CN111084096B - Breeding method of rice variety with high-resistance starch and low-gluten polymerization - Google Patents

Breeding method of rice variety with high-resistance starch and low-gluten polymerization Download PDF

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CN111084096B
CN111084096B CN201911405701.2A CN201911405701A CN111084096B CN 111084096 B CN111084096 B CN 111084096B CN 201911405701 A CN201911405701 A CN 201911405701A CN 111084096 B CN111084096 B CN 111084096B
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杨瑞芳
白建江
朴钟泽
万常照
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Shanghai Academy of Agricultural Sciences
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Abstract

The invention discloses a breeding method of a rice variety polymerized by high resistant starch and low glutelin, which comprises the steps of taking the rice variety with high resistant starch content as a parent I and the rice variety with low glutelin content as a parent II, and hybridizing to obtain F1 generation seeds; backcrossing the F1 generation with the parent I to obtain BC1F1 generation seeds, and selecting BC1F1 seeds with chalkiness degree of more than 70%; detecting the BC1F1 generation with resistant starch molecular marker and low glutelin molecular marker after sowing, screening to obtain high resistant starch genotype homozygous and low glutelin genotype heterozygous single plants, and selfing to obtain BC1F2 generation seeds; and planting BC1F2 generations, screening to obtain a strain line homozygous for both the resistant starch genotype and the low gluten genotype by adopting low gluten molecular marker detection, detecting and screening a strain line with the resistant starch content of more than 10% and the gluten content of less than 3% by using the obtained seeds, and continuously selfing the strain line of the screened BC1F3 generations to BC1F5 generations to obtain a new rice variety with high resistant starch content, low gluten content and high yield and low gluten.

Description

Breeding method of rice variety with high-resistance starch and low-gluten polymerization
Technical Field
The invention relates to the technical field of rice breeding, in particular to a rice variety which is suitable for being eaten by diabetics and nephropathy patients and is polymerized with high-resistance starch and low-gluten protein and a breeding method thereof.
Background
The latest data show that the number of Chinese diabetic patients reaches 1.14 hundred million, which accounts for about one third of the total number of the global diabetic patients, and China becomes the country with the largest number of the global diabetic patients. Even more alarming is that the prevalence of diabetes in our country is only about 40%. In addition to the inconvenience of diabetes itself to the life of the patient, diabetes may also cause various complications, such as cataract, nephropathy, diabetic foot, peripheral neuropathy, etc.
The resistant starch has important physiological functions, can reduce postprandial blood sugar and insulin response, improve the sensitivity of the body to insulin, prevent constipation and colon cancer, reduce the content of cholesterol and triglyceride in serum, reduce and control body weight, promote mineral absorption and the like. Compared with low-resistance starch drinkers, high-resistance starch drinkers have less insulin response, which has great influence on the control of postprandial blood glucose values of diabetics, and especially for noninsulin-dependent patients, the high-resistance starch food can delay the rise of postprandial blood glucose and effectively control the conditions of diabetes mellitus.
As a main staple food, the rice has attracted great attention of rice breeding experts at home and abroad to improve the resistant starch content. In addition to starch, rice contains about 8% to 10% protein, and is classified into four types according to solubility: the rice protein comprises alkali-soluble gluten, alcohol-soluble prolamin, water-soluble albumin and salt-soluble globulin, wherein the gluten accounts for 80 percent of the protein in rice and is the protein with the highest content in rice seeds and the protein which is most easily absorbed by a human body. However, although rice having a higher gluten content has a higher nutritional value, it is necessary to strictly control protein intake because excessive protein intake increases the burden on the kidney due to protein metabolism disorders in renal disease patients or diabetic patients who suffer from renal dysfunction. Therefore, the cultivation of the low-gluten rice as a dietary therapy auxiliary product has great significance for special people such as nephropathy patients or diabetes patients with renal dysfunction.
Therefore, there is a need to provide a functional rice variety suitable for diabetic patients and renal disease patients, particularly for diabetic patients who have renal complications or renal dysfunction.
Disclosure of Invention
In view of the above technical problems, the present invention aims to provide a method for breeding functional rice varieties suitable for diabetic patients and renal patients, which aims to aggregate 2 excellent characteristics of high-content resistant starch and low-content gluten, thereby breeding novel functional rice varieties particularly suitable for renal patients, diabetic patients and diabetic patients with renal complications or renal dysfunction.
The technical scheme of the invention is as follows.
The invention provides a breeding method of a rice variety polymerized by high resistant starch and low glutelin, which comprises the following steps:
(1) taking No. 1 hypoglycemic rice or other derivative varieties containing sbe3-rs genes as a parent I, taking a rice variety containing a glutelin Lgc-1 gene as a parent II, and hybridizing to obtain F1 generation seeds;
(2) backcrossing the F1 generation with the parent I to obtain BC1F1 generation seeds, polishing the seeds into brown rice, and screening the seeds with the chalkiness degree of more than 70 percent;
(3) planting the BC1F1 generation seeds obtained in the step (2), sampling and extracting DNA, screening single plants homozygous for sbe3-rs genotype and heterozygous for Lgc-1 genotype through high-resistance starch CAPS molecular marker detection and low-gluten KASP molecular marker detection, and obtaining BC1F2 generation seeds;
(4) planting BC1F2 generation seeds, sampling and extracting DNA, screening to obtain single plants homozygous for sbe3-rs genotype and Lgc-1 genotype through low glutelin KASP molecular marker detection to obtain BC1F3 generation seeds, polishing the seeds into polished rice, measuring resistant starch content and glutelin content after grinding, and screening to obtain single plant reserved seeds with the resistant starch content of more than 10 percent and the glutelin content of less than 3 percent;
(5) planting the BC1F3 seeds obtained in the step (4), and continuously selfing until BC1F5 seeds are obtained;
(6) and (3) planting BC1F5 seeds, and selecting a variety with the yield per mu of more than 400 kg.
In the breeding method provided by the invention, the parent I in the step (1) has rice high resistant starch content major gene SBE3-rs, namely mutation of base T- > C at 105 th position of 16 th exon of SBE3 gene, see Chinese patent application CN 201210266649.9. Typically, parent I has a gluten content of about 5.6%.
Preferably, the resistant starch content of said parent I is more than 10%. In the present invention, the percentage of resistant starch content is weight percent, calculated on the dry weight of the sample. In the text below, a specific determination of the resistant starch content is provided.
According to a specific embodiment of the present invention, the derivative variety of the parent I is oryza sativa No. 2 (plant New variety weight certificate No. 2018010533, its variety weight No. CNA20150659.3) or oryza sativa No. 3 (plant New variety weight certificate No. 2018011367, its variety weight No. CNA 20150658.4).
In the breeding method provided by the invention, the parent II in the step (1) comprises Lgc-1 gene, which is deleted in 3' end of GluB5 gene and 3.5kb DNA sequence downstream thereof (refer to Tian Meng et al, design and verification of two low glutelin gene insertion deletion markers, molecular plant breeding, 2010, 8 th vol, 2 nd, 340 nd page 344). Typically, the parent II has a resistant starch content of about 0.45%.
Preferably, the gluten content of the parent II is less than 3%. In the present invention, the percentage of gluten content is in weight percent, calculated on the dry weight of the sample. In the text below, specific assays for gluten content are provided.
According to a particular embodiment of the invention, the parent II is the Japanese low gluten variety LGC-1.
Preferably, in the breeding method provided by the present invention, genomic DNA is extracted from leaves of an individual plant.
Preferably, in the breeding method provided by the invention, high-resistance starch CAPS molecular marker detection is also carried out in the step (4) so as to verify sbe3-rs genotype homozygosis of the screened individual plant homozygous for Lgc-1 genotype. Alternatively, preferably, each generation of individual plants planted in step (5) is subjected to high-resistance starch CAPS molecular marker detection and low-gluten KASP molecular marker detection to verify that both sbe3-rs genotype and Lgc-1 genotype are homozygous.
Preferably, in the breeding method provided by the invention, the detection of the CAPS molecular marker of the high-resistance starch comprises the following steps:
the extracted DNA was PCR amplified with the following primers and then genotyped based on detection of the amplified product:
SpeI F(SEQ ID NO:1)：5’-ATGTGATGTGCTGGATTTGG-3’；
SpeI R(SEQ ID NO:2)：5’-TGTGGTTTTCATACCGTTCTTA-3’。
preferably, the obtained amplification product can be identified enzymatically by using a restriction enzyme SpeI, which comprises: PCR amplification yielded a 571bp fragment, which, if it had a T → C base mutation at position 105 of the 16 th exon corresponding to the rice starch branching enzyme SBE3 gene, resulted in the loss of the restriction site for the restriction enzyme SpeI at this position (due to the mutation of ACTAGT to ACCAGT). Therefore, SpeI is adopted for enzyme digestion, two bands of 375bp and 196bp are obtained as the enzyme digestion result, and the plant has low resistant starch content; only 571bp of one band, the plant is homozygous for the sbe3-rs genotype; there were 3 bands, and the plants were heterozygous for the sbe3-rs genotype.
Preferably, in the breeding method provided by the invention, the detection of the low glutelin KASP molecular marker comprises:
the extracted DNA was PCR amplified with the following primers and then genotyped based on detection of the amplified product:
primer 1(SEQ ID NO: 3): 5'-TGCTGGCATGGTAGGACAAAATTG-3', respectively;
primer 2(SEQ ID NO: 4): 5'-CTTGCTGGCATGGTAGGACAAAATTT-3', respectively;
universal primer 3(SEQ ID NO: 5):
5’-AACGGTTTAGATGAGAACTTCTGCACAAT-3’；
universal primer 4(SEQ ID NO: 6):
5’-AAAACAATACTGACACGCCACACACAAT-3’。
wherein, the universal primer 3 is positioned on the deletion fragment of Lgc-1 genotype, and the universal primer 4 is positioned on the downstream sequence.
Preferably, the 5 'end of primer 1 is connected with FAM fluorescent label, the 5' end of primer 2 is connected with HEX fluorescent label, and then identification is carried out according to the fluorescent signal of the amplification product, which comprises: reading the PCR reaction result on a microplate reader, analyzing the fluorescent signal by adopting SNPviewer2 software, showing red, wherein the plant is Lgc-1 genotype homozygous; blue in color, plants do not contain Lgc-1 genotype; green in color, and the plants are heterozygous for Lgc-1 genotype; pink appeared as an invalid result.
Preferably, in the breeding method provided by the invention, the step (5) or the step (6) may further comprise screening individuals with excellent agronomic traits. The agronomic trait being superior may include one or more of: compact plant type, high yield, strong disease resistance and lodging resistance. For example, according to an embodiment of the present invention, in step (5), a single plant with a compact plant size and resistance to lodging can be screened; in the step (6), single plants with compact plant types, high yield, strong disease resistance and lodging resistance can be screened.
Specifically, preferably, the plant type is compact, the included angle between the stems of the plant is less than 20 degrees, the yield is more than 30 g per plant, the disease resistance is strong, the rice blast resistance and the stripe disease resistance are achieved, and the lodging resistance is that the stems are upright. Optionally, the agronomic trait being superior further comprises one or more of: moderate plant height and good tillering force. Specifically, the plant height is moderate and is 90-110cm, and the tillering force is better than 7 effective tillers.
The invention discloses a breeding method of a rice variety polymerized by high resistant starch and low glutelin, which comprises the steps of taking the rice variety with high resistant starch content as a parent I and the rice variety with low glutelin content as a parent II, and hybridizing to obtain F1 generation seeds; backcrossing the F1 generation with the parent I to obtain BC1F1 generation seeds, and selecting BC1F1 seeds with chalkiness degree of more than 70%; detecting the BC1F1 generation with resistant starch molecular marker and low glutelin molecular marker after sowing, screening to obtain high resistant starch genotype homozygous and low glutelin genotype heterozygous single plants, and selfing to obtain BC1F2 generation seeds; planting BC1F2 generation, screening to obtain strain with homozygous resistant starch genotype and low gluten genotype by using low gluten molecular marker detection, and screening strain with resistant starch content higher than 10% and gluten content lower than 3% by using the obtained seed detection; the screened strains are continuously selfed to BC1F5 generation to obtain a new rice variety with high resistant starch content, low glutelin content and high yield and low glutelin. The rice variety bred by the invention has the characteristics of high resistant starch content and low glutelin content, and is suitable for diabetic patients and nephrotic patients needing to control protein intake.
Compared with the prior art, the invention provides a breeding method of a rice variety polymerized by high-resistant starch and low-gluten, and experiments prove that the breeding method obtains a novel rice variety with 2 excellent characteristics of polymerized high-resistant starch character and low-gluten, the rice variety is a stable variety doubly homozygous by high-resistant starch gene and low-gluten gene, the content of seed resistant starch is more than 10 percent, and the rice variety is about 20 times of that of a common rice variety; meanwhile, the gluten content is less than 3%, which is about half of common rice varieties. The novel rice variety is particularly suitable for people who need to limit protein intake and control blood sugar, such as diabetics, nephropathy patients and diabetic patients with nephropathy complications, can be used as a very effective dietary therapy auxiliary product, can become an important rice variety resource, and has important application value.
In addition, in the process of establishing the breeding method of the present invention, the inventors of the present invention also made other breeding operations, including backcrossing the seeds of the F1 generation with the parent II and screening to obtain individuals heterozygous for the sbe3-rs genotype of resistant starch and homozygous for the glutelin Lgc-1 genotype, and then screening to obtain individuals homozygous for both the sbe3-rs genotype and the glutelin Lgc-1 genotype, as described in example 1.
It was found that although the final objective of this conventional breeding method was also to obtain stable individuals homozygous for both resistant starch and glutelin genes, in the actual breeding process, the breeding method of the present invention that preferentially selects stable resistant starch genotypes more easily obtains stable individuals homozygous for both genes than the breeding method that preferentially selects stable glutelin genotypes: when the preference selects stable low glutelin genotype, only 5 strains realize double-gene homozygous, and the early generation has low fruiting rate, and the selected population is too small to be directly eliminated; when the resistant starch genotype is preferably stabilized, the number of individual plants is at most 4 times larger. Therefore, the breeding method is more beneficial to the continuous selfing in the later period and is more beneficial to the breeding of stable double-gene stable homozygous varieties with excellent comprehensive agronomic characters from the offspring groups. Therefore, compared with the conventional breeding method, the breeding method provided by the invention has the advantages of strong culture capacity, favorable passage of favorable characters and higher breeding efficiency, and provides a research direction for further improvement of rice varieties.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.
The following specific methods are used in the examples and may be applied to the corresponding descriptions of other parts of the present invention.
Determination of the content of resistant starch
The method for measuring the content of the resistant starch is slightly improved by using a Resistant Starch (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 650 ℃ water bath for incubation for 1h 20min, finally determining the glucose content with D-glucose oxidase/peroxidase reagent (GOPOD), and calculating the RS content.
(II) measurement of gluten content
1) Extraction of gluten
a) A0.5 g sample of rice flour was weighed into a 15mL centrifuge tube and 10mL distilled water was added. Placing the centrifuge tube into a constant temperature oscillation water bath shaker at 50 deg.C, extracting in water bath oscillation for 30min, cooling to room temperature, and centrifuging at 4000r/min for 20 min. Washing the residue with distilled water for 2 times, adding 10mL of water each time, shaking in water bath for 10min, centrifuging for 10min, and removing the supernatant;
b) adding 10mL of 10% NaCl solution into the residue, placing the centrifuge tube into a constant-temperature oscillation water bath shaking table at 50 ℃, oscillating and extracting in a water bath for 30min, taking out and cooling to room temperature, centrifuging at 4000r/min for 20min, and washing the residue with 10% NaCl solution for 2 times;
c) adding 10mL of 75% ethanol into the above residue, placing into 50 deg.C water bath, extracting under shaking in water bath for 5min, and extracting under shaking in water bath at room temperature for 30 min. Taking out, cooling to room temperature, centrifuging at 4000r/min for 20min, washing residue with 75% ethanol for 2 times, and shaking for washing residue at room temperature for 5min each time;
d) adding 10mL of 0.2% NaOH solution into the residue, shaking in a water bath shaker at constant temperature of 50 deg.C, shaking in water bath for 30min, taking out, cooling to room temperature, centrifuging at 4000r/min for 20min, and washing the residue with 0.2% NaOH solution for 2 times. The washing solution and the extracting solution are merged into a 25mL volumetric flask, and 0.2% NaOH solution is added to the volumetric flask to reach 25 mL.
2) Determination of the content
The gluten content was an average of 3 replicates as determined by Coomassie Brilliant blue G-250.
Making standard curve
Taking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of 100 mu g/mL standard protein (BSA) solution, supplementing the solution to 1mL by distilled water, adding 5mL of Coomassie brilliant blue solution, uniformly mixing, standing for 2min, measuring the absorbance of the blank at the wavelength of 595nm by taking the absorbance as a horizontal coordinate and the protein content as a vertical coordinate, drawing a standard curve, and solving a standard linear equation.
Measurement of samples
Taking 0.1-1mL (supplementing water to 1mL if less than 1mL) of sample extract, operating with the same standard curve, and determining the absorbance value of the sample. And if the content of the sample extracting solution is higher, diluting the sample extracting solution to a proper concentration and then measuring the sample extracting solution.
(III) extraction of DNA
And (3) extracting the leaf DNA of the rice plant to be detected by using a CTAB method for later use.
Diluting DNA, preparing a set of DNA working solution with the concentration of about 50-100 ng/microliter, and storing in a refrigerator at 4 ℃ for later use.
(IV) high-resistance starch CAPS molecular marker detection
The following CAPS marker primers were used to perform PCR amplification on leaf DNA extracted from the plants to be tested for genotyping:
SpeI F：5’-ATGTGATGTGCTGGATTTGG-3’；
SpeI R：5’-TGTGGTTTTCATACCGTTCTTA-3’。
the PCR amplification can obtain a 571bp fragment. After the PCR product was digested with SpeI at 37 ℃ for 5 hours, the digested product was analyzed by 1.5% agarose gel electrophoresis. If there is a base mutation of T → C at the 105 th exon corresponding to the 16 th exon of the rice starch branching enzyme SBE3 gene, this point mutation results in the loss of the restriction site for the restriction enzyme SpeI at this position (due to the mutation of ACTAGT to ACCAGT). Therefore, the enzyme digestion result has two spectral bands of 375bp and 196bp, and the plant is judged to have low resistant starch content; only 571bp of a band, the plant is judged to be homozygous for the sbe3-rs genotype; there were 3 bands, and the plants were judged to be heterozygous for the sbe3-rs genotype.
(V) detection of Low glutelin KASP molecular marker (provided by Shanghai shellfish Crystal biotechnology Co., Ltd.)
The following KASP marker primers are adopted to carry out PCR amplification on leaf DNA extracted from a plant to be detected so as to carry out genotyping, and the specific operation steps are as follows:
a) transferring the sample DNA from the 96-well plate to a 384-well plate by Replikator, and finally to a 1536-well plate, ensuring a final concentration of sample DNA of about 10 ng/. mu.l;
b) placing the pore plate filled with the sample DNA in a 65 ℃ oven for drying for 30 min;
c) after dryingThe DNA of (1) was subjected to PCR system construction, and 1. mu.l of each reaction system: 2 XKASP master mix 0.5. mu.l, 72 XAssay Primer mix 0.014. mu.l, H2O0.486 mul, preparing PCR reaction solution and subpackaging into the dried sample DNA pore plate;
d) sealing the membrane of the pore plate added with the reaction system, and quickly centrifuging at a low speed;
e) after centrifugation, PCR reaction was performed: first step pre-denaturation at 94 ℃ for 15 min; a second denaturation, renaturation/elongation step at 94 ℃ for 20s, from 61 ℃ to 66 ℃ (0.6 ℃ per cycle) for 60s, for 10 cycles; and the third step of denaturation, renaturation/extension, at 94 ℃ for 20s and 55 ℃ for 60s, and 26 cycles.
After the reaction is finished, reading the plate on a microplate reader Pheastar, analyzing the scanning data by adopting SNPviewer2 software, determining the Lgc-1 genotype of the rice sample according to the analysis result, and if the fluorescence signal data of the rice plant amplification product to be detected is analyzed to be red by SNPviewer2, the plant is Lgc-1 genotype homozygous (indicated as B in the embodiment); if the fluorescence signal data of the amplification product of the rice plant to be detected is blue after being analyzed by SNPviewer2, the plant does not contain Lgc-1 gene (in the embodiment, A represents); if the fluorescence signal data of the amplification product of the rice plant to be detected is analyzed to be green by SNPviewer2, the plant is heterozygous for the Lgc-1 genotype (expressed by H in the embodiment); and if the fluorescent signal data of the rice plant amplification product to be detected is pink after being analyzed by SNPviewer2, the result is invalid.
Example 1Breeding method-preferential selection of low glutelin genotype homozygous
(1) In 2016 winter, Hainan, a variety F1 seeds were obtained by crossing with oryza sativa No. 2 (resistant starch content about 13%; gluten content about 5.6%) as the parent I and with a Japanese low-gluten variety LGC-1 (gluten content about 2.6%, resistant starch content about 0.46%) as the parent II.
(2) In 2017 spring, Shanghai, the F1 generation was backcrossed with the parent II to obtain BC1F1 generation seeds.
(3) In 2017, in the south of the sea in winter, BC1F1 is sown in the field, sampling is carried out, DNA is extracted, single plants with heterozygous resistant starch sbe3-rs genotypes and homozygous low gluten Lgc-1 genotypes are obtained by screening through high resistant starch CAPS molecular marker detection and low gluten KASP molecular marker detection, and BC1F2 seeds are obtained by mixed harvesting.
(4) And in spring of 2018, in Shanghai, BC1F2 generations are planted, sampling is carried out, DNA is extracted, and a single plant homozygous for both resistant starch sbe3-rs genotype and low gluten Lgc-1 genotype is obtained by screening through high resistant starch CAPS molecular marker detection and low gluten KASP molecular marker detection.
Only 5 of 100 individuals are detected to be two completely homozygous individuals with genotypes, the fructification rate is very poor, and the selected population is too small to be directly eliminated.
Example 2The breeding method of the present invention, preferably selecting resistant starch genotype homozygous
(1) In 2016 winter, Hainan, Youngza 2 (same as example 1) was used as parent I, and Japanese low-gluten variety LGC-1 (same as example 1) was used as parent II, and were crossed to obtain seeds of generation F1 (same as step 1 of example 1).
(2) In winter and spring in 2017, in Shanghai, the F1 generation is backcrossed with a parent I to obtain seeds of the BC1F1 generation, the seeds of the BC1F1 generation are unpolished rice, seeds with the chalkiness degree of more than 70% are selected according to the appearance, Hainan is planted, a sample is taken, DNA is extracted, single plants which are homozygous for sbe3-rs genotype and heterozygous for Lgc-1 genotype are obtained by high-resistance starch CAPS molecular marker detection and low-gluten KASP molecular marker detection, and the seeds of the BC1F2 generation are obtained by mixed harvest.
(3) In 2017, in the south of the Hainan province, BC1F2 generations are planted, sampling is carried out, DNA is extracted, and by detecting a low glutelin KASP molecular marker, a single plant homozygous for both the sbe3-rs genotype and the Lgc-1 genotype is obtained through screening.
21 of the 92 individuals were tested as being completely homozygous for two genotypes and the results are shown in Table 1.
Table 1: genotyping results of KASP molecular marker detection
Figure BDA0002348576350000091
Figure BDA0002348576350000101
And (3) for the individual plant with the low glutelin KASP marker genotype B, simultaneously detecting by using a high-resistance starch CAPS molecular marker, verifying that the individual plant is homozygous for the resistant starch sbe3-rs genotype and homozygous for the low glutelin Lgc-1 genotype, and obtaining BC1F3 seeds by dividing the individual plant. Polished rice is milled from the seed part of BC1F3, the milled rice is respectively used for measuring the resistant starch content and the gluten content, and the seeds with the resistant starch content more than 10 percent and the gluten content less than 3 percent are reserved.
(4) In winter and spring in 2018, a BC1F3 strain is planted in the Shanghai, and a single plant with compact plant type and lodging resistance is screened to obtain a BC1F4 generation. During the period, the verification is carried out by combining the detection of the resistant starch molecular marker and the detection of the low glutelin molecular marker.
(5) And in summer and winter of 2018, growing BC1F4 generations according to the plant lines, screening single plants with compact plant types and lodging resistance, and continuously selfing to obtain BC1F5 generations. During the period, the verification is carried out by combining the detection of the resistant starch molecular marker and the detection of the low glutelin molecular marker.
(6) In 2019 spring, in Shanghai, BC1F5 is planted according to plant lines, 5 excellent varieties with compact single plant type, high yield, strong disease resistance, lodging resistance and yield of more than 400 kg per mu are selected, and the varieties are named as Hu rice RDL1 to Hu rice RDL 5.
The seeds of these 5 elite varieties were tested for resistant starch content and gluten content and the results are shown in tables 2 and 3.
Table 2: content determination of resistant starch of different varieties
Figure BDA0002348576350000102
Figure BDA0002348576350000111
The resistant starch content of 5 stable varieties was 11.03% to 15.9%.
Table 3: gluten content determination results for different varieties
Variety of (IV) C Gluten content (%)
Shanghai rice RDL1 2.55
Shanghai rice RDL2 2.69
Shanghai rice RDL3 2.63
Shanghai rice RDL4 2.31
Shanghai rice RDL5 2.34
The gluten content of 5 stable varieties is 2.31% -2.69%.
As shown above, stable high resistant starch gene and low glutelin gene double homozygous stable variety has been obtained, and resistant starch content is greater than 10%, glutelin content is less than 3%, the excellent characteristics of high resistant starch content and low glutelin content of parent are maintained, and the food is very suitable for diabetic patients and patients with bad renal function.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
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Claims (9)

1. A breeding method of a rice variety polymerized by high-resistance starch and low-gluten protein comprises the following steps:
(1) to hypoglycemic rice No. 1 or othersbe3-rsThe derived variety of the gene is a parent I, the resistant starch content of the parent I is more than 10 percent, so as to contain the low glutelinLgc-1The genetic rice variety is a parent II, the glutelin content of the parent II is less than 3 percent, and F1 generation seeds are obtained by hybridization;
(2) backcrossing the F1 generation with the parent I to obtain BC1F1 generation seeds, polishing the seeds into brown rice, and screening the seeds with the chalkiness degree of more than 70 percent;
(3) planting the BC1F1 generation seeds obtained in the step (2), sampling and extracting DNA, detecting by high-resistance starch CAPS molecular markers and low-gluten KASP molecular markers, and screening to obtainsbe3-rsGenotype homozygous andLgc-1obtaining a single plant with heterozygous genotype to obtain BC1F2 generation seeds;
(4) planting BC1F2 generation seeds, sampling and extracting DNA, detecting by low glutelin KASP molecular marker, screening to obtainsbe3-rsGenotype andLgc-1obtaining BC1F3 generation seeds from a single plant with homozygous genotype, polishing the seeds into polished rice, grinding the polished rice into powder, measuring the resistant starch content and the gluten content, and screening to obtain a single plant with the resistant starch content of more than 10 percent and the gluten content of less than 3 percent for seed reservation;
(5) planting the BC1F3 seeds obtained in the step (4), and continuously selfing until BC1F5 seeds are obtained;
(6) planting BC1F5 seeds, and selecting a variety with yield per mu of more than 400 kg;
wherein the low glutelin KASP molecular marker detection comprises: the extracted DNA was PCR amplified and detected using the following primers, and then genotyped based on detection of the amplified product:
primer 1: 5'-TGCTGGCATGGTAGGACAAAATTG-3', respectively;
primer 2: 5'-CTTGCTGGCATGGTAGGACAAAATTT-3', respectively;
and (3) a universal primer: 5'-AACGGTTTAGATGAGAACTTCTGCACAAT-3', respectively;
and (3) a universal primer 4: 5'-AAAACAATACTGACACGCCACACACAAT-3' the flow of the air in the air conditioner,
connecting the 5 'end of the primer 1 with a FAM fluorescent label, connecting the 5' end of the primer 2 with a HEX fluorescent label, then identifying according to the fluorescent signal of the amplified product, and selecting a plant showing green.
2. The breeding method according to claim 1, wherein the step (1) comprisessbe3-rsThe derived variety of the gene is the oryza sativa No. 2 or the oryza sativa No. 3.
3. The breeding method according to claim 1 or 2, wherein the parent II is the japanese low-gluten variety LGC-1.
4. The breeding method according to claim 1 or 2, wherein the genomic DNA is extracted from leaves of an individual plant.
5. The breeding method according to claim 1 or 2, characterized in that, in the step (4), high-resistant starch CAPS molecular marker detection is also carried out;
or, carrying out high-resistance starch CAPS molecular marker detection and low-gluten KASP molecular marker detection on each generation of individual plants planted in the step (5).
6. The breeding method according to claim 1 or 2, wherein the high-resistance starch CAPS molecular marker detection comprises:
the extracted DNA was PCR amplified with the following primers and then genotyped based on detection of the amplified product:
PCR-SpeI F：5’-ATGTGATGTGCTGGATTTGG-3’；
PCR-SpeI R：5’-TGTGGTTTTCATACCGTTCTTA-3’。
7. the selective breeding method according to claim 6, characterized in that restriction endonuclease is adoptedSpeI, carrying out enzyme digestion identification on the obtained amplification product.
8. The breeding method according to claim 1 or 2, wherein the step (5) or the step (6) further comprises screening individuals with superior agronomic characteristics.
9. The breeding method according to claim 8, wherein the agronomic trait being superior comprises one or more of: compact plant type, high yield, strong disease resistance and lodging resistance.
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