging a label and noting the treatment concentration and the treatment time.
(4) And continuously culturing the plant treated by the EMS in a nutrient solution, and respectively harvesting seeds after the young ears of the rice are developed and fructified according to the treatment. The plant culture conditions are 30 ℃, natural illumination conditions and the nutrient solution is changed every week.
(5) And harvesting seeds after the plants are mature, randomly selecting 50 seeds for each treatment, mixing the young embryos of each seed after 48h of germination, carrying out Illumina whole genome sequencing analysis on the mixed sample, and selecting a Nipponbare sequence of the japonica rice variety with the whole genome being determined as a reference genome sequence. M0.01M 0.025M 0.05M 0.1 sequencing depth of 100X per sample, sequencing data volume of 40G; CK sample sequencing depth is 10 ×, sequencing data volume is 4G.
EMS treatment of M after young panicle0Plant phenotype generation survey
EMS mutagenesis treatment is carried out on young ears of rice variety at the booting stage of Nippon sunny days by using an injection method, and M is investigated0The generation plant spike length, total grain number, solid grain number, ten grain length, ten grain width and seed setting rate phenotype. Compared with the control, the setting rate of the plant after EMS mutagenesis treatment is gradually reduced along with the increase of EMS concentration, and the result is shown in figure 1, which shows that the higher the EMS concentration is, the more damage is caused to the rice plant, and the harvested seed is reduced. The germination rates and germination indexes of the seeds harvested under different concentrations are measured, and the results are shown in fig. 2, the germination rates and the germination indexes of the seeds obtained through low-concentration EMS mutagenesis treatment are high, and the EMS treatment young ears have no significant influence on the vitality of the seeds. After EMS treatment at different concentrations, the results are shown in FIG. 3, the panicle length, ten grain length and ten grain width of rice are almost the same, and the EMS treatment of young panicles has no significant influence on the three traits.
4. Quality control analysis of rice whole genome sequencing data
According to the experimental design, 50 seeds are randomly selected for each treatment, young embryos of each seed are taken to be mixed after germination for 48 hours, Illumina whole genome sequencing analysis is carried out on a mixed sample, and a japonica rice variety Nipponbare sequence which is subjected to whole genome determination is selected as a reference genome sequence. In order to ensure the data quality, the raw data of rice sequencing was subjected to quality control, and data noise was reduced by data filtering, and the results are shown in table 1.
TABLE 1 statistical Table of base information before and after filtration
Note: m0.01, M0.025, M0.05 and M0.1 respectively indicate EMS concentrations of 0.01%, 0.025%, 0.05% and 0.1%.
As can be seen from Table 1, each datum of Q20 is greater than 97.34%, indicating that the data accuracy is very high and the sequencing results are reliable.
5. Variation condition of rice genome after EMS treatment at different concentrations
After the sequencing data are subjected to quality control, the published Nipponbare genome is taken as a reference sequence, and mutation treatment and variation of the control CK in the whole genome are compared. FIG. 4(circos plot) is the distribution of all variants across the genome.
Fig. 4 is, from outside to inside: length of each chromosome (unit: Mb). The frequency histogram is used to represent the gene density in different region windows of the chromosome. The SNP density of different region windows of the chromosome is represented by scattered points. The window size is set to 10 k. If the window SNP density is >0.0015, the dot is a red square; if the SNP density of the window is more than or equal to 0.0005 and less than or equal to 0.0015, the dots are gray circles; if the SNP density of the window is less than or equal to 0.0005, the dots are green triangles. The orange line indicates the location of DUP (tandem repeat) on the chromosome. Purple lines indicate the location of the DEL (large fragment deletion) on the chromosome. The green line indicates the position of the INS (insertion-type structural variation) on the chromosome. The blue line indicates the position of INV (inverted type structural variation) on the chromosome. The line in the inner circle indicates the position of the BND (structural variation of the translocation type between chromosomes) on each pair of chromosomes.
As can be seen from FIG. 4, SNP mutations occurred in each chromosome, including tandem repeats, large fragment deletions, insertion-type structural mutations and inversion-type structural mutations (SNP density 0.0005 < SNP density >0.0015), and more SNP mutations occurred at some positions of chr2, chr4, chr6, chr10 and chr12 (SNP density > 0.0015). The variation on chr1 is mainly structural variation of large fragment deletion and insertion types; the variation on chr2 is mainly a large fragment deletion; the variation on chr3 is mainly structural variation of tandem repeat and insertion type; the chr4 has more variation, but mainly has structural variation of tandem repeat and large fragment deletion insertion type; in addition, chr9, chr10, chr11 and chr12 are prone to structural variation of the type of interchromosomal translocation with other chromosomes.
6. SNV (SNPs, In Del) analysis In rice samples after sexual cell mutagenesis
(1) General case of different mutagenized SNPs, InDel
The variation data of SNP and InDel under EMS treatment of four concentrations are obtained by sorting and analysis, and are shown in Table 2.
TABLE 2 statistics of SNP and InDel variations
Note: m0.01, M0.025, M0.05 and M0.1 respectively indicate EMS concentrations of 0.01%, 0.025%, 0.05% and 0.1%.
As can be seen from table 2, M0.01 has 478 mutation sites, of which 66.95% are SNP mutations and 33.05% of InDel mutations; m0.025 has 735 mutation sites, including 71.16% SNP mutation and 28.84 InDel mutation; m0.05 has 804 mutation sites, wherein 72.39 percent of the mutation sites are SNP mutations, and 27.61 percent of the mutation sites are InDel mutations; of the four treatments, M0.1 had the most mutation sites, totaling 865 mutation sites, of which SNP accounted for 72.37% and InDel was only 27.63%. The phenomenon that SNP accounts for more than 60% of the total amount of mutation occurs in all four treatments, which indicates that single base mutation is the main mutation type of EMS-induced degeneration cells, and the SNP mutation ratio of M0.05 is the highest.
(2) Characteristic of EMS mutagenesis treatment for inducing SNP variation
SNP (single nucleotide polymorphism) in the young ear induced mutation processed by EMS is the most found mutation type. SNPs include transitions (A: G; T: C) and transversions (A > C/T; G > C/T; C > A/G; T > A/G), with ratios of transitions to transversions (Ti/Tv) of M0.01, M0.025, M0.05, M0.1 being 1.818, 1.433, 1.564, 1.687, respectively (FIG. 2). Generally, the SNP with conversion type accounts for about 2/3 of the total amount of SNP, and the conversion is high because cytosine (C) on CpG dinucleotide is the most easily mutated site in genome, most of which is methylated, and amino groups can be spontaneously removed to form thymine (T) (Zhu Yuxian, 2013). In the SNPs induced by the two treatments, the contents of A > G and T > C are higher. There were 100 a > G mutations in M0.01, 100T > C mutations, 155 a > G mutations in M0.025, 153T > C mutations, 167 a > G mutations in M0.05, 188T > C mutations, 177 a > G mutations in M0.1, 216T > C mutations, 1214 a > G mutations in M1, 1312T > C mutations. From the above results, it can be seen that the rate of transversion SNP induced by EMS-treated cells was higher, while induction of transversion SNP was relatively difficult, indicating that EMS-treated cells had higher induction efficiency.
(3) Distribution of EMS-induced SNP variation
The positions where SNP mutations occur are mostly concentrated in endogenous regions (Table 3). In 0.01% of the treatments 69.06% of the SNPs were in intergenic, 8.75% in intron, 6.88% in uppstream, 3.75% in downstream, 5% in exonic. 0.025% of the treatments had 83.37% of SNPs in intergenic, 3.44% in intron, 4.21% in upplow, 2.1% in downstream, 2.1% in exonic. 0.05% of the treatments had 71.13% of SNPs in intergenic, 8.59% in intranic, 6.7% in updam, 3.95% in downstream, 3.95% in exonic. 0.1% of the treatments had 71.25% of SNPs in intergenic, 8.31% in intron, 6.39% in updam, 4.63% in downstream, and 4.31% in exonic. Comparative analysis shows that the SNP of intergenic in the four groups reaches more than 60 percent, and the SNP of 0.025 percent reaches 80 percent.
TABLE 3 statistics of SNP variation distribution
7. Selection of appropriate mutagenesis dosage and mutagenesis population planting quantity calculation
Four different concentrations of EMS-treated young ears of rice can produce genome variation, but the number of variation is highly related to the treatment concentration. The number of genomic variations increased significantly with increasing treatment concentration, with the numbers of variations induced by M0.05 and M0.1 approaching and being significantly higher than M0.01 and M0.025. Comparing the effect of M0.05 and M0.1 treatments on young ears of rice, the ear length, total grain number, ten grain length and ten grain width of the M0.05 treatment are not significantly different from those of the M0.1 treatment, but the maturing rate of the M0.05 treatment is significantly higher than that of the M0.1 treatment, which indicates that the biological toxicity of the M0.05 treatment is lower. By combining the above analysis, M0.05 treatment has high induced genome mutation frequency and low biological toxicity, and is a suitable method for inducing rice sexual cell genome variation.
The scale of the mutagenic population planting directly influences the selection efficiency of mutation, the existing EMS-induced seeds generate mutation, and the planting scale of the offspring population is generally judged according to experience, so that a great amount of manpower and material resources are wasted. After the young ears of rice are treated by the method with M0.05, 804 variations are generated on the genome of the seeds, wherein SNP is the main variation type. Of the 582 SNP variations, 168 were located in the gene region, and these variations may result in altered gene function. Since about 40000 predicted genes of rice correspond to a mutation probability of 168/40000-0.42% for a single gene, the progeny need to plant at least 238 individuals if a mutation of any one gene is expected. In the M0.05 treatment, the nonsynonymous mutation accounted for 59.26% of the total variation, and therefore the probability of any gene having a functional mutation was 0.42% × 59.26% ═ 0.25%. According to the above calculation, if a mutant with altered function of any one gene is expected, the progeny need to be planted with at least 400 strains. For breeding, at least 20 individuals are needed for breeding evaluation, so that 20 × 400 to 8000 individuals are planted in the mutagenized population.
The mutagenic population scale calculation formula is:
in the formula: probability of single gene variation X1, number of SNPs located in gene region/40000
Nonsynonymous mutation probability X2 ═ nonsynonymous mutation SNP number/total SNP mutation number
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
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.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115372554A (en) * | 2022-07-29 | 2022-11-22 | 江西农业大学 | Research method for influence of different treatment methods on number of pueraria lobata chromosomes |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102791880A (en) * | 2009-12-18 | 2012-11-21 | 凯津公司 | Improved bulked mutant analysis |
| CN104024438A (en) * | 2012-09-28 | 2014-09-03 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Snp loci set and usage method and application thereof |
| CN108617502A (en) * | 2018-04-26 | 2018-10-09 | 贵州省蚕业研究所 | A method of building capsicum mutant library using ethylmethane sulfonate |
-
2019
- 2019-10-18 CN CN201910995181.9A patent/CN110583480A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102791880A (en) * | 2009-12-18 | 2012-11-21 | 凯津公司 | Improved bulked mutant analysis |
| CN104024438A (en) * | 2012-09-28 | 2014-09-03 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Snp loci set and usage method and application thereof |
| CN108617502A (en) * | 2018-04-26 | 2018-10-09 | 贵州省蚕业研究所 | A method of building capsicum mutant library using ethylmethane sulfonate |
Non-Patent Citations (3)
| Title |
|---|
| 杨学荣等: ""水稻花培育种技术操作和无性系变异体选择"", 《华中师范大学学报 (自然科学版)》 * |
| 穆平等: "《作物育种学》", 31 July 2017, 中国农业大学出版社 * |
| 赵丽梅等: ""大麦减数分裂早前期诱变效应的研究"", 《吉林农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115372554A (en) * | 2022-07-29 | 2022-11-22 | 江西农业大学 | Research method for influence of different treatment methods on number of pueraria lobata chromosomes |
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