CN117467787A - Molecular marker for identifying mycorrhizal efficient symbiotic rice, universal amplification primer and application - Google Patents
Molecular marker for identifying mycorrhizal efficient symbiotic rice, universal amplification primer and application Download PDFInfo
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- 239000003147 molecular marker Substances 0.000 title claims abstract description 28
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- 239000002773 nucleotide Substances 0.000 claims description 19
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- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 101000715709 Oryza sativa subsp. japonica Chitin elicitor receptor kinase 1 Proteins 0.000 abstract description 17
- 240000002582 Oryza sativa Indica Group Species 0.000 abstract description 4
- 240000008467 Oryza sativa Japonica Group Species 0.000 abstract description 4
- 239000000047 product Substances 0.000 description 22
- 108020004414 DNA Proteins 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 241000746966 Zizania Species 0.000 description 9
- 235000002636 Zizania aquatica Nutrition 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- 238000001962 electrophoresis Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 101100005922 Oryza sativa subsp. japonica CERK1 gene Proteins 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a molecular marker for identifying mycorrhiza efficient symbiotic rice, a universal amplification primer and application thereof. The molecular marker is rice OsCERK1 DY The invention also provides an amplification primer for identifying the molecular marker, the specific sequence of which is shown as SEQ ID NO.4 and SEQ ID NO.5, the amplification primer can be suitable for indica rice and japonica rice, is a universal amplification primer with high universality, can be used for identifying whether rice is mycorrhizal high-efficiency symbiotic rice or not, can be applied to the rice breeding field, comprises germplasm resource identification, purity identification and the like, and has remarkable application value.
Description
Technical Field
The invention relates to the field of molecular biology genetic breeding, in particular to a molecular marker for identifying mycorrhiza efficient symbiotic rice, a universal amplification primer and application thereof.
Background
PCR (polymerase chain reaction) is a commonly used molecular marker identification technology and is widely applied to the breeding field. The principle is that primers are used for amplifying specific sections of DNA for multiple rounds so as to generate a large number of replications, and specific genes or sequence variations are detected by the method. For example, PCR molecular markers can be used to screen for specific genotypes or traits, such as disease resistance, drought resistance, etc., and can be used to create molecular genetic maps or to conduct genetic diversity analysis. The PCR method comprises the following steps: first, a DNA template is mixed with a primer, dNTPs and a polymerase, and a plurality of rounds of cyclic reactions are performed to gradually amplify a DNA sequence. Then, the amplified product is detected and separated by gel electrophoresis or the like to determine the presence and size of the target DNA sequence.
OsCERK1 from wild rice in Dongxiang DY The gene can greatly improve mycorrhizal symbiotic capacity of rice and achieve the effects of losing weight and increasing yield. However, there is no simple and effective molecular marker for assisting breeding process in the process of cultivating mycorrhiza efficient symbiotic rice, and the existing molecular marker or applicationThe range is limited, or restriction enzyme treatment is needed when the PCR primer is used, as described in CN112980936A, when the amplification of the provided primer is finished, the PCR reaction product is digested by restriction enzyme RsaI, the PCR reaction product is digested for 1h at 37 ℃, the electrophoresis result is identified, the identification result can be obtained, and the result can be obtained after the amplification is carried out by enzyme digestion and electrophoresis, so that the identification process is complicated, the identification period is long, and the breeding progress is influenced.
Disclosure of Invention
The invention aims to provide a molecular marker for identifying mycorrhizal efficient symbiotic rice, a universal amplification primer and application thereof, wherein the molecular marker and the amplification primer can be used for identifying whether the rice is mycorrhizal efficient symbiotic rice or not, can be applied to the field of rice breeding, comprises germplasm resource identification, purity identification and the like, and has remarkable application value.
In order to achieve the aim, the invention provides a molecular marker for identifying mycorrhizal high-efficiency symbiotic rice, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the molecular marker can be used for identifying mycorrhizal high-efficiency symbiotic rice, when the rice OsCERK1 DY The insertion of the molecular marker sequence exists at the upstream 1611bp of the gene ATG locus, which shows that the rice is mycorrhizal high-efficiency symbiotic rice; if the molecular marker is not inserted, the non-mycorrhizal high-efficiency symbiotic rice is obtained.
The molecular marker provided by the invention can be used for identifying mycorrhizal efficient symbiotic rice.
The invention also provides an amplification primer for identifying the molecular marker, the nucleotide sequences of the amplification primer are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, and the amplification primer can be used for identifying mycorrhizal high-efficiency symbiotic rice. Specifically, the genome DNA of the rice to be detected is amplified by the amplification primer, and when the amplified product is a 97bp band or two bands shared by 97bp and 89bp, the rice to be detected is shown to be mycorrhizal high-efficiency symbiotic rice; when the amplified product is a strip only containing 89bp, the rice is indicated to be non-mycorrhizal high-efficiency symbiotic rice.
Preferably, the amplification conditions of the above amplification primers are specifically as follows:
the amplification system is 10 mu L, comprising: 1.0. Mu.L of 10 Xbuffer, 1.0. Mu.L of 2mM dNTP, 1. Mu.L of 10. Mu.M primer F, R each, 1U of Taq DNA polymerase and 1.0. Mu.L of genomic DNA, the remaining volume being ddH 2 Supplementing O;
the PCR reaction procedure is as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 58℃for 30sec, elongation at 72℃for 6sec for 32 cycles; extending at 72℃for 5min.
The invention also provides a kit for identifying the mycorrhizal efficient symbiotic rice, which comprises amplification primers with nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 respectively.
The kit provided by the invention can be used for rice breeding, rice germplasm resource identification, rice purity identification and the like, the genome of the rice to be detected is subjected to PCR amplification through the amplification primer in the kit, and the aim can be achieved through the amplification result.
The molecular marker and the amplification primer for identifying the mycorrhizal efficient symbiotic rice solve the problem of rapid identification of the mycorrhizal efficient symbiotic rice, and have the following advantages:
the invention provides a high-efficiency symbiotic rice gene OsCERK1 with mycorrhiza DY And (3) the closely linked molecular markers, and whether the rice to be detected is mycorrhizal high-efficiency symbiotic rice can be judged through the existence of the molecular markers.
The invention provides an amplification primer, and the rice can be judged whether to be mycorrhizal high-efficiency symbiotic rice or not according to the electrophoresis result of the amplification primer, and processes such as enzyme digestion or sequencing are not needed, so that the method is simple in steps, low in cost, high in identification efficiency and high in practicability.
The molecular marker and the amplification primer provided by the invention are suitable for various rice resources, not only comprise indica rice and japonica rice, but also have wide application variety range and strong universality.
Drawings
FIG. 1 shows the result of electrophoresis of the products of PCR amplification of rice by the amplification primers provided by the invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 development of molecular markers and general amplification primers
OsCERK1 of' Dongxiang wild rice DY There are two mutations in the gene that result in the presence of I118T, S121T in the protein amino acid sequence that it expresses, which is not present in the existing 4660 cultivars, whereas the mutation makes 'Dongxiang wild rice' a mycorrhizal high efficiency symbiotic rice, whereas the mutation is not present in the OsCERK1 gene of 'flower 11'.
Obtaining sequences of flower 11' genome (GCA_ 014526345.1) in rice ' and wild rice ' genome (GCA_ 000817225.1) in Dongxiang as template DNA, comparing sequences near OsCERK1 gene (LOC_Os08g42580) of two genome sequences, finding OsCERK1 in wild rice genome in Dongxiang DY An 8bp nucleotide is inserted at the upstream 1611bp of the ATG locus of the gene, the inserted nucleotide sequence is shown as SEQ ID NO.1, and the specific sequence is GGCGCCGC; wild rice genome OsCERK1 in Dongxiang DY The insertion of 19bp nucleotide exists at 47736bp upstream of the ATG locus of the gene, the inserted nucleotide sequence is shown as SEQ ID NO.2, and the specific sequence is GTCACACGTCATTGAGTTT; wild rice genome OsCERK1 in Dongxiang DY A deletion of 16bp nucleotide exists at 62072bp upstream of the ATG locus of the gene, the deleted nucleotide sequence is shown as SEQ ID NO.3, and the specific sequence is GCTAGCTAGCTAGCTA.
An amplification primer (Ch 8-1F, ch 8-1R) was designed for the above-described inserted 8bp nucleotide sequence, and the specific sequence was as follows:
Ch8-1F(SEQ ID NO.4)：5’-TCCGCCTCGAATGGCTCCAC-3’
Ch8-1R(SEQ ID NO.5)：5’-GGGAGGTGGAAGCTTCCATG-3’
when the 8bp nucleotide is inserted into the corresponding position of the template DNA, the amplified product is 97bp; when the 8bp nucleotide is not inserted, the amplified product is 89bp; when the amplified product has two bands of 97b and 89bp, the rice corresponding to the lane is heterozygous.
The amplification primer (Ch 8-2F, ch 8-2R) was designed for the above-described inserted 19bp nucleotide sequence, and the specific sequence is as follows:
Ch8-2F(SEQ ID NO.6)：5’-CGGAGCTCACCGTGCATGTG-3’
Ch8-2R(SEQ ID NO.7)：5’-CCGACGGCTGCATGTTGCTC-3’
when the 19bp nucleotide is inserted into the corresponding position of the template DNA, the amplified product is 188bp; when the 19bp nucleotide is not inserted, the amplified product is 169bp; when there are two bands of 188bp and 169bp in the amplified product, it is indicated that the rice corresponding to this lane is heterozygous.
The amplification primer (Ch 8-3F, ch 8-3R) was designed for the deleted 16bp nucleotide sequence as follows:
Ch8-3F(SEQ ID NO.8)：5’-TGTGATTGGAGCATGCATGCAG-3’
Ch8-3R(SEQ ID NO.9)：5’-CTCTCTGAGGAGTAGAGACGACTG-3’
when the 16bp nucleotide is deleted in the corresponding position of the template DNA, the amplified product is 130bp; when the 16bp nucleotide is not deleted, the amplified product is 146bp; when two bands of 130b and 146bp exist in the amplified product, the rice corresponding to the lane is shown to be heterozygous.
The 3 pairs of amplification primers designed by the method are used for carrying out amplification identification on a plurality of rice materials to explore whether the 3 inserted or deleted sequences can be used as a molecular marker for identifying mycorrhiza efficient symbiotic rice, and the specific process is as follows:
1. rice genome DNA extraction
The number of rice materials to be detected is 12, namely Ganjia rice No.1, wild incense B, taifeng B, wanzhen B, jinggui, hui incense No.2, KM102, daohuaxiang No.2, sanjiang No. 6, WJ17 and WJ126. Gangxi germ rice 1 is a single-segment substitution line of Dongxiang wild rice with middle and early 35 as background, and has been proved to carry homozygous OsCERK1 DY The gene has the same mutation as 'Dongxiang wild rice' on the OsCERK1 gene, so that the Ganjiao rice No.1 is also mycorrhizal high-efficiency symbiotic rice; the other 11 rice is a commercially available homozygous rice, and the above-mentioned mutation is not present in the OsCERK1 gene. The genome DNA of the 12 kinds of rice is extracted by adopting a CTAB method, and the specific steps are as follows:
(1) Cutting a small amount of leaves, placing into a 2.0mL centrifuge tube, adding 600 mu L of 1.5 XCTAB and steel balls, and oscillating at 60Hz for 1min on a tissue grinder;
(2) Placing the centrifuge tube into 65 ℃ water for warm bath for 30min, adding 600 mu L of chloroform (volume ratio is 24:1), and mixing for 5min by light shaking;
(3) Centrifuging the centrifuge tube 12000r/min for 10min, sucking 400 mu L of supernatant, and transferring the supernatant into a new centrifuge tube;
(4) Adding 800 μL of frozen 95% ethanol, mixing thoroughly, standing at-20deg.C for 20min;
(5) Centrifuging the centrifuge tube 12000r/min for 15min, and pouring out supernatant;
(6) Adding 500 μl of 75% ethanol, centrifuging at 12000r/min for 5min, and removing supernatant;
(7) Air-dry 75% ethanol and add 100. Mu.L ddH 2 O dissolves DNA for later use.
2. PCR amplification
PCR amplification was performed using the above-mentioned extracted genomic DNA as a template by 3 pairs of amplification primers (Ch 8-1F, ch8-1R; ch8-2F, ch-2R; ch8-3F, ch 8-3R) designed as described above;
wherein the amplification system is 10 mu L, comprising: 1.0. Mu.L of 10 Xbuffer, 1.0. Mu.L of 2mM dNTP, 1. Mu.L of 10. Mu.M forward and reverse primers (F, R) each, 1U of Taq DNA polymerase and 1.0. Mu.L of genomic DNA, the remainder being ddH 2 And supplementing O.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, tm annealing for 30sec, elongation at 72℃for 6sec for 32 cycles; extending at 72 ℃ for 5min; wherein, the Tm of the 3 pairs of primers in the order (Ch 8-1, ch8-2, ch 8-3) is 58 ℃, 60 ℃ and 58 ℃, respectively.
3. Polyacrylamide gel electrophoresis
Due to the inserted 8bp nucleusGlucuronide and OsCERK1 described above DY The physical distance of the mutation on the gene is very close, and the probability of gene exchange is very low, so that the inserted 8bp nucleotide is considered to be linked with the mutation. While the other two inserted and deleted fragments were compared with OsCERK1 DY The mutation in the gene is physically located relatively far apart and the probability of gene exchange is relatively high, so that the probability of linkage of the two markers to the mutation is low. The verification is carried out by combining the electrophoresis result, and the specific steps are as follows:
respectively performing polyacrylamide gel electrophoresis on the amplified products, performing sample application on 2 mu L of each amplified product by adopting 8% polyacrylamide gel, and performing electrophoresis for 2 hours at 100V.
The electrophoresis result is shown in figure 1, wherein lane M is DNA ladder, lanes 1-12 are respectively the products of Ganyuan rice No.1, wild Xiang B, taifeng B, wanzhen B, beijing Zhenzhu, hui Zhan, ganzhen No.2, KM102, daohuaxiang No.2, sanjiang No. 6, WJ17 and WJ126, ch8-1 in the figure is the product of an amplification primer Ch8-1F, ch8-1R, ch8-2 is the product of an amplification primer Ch8-2F, ch8-2R, and Ch8-3 is the product of an amplification primer Ch8-3F, ch 8-3R; as shown by the electrophoresis result, a 97bp band appears in Ganjiao rice No.1, which represents a band containing OsCERK1 DY The method comprises the steps of carrying out a first treatment on the surface of the The other species showed a 89bp band, representing the absence of OsCERK1 DY This result matches the background of all rice; while the other 19bp insert and 16bp
The missing molecular markers, primers and amplification products thereof do not accord with the background of all rice, and cannot be used as molecular markers for identifying whether the rice is mycorrhizal high-efficiency symbiotic rice. It can be known that the 8bp inserted molecular marker can be used for identifying whether the rice is mycorrhizal high-efficiency symbiotic rice, and an amplification primer designed for the molecular marker can be used for identifying the molecular marker; according to the amplification test results of a plurality of different varieties, the representative indica rice varieties and japonica rice varieties can be distinguished by means of the molecular marker, meanwhile, the amplification primer is also applicable to indica rice and japonica rice, the amplification primer is a universal amplification primer with high universality, the universal amplification primer can also be used for identifying whether rice is mycorrhizal high-efficiency symbiotic rice or not, and the amplification primer has obvious application value in the field of rice breeding, including germplasm resource identification, purity identification and the like.
Meanwhile, the amplification primer can be used for preparing an identification kit for identifying whether the rice to be detected is mycorrhizal high-efficiency symbiotic rice, and when the amplification product is a 97bp band or 97bp and 89bp shared two bands, the rice to be detected is the mycorrhizal high-efficiency symbiotic rice; when the amplified product is a strip only containing 89bp, the rice is indicated to be non-mycorrhizal high-efficiency symbiotic rice, and the kit can also be used for rice germplasm resource identification, purity identification and the like.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (7)
1. A molecular marker for identifying mycorrhizal high-efficiency symbiotic rice is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.
2. The use of the molecular marker of claim 1 for identifying mycorrhizal high-efficiency symbiotic rice.
3. The molecular tagged amplification primer of claim 1, wherein the amplification primer has a nucleotide sequence shown in SEQ ID No.4 and SEQ ID No.5, respectively.
4. Use of the amplification primer of claim 3 for identifying mycorrhiza efficient symbiotic rice.
5. The use according to claim 4, wherein the PCR reaction procedure for the amplification primers is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 58℃for 30sec, elongation at 72℃for 6sec for 32 cycles; extending at 72℃for 5min.
6. A kit for identifying mycorrhizal efficient symbiotic rice is characterized by comprising amplification primers with nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 respectively.
7. Use of the kit of claim 6 in any one of the following;
1) Breeding rice;
2) Identifying rice germplasm resources;
3) And (5) identifying the purity of the rice.
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