CN114946649A - Method for improving low-generation breeding efficiency of high-resistance starch rice - Google Patents
Method for improving low-generation breeding efficiency of high-resistance starch rice Download PDFInfo
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Abstract
A method for improving the low-generation breeding efficiency of high-resistance starch rice uses the high-resistance starch rice containing sbe3-rs gene as donor parent to cross-breed F 1 Mixed and harvested to obtain F 2 Seeds; water separation is carried out by utilizing the high chalkiness characteristic of high resistant starch rice grains, suspended grains are separated out, and F is obtained 2 Seeds; after the water-separated medium-selected seeds are subjected to genotyping detection, the ratio of the obtained high-resistance starch homozygous single plant is averagely improved by 4-8 times compared with the seeds which are not subjected to water separation, and the high-whiteness degree (with artificially selected high chalkiness degree)>50%) of the seeds, and the breeding efficiency of the seeds is improved by about 2 times compared with 30.6%. The method realizes the low-generation rapid screening of the homozygous single plants with high resistant starch content, effectively improves the breeding efficiency of breeding the rice variety containing the sbe3-rs genotype high resistant starch, saves the breeding cost, can rapidly and efficiently carry out mass operation, and obtains the high-quality high resistant starch rice germplasm.
Description
Technical Field
The invention belongs to the field of rice breeding, and particularly relates to a method for improving the low-generation breeding efficiency of high-resistance starch rice.
Background
Resistant Starch (RS) refers to "Starch or Starch degradation products that cannot be absorbed in the small intestine of healthy individuals" (defined by EURESTA, european RS). The resistant starch has important physiological functions, can reduce postprandial blood sugar and insulin response, improve the sensitivity of organisms to insulin, prevent constipation and colon cancer, reduce the contents of cholesterol and triglyceride in serum, reduce and control body weight and promote mineral absorption. The method for improving the dietary structure of people by screening and identifying the rice genetic resources with high resistant starch content and providing rice varieties with high resistant starch content is one of the most economic and effective means for treating and preventing the onset of civilized diseases such as diabetes mellitus.
The research institute of crop cultivation and breeding of agricultural academy of sciences of Shanghai city utilizes chemical mutagenesis and microspore culture technology in combination with conventional breeding technology to cultivate a new variety 'Jiangtao 1' of high-resistance starch japonica rice, and cooperates with related enterprises to carry out industrialized development, thereby relieving the difficult problems of eating difficulty and eating insufficiency of diabetes patients. The 'Jiangtao No. 1' is a research object, a major gene SBE3-RS for controlling the content of rice RS is positioned, and functional complementation verification is carried out, wherein the mutant gene has a base mutation of T → C at the 105 th position corresponding to the 16 th exon of a rice starch branching enzyme SBE3 gene, and a plurality of functional marker molecular markers are developed aiming at the site.
According to the genetic characteristics of the high resistant starch character gene and the high correlation relationship between the high resistant starch character gene and characters such as chalkiness rate, chalkiness degree and the like and by utilizing the developed CAPS/SpeI molecular marker, a rice molecular breeding technical system with high resistant starch content is established. The group successfully breeds new rice strains 'Yougao No. 2' and 'Yougao No. 3' with high RS content and both functionality and high yield and high quality by taking 'Jiangtao No. 1' as a high resistant starch character working parent. However, the high-quality sugar rice still has the problems of poor taste, poor stress resistance and the like, and the method has important significance for continuously improving the taste of the high-quality sugar rice and improving the comprehensive agronomic characters such as stress resistance and the like by a genetic breeding method.
Yanghuang et al (nuclear agriculture report, 2015, 29 (12): 2259-2267) firstly propose to select and eliminate most of low-resistant starch single plants by utilizing chalkiness characteristics of seeds of early generation of hybrid population, and simultaneously improve the breeding efficiency of high-resistant starch rice varieties by combining molecular marker-assisted selection. F 1 Plant harvest trait segregated F 2 The chalkiness degree of seeds and high-resistance starch grains is higher, the chalkiness grains are selected to be planted into cells, the probability that offspring obtains homozygous high-resistance starch genotype is greatly improved, and the chalkiness degree is selected manually>50% of F 2 The individual plant ratio of homozygous high resistant starch in the kernel was 30.6%.
The method for measuring the content of the resistant starch is slightly improved by mainly using an RS content measuring kit (Megazyme, Co. Wicklow, Ireland) provided by Megazyme company. The method comprises the following specific steps: accurately weighing 100mg of rice flour sample, carefully placing the rice flour sample into a plastic test tube with a screw cap, sequentially adding alpha-pancreatic amylase reaction solution and Amyloglucosidase (AGM), shaking and incubating for 16 hours at 37 ℃, dissolving non-RS, and hydrolyzing into D-glucose; after the incubation is finished, adding 99% ethanol to terminate the reaction; centrifuging the solution, discarding the supernatant to obtain a floccule at the bottom, namely RS in the sample, and washing the precipitate with 50% ethanol; inverting the centrifuge tube, dissolving the precipitate with 2 mol.L-1 potassium hydroxide after drying the precipitate, adding AGM, placing in a water bath at 60 ℃ for incubation for 1h, finally determining the glucose content with a glucose oxidase/peroxidase reagent (GOPOD) for D-glucose, and calculating the RS content. The determination process of the resistant starch is complex, long in time consumption and high in cost, and becomes highA limiting factor of resistant starch rice breeding. F 1 F is obtained by mixed harvest of low generation (10-18 plants) 2 The seeds are replaced, high chalky grains are manually selected, one combination needs to be selected for 1-2 hours, and the manual selection of the high chalky grains is time-consuming, time-consuming and high in cost.
Disclosure of Invention
The invention aims to provide a method for improving the low-generation breeding efficiency of high-resistance starch rice, overcomes the defects that the low-generation isolated population high-resistance starch homozygous single plant proportion is low in the breeding process of the existing high-resistance starch rice new variety, and the method for manually selecting high-chalky grains to improve the homozygous single plant proportion is time-consuming, labor-consuming and high in cost, realizes the low-generation rapid screening of high-resistance starch content homozygous single plants, effectively improves the breeding efficiency of breeding of sbe3-rs genotype-containing high-resistance starch rice varieties, saves the breeding cost, can be rapidly and efficiently operated in large batch, and obtains high-quality high-resistance starch rice germplasm.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for improving the low-generation breeding efficiency of high-resistance starch rice comprises the following steps:
1) the rice variety containing the high-resistance starch homozygous with the sbe3-rs gene is taken as a donor parent and is hybridized with another high-quality disease-resistant rice variety to obtain F 1 Seed generation; the high-quality disease-resistant rice refers to rice with compact plant type, robust plant, high yield, more panicle and large grain size and excellent rice quality, and the disease resistance refers to rice blast resistance;
2) planting F 1 Seed generation and harvest according to combination to obtain F 2 Generation of seed, pair F 2 Water dressing the seeds, that is, putting the seeds to be dressed in water, keeping the suspended seeds, drying in the sun until the water content reaches 12-14%, F 2 The ratio of homozygous high-resistance starch single plants in the grains is 50-70 percent;
3) planting F selected by water selection 2 Seeds, selecting individual plants with excellent comprehensive agronomic characters, harvesting according to the individual plants to obtain F 3 Seeds;
4) f to be obtained by dividing individual plants 3 Removing brown rice from seeds, and screeningKeeping single plant seeds with the brown rice chalkiness rate of more than 70 percent without molecular marker detection, planting the seeds into cells, carrying out rice blast field resistance identification on 50-100 plants in each cell, selecting a plant line with excellent comprehensive agronomic characters according with a breeding target, selecting excellent single plants from the selected plant lines, and harvesting according to the single plants to obtain F 4 Seed generation;
5) continuously planting in the way of step 4) until F is obtained 7 Seed generation, selection standard and step 4), planting F 7 And (3) carrying out KASP molecular marker detection of sbe3-rs genes at the seedling stage after the seeds, mixing and harvesting single seeds, and simultaneously determining the content of resistant starch to obtain the stable high-resistance starch improved variety meeting the breeding target.
Preferably, in the step 1), the high-quality disease-resistant rice refers to rice with compact plant type, robust plant, high yield, more panicle and large grain, good rice quality and rice blast resistance.
In step 1), the high-resistant starch rice variety is 'high-quality rice', and the high-quality disease-resistant rice is 'S08-18', 'SL-24', or 'Hu rice 89'.
Preferably, in the step 4), when the field resistance identification of the rice blast is carried out, artificial inoculation is mainly used, and natural induction is used as assistance.
And in the step 5), the content of the resistant starch of the obtained high-resistant starch rice variety is more than 10 percent.
In the planting process of conventional rice varieties, seeds with full seeds are usually selected by a saline water seed selection method and sown, so that seedlings are developed perfectly and grow into strong seedlings. The inventor researches and discovers that most of suspended grains are reserved by clean water for improving F by the characteristics that grains containing high resistant starch homozygous with sbe3-rs gene have loose structure among grains due to the starch structure of the grains, poor light transmittance and small specific gravity and have high chalkiness 2 High resistant starch gene homozygous ratio of the kernel.
The invention obtains high chalky grain in outlier with high efficiency by a low-generation water selection method, does not need molecular marker detection and resistant starch content determination in the low generation of breeding, only selects according to comprehensive agronomic characters in the field, investigates chalky grain rate indoors, and finally performs molecular marker detection and resistant starch content determination in high-generation stable lines, thereby greatly improving the breeding efficiency of breeding new varieties of high-resistant starch rice, and obtaining high-purity-closing ratio in the low-generation breeding process, wherein the homozygotic ratio is 54.65-63.16%.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses high-resistance starch rice containing sbe3-rs gene as donor parent to breed high-quality high-resistance starch rice variety, and the filial generation F is hybridized by a hybridization breeding method 1 Mixed and harvested to obtain F 2 Seeds; fully utilizing the high chalkiness characteristic of high resistant starch rice grains, having gaps in the starch grains of the grains and low specific gravity, carrying out water separation, separating suspended grains, airing to obtain F with the water content of 12-14% 2 The seeds do not need molecular marker detection, and the obtained high-resistance starch homozygous single plant is improved by 4-8 times in average in ratio compared with seeds not subjected to water selection.
In the present invention, in the pair F 2 Generation to F 6 In the seed generation breeding process, phenotype is utilized for screening, brown rice is removed by separating individual plants, the single plant with brown rice chalkiness rate of more than 70% is kept without molecular marker detection until F is obtained 7 The KASP molecular marker is used for detection after seed generation, and compared with F in the prior art 2 Compared with the screening method combining manual screening and molecular marker detection, the method greatly reduces the use of molecular marker detection, and saves manpower and breeding time.
Three hybrid combinations of the invention F 2 Seed selection by water F 2 The ratio of homozygous high-resistance starch in grains averagely reaches 58.65 percent, and the grain is manually selected to have high chalkiness degree (A)>50%) compared with the breeding efficiency of 30.6%, the breeding efficiency of the seeds is improved by about 2 times, and meanwhile, the selection method is simpler and quicker, the water selection efficiency is high, one combination can be selected in 10 minutes on average, the labor is greatly saved, and the cost is low.
The breeding method of the invention greatly improves the acquisition rate of the homozygous single plant of the low-generation resistant starch genotype of the high-resistant starch rice and the resistant starch F 2 Compared with the large population and low selection rate of the screened generations of the physical field, the homozygous rate obtained by low generations is greatly improved, the low-generation rapid screening of the homozygous single plants with high resistant starch content is realized, the breeding efficiency of the rice variety containing the sbe3-rs genotype high resistant starch is effectively improved, and the breeding cost is saved.
Drawings
FIG. 1 is an appearance diagram of the water-separated suspended medium seeds of example 1 of the present invention.
FIG. 2 is an appearance diagram of the bottom-sinking seed selection and discarding seed separation of the water separation in example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1 separate Water selection of different hybridization combinations F 1 Mixed harvested F 2 Seed of corn
High-resistance starch rice variety 'high-quality rice' containing sbe3-rs homozygous genotype is taken as a donor parent to be hybridized with 3 conventional rice varieties with good taste, and the 3 varieties are respectively 'S08-18', 'SL-24', 'Hu rice 89', and F is planted 1 Respectively mixing and harvesting in the mature period and the generation period F 1 Seed acquisition F 2 And (5) seed generation.
Handle F 2 The seeds are put into clear water, the plump seeds with large specific gravity all sink to the water bottom, the high-resistant starch homozygotic seeds can suspend because of low density, and the suspended seeds and the corresponding seeds which sink to the water bottom are respectively collected and reserved for later use.
Suspended seeds and seeds sinking to the bottom of the water are subjected to brown rice removal for chalky rate investigation, the appearance is shown in fig. 1-2, the chalky grain rate of the suspended seeds exceeds 80 percent, the suspended seeds simultaneously contain some shriveled grains and immature green grains, the shriveled grains and the immature green grains have low germination rate or are not grown into seedlings, and the suspended seeds and the seeds are naturally eliminated in the field planting process. The seeds at the bottom of the submerged water have good transparency and full seeds. And (4) drying the suspended seeds after water separation, reserving the seeds for later use, and discarding the settled seeds.
Example 2 No and Water beneficiation F 2 Generation seeds for KASP molecular marker detection genotyping
Using 'high-quality sugar rice' as donorThe parent is respectively F combined with three hybrids of ' S08-18 ', ' SL-24 ' and ' Hu rice 89 1-1 ，F 1-2 And F 1-3 And the gene typing of the sbe3-rs is detected by adopting an autonomously developed KASP molecular marker of the sbe 3-rs.
Designing a KASP primer according to the SNP mutation information site information of the high-resistance starch genotype sbe3-rs gene, wherein the KASP primer consists of two upstream primers (primer 1 and primer 2) and a downstream primer (primer 3), the 5 ' ends of the two upstream primers are respectively connected with a fluorescent tag sequence, the 5 ' end of the primer 1 is connected with a FAM fluorescent tag sequence 5'-GAAGGTGACCAAGTTCATGCT-3', and the 5 ' end of the primer 2 is connected with a HEX fluorescent tag sequence: 5'-GAAGGTCGGAGTCAACGG-3' are provided.
The specific nucleotides of the primers are as follows:
primer 1: 5'-TATGCTGAAAGTCATGATCAAGCACT-3', respectively;
primer 2: 5'-ATGCTGAAAGTCATGATCAAGCACC-3', respectively;
primer 3: 5'-CAACCAGAATGCAATAGTTTTGTCACCAA-3' are provided.
The 3' terminal allelic variant base T/C of the two upstream primers is used for distinguishing alleles, 1 and 3 amplification reads wild type, 2 and 3 amplification reads mutant type, if heterozygote type, 1 and 2 can be amplified with 3, and the sequence amplification of the downstream primer is 60-100 bp.
The specific KASP molecular marker detection method is as follows:
1. sample preparation: the DNA of a leaf sample is extracted by adopting a CTAB method, and the method for extracting the small-amount DNA of rice mainly refers to the report of McCouch et al (1988), and the method is briefly described as follows:
1) shearing a small piece of leaf 4-5cm, adding 700 μ L of 1.5 × CTAB (containing 1.5% CTAB, 75mM Tris-HCl, 15mM EDTA, 1.05M NaCl), and grinding thoroughly;
2) transferring the homogenate into a centrifugal tube of 1.5ml, carrying out water bath at 56 ℃ for 20min, and cooling to room temperature;
3) equal volume of chloroform was added: isoamyl alcohol (24: 1), shaking up;
4) centrifuging at the highest speed (13200rpm) for 10 min;
5) transferring the supernatant into a new centrifuge tube, adding precooled 100% alcohol with twice volume, standing for 20min, and centrifuging to collect DNA;
6) removing supernatant, air drying DNA, adding 50-100 μ L double distilled water for dissolving, and detecting in ultraviolet spectrophotometer.
7) Diluting DNA, preparing a set of DNA working solution with the concentration of about 50-100 ng/microliter, preserving in a refrigerator at 4 ℃ for later use,
2. KASP marker detection
PCR systems were constructed, 15. mu.l per reaction: 2 XKASP master mix 7.5. mu.l, primer 1-FAM: 0.25. mu.l, primer 2-HEX: 0.25. mu.l, universal primer 3: 0.5. mu.l, DNA template 2. mu.l, H2O: 4.5. mu.l.
And (3) PCR reaction conditions: first step pre-denaturation at 94 ℃ for 15 min; a second step of denaturation, renaturation and extension, wherein the temperature is 94 ℃ for 20s, and the temperature is reduced from 61 ℃ to 55 ℃ (0.6 ℃ per cycle) for 60s, and the number of cycles is 10; third, denaturation, renaturation and extension are carried out for 26 cycles at 94 ℃ for 20s and 55 ℃ for 60 s; and fourthly, after the reaction is finished, reading the plate on a microplate reader Pheastar.
Adopting SNPviewer2 software to analyze the scanning data, determining the sbe3-rs genotype of the rice sample according to the analysis result, and respectively carrying out water sorting and non-water sorting on 3 hybridization combinations (F) 1-1 、F 1-2 And F 1-3 ) F of (A) 2 The genotype of the population was analyzed and the results are shown in table 1.
TABLE 1 genotyping results of sbe3-rs Gene KASP assay
As can be seen from the table, F of 3 hybridization combinations without water selection 2 After germination of seeds, the detection shows that the ratio of the homozygous sbe3-rs genes is 7.69-12.36%, the average is only 9.98%, and F subjected to water selection by the method is 2 The homozygous ratio of generation gene is 54.65% -63.16%The homozygote type is improved by 4-8 times compared with the proportion without water selection, thus the homozygote type ratio obtained by low generation is greatly improved, and the acquisition rate of the low generation resistant starch genotype homozygous single plants of the high resistant starch rice is greatly improved.
Example 3
Planting of F sorted by Water sorting in example 1 2 Seeds, selecting individual plants with excellent comprehensive agronomic characters, harvesting according to the individual plants to obtain F 3 Seeds; removing brown rice from single plant indoors, screening single plant with chalkiness rate of over 70 percent of the brown rice, reserving the single plant without molecular marker detection, planting the single plant into cells, carrying out rice blast field resistance identification in the high rice blast period of 50-100 plants in each cell, selecting a plant line with excellent comprehensive agronomic characters according with breeding targets, selecting excellent single plant from the selected plant line, harvesting according to the single plant to obtain F 4 Seed generation;
according to F 4 The seed generation is continuously planted in the obtaining mode until F is obtained 7 Seed generation, selection criteria are same as F 4 Selection criteria for generations, planting F 7 After seed generation, KASP molecular marker detection of sbe3-rs gene is carried out in seedling stage to verify genotype, the KASP detection method refers to example 2, single plant seeds are harvested in a mixed mode, and meanwhile resistant starch content is measured, namely, stable high-resistant starch improved varieties which accord with breeding targets are obtained, and the resistant starch content of the obtained high-resistant starch rice varieties is more than 10%.
Claims (6)
1. A method for improving the low-generation breeding efficiency of high-resistance starch rice comprises the following steps:
1) the rice variety containing the high-resistance starch homozygous with the sbe3-rs gene is taken as a donor parent and is hybridized with another high-quality disease-resistant rice variety to obtain F 1 Seed generation;
2) planting F 1 Seed generation and harvest according to combination to obtain F 2 Generation of seed, pair F 2 Water dressing the seeds, that is, putting the seeds to be dressed in water, keeping the suspended seeds, drying in the sun until the water content reaches 12-14%, F 2 The ratio of homozygous high-resistance starch single plants in the grains is 50-70 percent;
3) planting water-sorted F 2 Seeds, selecting individual plants with excellent comprehensive agronomic characters, harvesting according to the individual plants to obtain F 3 Seeds;
4) f to be obtained by dividing individual plants 3 Removing brown rice from seeds, screening single plant seeds with the brown rice chalkiness rate of more than 70%, reserving, planting into cells with 50-100 plants in each cell without molecular marker detection, identifying rice blast field resistance, selecting plant lines with excellent comprehensive agronomic characters according with breeding targets, selecting excellent single plants from selected plant lines, and harvesting according to the single plants to obtain F 4 Seed generation;
5) continuously planting in the way of step 4) until F is obtained 7 Seed generation, selection standard is same as step 4), planting F 7 And (3) carrying out KASP molecular marker detection of sbe3-rs genes at the seedling stage after the seeds, mixing and harvesting single seeds, and simultaneously determining the content of resistant starch to obtain the stable high-resistance starch improved variety meeting the breeding target.
2. The method for improving the low-generation breeding efficiency of the high-resistant starch rice according to claim 1, wherein in the step 1), the high-quality disease-resistant rice refers to rice with compact plant type, robust plant, high yield, more panicle and large grain, excellent rice quality and rice blast resistance.
3. The method for improving the low-generation breeding efficiency of high-resistance starch rice according to claim 1, wherein in the step 1), the high-resistance starch rice variety is 'D-oryza sativa'.
4. The method according to claim 1, wherein the high-quality disease-resistant rice in step 1) is selected from the group consisting of 'S08-18', 'SL-24' and 'Hu rice 89'.
5. The method for improving the low-generation breeding efficiency of high-resistance starch rice as claimed in claim 1, wherein in the step 4), the artificial inoculation is mainly used and the natural induction is used as the auxiliary for the identification of the rice blast field resistance.
6. The method for improving the low-generation breeding efficiency of high-starch-resistance rice according to claim 1, wherein the resistant starch content of the obtained high-starch-resistance rice variety in the step 5) is more than 10%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105993931A (en) * | 2016-05-19 | 2016-10-12 | 上海市农业科学院 | Method for breeding of high resistant starch rice two-line sterile line |
| CN108901826A (en) * | 2018-07-03 | 2018-11-30 | 安徽荃银高科种业股份有限公司 | A kind of method of fast accurate breeding High quality and diseases resistance rice varieties |
| CN111084096A (en) * | 2019-12-31 | 2020-05-01 | 上海市农业科学院 | Breeding method of rice variety with high-resistance starch and low-gluten polymerization |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105993931A (en) * | 2016-05-19 | 2016-10-12 | 上海市农业科学院 | Method for breeding of high resistant starch rice two-line sterile line |
| CN108901826A (en) * | 2018-07-03 | 2018-11-30 | 安徽荃银高科种业股份有限公司 | A kind of method of fast accurate breeding High quality and diseases resistance rice varieties |
| CN111084096A (en) * | 2019-12-31 | 2020-05-01 | 上海市农业科学院 | Breeding method of rice variety with high-resistance starch and low-gluten polymerization |
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| 袁隆平主编, vol. 1, 山东科学技术出版社, pages: 255 * |
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