CN111676275A

CN111676275A - Method for detecting existence of T1510G mutation in rice HIS1 gene and dCAPS labeled primer

Info

Publication number: CN111676275A
Application number: CN202010523094.6A
Authority: CN
Inventors: 吕启明; 赵炳然; 张秀丽; 唐丽; 黄志远
Original assignee: Hunan Hybrid Rice Research Center
Current assignee: Hunan Hybrid Rice Research Center
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2020-09-18

Abstract

The invention discloses a method for detecting whether a T1510G mutation exists in a rice HIS1 gene, which comprises the following steps: carrying out PCR amplification on a DNA fragment containing a 1510 th nucleotide downstream of an ATG (initiation codon) of a HIS1 gene by using a specific dCAPS (dCAPS) labeled primer, carrying out enzyme digestion on an amplification product by using a restriction enzyme Dde1, judging whether a nucleotide sequence of the amplification product is cut, and if the nucleotide sequence is cut, indicating that a T1510G mutation exists; otherwise, the T1510G mutation was not present. The presence of the T1510G mutation in the rice HIS1 gene can be quickly and accurately identified. The invention also discloses a method for detecting whether the rice HIS1 gene has beta-triketone herbicide resistance and a method for producing hybrid rice seeds, and provides a corresponding dCAPS labeled primer, thereby achieving the purpose of purifying the seeds of the two-line hybrid rice by using the beta-triketone herbicide.

Description

Method for detecting existence of T1510G mutation in rice HIS1 gene and dCAPS labeled primer

Technical Field

The invention belongs to the technical field of rice gene mutation, and particularly relates to a method for detecting whether a T1510G mutation exists in a rice HIS1 gene, and detecting whether beta-triketone herbicide resistance exists in a rice HIS1 gene, a method for producing hybrid rice seeds, and a dCAPS marker primer.

Background

The hybrid rice provides important guarantee for food safety in China and the world, and the three-line hybrid rice and the two-line hybrid rice are mainly utilized in the current production. The three-line hybrid rice mainly utilizes a cell nucleus and cytoplasm interactive sterile line which has more stable fertility and relatively safe seed production, but needs a corresponding maintainer line for reproduction and is limited by a restoring and protecting relationship, and the breeding of a restorer line is difficult; the two-line hybrid rice mainly utilizes photo-thermo-sensitive genic male sterile lines, the male sterile lines can be propagated by themselves under the condition of low temperature in a short day, and normal materials can recover the fertility, so that the pairing is relatively free, but the fertility of the two-line male sterile lines is greatly influenced by the air temperature, and the breeding meets abnormal low temperature to cause the self-fructification of the male sterile lines, thereby influencing the seed purity of the two-line hybrid rice. At present, some attempts are made to solve the problem of purity of two-line hybrid rice seeds, for example, glyphosate-resistant genes are transferred into a restoring line of the two-line hybrid rice by a transgenic means to create a seed production system of the restoring line and the hybrid rice, wherein the restoring line and the hybrid rice are glyphosate-resistant, and a sterile line is glyphosate-resistant, and the sterile line is not glyphosate-resistant, so that the aim of purifying the hybrid rice is achieved by spraying glyphosate in a seedling stage and selectively killing the sterile line selfing seeds which are not glyphosate-resistant under the condition that the sterile line selfing fructification exists in the two-line hybrid rice seeds; the sterile line which is originally resistant to herbicide bentazon is transformed into a sensitive type by a physical and chemical mutagenesis method, so that the aim of purification is fulfilled by spraying bentazon in the seedling stage; and mutating Acetolactate Synthase (ALS) which is a target enzyme of the imidazolinone herbicide in the restorer line by a physical and chemical mutagenesis method to create two lines of restorer lines of the imidazolinone herbicide resistance, so that the aim of purification is achieved by spraying the imidazolinone at the seedling stage. However, transgenic rice is not openly planted in China at present, mutagenesis can only be performed on specific parents, transformation is limited by a plurality of factors, and practical application and popularization are slow.

The beta-triketone herbicide takes plant p-hydroxyphenylpyruvate dioxygenase (HPPD) as a target, blocks the normal synthetic pathway of plastoquinone and tocopherol by inhibiting the HPPD, causes the reduction of the biosynthesis of carotenoid, the obstruction of the electron transfer of a photosynthesis chain, the photooxidation of plastid and the like, leads chlorophyll molecules in plants to be damaged, and promotes the occurrence of albinism symptoms and even death of leaves. The beta-triketone herbicide has the characteristics of high efficiency, low toxicity, difficult resistance generation, good environmental compatibility, safe use and the like, and the currently developed varieties mainly comprise mesotrione, bicyclosulfulone (BBC), furansulcotrione and the like. Recently, the beta-triketone herbicide resistance gene HIS1, which encodes an Fe (II)/2-oxoglutarate-dependent oxygenase that detoxifies beta-triketone herbicides by catalyzing them for hydroxylation, has been cloned in rice.

Aiming at the seed production safety requirement of the two-line hybrid rice and different resistant characteristics of different indica rice varieties to beta-triketone herbicides, the HIS1 gene non-functional haplotype sterile line and the functional haplotype restorer line are selected, so that the aim of purifying the two-line hybrid rice seeds by using the beta-triketone herbicide is fulfilled, and the method has very important significance to the technical field.

Disclosure of Invention

Based on the findings, the technical problems to be solved by the invention are to provide a method for quickly and efficiently detecting whether the rice HIS1 gene has a T1510G mutation, a method for detecting whether the rice HIS1 gene has beta-triketone herbicide resistance, a method for producing hybrid rice seeds and dCAPS labeled primers suitable for the methods and the applications, so that the aim of purifying the seeds of the two-line hybrid rice by using the beta-triketone herbicide is fulfilled.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a method for detecting whether a T1510G mutation exists in a rice HIS1 gene, wherein the T1510G mutation refers to a T-to-G mutation existing at a 1510 th base position downstream of an initiation codon ATG of an indica rice HIS1 gene (hereinafter referred to as "T1510G mutation"), and the method comprises the following steps:

s1, taking a DNA sample of the rice to be detected as a template, and carrying out PCR amplification on a certain DNA fragment containing a site of 1510 th nucleotide downstream of an initiation codon ATG on a HIS1 gene by using a specific dCAPS labeled primer;

s2, performing enzyme digestion treatment on the amplification product obtained in the step S1 by using a restriction enzyme Dde1, and judging whether the nucleotide sequence of the amplification product is cut or not by an agarose gel electrophoresis method or a DNA sequencing method; if the gene is cut, the T1510G mutation exists in the rice HIS1 gene; if not cut, it indicates that the rice HIS1 gene has no T1510G mutation.

In the method, in step S1, the DNA sample is preferably obtained by extracting leaf DNA of the rice to be detected by a CTAB method.

In the above method, preferably, the DNA fragment is from 1510 th nucleotide downstream of the ATG start codon of the HIS1 gene to 50 th to 192 th nucleotides downstream of the 1510 th nucleotide; based on this, a specific dCAPS labeled primer is designed such that the recognition site of the forward primer is located 1510 th nucleotide downstream of the ATG start codon of HIS1 gene, and the recognition site of the reverse primer is located between 50 th and 192 th nucleotides downstream of the 1510 th nucleotide.

More preferably, the specific dCAPS labeled primers include the forward primer CCJ-3F and the reverse primer CCJ-3R of the following sequences:

forward primer CCJ-3F: 5'-CATCTTCAGGAGCCCGGTGCACAGGCTGA-3' (shown in SEQ. ID NO. 1);

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' (shown in SEQ. ID NO. 2).

More preferably, the specific operation of determining whether the nucleotide sequence of the amplification product is cut into two segments by agarose gel electrophoresis in step S2 includes the following steps: carrying out agarose gel electrophoresis on the amplification product subjected to enzyme digestion treatment by restriction enzyme Dde1, reading the length of a DNA fragment of the amplification product, and judging that the nucleotide sequence of the amplification product is cut if a band with the length of 192bp appears in the DNA fragment of the amplification product; if the DNA fragment of the amplification product has no band with the length of 192bp and only one nucleotide sequence with the length of 221bp is obtained, judging that the nucleotide sequence of the amplification product is not cut. The method can quickly, efficiently, accurately and conveniently identify whether the T1510G mutation exists in the rice strain HIS1 gene.

More preferably, the specific operation of determining whether the nucleotide sequence of the amplification product is cleaved into two segments by the DNA sequencing method in step S2 includes the following steps: carrying out DNA sequencing on the amplification product subjected to enzyme digestion treatment by restriction enzyme Dde1, and if the nucleotide sequence shown in SEQ.ID NO.13 and/or SEQ.ID NO.14 is obtained, judging that the nucleotide sequence of the amplification product is cut; and if the nucleotide sequence shown as SEQ.ID NO.11 is obtained, judging that the nucleotide sequence of the amplification product is not cut. The method can efficiently and accurately identify the mutation type of 1510 th base downstream of the initiation codon ATG on the rice line HIS1 gene.

Previous studies suggest that there is a difference in the resistance of different indica varieties to β -triones and that this difference is due to the presence of a 28bp deletion in the fourth exon of the HIS1 gene. However, later studies found that, in addition to the variation caused by the 28bp deletion, other variations may exist in the HIS1 gene, which may also cause loss of its function, and thus may also exhibit sensitivity to β -triones. Through further research, a T-to-G mutation, namely a T1510G mutation, exists at the 1510 th base position downstream of the ATG (initiation codon) of the indica rice HIS1 gene, and the T1510G mutation causes rice to be sensitive to beta-triketone herbicides. And in the T1510G mutation and the 28bp deletion, as long as any one or two of the mutations exist, the rice can be sensitive to the expression of the beta-triketone herbicide. And the beta-triketone herbicide resistant phenotype of the rice HIS1 gene is dominant, and the beta-triketone herbicide sensitive phenotype is recessive.

Based on a general inventive concept, the present invention also provides a method for detecting the presence of beta-triketone herbicide resistance in rice HIS1 gene, comprising the steps of:

SS1, detecting whether deletion variation with the length of 28bp exists in the HIS1 gene of the rice sample, wherein the deletion variation with the length of 28bp refers to the deletion variation of 28bp fragments existing in the fourth exon (829-856 nucleotides on a coding region) of the HIS1 gene of rice, and is hereinafter referred to as 28bp deletion; detecting whether the HIS1 gene of the rice sample has T1510G mutation or not according to the method for detecting whether the HIS1 gene of the rice has T1510G mutation or not;

SS2, if the detection shows that the HIS1 gene of the rice sample has deletion variation with the length of 28bp, or shows that the HIS1 gene of the rice sample has T1510G mutation, the detection shows that the HIS1 gene of the rice sample does not have beta-triketone herbicide resistance; and if the detection shows that the HIS1 gene of the rice sample does not have deletion variation with the length of 28bp and the HIS1 gene of the rice sample does not have T1510G mutation, the HIS1 gene of the rice sample has beta-triketone herbicide resistance.

Preferably, the operation of step SS1 specifically includes the following steps: taking a DNA sample of rice to be detected as a template, carrying out PCR amplification by using a specific molecular marker primer, taking an obtained amplification product, carrying out agarose gel electrophoresis, and observing the length of a DNA fragment of the amplification product; if the DNA fragment of the amplified product is a single 182bp strip, the deletion variation of 28bp length of the rice HIS1 gene is shown; if the DNA fragment of the amplified product is a single 210bp band or comprises two bands with the lengths of 182bp and 210bp respectively, the deletion variation with the length of 28bp does not exist in the rice HIS1 gene;

the specific molecular marker primer comprises a forward primer CCJ-1F and a reverse primer CCJ-1R with the following sequences:

forward primer CCJ-1F: 5'-GTACAACTTGACGATGTATCAAG-3' (shown in SEQ. ID NO. 3);

reverse primer CCJ-1R: 5'-GTTGAATCTTGCAAATGCAGGAGC-3' (shown in SEQ. ID NO. 4).

Based on a general inventive concept, the invention also provides a hybrid rice seed production method, which comprises the steps of firstly adopting the method for detecting whether the rice HIS1 gene has beta-triketone herbicide resistance to detect and screen the rice male parent and the rice female parent for hybrid seed production, and then adopting a mixed sowing and mixed harvesting mode or a photo-thermo-sensitive male sterile line two-line method to carry out hybrid rice seed production.

The method preferably adopts a mixed sowing and mixed harvesting mode to carry out hybrid rice seed production, and specifically comprises the following steps: firstly, detecting whether the HIS1 genes of male parent and female parent of rice have beta-triketone herbicide resistance, selecting rice varieties of which the HIS1 gene of female parent has beta-triketone herbicide resistance and the HIS1 gene of male parent does not have beta-triketone herbicide resistance to carry out mixed sowing, wherein the female parent is sterile line, after the rice is naturally pollinated, uniformly spraying the beta-triketone herbicide on the rice plants of the male parent and the female parent to remove the male parent which has no beta-triketone herbicide resistance, and obtaining the offspring of the true hybrid on the surviving rice plants of the female parent.

More preferably, when hybrid rice seed production is carried out by a mixed sowing and mixed harvesting mode, the adopted female parents comprise one or more of 033S, 1892S, 33S, 66S, 99S, GD-1S, GD-7S, N111S, N5088S, Y58S, 5S, DesS, Phoenix S, Fulong S2, Guangxian 24S, Guangzhan 63-4S, Hua 68S, Jian S, Jing 4155S, Longke 638S, Lvmin S, Meng S, Petasin 64S, Shen 08S, Tannong S, Tianan S, Tianyuan 6S, Wan S, Xinhua S, starlight S, Xuan 69S and strain 1S;

three-line hybrid rice female parent: II-32A, D62A, F32A, K17A, Q1A, Q4A, Q6A, T98A, V20A, Anfeng A, Oufuan A, Gong 1A, Bo II A, Bo III A, Bo A, Changfeng A, Chuan 106A, Chuan Gu A, Chuan nong 1A, Chuan nong 2A, Chuan nong 3A, Chuan nong 4A, Chuan Xiang 29A, Chuan Jiang 12A, Chuan 1A, Fengyuan A, Fuyi A, Jiangxiang A, gang 46A, gang 48A, Gonggang 901A, Jinggang Xiangjing 1A, Guang 8A, Lu and A, Guangdong 13A, Hengfeng A, Hongguan 1A, Jiangu 645A, Jin23A, Jinjingujing 3A, Gugu 7A, Luzhou 1A, Luzhou A, Guangxiang 1A, Guangdong A, Guangxiang 1A, Guang 1A, Jinba, Jinluo 23A, Jinyujin 3A, Jingjing 3A, Luzhou 1A, Luzhou A, Luzho, Any one or more of inner incense 3A, inner incense 5A, inner incense 7A, inner incense 8A, Quan9311A, Rongfeng A, Rong18A, Shen 97A, Shen 9A, Shennong 2A, Shu 21A, Shu 8A, Tai 3A, Taifeng A, Tianfeng A, Wan 23A, Wan 73A, Wan 8A, Wan 9A, Wanjin A, Wufeng A, Xiang 8A, Xiangfeng 70A, Xiuqingzao A, Xinrong A, Xiniia, Yixiang 1A, Yufeng A, Yuetai 4A, Yuntai A, Yun 109A, Zhenshan 97A, Zhong2A, Zhong3A and 9A;

the adopted male parents comprise Sihui No. 18, Zhenzhen No.2, De Hui 381, Fu Hui 2098, Zhonggang No.3, IR64, Yanhui 559, Gui 582, Yi Hui 3551, Chenghui 727, Minghui 73, HR1128, Suhui 728, Zhenzui 2308, Zhenhui 2308, Quanhui 039, Jinboat silk sprout, Huahui 352, Huahui 118, Yeqinglun, Yi 1313, Jinghui 838, Jinzao 47, Zhenhui 084, Zhenhui 42, Luhui H103, Neihui 94-11, quan 131, Minghui 100, Mihui 725, Hangzui 959, Nanfeng glutinous, Neihui 2539, Fu Hui 5138, Mihui 2040, Mihui 9939, Nante No. 202, Shuhao No.1, Minghui 707, Guanghui 3550, Jianhui 146, Xiang 13, Shuhao 204, Shuhui 1577, Minghui 312, Mianna 2115, Mianlong-Hai-Miao, Chunhui-Hai-122, Xinhui-Hai-Miao-Hai-122, Yanhui-Hai, Ciligonmi, nan hui 445, Minghui 2155, Yuchi 231-8, Wenhui 689, Jun Jie No.1, R527, Shu hui 203, Minghui 82, Miyang 23, ao R15, Chengdu dwarf No.8, Fuhui 9801, Chenghui 19, Minghui 78, Xiangzai long-shaped No.13, AoR 69, Gui Dynasty 13, Mihui 9937, Lehui 188, Minhui 1273, Shengyou No.2, Yuhui 310, Guang-Er104, De-Hui 3485, Chuan airline Hui 1618, Nanhui 115, Chuan nong 422, Yunhui 68, Shengli long-shaped, Yihui 3003, Luhui 615, Yahui 627, Guangdui stone, Changfeng B, Xiang late-restoring No.13, Qianhui 1385, Nanhui 716, Feihui 6, Hongnan, Digu, Qigui Zai early 25, Qigui 489, Qian Hui 3309, Qihui 3301, Yunqui late-Hui 676, Shi 7, Shi 7, Shi 7, Shi 7, Shi 7, Shi, Shi 7.

The traditional method is to mix and sow the male and female parents in the same growth period according to a certain proportion, then separate the hybrid seeds from the male parent seeds by physical or chemical methods, or directly remove the male parent in the field. However, as the rural labor is reduced, the main labor for producing hybrid rice seeds is limited, so that the labor investment is insufficient at the key moment of seed production, impurity removal is not timely, the difficulty in identifying the hybrid plants is high, and the seed quality is influenced. The invention firstly adopts the dCAPS labeled primer to identify whether the rice father and mother parent HIS1 gene has T1510G mutation, and then judges whether the rice HIS1 gene has beta-triketone herbicide resistance by combining with whether the rice HIS1 gene has 28bp deletion, when the male parent HIS1 gene has 28bp deletion variation or T1510G mutation and the female parent HIS1 gene is normal, the father and the female parents can be sowed in a hybrid rice seed production process in a mixed mode, and after pollination, the beta-triketone herbicide is sprayed in a rice field, so that the male parents which do not resist the herbicide are removed, only the female parent true hybrid is left, the seed production purity is greatly improved, and labor force is greatly liberated.

The method preferably adopts a two-line method of photo-thermo-sensitive male sterile line to carry out hybrid rice seed production, and specifically comprises the following steps: firstly, detecting whether beta-triketone herbicide resistance exists in male parent and female parent HIS1 genes of rice, selecting rice varieties of which the female parent HIS1 gene does not have beta-triketone herbicide resistance and the male parent HIS1 gene has beta-triketone herbicide resistance for hybridization, wherein the HIS1 genes of the male parent and the female parent are homozygotes, normally harvesting F1 generation seeds for planting, and uniformly spraying the F1 generation plants with the beta-triketone herbicide to remove progeny of female parent self-inoculation caused by temperature instability, namely obtaining true hybrid progeny.

More preferably, when the photo-thermo-sensitive male sterile line is adopted for hybrid rice seed production by a two-line method, the adopted female parent comprises any one or more of Annong S-1, Biao 810S, KT S, Hua1037S, Xiangling 628S, quasiss, Longs, Orlon 1S, Yannong S, Jinjin 4128S, 360S, Huayu 4127S, Z S, H175S, 885S, C815S, SE21S, Mingxiang 10S and Ming S, and the adopted male parent comprises any one or more of Yuxiang oil, R402, P143, Yuexian silk sprout, Zhongzao 22, R534 and Huazhan.

More preferably, when the hybrid rice is produced by a photo-thermo-sensitive male sterile line 'two-line method', the adopted male and female parent combination comprises the following steps: the quanliangyou sesame oil account combination comprises a quanliangyou sesame oil account combination consisting of a quanliangyou female parent and a Jade oil account male parent, a quanliangyou 402 combination consisting of a quanliangyou female parent and an R402 male parent, a Miniangyou 143 combination consisting of a Miniangyou S female parent and a P143 male parent, a Longiangyou Yueyao Si Miao combination consisting of a Longiangyou S female parent and a Yueyao Si Miao male parent, a Jinliangyou 22 combination consisting of a jin 4128S female parent and a Zhongzao 22 male parent, a jin liangyou 534 combination consisting of a jin 4128S female parent and a Huazhan male parent, a Yuliangyou 22 combination consisting of a Huayu 4127S female parent and a Zhongzao 22 male parent, or a Yunliangyou 22 combination consisting of.

In the above method, preferably, the β -triketone herbicide is a tembotrionate herbicide or a mesotrionate herbicide, and the application is carried out by diluting the concentration of the tembotrionate herbicide to 0.05g/L-0.6g/L and then spraying the target plant, or by diluting the concentration of the mesotrionate herbicide to 0.01g/L-0.1g/L and then spraying the target plant.

In part of indica rice varieties, 28bp fragment deletion mutation (28bp deletion) exists in the fourth exon of the HIS1 gene or T-G mutation (T1510G mutation) exists at the 1510 th base position downstream of the initiation codon ATG, so that the resistance of the rice to the beta-triketone herbicide is lost, and by utilizing the characteristic, the male parent and the female parent can be mixedly sown and mixedly harvested in the hybrid seed production process, after pollination, the beta-triketone herbicide is sprayed, the male parent which does not resist the herbicide is removed, the female parent true hybrid is left, and the labor force can be greatly liberated; meanwhile, the condition of 'swinging seed' in the seed production process of the two-line sterile line can be effectively relieved, and the purity and the quality of the seed production are improved.

Based on a general inventive concept, the present invention also provides a dCAPS marker primer for detecting the presence of a T1510G mutation in rice HIS1 gene or detecting the presence of β -triketone herbicide resistance in rice HIS1 gene, comprising a forward primer CCJ-3F and a reverse primer CCJ-3R of the following sequences:

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' (shown in SEQ. ID NO. 2).

Compared with the prior art, the invention has the beneficial effects that:

1. the method for detecting whether the rice HIS1 gene has the T1510G mutation can quickly, efficiently, accurately and conveniently identify whether the rice line HIS1 gene has the T1510G mutation, and judges whether the rice HIS1 gene has the beta-triketone herbicide resistance by combining the fact that whether the rice HIS1 gene has 28bp deletion, thereby providing a certain theoretical basis for the use of the beta-triketone herbicide in part of indica rice fields and laying a good foundation for the subsequent production and purification of hybrid rice seeds by adopting the beta-triketone herbicide.

2. The method for detecting whether the rice HIS1 gene has beta-triketone herbicide resistance can quickly, efficiently, accurately and conveniently identify whether the rice strain HIS1 gene has beta-triketone herbicide resistance.

3. The invention is applied to hybrid rice seed production, can greatly improve seed production purity, quickly, accurately and efficiently obtain true hybrid progeny, has simple operation, greatly liberates labor force, can relieve the phenomenon of seed swinging in the seed production process of a two-line method, and has important significance in improving the purity of hybrid rice seeds and improving germplasm resources.

4. The dCAPS labeled primer can quickly, efficiently, accurately and conveniently identify whether the T1510G mutation exists in the rice strain HIS1 gene.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is an electrophoretogram of HIS1 gene of 24 of the rice materials tested in example 1;

FIG. 2 shows the results of the sensitivity of indica rice material IR64 to beta-trione herbicides in example 1;

FIG. 3 shows the results of the sensitive identification of Nipponbare, an indica rice material, to a beta-triketone herbicide in example 1;

FIG. 4 is a graph showing the gradation of the sensitivity of indica rice material to beta-triketone herbicides in example 1 (6 grades, from left to right: 0 and 1 grades show high resistance, 2 and 3 grades show moderate resistance and high sensitivity, and 4 and 5 grades show high sensitivity);

FIG. 5 shows the results of herbicide application of 493 parts of indica rice material in example 1 (from left to right: grade 0 and grade 1 show high resistance, grade 2 and grade 3 show moderate resistance and feel, and grade 4 and grade 5 show high feel);

FIG. 6 is a haplotype comparison of HIS1 gene in example 1;

FIG. 7 is a graph showing the amino acid differences and key domain prediction of each haplotype of HIS1 in example 1;

FIG. 8 is a three-dimensional structural diagram of HIS1 protein in example 1;

FIG. 9 is a result of measuring the expression levels of haplotypes H2 and H3 of HIS1 in example 1;

FIG. 10 is an electrophoretogram of sequences amplified by dCAPS detection marker pairs H2, H3 and H5 in example 1;

FIG. 11 is the sequence electrophoresis of the products of the amplification of H2, H3 and H5 in example 1 after digestion;

FIG. 12 shows the results of detection of the F2 populations of Tungxiang 42 and Xianhui 207 and their constructs using dCAPS detection markers in examples 1 and 2;

FIG. 13 shows the results of BBC spraying experiments of 8 two-line sterile lines in example 4 at a concentration of 0.4 g/L;

FIG. 14 is the BBC concentration gradient experiment results of Y58S, SE21S and Ming S in example 4;

FIG. 15 shows the BBC concentration gradient experiment results of the Ming S, P143 and its hybrid Ming Liangyou 143 in example 4;

FIG. 16 is the results of a mesotrione concentration gradient experiment with IR64 from example 5;

FIG. 17 is the results of a mesotrione concentration gradient experiment for Nipponbare in example 5;

FIG. 18 is the results of the mesotrione concentration gradient experiment for Y58S from example 5;

FIG. 19 is the results of a mesotrione concentration gradient experiment with SE21S from example 5;

FIG. 20 shows the results of the mesotrione concentration gradient experiment of Ming S, P143 and its hybrid Ming Liangyou 143 of example 5.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

Example 1:

the invention relates to a method for detecting whether a T1510G mutation exists in a rice HIS1 gene, which specifically comprises the following steps:

s1, extracting DNA sample from fresh young leaf of rice by CTAB method, namely, putting a proper amount of fresh leaf of rice into a 2mL centrifuge tube, adding steel balls, quickly freezing in liquid nitrogen, putting into a high-throughput tissue grinder (Ningbo Xinzhi Scientz-48) to break tissue, immediately adding 650 mu L of 2 × CTAB solution preheated at 65 ℃, putting into a water bath kettle at 65 ℃ for constant temperature 45min, uniformly reversing the upper part and the lower part every 10min, ending 45min, putting into a ventilation cabinet after the DNA sample is cooled to room temperature, adding 650 mu L of chloroform/isopropanol (24: 1) solutionMixing the above materials by up-down reversal, centrifuging at 12000rmp for 10min, transferring 500 μ l of supernatant into 1.5mL centrifuge tube, adding 500 μ l of isopropanol solution, mixing, precipitating at-20 deg.C for 2h, centrifuging at 12000rmp for 10min, discarding supernatant, adding 1mL 70% ethanol solution to remove impurities, centrifuging at 12000rmp for 5min, pouring out supernatant, adding 200 μ l ddH₂And dissolving the obtained product in a refrigerator at 4 ℃ overnight to obtain the extracted rice sample DNA template.

S2, using the DNA sample of the step S1 as a template, and carrying out PCR amplification by using dCAPS labeled primers: the synthesized primers CCJ-3F and CCJ-3R are diluted to 10 mM; preparing a PCR reaction system: a20. mu.L PCR reaction system contained 2. mu.L (50 ng/. mu.L) of genomic DNA, 1. mu.L (5. mu.M/mL) of each of the primers, and 16. mu.L of 1.1 XPCR Mix (Opticalke Biotechnology Co., Ltd.). The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 4 min; 35 cycles of 95 ℃ for 20s, 58 ℃ for 20s and 72 ℃ for 20 s; extension at 72 ℃ for 5 min.

The dCAPS labeled primers include a forward primer CCJ-3F and a reverse primer CCJ-3R of the following sequences:

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' (shown in SEQ. ID NO. 2);

the dCAPS labeled primer is designed such that the recognition site of the forward primer is located 1510 th nucleotide downstream of the ATG start codon of HIS1 gene, and the recognition site of the reverse primer is located between 50 th and 192 th nucleotides downstream of the 1510 th nucleotide.

S3, carrying out enzyme digestion on the amplification product obtained in the step S2 by using a restriction enzyme Dde1, carrying out agarose gel electrophoresis, reading the length of the DNA fragment, and judging that the nucleotide sequence of the amplification product is cut if a band with the length of 192bp appears in the DNA fragment of the amplification product; through a DNA sequencing method, two nucleotide sequences with the lengths of 29bp and 192bp are obtained after the DNA sequencing method is cut by a restriction enzyme Dde1, wherein the nucleotide sequence with the length of 29bp is shown as SEQ ID NO.13, the nucleotide sequence with the length of 192bp is shown as SEQ ID NO.14, and the T1510G mutation of the rice HIS1 gene is shown; if the DNA fragment of the amplified product has no band with the length of 192bp, and only one nucleotide sequence with the length of 221bp is obtained and is shown in SEQ.ID NO.11, the nucleotide sequence of the amplified product is judged not to be cut, which indicates that the rice HIS1 gene has no T1510G mutation.

To verify that the variation of T1510G is critical to the loss of function of HIS1 gene against β -triketone herbicides, and the accuracy of the dCAPS marker primers and detection method of this example, we performed the following experiments:

1. molecular marker designed for judging whether 28bp deletion exists in HIS1 gene or not and detection of Chinese indica rice material

Aiming at the reported 28bp deletion mutation causing the loss of the function of the beta-triketone herbicide broad-spectrum resistance gene HIS1, a molecular marker CCJ-1F/CCJ-1R is designed, and the molecular marker is adopted to detect 631 parts of Chinese main indica rice materials (comprising 221 parts of conventional rice, 213 parts of three-line restoring line, 59 parts of two-line restoring line, 87 parts of three-line sterile line and 51 parts of two-line sterile line), the detection result is shown in Table 1, and the HIS1 gene electrophoresis pattern of 24 parts of rice materials is shown in figure 1. As a result, the 28bp deletion was found to be widely present in Chinese indica rice material (Table 1), and we also found 5 deletions in the two-line sterile line and a large amount of non-mutated material in the restorer line, so that it was possible to purify two-line hybrid rice seeds based on the presence or absence of the HIS1 gene function.

Forward primer CCJ-1F: 5- 'GTACAACTTGACGATGTATCAAG-' 3 (shown in SEQ. ID NO. 3);

reverse primer CCJ-1R: 5- 'GTTGAATCTTGCAAATGCAGGAGC-' 3 (shown in SEQ. ID NO. 4).

2. Verification of relation between indica rice material 28bp deletion and beta-triketone herbicide sensitive phenotype

In order to verify whether the HIS1 gene deletion 28bp and the materials sensitive to the beta-triketone herbicide are completely consistent, a beta-triketone herbicide spraying experiment (comprising 221 parts of conventional rice, 213 parts of three-line restorer lines and 59 parts of two-line restorer lines, and the sterile line seeds are limited and cannot be subjected to spraying identification) is carried out on 493 parts of indica rice materials, and the experiment results are shown in table 1.

Firstly, carrying out a beta-triketone herbicide concentration gradient experiment by using indica rice variety IR64 (in which 28bp deletion exists in HIS1 gene) which is known to be sensitive to the beta-triketone herbicide and japonica rice variety Nipponbare (HIS1 gene is normal) which shows resistance to the beta-triketone herbicide, and screening the appropriate concentration of the beta-triketone herbicide for identification. As a result, as shown in FIGS. 2 and 3, it was found that when the β -triketone herbicide, mesotrione (BBC), was applied at a concentration of 0.15g/L, the two showed a significant difference, and that when the BBC concentration reached 1.2g/L, Nipponbare also started to show a little sensitive character. Finally, the BBC concentration of 0.4g/L is adopted to carry out the spraying and identification of the indica rice material.

Due to the subtle differences in BBC phenotype of the different materials, we divided rice material into six grades for BBC phenotype (fig. 4), where

grades

0 and 1 indicated high resistance,

grades

2 and 3 indicated moderate resistance and moderate sensitivity, and

grades

4 and 5 indicated high sensitivity. It can be seen from the results of spraying 493 parts of indica rice material (fig. 5) that most of the rice material has high resistance or high sense to BBC, which indicates that the resistance to BBC in rice is a quality trait controlled by major genes, which is identical to that the HIS1 gene is a major resistance gene of beta-triketone herbicide. Through the control analysis of the 28bp deletion and BBC sensitive materials, the two are found to be associated in three different types of rice materials (see table 1), but the BBC sensitive materials are obviously more than 28bp deletion materials, which indicates that in the indica rice materials, besides the 28bp deletion, other variations may exist in the HIS1 gene, so that the function of the HIS1 gene is lost, and the BBC sensitive materials are shown.

Table 1: indica rice material HIS1 gene function mutation and beta-triketone herbicide sensitive phenotype quantity

3. Determination of HIS1 Gene haplotype in indica Rice Material

In order to determine whether the HIS1 gene has other variation, the genome sequence from the initiation codon to the termination codon of the HIS1 gene is amplified by high fidelity PCR through primers CCJF-F/CCJF-R and then sequenced, so that 493 HIS1 gene sequences in the indica rice material with the BBC phenotype determined are obtained. Through comparison, 493 indica rice have only 5 mutation sites on the coding region of HIS1 gene, namely 9 th, 61 th, 829 th, 856 th, 1055 th and 1510 th bases. These mutation sites divided the HIS1 gene into 5 haplotypes, as shown in FIG. 6, in which haplotype 2(H2) is identical to Nipponbare and haplotype 5(H5) is identical to IR 64. Compared to H2, H1 has two single base variations at

positions

9 and 1055; h3 only had one T to G variation at position 1510 (T1510G mutation); h4 has two base variations at

positions

61 and 1055; h5 has mainly a 28bp deletion. After correlating each haplotype of the HIS1 gene with the BBC sensitive phenotype of the material, we found that only H3 and H5 appeared sensitive, where H5 was due to a 28bp deletion and H3 was most likely due to the T1510G variation, since only this one variation was present for H3 compared to BBC resistant H2.

Forward primer CCJF-F: 5'-AAGCAAGAACAAAGTTAAGCAAG-3' (shown in SEQ. ID NO. 5);

reverse primer CCJF-R: 5'-AAGGAAATACTTGCTACTGAAAGC-3' (shown in SEQ. ID NO. 6).

4. It was determined that the T1510G mutation caused the loss of function of the HIS1 gene against beta-triketone herbicides

First, we aligned the amino acid sequences of 5 haplotypes of the HIS1 gene, and as shown in FIG. 7, found that H3 has only one amino acid variation (V286G) at position 286, which is located in the key structural domain 2OG-Fell _ OXy of HIS1 protein, suggesting that this site may play an important role in the function of HIS1 protein. Next, we analyzed the three-dimensional structure of HIS1 protein by homology modeling, and found that V286G is present in the pocket structure of HIS1 protein interacting with iron ions (fig. 8), and the amino acid at this position is very important for the function of HIS1 protein, so that V286G variation may affect the anti-BBC function of HIS1 protein. In order to eliminate the fact that H3 is sensitive to BBC caused by different expression levels of HIS1 genes, 5 parts of materials are randomly selected from H2 and H3 respectively, a primer CCJ-2F/CCJ-2R is used for detecting the expression level of the HIS1 gene, Nipponbare and IR64 are used as controls, a rice gene Actin is used as an internal reference, and the primer is Actin-F/Actin-R. As shown in FIG. 9, the HIS1 gene was normally expressed in the H3 material, and was not significantly different from H2, so that it could be excluded that the loss of the function of H3 against BBC was caused by the difference in the expression level of the HIS1 gene.

Forward primer CCJ-2F: 5'-GGTAATGTGCAATGGCATCTTC-3' (shown in SEQ. ID NO. 7);

reverse primer CCJ-2R: 5'-CAGATCCTCAGGGAGTCGATGTAC-3' (shown in SEQ. ID NO. 8);

the forward primer Actin-F: 5'-CCTCGTCTCGACCTTGCTGGG-3' (shown in SEQ. ID NO. 9);

reverse primer Actin-R: 5'-GAGAACAAGCAGGAGGACGGC-3' (shown in SEQ. ID NO. 10).

To further verify that the function loss of the H3 resistant beta-triketone herbicide is caused by mutation of the HIS1 gene T1510G, a F2 group which is constructed by taking rice variety Nongxiang 42 (containing H2 and resistant to BBC) and Xianhui 207 (containing H3 and sensitive to BBC) as parents is constructed. The result of spraying 0.4g/L of BBC on the population shows that 737 strains which can resist to the BBC and 231 strains which are sensitive to the BBC in 968 single strain F2 populations in total have a sensitivity ratio of 3:1(X2 is 0.607 (X (0.05)2 is 3.84), which indicates that only one dominant major anti-BBC gene exists in F2 populations constructed by H2 and H3.

Furthermore, aiming at the T1510G mutation of H3 specific to other haplotypes, a dCAPS labeled primer is designed, the front primer and the rear primer are CCJ-3F and CCJ-3R respectively, and a 221bp sequence 1 (shown as SEQ.ID NO. 11) is amplified in other haplotypes such as Nipponbare (H2) and IR64(H5) by utilizing the pair of primers; a221 bp sequence 2 (as shown in SEQ. ID NO.12) was amplified from H3-containing rice material such as Michelia cordata 207. The amplified sequences were electrophoresed and observed, as shown in FIG. 10. Because the T1510G mutation causes that only one restriction enzyme Dde1 specific recognition site (CTNAG) is contained in the sequence 2 amplified by H3, the PCR product can be subjected to enzyme digestion by Dde1 to obtain two sequences of 29bp and 192bp in length, namely a sequence 3 (shown as SEQ. ID NO. 13) and a sequence 4 (shown as SEQ. ID NO. 14), the 221bp sequence can be observed to be subjected to enzyme digestion through electrophoresis (shown as FIG. 11), a 192bp band is generated, and the Dde1 cannot be subjected to enzyme digestion in other haplotype materials. According to the characteristics of agarose gel electrophoresis, if T1510G mutation exists, a 29bp band obtained by enzyme digestion is not observed, only a 192bp band can be observed, and generally a 221bp sequence and a 192bp band generally appear in an electrophoresis result after the 221bp sequence is digested, so that the nucleotide sequence of an amplification product can be judged to be cut by the Dde1 as long as a 192bp band is observed; if the T1510G mutation was not present, a 192bp band was never present.

Using the dCAPS labeled primers and detection method of this example, we performed detection on F2 population constructed from Tungxiang 42 and Xianhui 207, and the results are shown in FIG. 12, and found that the T1510G mutation is completely co-isolated with BBC sensitive phenotype of each individual strain.

Taken together, we determined that the T1510G mutation is critical to the loss of function of the HIS1 gene against β -triketones. The dCAPS marker primer and the detection method can quickly, efficiently, accurately and conveniently identify whether the T1510G mutation exists in the rice strain HIS1 gene, and further lay a good foundation for judging whether the rice HIS1 gene has beta-triketone herbicide resistance.

Example 2:

a method for detecting whether the rice HIS1 gene has beta-triketone herbicide resistance comprises the following steps:

SS1, extracting DNA sample from fresh young leaf of rice by CTAB method, loading appropriate amount of fresh leaf of rice into 2mL centrifuge tube, adding steel ball, quickly freezing in liquid nitrogen, breaking tissue with high-throughput tissue grinder (Ningbo Xinzhi Scientz-48), immediately adding 650 μ L of preheated 2 × CTAB solution at 65 deg.C, placing in 65 deg.C water bath pan, keeping constant temperature for 45min, uniformly reversing every 10min, finishing 45min, cooling DNA sample to room temperature, placing in ventilation cabinet, adding 650 μ L of chloroform-isopropanol (24: 1) solution, uniformly reversing, centrifuging at 12000rmp for 10min, transferring 500 μ L of supernatant into 1.5mL centrifuge tube, adding 500 μ L of isopropanol solution, mixing, precipitating at-20 deg.C for 2h, centrifuging at 12000rmp for 10min, discarding supernatant, adding 1mL of 70% ethanol solution, removing impurities, centrifuging at 12000rmp for 5min, pouring out supernatant,add 200. mu.l ddH₂And dissolving the obtained product in a refrigerator at 4 ℃ overnight to obtain the extracted rice sample DNA template.

SS2, using the DNA sample of step SS1 as a template, and performing PCR amplification by using the dCAPS labeled primers: the synthesized primers CCJ-3F and CCJ-3R are diluted to 10 mM; preparing a PCR reaction system: a20. mu.L PCR reaction system contained 2. mu.L (50 ng/. mu.L) of genomic DNA, 1. mu.L (5. mu.M/mL) of each of the primers, and 16. mu.L of 1.1 XPCR Mix (Opticalke Biotechnology Co., Ltd.). The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 4 min; 35 cycles of 95 ℃ for 20s, 58 ℃ for 20s and 72 ℃ for 20 s; extension at 72 ℃ for 5 min. Taking the obtained amplification product, carrying out enzyme digestion by using a restriction enzyme Dde1, then carrying out agarose gel electrophoresis, reading the length of a DNA fragment of the amplification product, and if a band with the length of 192bp appears in the DNA fragment of the amplification product, judging that the nucleotide sequence of the amplification product is cut; through a DNA sequencing method, two nucleotide sequences with the lengths of 29bp and 192bp are obtained after the nucleotide sequences are cut by restriction endonuclease Dde1, wherein the nucleotide sequence with the length of 29bp is shown as SEQ ID NO.13, the nucleotide sequence with the length of 192bp is shown as SEQ ID NO.14, T1510G mutation exists in the rice HIS1 gene, and the fact that the rice HIS1 gene does not have beta-triketone herbicide resistance can be further determined;

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' (shown in SEQ. ID NO. 2).

SS3, using the DNA sample obtained in the step SS1 as a template, and carrying out PCR amplification by using a molecular marker CCJ-1F/CCJ-1R for detecting whether 28bp deletion exists on the fourth exon of the rice HIS1 gene: the synthesized primer CCJ-1F, CCJ-1R was diluted to 10 mM; preparing a PCR reaction system: a20. mu.L PCR reaction system contained 2. mu.L (50 ng/. mu.L) of genomic DNA, 1. mu.L (5. mu.M/mL) of each of the primers, and 16. mu.L of 1.1 XPCR Mix (Optimalaceae Biotechnology Co., Ltd.). The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 4 min; 35 cycles of 95 ℃ for 20s, 58 ℃ for 20s and 72 ℃ for 20 s; extension at 72 ℃ for 5 min. Carrying out agarose gel electrophoresis on the obtained amplification product, and observing the length of the DNA fragment of the amplification product; if the DNA fragment of the amplified product is a single 182bp strip, the deletion variation of 28bp length of the rice HIS1 gene is shown, and the fact that the rice HIS1 gene does not have beta-triketone herbicide resistance can be further determined. If the DNA fragment of the amplified product is a single 210bp strip or comprises two strips with the lengths of 182bp and 210bp respectively, the deletion variation of the rice HIS1 gene with the length of 28bp does not exist, in this case, if the DNA fragment of the amplified product obtained according to the step SS2 does not have a strip with the length of 192bp, only one nucleotide sequence with the length of 221bp (shown as SEQ. ID NO. 11) is obtained, the HIS1 gene of the rice to be detected does not have T1510G mutation, and the fact that the rice HIS1 gene has beta-triketone herbicide resistance can be further determined;

forward primer CCJ-1F: 5'-GTACAACTTGACGATGTATCAAG-3' (shown in SEQ. ID NO. 3);

reverse primer CCJ-1R: 5'-GTTGAATCTTGCAAATGCAGGAGC-3' (shown in SEQ. ID NO. 4).

The method of the embodiment is adopted to detect and identify the function of 493 indica rice material HIS1 genes, and the specific experimental results are shown in Table 1. As can be seen from Table 1, the sum of the amounts of rice material with 28bp deletion and T1510G mutation was the same as the sum of the number of BBC-sensitive phenotypes, indicating that the presence of 28bp deletion or T1510G mutation resulted in rice sensitive to BBC expression; the sum of the amounts of rice material with neither the 28bp deletion nor the T1510G mutation was the same as the sum of the amounts of resistant phenotypes to BBC, indicating that the function of the HIS1 gene for resistance to beta-triketones is essentially completely controlled by both the 28bp deletion and the T1510G mutation types. As long as 28bp deletion or T1510G mutation is detected in the rice material, the HIS1 gene of the rice material can be identified to have no beta-triketone herbicide resistance; on the contrary, if the rice material is detected to have neither 28bp deletion nor T1510G mutation, the rice material HIS1 gene can be identified to have beta-triketone herbicide resistance.

The dCAPS marker primer can quickly, efficiently, accurately and conveniently identify whether the T1510G mutation exists in the rice line HIS1 gene, and judge whether the rice HIS1 gene has beta-triketone herbicide resistance by combining the fact that whether the rice HIS1 gene has 28bp deletion, thereby providing a certain theoretical basis for the use of the beta-triketone herbicide in part of indica rice fields and laying a good foundation for the subsequent hybrid rice seed production by adopting the beta-triketone herbicide.

Example 3:

a method for producing hybrid rice seeds, which adopts a mixed sowing and mixed harvesting mode to produce the hybrid rice seeds, comprises the following steps:

SSS1, and the adoption of the dCAPS marker primer CCJ-3F/CCJ-3R and the molecular marker primer CCJ-1F/CCJ-1R for detecting whether the HIS1 gene of the male parent and the female parent of the rice has beta-triketone herbicide resistance, wherein the specific operation method is shown in example 2;

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' (shown in SEQ. ID NO. 2);

forward primer CCJ-1F: 5'-GTACAACTTGACGATGTATCAAG-3' (shown in SEQ. ID NO. 3);

reverse primer CCJ-1R: 5'-GTTGAATCTTGCAAATGCAGGAGC-3' (shown in SEQ. ID NO. 4).

SSS2, selecting rice variety Longke 638S with female parent HIS1 gene having beta-triketone herbicide resistance and rice variety HR1128 with male parent HIS1 gene having no beta-triketone herbicide resistance for mixed sowing;

after natural pollination of SSS3, 638S of female parent Longke and HR1128 of male parent is completed, the concentration of active ingredients of the biciflavone herbicide is diluted to 0.4g/L, and male parent and female parent rice plants are uniformly sprayed; the male parent HR1128, because it does not have the resistance of the bifenox herbicide, begins to whiten and then gradually withers, and finally only leaves the seeds produced after pollination by 638S of the female parent family.

In this example, 100 stifles of maternal clone 638S and 20 stifles of paternal HR1128 were co-sown, and finally, the complete death of the 20 stifles of paternal was counted. The purity of the harvested seeds is identified, and 100% of the seeds are hybrid seeds. Compared with the traditional hybrid rice seed production process, the male parent and the female parent need to be separately planted, and the male parent material is cut off after pollination, so that the scheme realizes the mixed sowing and mixed harvesting of the male parent and the female parent and greatly reduces the production cost of hybrid rice seeds. The invention utilizes the characteristic that the biciflavone herbicide causes male parent albino death of beta-triketone herbicide resistance gene, can quickly and accurately realize the purpose of impurity removal, greatly liberates labor force, and promotes and optimizes rice germplasm resources.

Example 4:

a hybrid rice seed production method adopts a photo-thermo-sensitive male sterile line two-line method to carry out hybrid rice seed production, and comprises the following steps:

SSS1, detecting whether the HIS1 genes of male parent and female parent of rice have beta-triketone herbicide resistance: by adopting the dCAPS labeled primer CCJ-3F/CCJ-3R and the molecular labeled primer CCJ-1F/CCJ-1R, 51 sterile lines of the two-line hybrid rice in example 1 are further detected, and the detection results are shown in Table 1; the result shows that T1510G mutation exists in 3 sterile lines, 28bp deletion mutation exists in 5 sterile lines, and HIS1 gene function deletion mutation exists in 8 rice materials in two sterile lines, which are Xiangling 628S, C815S, Hua 1037S, Ming S, quan S, Hua 68S, SE21S and Ming Xiang 10S respectively;

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' (shown in SEQ. ID NO. 2);

forward primer CCJ-1F: 5'-GTACAACTTGACGATGTATCAAG-3' (shown in SEQ. ID NO. 3);

reverse primer CCJ-1R: 5'-GTTGAATCTTGCAAATGCAGGAGC-3' (shown in SEQ. ID NO. 4);

to further verify the result, the 8 two-line sterile lines are subjected to a 0.4g/L concentration biciflavone herbicide (BBC) spraying experiment, and the result is shown in FIG. 13, and the sterile lines are found to be sensitive to BBC; furthermore, BBC concentration gradient experiments are carried out on three representative types of two-line sterile lines Y58S (HIS1 is normal), SE21S (H3) and Ming S (H5), and as shown in FIG. 14, SE21S and Ming S are found to show obvious phytotoxicity when the BBC is 0.05g/L, while Y58S begins to show phytotoxicity when the concentration is 1.2 g/L;

SSS2, selecting a rice variety Ming S without beta-triketone herbicide resistance in a female parent HIS1 gene and a rice variety P143 with beta-triketone herbicide resistance in a male parent HIS1 gene for hybridization, wherein the beta-triketone herbicide resistance/sensitive phenotypes of the male parent HIS1 gene and the female parent HIS1 gene are homozygotes, and normally harvesting seeds of F1 generation for planting to obtain a hybrid Mingliangyou 143 (showing resistance to the beta-triketone herbicide); the BBC herbicide with different concentration gradients is uniformly sprayed on the hybrid Mingliangyou 143, and the result is shown in figure 15, when the concentration of the BBC is in the range of 0.05g/L-0.6g/L, the hybrid Mingliangyou 143 shows resistance, and the female parent Mingliangyou shows sensitivity; finally, the impurity removal and purification experiment is carried out on the hybrid seeds of the Mingliangyou 143 by using BBC with the concentration of 0.4g/L, and the result shows that the hybrid sterile line Mingliangyou can kill 100 percent, but no obvious phytotoxicity is generated on the hybrid seeds of the Mingliangyou 143 (see table 2), so that the progeny of the female parent self-inoculation caused by temperature instability is removed, and the true hybrid progeny is obtained.

Table 2: impurity removal and purification results of 0.4g/L concentration BBC in Mingliangyou 143

Type (B)	Total number of	Number of deaths	Number of normal growth	Mortality rate
					Mingliangyou 143	2000	0	2000	0
Ming S	20	20	0	100％

Example 5:

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' (shown in SEQ. ID NO. 2);

reverse primer CCJ-1R: 5- 'GTTGAATCTTGCAAATGCAGGAGC-' 3 (shown in SEQ. ID NO. 4);

in order to verify the function of mesotrione in the seed purification of two-line hybrid rice, a mesotrione concentration gradient experiment is carried out on Nipponbare (HIS1 normal), Y58S (HIS1 normal), SE21S (H3) and IR64(H5), and the results are shown in a graph of 16-19, and the results show that when the concentration is 0.01g/L, the IR64 and SE21S show obvious phytotoxicity, while the Nipponbare and Y58S show obvious phytotoxicity when the concentration reaches 0.45g/L, and prove that the mesotrione and BBC have similar phenotypes to rice;

SSS2, selecting a rice variety Ming S without beta-triketone herbicide resistance in a female parent HIS1 gene and a rice variety P143 with beta-triketone herbicide resistance in a male parent HIS1 gene for hybridization, wherein the beta-triketone herbicide resistance/sensitive phenotypes of the male parent HIS1 gene and the female parent HIS1 gene are homozygotes, and normally harvesting seeds of F1 generation for planting to obtain a hybrid Mingliangyou 143 (showing resistance to the beta-triketone herbicide); the hybrid Mingliangyou 143 was uniformly sprayed with mesotrione herbicide at different concentration gradients, and the results are shown in FIG. 20, where mesotrione concentration was in the range of 0.01g/L to 0.1g/L, mesotrione herbicide was safe (performance-resistant) to hybrid Mingliangyou 143, while maternal Mingliang showed severe injury (performance-sensitive); finally, the mesotrione herbicide with the concentration of 0.03g/L is used for carrying out impurity removal and purification experiments on the hybrid seeds Mingliangyou 143, and the results show that the mesotrione herbicide can kill sterile line Mingliangyou self-bred fructification seeds by 100 percent, and has no influence on the growth of the hybrid seeds Mingliangyou 143 (see table 3), so that progeny of female parent self-breeding caused by temperature instability is removed, and true hybrid progeny is obtained. Because the HIS1 gene is a broad-spectrum resistance gene of the beta-triketone herbicide, the beta-triketone herbicide (including BBC, mesotrione, fursulcotrione and the like) can be used for removing impurities and purifying the two-line hybrid rice seeds.

Table 3: impurity removal and purification result of mesotrione with concentration of 0.03g/L in Mingliangyou 143

Type (B)	Total number of	Number of deaths	Number of normal growth	Mortality rate
					Mingliangyou 143	3000	0	3000	0
Ming S	30	30	0	100％

In general, aiming at the seed production safety requirements of two-line hybrid rice and different indica rice varieties with different anti-infection characteristics to beta-triketone herbicides, the invention systematically analyzes the haplotypes of the HIS1 gene in main indica rice in China, determines whether each haplotype has the anti-herbicide function, identifies a new HIS1 non-functional haplotype and designs a corresponding detection molecular marker. By selecting the HIS1 gene non-functional haplotype sterile line and functional haplotype restorer line, the aim of purifying the hybrid rice seeds by the two-line method by using the beta-triketone herbicide is fulfilled.

Sequence listing

<110> research center for hybrid rice in Hunan province

<120> method for detecting presence or absence of T1510G mutation in rice HIS1 gene and dCAPS labeled primer

<160>14

<170>SIPOSequenceListing 1.0

<210>1

<211>29

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<213> Artificial Sequence (Artificial Sequence)

<400>1

catcttcagg agcccggtgc acaggctga 29

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

tcagggagtc gatgtacctc 20

<210>3

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gtacaacttg acgatgtatc aag 23

<210>4

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gttgaatctt gcaaatgcag gagc 24

<210>5

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

aagcaagaac aaagttaagc aag 23

<210>6

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

aaggaaatac ttgctactga aagc 24

<210>7

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ggtaatgtgc aatggcatct tc 22

<210>8

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

cagatcctca gggagtcgat gtac 24

<210>9

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

cctcgtctcg accttgctgg g 21

<210>10

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gagaacaagc aggaggacgg c 21

<210>11

<211>221

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<213> Rice (Oryza sativa L.)

<400>11

catcttcagg agcccggtgc acaggctgat gacaaacgcc gagaaggaga ggatctccct 60

ggccatgtta tacagcgtga acgatgagaa agacattgag ccggcggctg gtttgctgga 120

tgagaatcgg cctgcaagat acaggaaagt gagcgtcgaa gagttcaggg ccgggatctt 180

tggaaaattc tctcgaggag agaggtacat cgactccctg a 221

<210>12

<211>221

<212>DNA

<213> Rice (Oryza sativa L.)

<400>12

catcttcagg agcccggtgc acaggctgag gacaaacgcc gagaaggaga ggatctccct 60

ggccatgtta tacagcgtga acgatgagaa agacattgag ccggcggctg gtttgctgga 120

tgagaatcgg cctgcaagat acaggaaagt gagcgtcgaa gagttcaggg ccgggatctt 180

tggaaaattc tctcgaggag agaggtacat cgactccctg a 221

<210>13

<211>29

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<213> Rice (Oryza sativa L.)

<400>13

catcttcagg agcccggtgc acaggctga29

<210>14

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<212>DNA

<213> Rice (Oryza sativa L.)

<400>14

ggacaaacgc cgagaaggag aggatctccc tggccatgtt atacagcgtg aacgatgaga 60

aagacattga gccggcggct ggtttgctgg atgagaatcg gcctgcaaga tacaggaaag 120

tgagcgtcga agagttcagg gccgggatct ttggaaaatt ctctcgagga gagaggtaca 180

tcgactccct ga 192

Claims

1. A method for detecting whether a T1510G mutation exists in a rice HIS1 gene comprises the following steps:

s1, using a DNA sample of the rice to be detected as a template, and carrying out PCR amplification on a DNA fragment containing a site of 1510 th nucleotide downstream of an initiation codon ATG on a HIS1 gene by using a specific dCAPS labeled primer;

2. The method according to claim 1, wherein in step S1, the DNA fragment is from 1510 th nucleotide downstream of the ATG start codon of the HIS1 gene to 50 th to 192 th nucleotide downstream of the 1510 th nucleotide;

the specific dCAPS labeled primer comprises a forward primer CCJ-3F and a reverse primer CCJ-3R with the following sequences:

forward primer CCJ-3F: 5'-CATCTTCAGGAGCCCGGTGCACAGGCTGA-3', respectively;

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' are provided.

3. The method according to claim 2, wherein the specific operation of determining whether the nucleotide sequence of the amplification product is cleaved into two pieces by agarose gel electrophoresis in step S2 comprises the following steps: carrying out agarose gel electrophoresis on the amplification product subjected to enzyme digestion treatment by restriction enzyme Dde1, reading the length of a DNA fragment of the amplification product, and judging that the nucleotide sequence of the amplification product is cut if a band with the length of 192bp appears in the DNA fragment of the amplification product; if the DNA fragment of the amplification product does not have a band with the length of 192bp, judging that the nucleotide sequence of the amplification product is not cut.

4. The method according to claim 2, wherein the specific operation of determining whether the nucleotide sequence of the amplification product is cleaved into two segments by DNA sequencing in step S2 comprises the following steps: carrying out DNA sequencing on the amplification product subjected to enzyme digestion treatment by restriction enzyme Dde1, and if the nucleotide sequence shown in SEQ.ID NO.13 and/or SEQ.ID NO.14 is obtained, judging that the nucleotide sequence of the amplification product is cut; and if the nucleotide sequence shown as SEQ.ID NO.11 is obtained, judging that the nucleotide sequence of the amplification product is not cut.

5. A method for detecting whether the rice HIS1 gene has beta-triketone herbicide resistance comprises the following steps:

SS1, detecting whether the HIS1 gene of the rice sample has deletion variation with the length of 28 bp; and detecting the presence of a T1510G mutation in the HIS1 gene of the rice sample according to the method of any one of claims 1 to 4;

6. The method according to claim 5, wherein the operation of step SS1 specifically comprises the steps of: taking a DNA sample of rice to be detected as a template, carrying out PCR amplification by using a specific molecular marker primer, taking an obtained amplification product, carrying out agarose gel electrophoresis, and observing the length of a DNA fragment of the amplification product; if the DNA fragment of the amplified product is a single 182bp strip, the deletion variation of 28bp length of the rice HIS1 gene is shown; if the DNA fragment of the amplified product is a single 210bp band or comprises two bands with the lengths of 182bp and 210bp respectively, the deletion variation with the length of 28bp does not exist in the rice HIS1 gene;

forward primer CCJ-1F: 5'-GTACAACTTGACGATGTATCAAG-3', respectively;

reverse primer CCJ-1R: 5'-GTTGAATCTTGCAAATGCAGGAGC-3' are provided.

7. A method for producing hybrid rice seeds is characterized in that the method of claim 5 or 6 is adopted to detect and screen male and female rice parents for hybrid seed production, and then a mixed sowing and mixed harvesting mode or a photo-thermo-sensitive male sterile line two-line method is adopted to carry out hybrid rice seed production on the male and female rice parents.

8. The method as claimed in claim 7, wherein the hybrid rice seed production by mixed seeding and mixed harvesting comprises the following steps: firstly, detecting whether the HIS1 genes of male parent and female parent of rice have beta-triketone herbicide resistance, selecting rice varieties of which the HIS1 gene of female parent has beta-triketone herbicide resistance and the HIS1 gene of male parent does not have beta-triketone herbicide resistance to carry out mixed sowing, wherein the female parent is sterile line, after the rice completes natural pollination, uniformly spraying the beta-triketone herbicide on the rice plants of the male parent and the female parent to remove the male parent which has no beta-triketone herbicide resistance, and obtaining the offspring of the true hybrid on the surviving rice plants of the female parent; the beta-triketone herbicide is a mesosulfuron herbicide or a pyroxsulazone herbicide, and is sprayed on a target plant after the concentration of the mesosulfuron herbicide is diluted to 0.05g/L-0.6g/L or the pyroxsulazone herbicide is diluted to 0.01g/L-0.1g/L when the beta-triketone herbicide is applied.

9. The method as claimed in claim 7, wherein the method for hybrid rice seed production by using photo-thermo-sensitive male sterile line 'two-line method' comprises the following steps: firstly, detecting whether beta-triketone herbicide resistance exists in male parent and female parent HIS1 genes of rice, selecting rice varieties of which the female parent HIS1 gene does not have beta-triketone herbicide resistance and the male parent HIS1 gene has beta-triketone herbicide resistance for hybridization, wherein the HIS1 genes of the male parent and the female parent are homozygotes, normally harvesting F1 generation seeds for planting, and uniformly spraying the F1 generation plants with the beta-triketone herbicide to remove progeny of female parent selfing inoculation caused by temperature instability, namely obtaining true hybrid progeny; the beta-triketone herbicide is a mesosulfuron herbicide or a pyroxsulazone herbicide, and is sprayed on a target plant after the concentration of the mesosulfuron herbicide is diluted to 0.05g/L-0.6g/L or the pyroxsulazone herbicide is diluted to 0.01g/L-0.1g/L when the beta-triketone herbicide is applied.

10. A dCAPS marker primer for detecting the existence of a T1510G mutation in rice HIS1 gene or detecting the existence of beta-triketone herbicide resistance in rice HIS1 gene, wherein the dCAPS marker primer comprises a forward primer CCJ-3F and a reverse primer CCJ-3R with the following sequences:

forward primer CCJ-3F: 5'-CATCTTCAGGAGCCCGGTGCACAGGCTGA-3', respectively;

reverse primer CCJ-3R: 5'-TCAGGGAGTCGATGTACCTC-3' are provided.