CN111118201B - Molecular marker closely linked with gene du13(t) for regulating and controlling low amylose content of rice and application thereof - Google Patents
Molecular marker closely linked with gene du13(t) for regulating and controlling low amylose content of rice and application thereof Download PDFInfo
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Abstract
The closely linked molecular marker is obtained by amplifying an Indel-176-F primer and an Indel-176-R primer, and the length of a target fragment is 80 bp. The molecular marker is applied to molecular marker-assisted selective breeding, so that materials containing the gene du13(t) for regulating and controlling the low amylose content of rice can be effectively predicted and screened, and the breeding of new rice varieties with low amylose content is accelerated.
Description
Technical Field
The invention belongs to the technical field of rice molecular marker assisted selective breeding, and particularly relates to a molecular marker closely linked with a gene for regulating and controlling the low amylose content of rice and application thereof, which are suitable for molecular assisted selective breeding by using the gene.
Background
Rice (Oryza sativa L.) is one of the major food crops in the world, and rice is the staple food for half of the world's population. China is a large country of rice consumption, and more than 60% of people use rice as staple food. For a long time, high yield is the primary index of rice breeding, and with the improvement of living standard of people, the demand of high-quality rice is increasing day by day. Rice is mainly composed of starch, which is classified into amylose and amylopectin. The high and low amylose contents are key factors for determining the quality of rice, the low amylose contents are good in taste of rice and can not be regenerated after being cooled, and the high amylose contents are opposite. Research shows that although the content of starch in rice is related to breeding, cultivation, fertilization and the like, the content is mainly controlled by corresponding genes. At present, molecular marker technologies such as sequence-tagged sites (STS), simple repeat sequences (SSR), nucleotide polymorphisms (SNP) and the like are adopted at home and abroad to discover and position genes controlling amylose such as wax (Wx), du, lam (t), Sug and the like, wherein Wx gene coding particles are combined with starch synthetase; the du gene is a recessive gene independent of Wx inheritance, and can reduce amylose content by influencing mRNA (messenger ribonucleic acid) of a Wx gene transcription product; the lam (t) gene is a carbohydrate gene which does not participate in the synthesis of amylose but can reduce the amylose content (research progress on the utilization of low amylose genes in rice, such as Zhuchanlan, J.Chinese agricultural science, 2004,37(2): 157-162; Kiswara et al, genetic analysis and molecular mapping of low amylose du12(t) in rice (Oryza sativa L.) [ J.Theotectical and Applied Genetics,2014,127(1): 51-57).
In recent years, some rice varieties or intermediate materials have been developed using some genes regulating low amylose content, such as a mikyqueen mutant material containing Wx gene regulating starch content obtained from Koshihikari by N-methyl-N-nitrosourea (MNU) mutagenesis; the inclusion of the Wx gene was also found in the naturally mutated materials ARC6622 and ARC10818 of Pokhareli Mashinode; the du (2035), du-2 and du (2120) genes with starch content regulation and control are obtained from Sasanishiki through Ethylene Imine (EI) mutagenesis, and mutant materials (Zhu Changlan et al, research on breeding and utilization of low amylose content genes of rice [ J ]. Chinese agricultural science, 2004,37(2): 157;) are obtained from 2035, 2057, 2083 and the like, but the detected number of the genes with low amylose content regulation and control is limited, so that the higher and higher requirements of people on high-quality rice are difficult to meet, and the international competition of the high-quality rice is increasingly aggravated, therefore, germplasm resource materials for regulating new low amylose content genes of rice are developed, and the germplasm resource materials are still an urgent task for breeding workers in the field.
Disclosure of Invention
In order to solve the technical problems that the existing low-amylose content rice gene resource material regulation and control is limited and breeding of new low-amylose content rice varieties is not facilitated, the offspring material of the new low-amylose content gene du13(t) is selected in a targeted manner, and the molecular marker Indel-176 closely linked with the new low-amylose content gene du13(t) is provided and is applied to breeding of new low-amylose content rice varieties in a molecular marker-assisted selection mode, so that the increasingly high requirements of people on high-quality rice and increasingly international competition on the high-quality rice are met.
The molecular marker Indel-176 closely linked with the gene du13(t) for regulating and controlling the low amylose content of rice provided by the invention is a fragment amplified by an Indel-176-F primer and an Indel-176-R primer, the length of a target fragment is 80bp, and the nucleotide sequence of the Indel-176-F primer is shown as SEQ ID NO: 1, the nucleotide sequence of Indel-176-R primer is shown as SEQ ID NO: 2, respectively.
The closely linked molecular marker Indel-176 is located at 29.22Mb on chromosome 6 of rice.
The invention also provides application of the molecular marker Indel-176 closely linked with the gene du13(t) for regulating and controlling the low amylose content of rice in the molecular marker-assisted selective breeding of a new rice variety with low amylose content.
The application comprises the step of amplifying a segment of 80bp in a target rice material by using an Indel-176-F primer and an Indel-176-R primer through PCR, wherein the target rice material contains a site for regulating and controlling the gene du13(t) with low amylose content.
In the application, the amplification system of the PCR amplification is as follows: in a 20-mu-L PCR reaction system, 10 mu L of 2 XPCR Mastermix, 1 mu L of 10 mu M Indel-176-F primer, 1 mu L of 10 mu M Indel-176-R primer and 1 mu L of 10 ng/mu L template DNA are added with sterilized ultrapure water to 20 mu L finally; the PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; the parameters for 32 PCR cycles were: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1 min; after the circulation is finished, the PCR product is finally extended for 5min at 72 ℃, and is stored at 4 ℃.
The closely linked molecular marker Indel-176 can be used for efficiently detecting and controlling the gene du13(t) with low amylose content in rice in a large scale at the seedling stage of the rice, has the characteristics of strong target property, low cost, simplicity, convenience, rapidness, high accuracy and no environmental influence, and is suitable for molecular marker-assisted breeding research work of new varieties of rice with low amylose content.
SEQ ID NO: 1 shows the nucleotide sequence of Indel-176-F primer.
SEQ ID NO: 2 shows the nucleotide sequence of the Indel-176-R primer.
Drawings
FIG. 1 shows appearance quality of rice, wherein A is Yunjuan No. 41, and B is IRAT 104.
FIG. 2 is a polyacrylamide gel electrophoresis diagram of 8% of amplification products of a main-cultivated rice variety in Yunnan province containing no gene du13(t) for regulating the low amylose content of rice under the condition of a tightly linked molecular marker Indel-176 of donor Yunja No. 41 and gene du13(t) for regulating the low amylose content of rice. Wherein M is a molecular weight marker of 2000bp (Takara DL2000), and 1 is a parent Yunjun No. 41; 2 is Yujing No. 24; 3 is Yunjing No. 26; no. 4 is Yunjing No. 39; 5 is Chujing No. 28; jing rice No. 1 is No. 6; 7 is Li Jing No. 9; 8 is Longke No. 16.
FIG. 3 is a drawing of the closely linked molecular marker Indel-176 expressed in Yunjing No. 41Hybridization F of IRAT1041Generation selfing F2Individuals and 30F3The amplified products of the population are electrophoresed on 8% polyacrylamide gel. Wherein M is a 2000bp molecular weight marker (Takara DL2000),1 is a parent IRAT104, 2 is a parent Yunjiu No. 41, and 3 is a Yunjiu No. 41 multiplied by IRAT104 hybrid F1Generation selfing F2Individual, No. 4-33 Yunjing No. 41 multiplied by IRAT104 selfing F3Randomly drawn individuals with low amylose content from the population.
Detailed Description
The invention is further described in detail below by way of specific examples in conjunction with the accompanying drawings. The specific techniques not described in the examples were carried out in accordance with the literature of the art (the protocols for cloning experiments in sections) or the specifications of the products.
Yunjing No. 41 is a new high-quality and high-yield japonica-type soft and fragrant rice strain bred by the institute of food crops, college of agricultural sciences in Yunnan province, and has the characteristics of oily and soft rice, good taste and no retrogradation after cooling. Yunyuan No. 41 is published in non-patent literature, "Dongyunwu et al. Yuxi City, analysis of yield of high-altitude japonica rice variety, Yunnan agricultural science and technology, 1 month and 25 days in 2017, and 1 st stage in 2017: 18 to 20 ".
IRAT104 is japonica type upland rice, and is published in non-patent literature "Hefengyi et al. New variety of upland rice" IRAT104 "physiological characteristics and cultivation strategy.Yunnan agricultural science and technology, 5.25.1995, 03: 29-30.
The Yunjing No. 41 and IRAT104 applicants have a reserve, and the applicants are guaranteed to be released to the public within 20 years from the filing date of the present patent. Applicant contact address: beijing Lou 2238 of Panlongdistrict of Kunming, Yunnan province, institute of agricultural environmental resources, academy of agricultural sciences, Yunnan province, zip code: 650205.
reagents or instruments used in each example and the remaining biological materials (rice material) except Yunjing No. 41 and IRAT104 were commercially available.
Example 1: appearance quality analysis and amylose determination
Respectively and naturally air-drying Yunjing No. 41 and IRAT104 seeds, and shelling the sample by a small-sized polished rice machine for Japanese Kate experiments to prepare the polished rice. The rice appearance quality of Yunjuan No. 41 and IRAT104 was investigated, and the results are shown in FIG. 1: a is the dark endosperm of the Yunju No. 41 rice, and B is the transparent IRAT104 rice. According to the method of spectrophotometry for determining rice amylose (NY/T2639-2014): the content of the amylose of Yunjing No. 41 is 8.96 percent; the amylose content of IRAT104 was 16.41%.
Example 2: DNA extraction, PCR amplification and PCR product detection
1. DNA extraction from rice leaves
Extracting DNA of a test material by adopting a CTAB method, and comprising the following steps: (1) taking about 1g of young and tender rice leaves, grinding the young and tender rice leaves by using liquid nitrogen, transferring the young and tender rice leaves into a 1.5ml centrifuge tube, and adding 800 mu L CTAB; (2) mixing, placing into 65 deg.C water bath for 30min (mixing once every 15 min); (3) adding 200 μ L chloroform (chloroform), and mixing; (4) centrifuging at 12000rpm for 10 min; (5) transferring the supernatant to another new 1.5ml centrifuge tube, adding 600. mu.L of isopropanol, and turning upside down until floc appears; (6) centrifuging at 12000rpm for 10 min; (7) discarding the supernatant, adding 75% ethanol about 800 μ L, mixing by turning upside down, and washing DNA; (8) centrifuging at 12000rpm for 3 min; (9) discarding the supernatant, air-drying the precipitate at room temperature, and dissolving with 200 μ L of 0.1 TE; (10) standing at 4 ℃ overnight, dissolving DNA, measuring the concentration of the extracted DNA by using a spectrophotometer, and preparing the DNA into working solution with the concentration of 10 ng/mu L; (11) transferring to-20 deg.C for storage.
2. PCR amplification
(1) PCR amplification System: the total reaction volume was 20. mu.L, with 2 XPCR Master mix 10. mu.L, 10. mu.M forward primer and 10. mu.M reverse primer each 1. mu.L, 10 ng/. mu.L template DNA 1. mu.L, and finally sterilized ultrapure water was supplemented to a final reaction volume of 20. mu.L.
(2) PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; the parameters for 32 PCR cycles were: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1 min; after the circulation is finished, the PCR product is finally extended for 5min at 72 ℃, and is stored at 4 ℃.
3. PCR product detection
mu.L of the PCR product was collected and electrophoresed on 8% polyacrylamide gel. The electrophoresis voltage is 180V, and the electrophoresis time is 180 min. After the electrophoresis is finished, dyeing and photographing are carried out by adopting a silver dyeing technology, and the test result is recorded.
Example 3: method for detecting common rice variety and low amylose rice variety Yunjiu No. 41 by using molecular marker Indel-176
1. DNA extraction, PCR amplification and PCR product detection
Selecting a low-amylose rice variety: yujing No. 41; among the common rice plants: yujing No. 24; yujing No. 26; yujing No. 39; chu Jing No. 28; jing Rice No. 1; no. 9 Lijing; clone No. 16, at seedling stage DNA was extracted as in example 2, and the DNA sequence shown in SEQ id no: 1 and SEQ ID NO: 2, performing PCR amplification on the primers, and detecting the PCR product, wherein the results are shown in FIG. 2: the length of the amplification product of Yunjing No. 41 is 80bp, and the length of the amplification product of the other 7 common rice varieties is 77 bp.
The above results indicate that the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 is a primer to carry out PCR amplification to obtain a segment with the length of 80bp which can be used as a marker for detecting whether the gene for regulating and controlling the low amylose content of the rice exists in the rice variety.
And (4) conclusion: the molecular marker Indel-176 can be used for reliably detecting whether genes for regulating and controlling the low amylose content of rice exist in rice varieties.
Example 4 use of molecular marker Indel-176 to localize and regulate the gene du13(t) with low amylose content in rice from the progeny of a cross
According to the invention, Yujing 41 with 8.96% of amylose content and IRAT104 with 16.41% of amylose content are hybridized in the research process, a genetic location population is constructed, a molecular marker technology is adopted, Nipponbare is taken as a reference sequence, genetic analysis is combined, and finally 1 new gene du13(t) for regulating and controlling low amylose content is located from Yujing 41, the gene is located in chromosome 6 from 29.22Mb to 29.40Mb, and no gene for regulating and controlling low amylose content is found in the region before, so that the gene is a new locus, the locus du is named 13(t), and a molecular marker Indel-176 tightly linked with the du13(t) gene is screened.
1、F3Population construction
Hybridizing Yunjing No. 41 multiplied by IRAT104 to obtain F1Instead, F is1Generation of normalityPlanting in greenhouse to obtain F2Selfing the population, and then breeding F2Planting in greenhouse, harvesting F3After rice is replaced, the rice is naturally dried and then is manually hulled.
2. Analysis of genetic regularities
Following the procedure in example 1, 60F pellets were randomly aligned2The selfed population was subjected to rice appearance quality analysis, wherein the appearance quality was 46 grains (X) and 14 grains (X) of the number of the clear endosperm and the dark endosperm20.0222 and 0.88) according to an expected separation ratio of 3:1, indicating that the Yunjing No. 41 contains a gene for regulating the low amylose content of the rice and is a single recessive gene; for 229 selfing F3Analysis of appearance quality of rice in the population, the number of the population with transparent, heterozygous and dark endosperm as appearance quality is 47 parts, 122 parts and 60 parts (chi)22.1769 and P0.1401), meets the expected segregation ratio of 1:2:1, and proves that the gene for regulating the low amylose content of the rice contained in Yunjing No. 41 is a single recessive genetic gene.
3、F3Population DNA extraction, PCR amplification and PCR product detection
F having a phenotype identical to A in FIG. 13Seeds were sown in the greenhouse and F was extracted at seedling stage as in example 23Population low amylose content individual DNA represented by SEQ ID NO: 1 and SEQ ID NO: 2, performing PCR amplification on the primers, and detecting the PCR product, wherein the results are shown in FIG. 3: the length of amplification products of Yungujing No. 41 is 80bp, the length of amplification products of IRAT104 is 77bp, and 30 randomly selected F2F obtained by selfing generation individual plant3The group shows that the lengths of the amplification products of the homozygous low amylose content single plants are all 80 bp.
The above results indicate that the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 is a primer to carry out PCR amplification to obtain a fragment with the length of 80bp, namely a molecular marker Indel-176, which can be used as a marker for detecting whether the site of the gene du13(t) for regulating the content of the low amylose starch of the rice in the filial generation of the rice is present or not.
And (4) conclusion: the molecular marker Indel-176 can be efficiently used for screening homozygous individuals for regulating and controlling the low amylose content gene du13(t) of rice in filial generations, has the characteristics of strong target, low cost, simplicity, convenience, rapidness, high accuracy and no environmental influence, and is suitable for molecular marker-assisted breeding research work of new varieties of low amylose content rice.
Sequence listing
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<120> and gene du13(t) closely linked molecular marker for regulating and controlling low amylose content of rice and application thereof
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Claims (2)
1. Gene for detecting and regulating low amylose content in ricedu13(t) application of a primer of a tightly linked molecular marker Indel-176 in molecular marker-assisted selective breeding of a new rice variety with low amylose content, which is characterized in that an Indel-176-F primer and an Indel-176-R primer are used for carrying out PCR amplification on a target rice material, and if an 80bp fragment is amplified, the target rice material contains a gene for regulating and controlling the low amylose contentdu13(t) a locus indicating that the rice material is a rice variety with low amylose content, and the nucleotide sequence of the Indel-176-F primer is shown as SEQ ID NO: 1, the nucleotide sequence of Indel-176-R primer is shown as SEQ ID NO: 2, respectively.
2. The use according to claim 1, wherein the amplification system of the PCR amplification is: in a 20-mu-L PCR reaction system, 10 mu L of 2 XPCR Master mix, 1 mu L of 10 mu M Indel-176-F primer, 1 mu L of 10 mu M Indel-176-R primer and 1 mu L of 10 ng/mu L template DNA are added with sterilized ultrapure water to 20 mu L finally; the PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; the parameters for 32 PCR cycles were: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1 min; after the circulation is finished, the PCR product is finally extended for 5min at 72 ℃, and is stored at 4 ℃.
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