CN116334287A - Primer group, primer pool and kit for detecting rice functional genes and application of primer group, primer pool and kit - Google Patents
Primer group, primer pool and kit for detecting rice functional genes and application of primer group, primer pool and kit Download PDFInfo
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- CN116334287A CN116334287A CN202310310608.3A CN202310310608A CN116334287A CN 116334287 A CN116334287 A CN 116334287A CN 202310310608 A CN202310310608 A CN 202310310608A CN 116334287 A CN116334287 A CN 116334287A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The invention discloses a primer group, a primer pool and a kit for detecting rice functional genes and application thereof, and belongs to the technical field of biology. The primer set comprises a1 st primer pair to a 250 nd primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair, the forward primer of the 250 nd primer pair and the reverse primer of the 250 nd primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO: 500. The primer group and the primer pool can be used for carrying out multiplex PCR amplification on a plurality of genes in the same reaction system, so that the operation steps are greatly simplified, the amplification efficiency is improved, and meanwhile, the higher success rate can be ensured, and the omission ratio of the genes is reduced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group, a primer pool, a kit and application thereof for detecting rice functional genes.
Background
Rice (Oryzasativa) is widely planted in the world, one of the most important food crops worldwide, and more than half of the world's population is taking rice as the staple food. The country is not only the largest paddy rice producing country in the world, but also the largest paddy rice consuming country. The safety supply of rice is directly related to the grain safety and social stability of China, so the development of the rice industry occupies an extremely important position in national economy and national safety.
Scientists have long focused on the discovery of important genes of rice and the research of molecular mechanisms thereof. Since the 90 s of the 20 th century, rice geneticists, breeders and molecular biologists are tightly cooperated to complete rice genome sequencing, analyze the genetic basis of a series of important agronomic traits of rice, clone a batch of key genes for controlling the important traits of rice such as plant type, yield, flowering period, disease resistance, insect resistance, stress resistance, nutrition efficiency, rice quality and the like, and clarify the molecular mechanism and genetic regulation network for controlling the traits. In recent years, molecular module design breeding concepts have been proposed in various crops. The important agronomic trait gene of each clone is one molecular module. The molecular module design breeding based on whole genome navigation is to diagnose and model important character genes of bred varieties and breeding backbone parents through genome scanning, which have excellent alleles and which have short necessary excellent isogenes, then design an optimal path for variety improvement according to the existing molecular module and interaction relation, and screen individuals polymerizing a plurality of excellent genotypes from offspring rapidly and accurately through hybridization, backcross and gene editing technology and molecular marking technology.
Although more than 200 important agronomic trait genes have been cloned in rice, the important agronomic trait genes "whole genome navigation map" have been mapped, and molecular markers useful for auxiliary selection have been developed for individual genes. However, the single-marker detection technology is used for detecting only one gene at a time, so that the gene locus sequences with multiple characters cannot be obtained at one time, and multiple times of detection are needed, so that the detection period is long, the cost is high, the operation is extremely complicated, and the detection of rice functional genes is not facilitated.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiment of the invention provides a primer set, a primer pool, a kit and application thereof for detecting rice functional genes. The technical scheme is as follows:
in one aspect, the invention provides a primer set for detecting rice functional genes, the primer set comprises a1 st primer pair to a 250 nd primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair, the forward primer of the 250 nd primer pair and the reverse primer of the 250 nd primer pair are sequentially shown as SEQ ID NOs in a sequence table: 1 to SEQ ID NO: 500.
In another aspect, an embodiment of the present invention provides a primer pool for detecting a rice functional gene, the primer pool comprising: at least one of primer pool a, primer pool B, primer pool C, primer pool D, primer pool E, primer pool F, primer pool G, primer pool H, and primer pool I, the primer pool a comprising primer pair 1 to primer pair 36, the primer pool B comprising primer pair 37 to primer pair 76, the primer pool C comprising primer pair 77 to primer pair 114, the primer pool D comprising primer pair 115 to primer pair 120, the primer pool E comprising primer pair 121 to primer pair 145, the primer pool F comprising primer pair 146 to primer pair 161, the primer pool H comprising primer pair 186 to primer pair 200, the primer pool I comprising primer pair 201 to primer pair 250, each of the primer pair comprising forward and reverse primer pairs 1, 250 to primer pair 250, and forward and reverse primer pairs of SEQ ID of the primers of SEQ ID No. 1 to 250: 1 to SEQ ID NO: 500.
Specifically, the primer pool includes: primer pool A, primer pool B, primer pool C, primer pool D, primer pool E, primer pool F, primer pool G, primer pool H and primer pool I.
In yet another aspect, an embodiment of the present invention provides a kit for detecting a rice functional gene, the kit comprising the primer set of claim 1.
In still another aspect, an embodiment of the present invention provides an application of a primer set for detecting a rice functional gene, the application including: the primer set according to claim 1 is used for detecting rice functional genes.
Specifically, the detection of rice functional genes comprises: the primer pool A is used for detecting abiotic stress resistance genes, the primer pool B is used for detecting rice stress resistance genes, the primer pool C is used for detecting rice heading stage genes, the primer pool D is used for detecting nitrogen fertilizer utilization rate genes, the primer pool E is used for detecting rice plant type genes, the primer pool F is used for detecting secondary metabolite genes, the primer pool G is used for detecting rice seed color and mango length genes, the primer pool H is used for detecting rice quality genes, and the primer pool I is used for detecting yield-related genes.
The technical scheme provided by the embodiment of the invention has the beneficial effects that: the primer group and the primer pool provided by the embodiment of the invention can carry out multiplex PCR amplification in the same reaction system, the primers can not interfere with each other during amplification, the primers are compared two by two, the primers capable of forming dimers are screened out, the formation of primer dimers is avoided, and the non-specific amplification is reduced. Meanwhile, the primer group and the primer pool provided by the embodiment of the invention can carry out multiplex PCR amplification on a plurality of genes in the same reaction system, thereby greatly simplifying the operation steps, improving the amplification efficiency, ensuring higher success rate and reducing the omission factor of the genes. The primer group and the primer pool provided by the invention can be used for detecting rice functional genes, and a method for rapidly, accurately and inexpensively detecting important plant types, yield, flowering period, biological stress resistance, non-biological stress resistance, fertility, nutrition efficiency and rice quality gene variation information of rice is provided for rice genetic and breeding workers, so that excellent alleles carried by existing varieties, backbone parents and strains can be rapidly and accurately diagnosed, guidance is provided for making breeding strategies, and breeding efficiency can be greatly improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
Example 1
The embodiment of the invention provides a primer group for detecting rice functional genes, which comprises a1 st primer pair to a 250 nd primer pair, wherein each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair, the forward primer of the 250 nd primer pair and the reverse primer of the 250 nd primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO:500, specifically as shown in table 1.
Table 1 shows the sequences of 250 primer pairs
The embodiment of the invention also provides a primer pool for detecting rice functional genes, which comprises the following components: primer pool a, primer pool B, primer pool C, primer pool D, primer pool E, primer pool F, primer pool G, primer pool H, and primer pool I, primer pool a including 1 st to 36 th primer pairs, primer pool B including 37 th to 76 th primer pairs, primer pool C including 77 th to 114 th primer pairs, primer pool D including 115 th to 120 th primer pairs, primer pool E including 121 th to 145 th primer pairs, primer pool F including 146 th to 161 th primer pairs, primer pool G including 161 th to 185 th primer pairs, primer pool H including 186 th to 200 th primer pairs, primer pool I including 201 st to 250 th primer pairs, each primer pair including forward and reverse primers, the 1 st primer pair forward primer, the 1 st primer pair forward to 250 th primer pair forward to 250 nd primer pair of SEQ ID of the reverse primer pair in sequence: 1 to SEQ ID NO: 500.
Specifically, the primer set includes: primer pool A, primer pool B, primer pool C, primer pool D, primer pool E, primer pool F, primer pool G, primer pool H and primer pool I.
On the other hand, the embodiment of the invention provides a kit for detecting rice functional genes, which comprises the primer group.
In still another aspect, an embodiment of the present invention provides an application of a primer set for detecting a functional gene of rice, where the application includes: the primer group is used for detecting rice functional genes.
Further, detecting rice functional genes includes: primer pool a is used to detect abiotic stress resistance genes, which in this example include: salt-tolerant gene Os-HKT1;5/SKC1 and OsHKT1;1 and OsHKT2;1, aluminium toxin resistance genes NRAT1, herbicide resistance genes CYP72A31/BST and HIS1, flooding resistance genes OsCBL10, drought resistance genes OsPP15 and DCA1, COLD and freezing resistance genes COLD1, hbd2/OsCKI1/LTG1/LTRPK1, qLTG3-1, osMYB2, CTB4a, bZIP73, osLTPL159 and HAN1, cadmium and heavy metal accumulation genes OsHMA4, CAL1, osCd1, osHMA3 and qGMN7.1/OsNRAMP5, boron potassium high efficiency genes BET1 and OsHKT2;1.
the primer pool B is used for detecting rice stress resistance genes, and the biological stress resistance genes comprise: rice blast resistance genes Pi35, bsr-d1, pi21, piPR1, pi9, pi2, pi50, piz-t, pi25 (Pid 3), pid3-A4, pid2Pi-i (Pi 5-1), pi56, pia/RGA4, pia/RGA5, pb1, pi54 (Pi-kh), pi-ta, ptr, osCERK and LHCB5, bacterial leaf blight resistance genes Xa1, xa5, xa13, xa21, xa26/Xa3, xa25 and OsSWEET13/Xa25, brown planthopper resistance genes Bph3 and Bph29, east-gray virus resistance genes eIF4G and OsSAP16, rice rust resistance genes STV11/OsSOT1.
The primer pool C is used for detecting rice heading stage genes, and the rice heading stage genes comprise: HESO1/OsHESO1, osMADS51, osCOL4, DTH2/Hd7, ehd4, ef-cd, osMADS50/Hd9/OsSOC1/DTH3, MIR528, hd6, EL1, hd16, hd17/Hd3b, RFT1, hd3a, hd1, ghd7/Hd4, LOC_Os07g49460, osPRR37/Hd2/Ghd7.1, hd18, ghd8, DTH8, ehd1, osMADS56 and RCN1.
The primer pool D is used for detecting nitrogen fertilizer utilization rate genes, and the rice nitrogen fertilizer utilization rate genes comprise: nitrogen-efficient genes osnpf6.1, osNR2, ARE1 and nrt1.1b.
The primer pool E is used for detecting rice plant type genes, wherein the rice plant type genes comprise: tillering angle, ear grain number, grain width, grain length, grain width, chalkiness, fragrance, gelatinization temperature and amylose, specific genes include: D2/CYP90D2/SMG11, D61/OsBRI1, sd1, ILI3/OsbHLH153, SCM3/OsTB1, SLR1/OsGAI/Slr1-D, TAC3, osGSK2, SBI/OsGA2ox4, SCM2/APO1, TIG1, osOTUB1/WTG1, osSPY, osTb2, qRT9, TAC1, osbHLH174, GATA28 and TIPS-11-9.
The primer pool F is used for detecting secondary metabolite genes, and the rice secondary metabolite genes comprise: osUGT706D1, osUGT706C2, osUGT707A2, osAT4C, os-TBT1 and Os-TBT2.
The primer pool G is used for detecting rice seed color and miscanthus sinensis length genes, wherein the rice seed color and miscanthus sinensis length genes comprise: rd/DFR/OsDfr, an-1, an-2/OsLOGL6/LABA1, dep/GLR1/GL1, osC1, rc and RAE2/GAD1.
The primer pool H is used for detecting rice quality genes, and the rice quality genes comprise: osAAP6, chalk5, osACS6/SSG6, wall/GBSSI, ALK/SSSII, fgr/Badh2 and OsGluA2.
Primer pool I was used to detect yield-related genes. Yield-related genes include: yield genes Gn1a/OsCKX2, NOG1, LAX1, GW2, qNGR2/GRF4, osLG3/OsERF62/OsRAF, qLGY3/OsMADS1/GW3p6/OsLG3b, GS3, GL3.2/CYP78A5, GL3.1/qGL3, GL3.3/qTGW3, GNP1, SPIKE/NAL1/LSCHL4/GP, PTB1, GS5, GW5/GSE5/qSW, GS6/DLT/OsGRAS-32/SMOS2/D62, TGW6, GW6a/OsglHAT1, osSNB, osSPL13/GLW7, GW7, GE/CYP78A13/BG2, SGDP7/FZP, GW8/OsSPL16 and GFR1; restoring genes Rf-1/Rf1a/Rf1/PPR791, RF1b, rf3/OsMADS3, rf2 and Rf4; hybrid sterility genes ESA1, saF, saM, hwi2, S28/mtRPL27a, DGS1/RPC4, DPL2, S5-3/ORF3, S5-4/ORF4, S5, S7, HSA1a and HSA1b, photo-thermo-sensitive sterility genes TMS5, pms1/PMS1T and P/TMS12-1/Pms3/LDMAR.
In this embodiment, the rice sample to be measured may be a tissue or organ such as a seed, seedling, leaf or spike of rice, preferably a rice seed or leaf.
In this example, 15 rice varieties were selected as the rice to be measured, and these 15 rice varieties were Q-22W180, Q-22W210, Q-22W262, Q-22W282, Q-22W287, Q-22W290, Q-22W292, Q-22W297, Q-22W330, Q-22W350, Q-22W386, Q-22W402, Q-22W538, Q-22W539 and Q-22W540, respectively. The 15 rice varieties are provided by food crop research institute of the national academy of agricultural sciences of Hubei province.
Extracting the whole genome DNA of the rice to be detected.
CTAB (cetyltrimethylammonium bromide) method, SDS (sodium dodecyl sulfate) method or existing commercial whole genome DNA extraction kit and extraction method thereof are adopted.
In this example, the CTAB method was used to extract whole genome DNA of the sample to be tested. Specifically, about 200 mg-300 mg of rice leaves to be measured are placed in a 2.0mL centrifuge tube, and are fully ground; adding 500 mu L of CTAB extract preheated at 65 ℃ into each centrifuge tube, fully and uniformly mixing, and slightly and reversely mixing each centrifuge tube during water bath at 65 ℃ for 30 min; adding mixed solution of chloroform/isoamyl alcohol (the volume ratio of the chloroform to the isoamyl alcohol is 24:1) into each centrifuge tube, fully mixing, standing for 10min, centrifuging at 12000rpm for 10min, and absorbing supernatant; the supernatant was transferred to a new centrifuge tube, and an equal volume of pre-chilled isopropyl alcohol was added, gently mixed upside down, left at-20℃for 30min, and centrifuged at 12000rpm for 10min at4 ℃. Discarding the supernatant, retaining the precipitate, adding 70% ethanol into the precipitate for washing, washing for 2 times in this example to obtain the whole genome DNA of the rice to be tested, drying the whole genome DNA of the rice to be tested at room temperature, adding 100 μl of water, and dissolving fully for standby.
The obtained whole genome DNA of the rice to be detected is subjected to multiplex PCR (PolymeraseChain Reaction ) amplification reaction by utilizing the primer group provided by the embodiment of the invention.
Specifically, the primer set for the multiplex PCR amplification reaction is a mixture of all 250 primer pairs. The multiplex PCR amplification reaction used KAPA2GFAST MultiplexPCRKits (available from Kapa biosystems). In this example, the multiplex PCR amplification reaction system was 20. Mu.L, 50ng of whole genome DNA of rice to be tested, 10. Mu.L of 2multiplex PCRmix were added to each reaction tube, 0.5. Mu.L of each primer pool (primer pools A to I) was added, and ddH was added 2 O was made up to 20. Mu.L.
The conditions for round 1 multiplex PCR amplification reactions were as follows: preheating for 3min at 95 ℃; (annealing at 95 ℃ for 20s, renaturation at 60 ℃ for 30s, and extension at 72 ℃ for 2 min). Times.25 cycles; maintained at 72℃for 5min.
After the multiplex PCR amplification reaction is finished, a multiplex PCR amplification reaction product is obtained, and the multiplex PCR amplification reaction product is purified. Specifically, the multiplex PCR amplification reaction product is purified by using an AgencourtAMPureXPkit purchased from Beckman company, and redundant primers are removed to obtain the purified multiplex PCR amplification reaction product. See the instructions of the kit for specific operations.
And (3) carrying out library construction and sequencing (detection of rice important character gene loci) on the multiple PCR amplification reaction products.
The purified multiplex PCR amplification reaction products are used for constructing a sequencing library by adopting illumine, MGI or other commercial kits for library establishment, and sequencing analysis is carried out.
In the present embodiment, use is made of
Ultra TM II DNA Library Prep Kit fo/>
(from New England BioLabs) a second generation sequencing library was constructed. Specifically, the purified multiplex PCR amplification reaction products are quantified. Taking 30ng of purified multiplex PCR amplification reaction product, carrying out DNA fragment end repair and A addition, then carrying out linker connection to obtain a connection product, and carrying out magnetic bead purification on the connection product to obtainObtaining a second generation sequencing library. And quantifying the constructed second-generation sequencing library by adopting qPCR, and sequencing by a second-generation sequencing platform after quality inspection.
Sequencing data bioinformatic analysis.
And obtaining the sequence of the rice yield related gene locus to be detected, sequencing, and analyzing the obtained biological information. Specifically, for the raw data obtained by sequencing, firstly removing low-quality reads, comparing the rest reads with the whole genome reference sequence of the rice variety Nipponbare by using BWA software, finding out the variation between different rice varieties and the whole genome reference sequence, and then analyzing by using Samstools software, wherein the analysis results are shown in Table 2.
Table 2 shows the detection of 250 gene loci of 15 rice varieties to be tested
As can be seen from table 2, the sequencing results of the 15 rice varieties to be tested provided in this example show that: the average detection efficiency for the target gene loci is over 93%, the average Coverage (Coverage) is over 94%, and the sequencing depth of each target gene locus is over 1000 times. This shows that the detection by using the primer set provided by the embodiment of the invention can effectively avoid the mutual interference between the primer pairs. Meanwhile, the primer group can efficiently detect the rice variety and the gene composition of the backbone parent and the hybrid progeny strain, and greatly improves the detection efficiency of rice hybridization breeding.
The excellent alleles of 15 rice varieties to be tested were diagnosed using multiplex PCR amplification and bioinformatic analysis procedures, and the allelic compositions of a part of the abiotic stress resistance genes (salt tolerance genes), the rice stress resistance genes (brown planthopper resistance genes, rice blast resistance genes, rice stripe rust resistance genes and bacterial leaf blight resistance genes), the yield-related genes (restorer genes) and the rice plant type genes are shown in Table 3.
Table 3 shows the partial locus allele compositions in 15 rice varieties to be tested
As can be seen from the combination of Table 3, the 15 rice varieties to be detected provided by the embodiment of the invention are consistent with the detection results provided by Table 3, and the primer set, the primer pool and the kit provided by the embodiment of the invention can be accurately detected.
The primer group and the primer pool provided by the embodiment of the invention can carry out multiplex PCR amplification in the same reaction system, the primers can not interfere with each other during amplification, the primers are compared two by two, the primers capable of forming dimers are screened out, the formation of primer dimers is avoided, and the non-specific amplification is reduced. Meanwhile, the primer group and the primer pool provided by the embodiment of the invention can carry out multiplex PCR amplification on a plurality of genes in the same reaction system, thereby greatly simplifying the operation steps, improving the amplification efficiency, ensuring higher success rate and reducing the omission factor of the genes. More importantly, the primer set and primer pool can be utilized to directly construct a sequencing library and match various sequencing platforms of existing mainstream second generation sequencing platforms, such as Illumina or MGI. The primer group and the primer pool provided by the invention can be used for detecting rice functional genes, and a method for rapidly, accurately and inexpensively detecting important plant types, yield, flowering period, biological stress resistance, non-biological stress resistance, fertility, nutrition efficiency and rice quality gene variation information of rice is provided for rice genetic and breeding workers, so that excellent alleles carried by existing varieties, backbone parents and strains can be rapidly and accurately diagnosed, guidance is provided for making breeding strategies, and breeding efficiency can be greatly improved.
The foregoing description of the preferred embodiments of the present invention is not intended to limit the invention, but rather, the invention is to be construed as limited to the appended claims.
Claims (6)
1. A primer set for detecting rice functional genes, wherein the primer set comprises a1 st primer pair to a 250 nd primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair, the forward primer of the 250 nd primer pair and the reverse primer of the 250 nd primer pair are sequentially shown as SEQ ID NOs in a sequence table: 1 to SEQ ID NO: 500.
2. A primer pool for detecting a rice functional gene, the primer pool comprising: at least one of primer pool a, primer pool B, primer pool C, primer pool D, primer pool E, primer pool F, primer pool G, primer pool H, and primer pool I, the primer pool a comprising primer pair 1 to primer pair 36, the primer pool B comprising primer pair 37 to primer pair 76, the primer pool C comprising primer pair 77 to primer pair 114, the primer pool D comprising primer pair 115 to primer pair 120, the primer pool E comprising primer pair 121 to primer pair 145, the primer pool F comprising primer pair 146 to primer pair 161, the primer pool H comprising primer pair 186 to primer pair 200, the primer pool I comprising primer pair 201 to primer pair 250, each of the primer pair comprising forward and reverse primer pairs 1, 250 to primer pair 250, and forward and reverse primer pairs of SEQ ID of the primers of SEQ ID No. 1 to 250: 1 to SEQ ID NO: 500.
3. The primer pool of claim 1, wherein the primer pool comprises: primer pool A, primer pool B, primer pool C, primer pool D, primer pool E, primer pool F, primer pool G, primer pool H and primer pool I.
4. A kit for detecting a rice functional gene, comprising the primer set according to claim 1.
5. An application of a primer group for detecting rice functional genes, which is characterized by comprising the following steps: the primer set according to claim 1 is used for detecting rice functional genes.
6. The use according to claim 5, wherein said detecting rice functional genes comprises: the primer pool A is used for detecting abiotic stress resistance genes, the primer pool B is used for detecting rice stress resistance genes, the primer pool C is used for detecting rice heading stage genes, the primer pool D is used for detecting nitrogen fertilizer utilization rate genes, the primer pool E is used for detecting rice plant type genes, the primer pool F is used for detecting secondary metabolite genes, the primer pool G is used for detecting rice seed color and mango length genes, the primer pool H is used for detecting rice quality genes, and the primer pool I is used for detecting yield-related genes.
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