CN112176098A - Primer, kit and method for screening tillering early-growing and fast-growing rice germplasm - Google Patents
Primer, kit and method for screening tillering early-growing and fast-growing rice germplasm Download PDFInfo
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Abstract
The invention discloses a primer, a kit and a method for screening tillering early-growing and fast-growing rice germplasm, which relate to the technical field of molecular markers and the technical field of crop genetic breeding, and comprise four groups of primers, wherein each group of primers comprises a forward primer and a reverse primer, and the sequences of the four groups of primers are shown as SEQ ID NO 1-8; the kit comprises the primer, a PCR reaction buffer solution and Taq DNA polymerase; the screening method comprises the following steps: extracting the total genome DNA of the rice germplasm to be detected, using the total genome DNA as a template, carrying out PCR amplification by using the primer or the kit, carrying out electrophoresis on the obtained PCR amplification product, and carrying out photographing and analysis in an agarose gel imaging system. The method has the advantages that the screening of the tillering early-growing and fast-growing rice germplasm can be realized through four groups of primers, the method is accurate and is not interfered by the outside, the detection of 4 molecular markers can be completed through 1 PCR amplification by adopting the kit, and the method has the advantages of simplicity and high efficiency.
Description
Technical Field
The invention relates to the technical field of molecular markers and crop genetic breeding, in particular to a primer, a kit and a method for screening early-tillering and fast-growing rice germplasm.
Background
The early growth and quick development of the rice means early tillering and quick tillering of a rice group at the early stage, and the theoretical effective tillering number is reached as early as possible, so that the earning rate and the uniformity of rice ears are improved, and the stable yield and the high yield of the rice are finally realized. Therefore, the screening of early-tillering and fast-growing rice germplasm has important significance for breeding stable-yield and high-yield rice varieties.
The early-growing and quick-growing character of rice tillering is the quantitative character regulated by multiple genes, but is easily influenced by factors such as planting density of rice, fertilizer and water management, local climatic conditions and the like. Firstly, in the process of screening early-tillering and fast-growing rice germplasm in a field, breeding workers are easily interfered by climate conditions, soil conditions and cultivation management measures of rice planting places, so that character identification results are possibly distorted. Secondly, observing or investigating the early-growing and quick-growing character of rice tillering needs to observe and investigate the tillering dynamic state of rice at fixed points and continuously when the rice enters the tillering stage, and has the advantages of large workload, long duration and large consumption of human resources. Finally, the differences of regional climate conditions, soil conditions and cultivation management measures of different research units and the differences of evaluation criteria of different researchers cause the lack of comparability between the identification results of different research units.
Therefore, the accuracy and the reliability of the identification result of the early and fast tillering property of the rice are improved, and the comparability of the identification result is improved, so that the method has important significance for improving the breeding effect of the early and fast tillering property of the rice, reducing the resource consumption in the identification process and reducing the resource waste caused by the repeated identification of different research units.
Disclosure of Invention
The invention aims to at least solve one of the technical problems in the prior art and provides a primer, a kit and a method for screening tillering early-growing and fast-growing rice germplasm.
The technical solution of the invention is as follows:
the invention provides a primer for screening tillering early-growing and fast-growing rice germplasm, which comprises four groups of primers, namely a primer 1, a primer 2, a primer 3 and a primer 4, wherein the four groups of primers respectively comprise a forward primer and a reverse primer, and the sequences of the four groups of primers are respectively as follows:
SEQ ID No.1F：5′-TGCAATGACATGCCTGAAGT-3′；
SEQ ID No.1R：5′-CTGGCAAGACGCAACTAACA-3′；
SEQ ID No.2F：5′-ACTCCCTCGTCTCTAGAGTTCTCC-3′；
SEQ ID No.2R：5′-GTTGGGAGTCTATCCCATCG-3′；
SEQ ID No.3F：5′-CACCAGCTGTTGCTTCCATAAGTACC-3′；
SEQ ID No.3R：5′-GTCGACGTCCACGATGACTACC-3′；
SEQ ID No.4F：5′-TAGCGATTGGATGGAGGCTGAGG-3′；
SEQ ID No.4R：5′-GGTCGCCTCGCCATTAGTTACG-3′。
the second aspect of the invention provides a kit for detecting the early-tillering and fast-growing rice germplasm, which comprises the primer.
Preferably, the mass ratio of the forward primer 1, the forward primer 2, the forward primer 3 and the forward primer 4 is 3:2:1:1, and the mass ratio of the reverse primer 1, the reverse primer 2, the reverse primer 3 and the reverse primer 4 is 3:2:1: 1.
Preferably, the kit further comprises a PCR reaction buffer and Taq DNA polymerase.
Preferably, the volume of the PCR reaction buffer is 12.5ul, and 3U of Taq DNA polymerase is used.
Preferably, the total volume of the forward primers of the four sets of primers is 3ul and the total mass is 150ng, and the total volume of the reverse primers of the four sets of primers is 3ul and the total mass is 150 ng.
Preferably, the kit also comprises a DNA Marker which shows bands with the sizes of 301bp, 244bp, 156bp and 123bp respectively after electrophoresis.
The third aspect of the invention provides a method for detecting the early-tillering and fast-growing rice germplasm, which is characterized by using the primer or the kit for detection.
Preferably, the method comprises:
s1, extracting the total DNA of the genome of the rice germplasm to be detected;
s2, taking the extracted total genomic DNA as a DNA template, and carrying out PCR amplification reaction on the primer or the kit and the DNA template to obtain a PCR amplification product;
s3, carrying out electrophoresis on the PCR amplification product in agarose gel;
s4, after electrophoresis, photographing and analyzing in an agarose gel imaging system.
Preferably, in step S2, the PCR amplification reaction includes: taking 12.5ul of PCR Mastermix buffer solution, 5.0ng of 2ul of DNA template, 150ng of 3ul of forward mixed primer, 150ng of 3ul of reverse mixed primer and 4.5ul of double distilled water to prepare a 25ul reaction system, wherein the mass ratio of the forward primer 1, the forward primer 2, the forward primer 3 and the forward primer 4 in the forward mixed primer is 3:2:1:1, and the mass ratio of the reverse primer 1, the reverse primer 2, the reverse primer 3 and the reverse primer 4 in the reverse mixed primer is 3:2:1: 1; the reaction program was pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 20 sec, extension at 72 ℃ for 30 sec, 35 cycles, and incubation at 72 ℃ for 5 min.
Preferably, in step S3, the PCR amplification product is electrophoresed in agarose gel with a concentration of 3% for 25 minutes.
Preferably, in step S4, comparing the electrophoresis strip of the rice germplasm to be detected with the strip of the DNA Marker in the gel photograph, the more the number of the electrophoresis strips of the rice germplasm to be detected is consistent with the number of the DNA Marker, the stronger the tillering early-growing fast-growing property of the rice germplasm to be detected is determined, and the less the number of the strips of the rice germplasm to be detected is consistent with the number of the DNA Marker, the weaker the tillering early-growing fast-growing property of the rice germplasm to be detected is determined, and the invention has at least one of the following beneficial effects:
the method can screen the germplasm of the early-growing and fast-growing tillering rice by four groups of primers, and has the advantages of accuracy and no external interference.
The kit can complete the amplification and detection of four SSR molecular markers by using one PCR amplification reaction, and has the advantages of simple, quick and efficient detection method and reliable result.
The screening method is not limited by seasons, environment and time, has reliable screening results, and has important significance for improving the breeding effect of the early-growing and quick-growing character of rice tillering, reducing the resource consumption in the identification process and reducing the resource waste caused by repeated identification of different research units.
Drawings
FIG. 1 is an agarose electrophoresis picture of a specific DNA Marker containing a rice germplasm genome amplification product with consistent size and high early tillering and fast growing property in a kit in a preferred embodiment of the invention;
FIG. 2 is an agarose electrophoresis image of rice to be detected by the screening method in the preferred embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
The embodiment provides a group of 4 SSR molecular markers associated with early-growing and fast-growing traits of rice tillering and four groups of primers corresponding to the 4 SSR molecular markers, wherein the four groups of primers are respectively primer 1, primer 2, primer 3 and primer 4, the four groups of primers comprise forward primers and reverse primers, and the nucleotide sequences of the forward primers and the reverse primers of the four groups of primers are shown in table 1:
TABLE 1
Firstly, extracting the genome DNA of the rice germplasm with early and rapid tillering, and carrying out PCR amplification by using the genome DNA as a template, wherein the sizes of products are 301bp, 244bp, 156bp and 123bp in sequence; and then extracting the rice germplasm genome DNA with early tillering and weak rapid germination, and performing PCR amplification by using the forward mixed primer and the reverse mixed primer of the SSR molecular marker, wherein the size of the amplified strip is not consistent with that of the molecular marker.
Therefore, the strength of the early-tillering and fast-growing character of the rice germplasm is in direct proportion to the quantity of the SSR molecular markers, namely, if a certain rice germplasm genome sequence carries the 4 SSR molecular markers, the early-tillering and fast-growing character of the rice germplasm is strong; if the SSR molecular markers of 2 or 3 of the 4 SSR molecular markers are carried, the tillering early and quick growing property is moderate; if the SSR molecular markers of 0 or 1 of the 4 SSR molecular markers are carried, the tillering early-growing and quick-growing performance of the SSR molecular markers is weak.
The embodiment also provides a kit for screening the tillering early-growing and fast-growing rice germplasm, which comprises 4 SSR molecular markers of forward mixed primers, reverse mixed primers, PCR reaction buffer solution, Taq DNA polymerase and other reagents, and specifically comprises the following steps: PCR Mastermix buffer solution 12.5ul, DNA template 2ul 5.0ng, Taq DNA polymerase 3U, forward primer 3ul 150ng, reverse primer 3ul 150ng, double distilled water 4.5ul, prepared into 25ul reaction system. Wherein, the mass ratio among the forward primer 1, the forward primer 2, the forward primer 3 and the forward primer 4 in the forward mixed primer is 3:2:1:1, and the mass ratio among the reverse primer 1, the reverse primer 2, the reverse primer 3 and the reverse primer 4 in the reverse mixed primer is 3:2:1: 1.
The kit further comprises a DNA Marker which is consistent with the PCR amplification products marked by the 4 SSR molecules in size, and the DNA Marker presents bands with the sizes of 301bp, 244bp, 156bp and 123bp after agarose electrophoresis, which is shown in figure 1, wherein a Lane 1 is the commercially purchased DNA Marker, and a Lane 2 is the DNA Marker of the kit in the embodiment.
The embodiment also provides a method for screening the germplasm of the early-growing and fast-growing tillering rice, which comprises the following steps:
s1, randomly selecting 15 lines from the backcross recombinant inbred line of Wuyujing 24/T461// Wuyujing 24 as the rice germplasm material to be detected in the embodiment, wherein the lines are respectively marked as WTL001, WTL023, WTL047, WTL051, WTL077, WTL103, WTL106, WTL143, WTL176, WTL221, WTL262, WTL279, WTL303, WTL318 and WTL327, after the rice germplasm of the 15 lines grows to 1 leaf and 1 heart through seed soaking and germination acceleration, respectively cutting 0.5 g of young leaves of each rice germplasm, extracting genome total DNA by adopting a CTAB method, detecting the extraction integrity of the genome total DNA by agarose electrophoresis, and determining the concentration of the extracted total DNA by adopting a spectrophotometer; in addition, the 15 strains are respectively selected to be uniform and orderly germinated, seeds are directly sown in a hole-based single-seed sowing mode in 2019 in south China, 4 months and 15 days, 2020 and 21 months in 5 months, the row spacing of the seeds is 16.7 multiplied by 23.3 centimeters, and the seeds are managed according to conventional cultivation management measures, wherein the number of days from sowing to 1 tillering and the number of days to 15 tillering of the 15 strains are shown in a table 2.
S2, taking the extracted total genomic DNA as a DNA template, and carrying out PCR amplification reaction by using the kit and the 5.0ng DNA template, wherein the reaction procedures are pre-denaturation at 95 ℃ for 3 minutes, denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 20 seconds, extension at 72 ℃ for 30 seconds, 35 cycles and heat preservation at 72 ℃ for 5 minutes to obtain a PCR amplification product;
s3, spotting 4ul of the PCR amplification product into a loading hole of 3% agarose gel, spotting a DNA Marker in the kit into the loading hole of 3% agarose gel, turning on a power supply of an electrophoresis apparatus, and stopping electrophoresis after electrophoresis for 25 minutes at a voltage of 5V/cm;
and S4, after the electrophoresis is finished, photographing in an agarose gel imaging system to obtain a gel photo, and comparing and analyzing an electrophoresis strip of the rice germplasm to be detected in the gel photo with a strip of a DNA Marker.
TABLE 2
The detection results are shown in fig. 2, wherein lanes 1 and 18 are commercially available DNA markers, lane 2 is the DNA Marker of the kit of the present invention, lanes 3 to 6 are stable lines WTL001, WTL023, WTL103 and WTL327 having strong early tillering and rapid germination in the rice backcross recombinant inbred line, lanes 7 to 13 are stable lines WTL047, WTL077, WTL106, WTL143, WTL221, WTL279 and WTL318 having medium early tillering and rapid germination in the rice backcross recombinant inbred line, and lanes 14 to 17 are stable lines WTL051, WTL176, WTL262 and WTL303 having weak early tillering and rapid germination in the rice backcross recombinant inbred line.
As can be seen from FIG. 2, the lanes 3-6 all include 4 bands of 301bp, 244bp, 156bp and 123bp, i.e., the genomes of 4 strains WTL001, WTL023, WTL103 and WTL327 carry 4 SSR molecular markers. The lane 7 includes 3 bands of 301bp, 156bp and 123bp, the lane 8 includes 2 bands of 244bp and 123bp, the lane 9 includes 2 bands of 156bp and 123bp, the lane 10 includes 2 bands of 301bp and 244bp, the lane 11 includes 2 bands of 301bp and 156bp, the lane 12 includes 2 bands of 244bp and 156bp, and the lane 13 includes 2 bands of 301bp and 123bp, i.e., 2 or 3 of the 4 SSR molecular markers are carried by 7 strains of WTL047, WTL077, WTL106, WTL143, WTL221, WTL279 and WTL 318. Lane 14 includes the band of 156bp, and lanes 15 to 17 do not include the above band, i.e., 0 or 1 of the 4 SSR markers are carried by the genomes of the 4 strains WTL051, WTL176, WTL262 and WTL 303. Therefore, it is found from the above-mentioned detection results that 4 lines WTL001, WTL023, WTL103 and WTL327 are rice germplasm with strong tillering early-onset property, 7 lines WTL047, WTL077, WTL106, WTL143, WTL221, WTL279 and WTL318 are rice germplasm with moderate tillering early-onset property and the like, and 4 lines WTL051, WTL176, WTL262 and WTL303 are rice germplasm with weak tillering early-onset property.
Comparing the detection result in fig. 2 with the early tillering fast-growing germplasm of each strain in table 2, it can be seen that the detection result of the method of the present embodiment is consistent with the result of the early tillering fast-growing germplasm of each strain in table 2, and thus, the method of the present embodiment is accurate in detection and can be used for detecting and screening the early tillering fast-growing germplasm of rice. In addition, the detection result further verifies that the strength of the early tillering and fast growing property of the rice strain is in direct proportion to the quantity of the 4 SSR molecular markers.
Therefore, the kit of the embodiment can complete the amplification and detection of four SSR molecular markers by using one PCR amplification reaction, the detection method is simple and rapid, the result is reliable, the method for screening the rice germplasm with the tillering early-growing and fast-growing character is not limited by seasons, environments and time, the screening result is reliable, and the kit has important significance for improving the breeding effect of the rice germplasm with the tillering early-growing and fast-growing character, reducing the resource consumption in the identification process and reducing the resource waste caused by the repeated identification of different research units.
The above are merely characteristic embodiments of the present invention, and do not limit the scope of the present invention in any way. All technical solutions formed by equivalent exchanges or equivalent substitutions fall within the protection scope of the present invention.
SEQUENCE LISTING
<110> university of agriculture in Jiangxi
<120> primer, kit and method for screening tillering early-growing and fast-growing rice germplasm
<130> Jiangxi university of agriculture
<160> 8
<170> PatentIn version 3.5
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<213> Artificial Sequence 1(Artificial Sequence1)
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actccctcgt ctctagagtt ctcc 24
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caccagctgt tgcttccata agtacc 26
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gtcgacgtcc acgatgacta cc 22
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ggtcgcctcg ccattagtta cg 22
Claims (10)
1. The primer for screening the tillering early-growing and fast-growing rice germplasm is characterized by comprising four groups of primers 1, 2, 3 and 4, wherein the four groups of primers respectively comprise a forward primer and a reverse primer, and the sequences of the four groups of primers are respectively as follows:
SEQ ID No.1F：5′-TGCAATGACATGCCTGAAGT-3′；
SEQ ID No.1R：5′-CTGGCAAGACGCAACTAACA-3′；
SEQ ID No.2F：5′-ACTCCCTCGTCTCTAGAGTTCTCC-3′；
SEQ ID No.2R：5′-GTTGGGAGTCTATCCCATCG-3′；
SEQ ID No.3F：5′-CACCAGCTGTTGCTTCCATAAGTACC-3′；
SEQ ID No.3R：5′-GTCGACGTCCACGATGACTACC-3′；
SEQ ID No.4F：5′-TAGCGATTGGATGGAGGCTGAGG-3′；
SEQ ID No.4R：5′-GGTCGCCTCGCCATTAGTTACG-3′。
2. a kit for screening tillering early-growing and fast-growing rice germplasm, which is characterized by comprising the primer of claim 1.
3. The kit for screening tillering early-growing and fast-growing rice germplasm according to claim 2, wherein the mass ratio of the forward primer 1 to the forward primer 2 to the forward primer 3 to the forward primer 4 is 3:2:1:1, and the mass ratio of the reverse primer 1 to the reverse primer 2 to the reverse primer 3 to the reverse primer 4 is 3:2:1: 1.
4. The kit for screening tillering early-growing and fast-growing rice germplasm according to claim 3, wherein the total volume of the forward primers of the four groups of primers is 3ul, and the total mass is 150ng, and the total volume of the reverse primers of the four groups of primers is 3ul, and the total mass is 150 ng.
5. The kit for screening tillering early-growing and fast-growing rice germplasm according to claim 2, characterized by further comprising 12.5ul of PCR reaction buffer and 3U of Taq DNA polymerase.
6. The kit for screening tillering early-growing and fast-growing rice germplasm according to claim 2, characterized by further comprising a DNA Marker, wherein the DNA Marker presents bands with the sizes of 301bp, 244bp, 156bp and 123bp respectively after electrophoresis.
7. A method for screening tillering early-growing fast-growing rice germplasm, which is characterized by comprising the step of detecting by using the primer of claim 1 or the kit of any one of claims 2 to 6.
8. The method for screening tillering early-growing and fast-growing rice germplasm according to claim 7, which comprises:
s1, extracting the total DNA of the genome of the rice germplasm to be detected;
s2, taking the extracted total genomic DNA as a DNA template, and carrying out PCR amplification reaction on the primer of claim 1 or the kit of any one of claims 2-6 and the DNA template to obtain a PCR amplification product;
s3, carrying out electrophoresis on the PCR amplification product and DNAmarker in the same agarose gel;
and S4, after the electrophoresis is finished, photographing in an agarose gel imaging system to obtain a gel picture, and analyzing an electrophoresis strip on the gel picture.
9. The method for screening tillering early-growing and fast-growing rice germplasm according to claim 7, wherein in step S2, the PCR amplification reaction comprises: taking 12.5ul of PCR Mastermix buffer solution, 5.0ng of 2ul of DNA template, 150ng of 3ul of forward mixed primer, 150ng of 3ul of reverse mixed primer and 4.5ul of double distilled water to prepare a 25ul reaction system, wherein the mass ratio of the forward primer 1, the forward primer 2, the forward primer 3 and the forward primer 4 in the forward mixed primer is 3:2:1:1, and the mass ratio of the reverse primer 1, the reverse primer 2, the reverse primer 3 and the reverse primer 4 in the reverse mixed primer is 3:2:1: 1; the reaction program was pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 20 sec, extension at 72 ℃ for 30 sec, 35 cycles, and incubation at 72 ℃ for 5 min.
10. The method for screening tillering early-growing and fast-growing rice germplasm according to claim 7, wherein the PCR amplification product is electrophoresed in 3% agarose gel at a voltage of 5V/cm for 25 minutes in step S3.
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| CN202011246990.9A CN112176098B (en) | 2020-11-10 | 2020-11-10 | Primer, kit and method for screening tillering early-growing and fast-growing rice germplasm |
| AU2021100768A AU2021100768A4 (en) | 2020-11-10 | 2021-02-08 | Primers for rice germplasm with early tillering vigour, kit, and method |
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| CN110692507A (en) * | 2018-07-09 | 2020-01-17 | 中国科学院遗传与发育生物学研究所 | Method for improving plant species |
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| CN110692507A (en) * | 2018-07-09 | 2020-01-17 | 中国科学院遗传与发育生物学研究所 | Method for improving plant species |
Non-Patent Citations (3)
| Title |
|---|
| DAISUKE FUJITA: "Fine mapping of a gene for low-tiller number, Ltn, in japonica rice (Oryza sativa L.) variety Aikawa 1", 《THEOR APPL GENET 》 * |
| M J THOMSON: "Mapping quantitative trait loci for yield, yield components and morphological traits in an advanced backcross population between Oryza rufipogon and the Oryza sativa cultivar Jefferson", 《THEOR APPL GENET》 * |
| MD NASHIR UDDIN: "Identification of a low tiller gene from a new plant type cultivar in rice ( Oryza sativa L.)", 《BREED SCI》 * |
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| AU2021100768A4 (en) | 2021-04-22 |
| CN112176098B (en) | 2021-05-04 |
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