CN114946649B - 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
Method for improving low-generation breeding efficiency of high-resistance starch rice by using high-resistance starch rice containing sbe3-rs gene as donor parent and crossing offspring F 1 Mixing and harvesting to obtain F 2 Seed; water separation is carried out by utilizing the high chalkiness characteristic of high-resistance starch rice grains, and suspended grains are separated to obtain F 2 Seed; the genotyping detection of the selected seeds after water selection shows that the average ratio of the obtained high-resistance starch homozygous individual strain is improved by 4-8 times compared with the seed without water selection, and the high chalkiness degree of manual selection is achieved>50%) the breeding efficiency of the seeds is improved by nearly 2 times compared with 30.6%. The method realizes the rapid screening of homozygous single plants with high resistant starch content in low generation, effectively improves the breeding efficiency of breeding rice varieties with high resistant starch containing sbe3-rs genotype, saves the breeding cost, and can rapidly and efficiently perform mass operation to obtain 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 low-generation breeding efficiency of high-resistance starch rice.
Background
Resistant Starch (RS for short) refers to "Starch or Starch degradation products that are not absorbable in the small intestine of healthy individuals" (defined by the european RS association, eurata). The resistant starch has important physiological functions, can reduce postprandial blood sugar and insulin response, improve the sensitivity of an organism to insulin, prevent constipation and colon cancer, reduce the content of cholesterol and triglyceride in serum, reduce and control weight, and promote mineral absorption. The rice genetic resource with high resistant starch content is screened and identified, so that the rice variety with high resistant starch content is provided, and the improvement of the dietary structure of people is one of the most economical and effective means for treating and preventing the onset of civilized diseases such as diabetes mellitus.
The research institute of crop cultivation and breeding in Shanghai agricultural sciences utilizes chemical mutagenesis and microspore culture technology and combines the conventional breeding technology to cultivate a new variety of high-resistance starch japonica rice No. 1' of hypoglycemic rice, and cooperates with related enterprises to carry out industrialized development, thereby relieving the difficult problem that diabetics eat food difficultly. The research of cultivation and breeding of crops in Shanghai national academy of agricultural sciences is that 'sugar-reducing rice No. 1' is taken as a research object, a main gene SBE3-RS for controlling the RS content of rice is positioned, functional complementation verification is carried out, the mutant gene has a base mutation of T-C at the 105 th position corresponding to the 16 th exon of the SBE3 gene of rice starch branching enzyme, and a plurality of functional marker molecular markers are developed for the locus.
According to the genetic characteristics of the high-resistance starch character gene and the high correlation relationship between the high-resistance starch character gene and characters such as chalkiness, chalkiness and the like, a developed CAPS/SpeI molecular marker is utilized to establish a rice molecular breeding technology system with high-resistance starch content. The team uses 'sugar-reducing rice No. 1' as a parent of a high-resistance starch character, and successfully breeds a new high RS content rice strain 'excellent sugar rice No. 2' and 'excellent sugar rice No. 3' which have both functions and high yield and high quality. However, the rice with excellent sugar still has the problems of poor taste, poor stress resistance and the like, and the genetic breeding method is of great significance in continuously improving the taste of the rice with excellent sugar, improving the stress resistance and other comprehensive agronomic characters.
Yang Ruifang et al (journal of nuclear agriculture, 2015, 29 (12): 2259-2267) firstly propose to select and eliminate most of low-resistance starch single plants by utilizing the chalky characteristic of early generation grains of hybrid populations, and simultaneously combine molecular marker assisted selection to improve the breeding efficiency of high-resistance starch rice varieties. F (F) 1 Planting to obtain separated charactersF 2 Seed, high resistant starch grain chalkiness is higher, and selecting chalkiness grain to plant into cell, the probability of obtaining homozygous high resistant starch genotype by offspring is greatly improved, and chalkiness degree is selected by manpower>50% F 2 The ratio of homozygous highly resistant starch individuals in the grain was 30.6%.
The resistant starch content assay was mainly carried out using an RS content assay kit (Megazyme, co. Wicklow, ireland) provided by Megazyme company, and the method was slightly improved. The method comprises the following specific steps: accurately weighing 100mg rice flour sample, carefully placing into a plastic test tube with a screw cap, sequentially adding alpha-pancreatic amylase reaction solution and Amyloglucosidase (AGM), vibrating at 37 ℃ and incubating for 16 hours, dissolving non-RS, and hydrolyzing into D-glucose; after the incubation is finished, 99% ethanol is added to terminate the reaction; centrifuging the solution, discarding the supernatant, obtaining residual floccules at the bottom, namely RS in the sample, and washing the precipitate with 50% ethanol; the centrifuge tube was inverted, the pellet was dried, dissolved in 2mol L-1 potassium hydroxide, AGM was added, incubated in a 60℃water bath for 1h, and finally glucose content was measured with D-glucose using glucose oxidase/peroxidase reagent (GOPOD) reagent, and RS content was calculated. The determination process of the resistant starch is complex, long in time consumption and high in cost, and becomes a limiting factor for breeding high-resistance starch rice. F (F) 1 F is obtained by mixed harvesting of low generation (10-18 plants) 2 Instead of seeds, the selection of the chalk-like grains by manpower, a combination of which takes 1-2 hours, takes time and is costly.
Disclosure of Invention
The invention aims to provide a method for improving the low-generation breeding efficiency of high-resistance starch rice, which overcomes the defects that the proportion of low-generation separation group high-resistance starch homozygous single plants 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 proportion of homozygous single plants is time-consuming, labor-consuming and high in cost, realizes the rapid screening of the high-resistance starch content homozygous single plants in the low generation, effectively improves the breeding efficiency of the high-resistance starch rice variety containing sbe3-rs genotype, saves the breeding cost, and can rapidly and efficiently perform mass operation to obtain high-quality high-resistance starch rice varieties.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for improving the low-generation breeding efficiency of high-resistance starch rice comprises the following steps:
1) The rice variety with high resistance and homozygous sbe3-rs gene is used as donor parent and hybridized with another rice variety with high disease resistance to obtain F 1 Seed generation; the high-quality disease-resistant rice has compact plant type, strong plants, high yield, large grains per spike and excellent rice quality, and the disease resistance refers to rice blast resistance;
2) Planting F 1 Seed generation, harvesting according to combination to obtain F 2 Seed generation, pair F 2 Water selecting the seed, namely placing the seed to be selected in water, retaining the suspended seed, airing until the water content reaches 12-14%, F 2 The ratio of the homozygous high-resistance starch single plants in the seeds is 50% -70%;
3) F selected in planting warp water selection 2 Seed, selecting single plant with excellent comprehensive agronomic characters, harvesting according to the single plant to obtain F 3 Seed;
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%, planting into cells without molecular marker detection, carrying out 50-100 plants in each cell, carrying out rice blast field resistance identification, selecting a plant line with good comprehensive agronomic characters meeting breeding targets, selecting a good single plant from the selected plant lines, and harvesting according to the single plant to obtain F 4 Seed generation;
5) Planting continuously according to the method of the step 4) until F is obtained 7 Seed generation, selection standard and step 4), F is planted 7 And (3) after the seeds are planted, carrying out KASP molecular marker detection of the sbe3-rs gene in the seedling stage, mixing and harvesting single seeds, and simultaneously measuring the content of resistant starch to obtain a stable high-resistance starch improved variety meeting the breeding target.
Preferably, in the step 1), the high-quality disease-resistant rice is rice with compact plant type, strong plants, high yield, large grains per spike, excellent rice quality and rice blast resistance.
In the step 1), the high-resistance starch rice variety is 'high-quality sugar rice', and the high-quality disease-resistant rice is 'S08-18', 'SL-24', 'Shanghai rice 89'.
Preferably, in the step 4), the rice blast field resistance is identified by using artificial inoculation as a main part and natural induction as an auxiliary part.
In step 5), the content of resistant starch in the obtained high-resistant starch rice variety is more than 10%.
In the conventional rice variety planting process, seeds with full seeds are usually selected by a saline water seed selection method and sowed down, so that seedlings develop perfectly and grow into strong seedlings further. The inventor researches find that the rice containing the high-resistance starch homozygous for the sbe3-rs gene has the characteristics of loose inter-grain structure, poor light transmittance, small specific gravity and high chalkiness due to the starch structure of the grains, and most suspended grains are reserved through clear water flotation so as to improve F 2 High resistant starch gene homozygous ratio of kernels.
According to the invention, gao E grains in the isolate are obtained efficiently by a low-generation water selection method, molecular marker detection and resistant starch content measurement are not required in the low-generation breeding, only the screening is carried out in the field according to comprehensive agronomic characters, the chalky grain rate investigation is carried out indoors, and finally the molecular marker detection and resistant starch content measurement are carried out on the high-generation stable strain, so that the breeding efficiency of the novel high-resistant starch rice variety breeding is greatly improved, and the high-homozygosity rate can be obtained in the low-generation breeding process, wherein the homozygosity rate 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 select and breed high-quality high-resistance starch rice variety, and the hybridization offspring F is hybridized by hybridization breeding method 1 Mixing and harvesting to obtain F 2 Seed; fully utilizing the high chalkiness characteristic of high-resistance starch rice grains, wherein the starch grains of the grains have gaps and low specific gravity, separating suspended grains by water separation, and airing until the moisture content reaches 12-14% to obtain F 2 Seed, no molecular marker detection is needed, and the obtained high-resistance starch homozygous single plant ratio is not availableThe average of the water-selected seeds is improved by 4-8 times.
In the present invention, in pair F 2 Substituted for F 6 In the seed selection process of the generation seeds, phenotype is used for screening, single plants are separated to remove brown rice, single plants with the brown rice chalkiness rate of more than 70% are reserved, molecular marker detection is not needed, and F is obtained 7 After seed generation, KASP molecular marker is used for detection, compared with F in the prior art 2 Compared with the screening method of combining manual screening with molecular marker detection, the method greatly reduces the use of molecular marker detection and saves manpower and breeding time.
F of the three hybridization combinations of the invention 2 Seed selection F by water 2 The average ratio of the homozygous high-resistance starch single plants in the grains reaches 58.65 percent, and the grain is manually selected to have high chalkiness degree>50%) seed breeding efficiency is improved by nearly 2 times compared with 30.6%, meanwhile, the selecting method is simpler and faster, water selecting efficiency is high, an average of 10 minutes can select one combination, labor is greatly saved, and cost is low.
The breeding method of the invention greatly improves the yield of the low-generation resistant starch genotype homozygous single plant of the high-resistant starch rice and the resistant starch F 2 Compared with large field screening population and low selection rate of the generation material, the homozygous rate obtained in the low generation is greatly improved, the low generation rapid screening of the homozygous single plant with high resistant starch content is realized, the breeding efficiency of the rice variety with high resistant starch containing sbe3-rs genotype is effectively improved, and the breeding cost is saved.
Drawings
FIG. 1 is an external view of the water-separated seed-in-suspension of example 1 of the present invention.
FIG. 2 is an external view of the water-separated seed of the bottom separator in example 1 of the present invention.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Example 1 Water selection of different hybridization combinations F respectively 1 F of Mixed harvest 2 Seed (seed)
Uses high-resistance starch rice variety 'excellent sugar rice' containing sbe3-rs homozygous genotype as a supplyThe parent strain is hybridized with 3 conventional rice varieties with good taste, the 3 varieties are respectively 'S08-18', 'SL-24', 'Shanghai rice 89', and F is planted 1 Generation and maturity are respectively mixed and harvested F 1 Seed harvesting F 2 Seed generation.
Handle F 2 The seeds of the generation are put into clear water, the seeds with relatively full specific gravity are all sunk in the water, the high-resistance starch homozygous seeds are suspended because of the relatively small density, and the suspended seeds and the corresponding seeds sunk in the water are respectively collected and reserved for standby.
The chalkiness rate is investigated by removing brown rice from suspended seeds and seeds sunk in the water, the appearance is shown in figures 1-2, the chalkiness rate of the suspended seeds exceeds 80%, the suspended seeds contain some shrunken grains and immature green grains, the germination rate of the shrunken grains and the immature green grains is very low or the green grains do not grow into seedlings, and the suspended seeds are naturally eliminated in the field planting process. The submerged seeds have good transparency and full seeds. And (5) airing the suspended seeds for seed reservation after water selection, and discarding the sinking seeds.
Example 2F without Water selection and Water selection 2 KASP molecular marker detection genotyping of seed generation
The 'you sugar rice' is used as donor parent, and the three hybridization combinations of the 'you sugar rice' and the 'S08-18', 'SL-24', 'Hu rice 89' are respectively F 1-1 ，F 1-2 And F 1-3 And detecting the KASP molecular marker of the sbe3-rs, which is independently developed, for the genotyping of the sbe 3-rs.
According to SNP mutation information locus information of high-resistance starch genotype sbe3-rs genes, designing KASP primers, wherein each KASP primer consists of two upstream primers (primer 1 and primer 2) and one downstream primer (primer 3), and fluorescent tag sequences are respectively connected to the 5' ends of the two upstream primers, wherein FAM fluorescent tag sequences 5'-GAAGGTGACCAAGTTCATGCT-3' are connected to the 5' ends of the primers 1, and HEX fluorescent tag sequences are connected to the 5' ends of the primers 2: 5'-GAAGGTCGGAGTCAACGG-3'.
The specific nucleotides of the primer are as follows:
primer 1:5'-TATGCTGAAAGTCATGATCAAGCACT-3';
primer 2:5'-ATGCTGAAAGTCATGATCAAGCACC-3';
primer 3:5'-CAACCAGAATGCAATAGTTTTGTCACCAA-3'.
The allelic variation base T/C at the 3' end of the two upstream primers is used for distinguishing alleles, 1 and 3 amplification read wild type, 2 and 3 amplification read mutant type, if heterozygous, 1 and 2 can be amplified with 3 amplification, and the sequence of the downstream primer can be amplified between 60 and 100bp.
The specific detection method of the KASP molecular marker is as follows:
1. sample preparation: the CTAB method is used for extracting leaf sample DNA, and the rice small-scale DNA extraction method is mainly referred to the report of McCouch et al (1988), and the method is briefly described as follows:
1) Cutting a small blade 4-5cm, adding 700 μL 1.5XCTAB (containing 1.5%CTAB,75mM Tris-HCl,15mM EDTA,1.05M NaCl), and grinding thoroughly;
2) Transferring the homogenate into a centrifuge tube with volume of 1.5ml, carrying out water bath at 56 ℃ for 20min, and cooling to room temperature;
3) An equal volume of chloroform was added: isoamyl alcohol (24:1), shaking uniformly;
4) Centrifuging at the highest speed (13200 rpm) for 10min;
5) Transferring the supernatant into a new centrifuge tube, adding twice the volume of precooled 100% alcohol, standing for 20min, and centrifuging to collect DNA;
6) Removing supernatant, air drying DNA, adding 50-100 μl of 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/mu L, storing in a refrigerator at 4 ℃ for standby,
2. kasp marker detection
PCR system construction, 15 μl system per reaction: 2 XKASP master mix 7.5. Mu.l, primer 1-FAM:0.25 μl, primer 2-HEX:0.25 μl, universal primer 3:0.5 μl, 2 μl of DNA template, H2O:4.5 μl.
PCR reaction conditions: the first step is to pre-denature at 94 ℃ for 15min; the second step of denaturation, renaturation and extension, wherein the temperature is 94 ℃ for 20s, the temperature is reduced from 61 ℃ -55 ℃ (0.6 ℃ for each cycle) for 60s, and the total time is 10 cycles; thirdly, denaturation, renaturation and extension are carried out, wherein the temperature is 94 ℃ for 20s, the temperature is 55 ℃ for 60s, and 26 cycles are carried out; and fourthly, reading the plate on a Pherastar of an enzyme label instrument after the reaction is finished.
The scan data were analyzed using SNPviewer2 software, and the sbe3-rs genotype of the rice samples was determined based on the analysis results, for each of the 3 hybridization combinations (F 1-1 、F 1-2 And F 1-3 ) F of (2) 2 The genotype of the population was analyzed and the results are shown in Table 1.
TABLE 1 genotyping results of the detection of the sbe3-rs Gene KASP
As can be seen from the table, F of 3 hybridization combinations without water selection 2 After germination of the seed, the detection shows that the ratio of the homozygous sbe3-rs gene is 7.69% -12.36%, the average is only 9.98%, and F is selected by the water selection method 2 The homozygous ratio of the generation gene is 54.65% -63.16%, the average is about 58.65%, the homozygous ratio of the water selection is improved by 4-8 times compared with the homozygous ratio of the water selection, and therefore, the homozygous ratio of the low generation is greatly improved, and the homozygous single plant yield of the low generation resistant starch genotype of the high resistant starch rice is greatly improved.
Example 3
Planting F selected in Water selection of example 1 2 Seed, selecting single plant with excellent comprehensive agronomic characters, harvesting according to the single plant to obtain F 3 Seed; separating the brown rice from the single plants indoors, screening the single plants with the brown rice chalkiness rate of more than 70%, planting the single plants into cells without molecular marker detection, carrying out 50-100 plants in each cell, carrying out rice blast field resistance identification in a rice blast high-incidence period, selecting a plant line with good comprehensive agronomic characteristics meeting breeding targets, selecting a good single plant from the selected plant lines, and 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 the seed is obtainedF 7 Seed generation, selection criteria are the same as F 4 Screening standard of generation, planting F 7 After seed generation, KASP molecular marker detection of the sbe3-rs gene is carried out in the seedling stage to verify the genotype, the KASP detection method is referred to in example 2, individual seeds are harvested in a mixed mode, and the resistant starch content is measured, so that a stable high-resistance starch improved variety meeting the breeding target is obtained, and the resistant starch content of the obtained high-resistance starch rice variety is more than 10%.
Claims (3)
1. A method for improving the low-generation breeding efficiency of high-resistance starch rice comprises the following steps:
1) The rice variety with high resistance and homozygous sbe3-rs gene is used as donor parent and hybridized with another rice variety with high disease resistance to obtain F 1 Seed generation; the high-resistance starch rice variety is 'high-quality sugar rice', and the high-quality disease-resistant rice is 'S08-18', 'SL-24', 'Shanghai rice 89';
2) Planting F 1 Seed generation, harvesting according to combination to obtain F 2 Seed generation, pair F 2 Water selecting the seed, namely placing the seed to be selected in water, retaining the suspended seed, airing until the water content reaches 12-14%, F 2 The ratio of the homozygous high-resistance starch single plants in the seeds is 50% -70%;
3) F selected in planting warp water selection 2 Seed, selecting single plant with excellent comprehensive agronomic characters, harvesting according to the single plant to obtain F 3 Seed;
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%, planting into cells without molecular marker detection, carrying out 50-100 plants in each cell, carrying out rice blast field resistance identification, selecting a plant line with good comprehensive agronomic characters meeting breeding targets, selecting a good single plant from the selected plant lines, and harvesting according to the single plant to obtain F 4 Seed generation;
5) Planting continuously according to the method of the step 4) until F is obtained 7 Seed generation, selection standard and step 4), F is planted 7 After seeds are planted, KASP molecular marker detection of the sbe3-rs gene is carried out in the seedling stage, individual seeds are harvested in a mixed mode,and meanwhile, the content of resistant starch is measured, so that a stable high-resistance starch improved variety meeting the breeding target is obtained, and the content of the resistant starch in the obtained high-resistance starch rice variety is more than 10%.
2. 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-quality disease-resistant rice is rice with compact plant type, strong plants, high yield, large grains per spike, 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 4), the rice blast field resistance is identified mainly by artificial inoculation and is induced naturally as an auxiliary.
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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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| 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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