CN114350841B - Polymorphic molecular marker based on whole genome sequencing, preparation method and application - Google Patents
Polymorphic molecular marker based on whole genome sequencing, preparation method and application Download PDFInfo
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Abstract
The application relates to the technical field of biological information, in particular to a polymorphic molecular marker based on whole genome sequencing, a preparation method and application; the molecular marker comprises 81 groups of primer pairs, and the sequences of the primer pairs are shown as SEQ ID NO. 1-SEQ ID NO. 162; the method comprises the following steps: establishing a library and sequencing the genomic DNA of the Wufeng B and the Guanghui 308 to obtain original data, and performing quality control screening to obtain effective data; comparing and assembling the effective data and the reference genome to obtain a comparison file; obtaining the consistency sequences of the Wufeng B and the Guanghui 308; comparing the consistent sequences to obtain an InDel locus; obtaining a designed primer sequence according to the InDel locus; searching and comparing the designed primer sequence in the genome to obtain a labeled primer; carrying out PCR amplification and electrophoresis detection to obtain an electrophoresis result picture; judging whether the labeled primer is a polymorphic molecular marker or not according to the electrophoresis result picture; the application comprises the following steps: the molecular marker is used for rice genetic map construction, QTL positioning, molecular marker assisted breeding and genetic diversity analysis.
Description
Technical Field
The application relates to the technical field of biological information, in particular to a polymorphic molecular marker based on whole genome sequencing, a preparation method and an application.
Background
Rice is one of the main grain crops in China and is also one of the first protective crops covered by the new plant variety protection regulations in China. The classification mode of rice is complicated, according to the type of rice, the rice can be divided into indica type rice and japonica type rice, early season rice and middle and late rice, glutinous rice and non-glutinous rice, and how to accurately judge which type of rice the rice seedling belongs to is an important index for measuring the quality of the seedling, and the yield, the quality and the economic benefit of the rice are directly influenced, so that various levels of crop variety approval mechanisms and seed seedling production departments pay increasing attention to the identification of the counterfeit and the inferior of the rice variety, and currently, the identification is carried out by adopting SSR markers or fingerprint spectrum construction and other modes. Meanwhile, molecular markers are also widely applied to linkage map construction, gene positioning, molecular assisted breeding and the like, but because the existing SSR markers are unevenly distributed on 12 chromosomes of rice, most SSR markers only have good polymorphism selection for the combination of indica rice and japonica rice, and for the combined lines of indica rice and indica rice occupying most planting areas, the SSR markers do not have polymorphism or have extremely small difference and are difficult to distinguish.
Therefore, how to provide a polymorphic molecular marker for indica rice combination is a technical problem which needs to be solved urgently at present.
Disclosure of Invention
The application provides a polymorphic molecular marker based on whole genome sequencing, a preparation method and application thereof, which aim to solve the technical problem of lack of polymorphic molecular markers of indica rice combination in the prior art.
In a first aspect, the present application provides a polymorphic molecular marker based on whole genome sequencing, wherein the molecular marker comprises 81 sets of primer pairs, the sequences of the primer pairs are shown as SEQ ID No.1 to SEQ ID No.162, and the molecular marker is prepared from the whole genome of hybrid rice of an indica maintainer line and hybrid rice of an indica restorer line.
Optionally, the hybrid rice of the indica type maintainer line is indica rice variety wufeng B, and the hybrid rice of the indica type restorer line is kuohui 308.
In a second aspect, the present application also provides a method for preparing a polymorphic molecular marker based on whole genome sequencing, the method being used for preparing the molecular marker of the first aspect, the method comprising:
respectively obtaining indica type maintainer line hybrid rice and indica type restorer line hybrid rice;
carrying out DNA library construction and sequencing on the hybrid rice of the indica type maintainer line and the hybrid rice of the indica type restorer line to obtain original data containing the genome of the hybrid rice of the indica type maintainer line and the genome of the hybrid rice of the indica type restorer line;
quality control screening is carried out according to the original data to obtain effective data after quality control;
comparing and assembling the effective data and the reference genome to obtain a comparison file;
respectively obtaining the consistency sequence of the hybrid rice of the indica type maintainer line and the consistency sequence of the hybrid rice of the indica type restorer line according to the comparison file;
comparing the consistency sequence of the hybrid rice of the indica type maintainer line with the consistency sequence of the hybrid rice of the indica type restorer line to obtain an InDel locus;
obtaining a plurality of pairs of designed primer sequences according to each InDel locus;
searching and comparing the designed primer sequence in the indica type maintainer line hybrid rice genome and the indica type restorer line hybrid rice genome to obtain a labeled primer with high specificity;
carrying out PCR amplification and electrophoresis detection on the marker primer by taking the genome of the indica type maintainer line hybrid rice and the genome of the indica type restorer line hybrid rice as templates to obtain an electrophoresis result graph;
judging whether the labeled primer is a polymorphic molecular marker or not according to the electrophoresis result picture;
if so, the labeled primer is a polymorphic molecular marker, and the labeled primer is output as the polymorphic molecular marker.
Optionally, the obtaining a plurality of pairs of designed primer sequences according to each InDel site specifically includes:
designing primers according to the InDel sites to respectively obtain a plurality of pairs of primer sequences and corresponding product fragment lengths;
judging whether the ratio of the difference length of a plurality of pairs of primer sequences to the length of the corresponding product fragment is more than or equal to 8 percent;
if so, outputting the designed primer sequence if the designed primer is the required designed primer sequence.
Optionally, the primer design comprises: and selecting a preset number of InDel sites with different lengths at each interval of preset length.
Optionally, the preset length is 2M to 10M, and the preset number is 2 to 3.
Optionally, the difference length is 15bp to 60bp.
Optionally, primer design is performed according to the InDel locus to obtain an InDel labeled primer, and the method specifically includes:
designing a primer according to the InDel locus to respectively obtain a designed primer and the estimated length of a product fragment;
judging whether the designed primer is a required InDel labeled primer or not according to the difference length and the estimated product fragment length;
if so, outputting the InDel labeled primer if the designed primer is the required InDel labeled primer.
Optionally, the electrophoresis detection includes agarose gel detection, and the agarose gel concentration of the electrophoresis detection is 2% to 4%.
Optionally, the electrophoresis detection time is 0.5 h-1.5 h.
In a third aspect, the present application also provides the use of a polymorphic molecular marker based on whole genome sequencing, said use comprising: the molecular marker of the first aspect is used for rice genetic map construction, QTL positioning, molecular marker assisted breeding and genetic diversity analysis.
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
the polymorphic molecular marker based on whole genome sequencing provided by the embodiment of the application is characterized in that the whole genome of indica type maintainer line hybrid rice and indica type restorer line hybrid rice is taken as a template, a library is firstly constructed and sequenced to obtain original data, then quality control screening and comparison assembly are carried out on the original data to obtain comparison files, respective consistency sequences are obtained through the comparison files, comparison is carried out by utilizing the consistency sequences to obtain InDel sites, primer design is carried out according to each InDel site to obtain a plurality of pairs of designed primer sequences, then the designed primer sequences are searched and compared in respective genomes to obtain marker primers, finally the marker primers are subjected to PCR amplification and electrophoresis detection, and judgment is carried out according to the obtained electrophoresis result, so that the molecular marker with polymorphism on indica type rice and indica type rice combination can be screened, and the purpose of obtaining the polymorphic molecular marker aiming at indica type rice combination is realized.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive labor.
Fig. 1 is a schematic flow chart of a method provided in an embodiment of the present application;
FIG. 2 is a schematic flow chart illustrating a method according to an embodiment of the present disclosure;
FIG. 3 is a continuation of FIG. 2;
FIG. 4 is a schematic diagram of the electrophoresis result of the primer pair 1 provided in the embodiment of the present application;
FIG. 5 is a schematic diagram of the electrophoresis result of primer pair 2 provided in the present embodiment;
FIG. 6 is a schematic diagram showing the electrophoresis result of primer pair 3 provided in the present embodiment;
FIG. 7 is a schematic diagram showing the electrophoresis result of primer pair 4 provided in the examples of the present application;
FIG. 8 is a schematic diagram showing the electrophoresis result of primer pair 5 provided in the present embodiment;
FIG. 9 is a schematic diagram showing the electrophoresis result of the primer pair 6 provided in the embodiment of the present application;
FIG. 10 is a schematic diagram showing the electrophoresis result of the primer pair 7 provided in the embodiment of the present application;
FIG. 11 is a schematic diagram showing the electrophoresis result of the primer pair 8 provided in the embodiment of the present application;
FIG. 12 is a schematic diagram showing the electrophoresis result of the primer pair 9 provided in the embodiment of the present application;
FIG. 13 is a schematic diagram showing the electrophoresis result of the primer pair 10 provided in the embodiment of the present application;
FIG. 14 is a schematic diagram showing the electrophoresis result of the primer pair 11 provided in the embodiment of the present application;
FIG. 15 is a schematic diagram showing the electrophoresis result of the primer pair 12 provided in the embodiment of the present application;
FIG. 16 is a schematic diagram showing the electrophoresis result of the primer pair 13 provided in the embodiment of the present application;
FIG. 17 is a schematic diagram showing the electrophoresis result of the primer pair 14 provided in the embodiment of the present application;
FIG. 18 is a schematic diagram showing the electrophoresis result of the primer pair 15 provided in the example of the present application;
FIG. 19 is a diagram illustrating the electrophoresis result of the primer pair 16 provided in the embodiment of the present application;
FIG. 20 is a diagram illustrating the electrophoresis result of the primer pair 17 provided in the present embodiment;
FIG. 21 is a diagram showing the electrophoresis result of the primer pair 18 provided in the example of the present application;
FIG. 22 is a schematic diagram showing the electrophoresis result of the primer pair 19 provided in the example of the present application;
FIG. 23 is a diagram illustrating the electrophoresis result of the primer pair 20 provided in the embodiment of the present application;
FIG. 24 is a schematic diagram showing the electrophoresis result of the primer pair 21 provided in the example of the present application;
FIG. 25 is a diagram illustrating the electrophoresis results of the primer pair 22 provided in the present embodiment;
FIG. 26 is a diagram illustrating electrophoresis results of a primer pair 23 provided in an embodiment of the present application;
FIG. 27 is a schematic diagram showing the electrophoresis result of the primer pair 24 provided in the example of the present application;
FIG. 28 is a diagram showing the electrophoresis result of the primer pair 25 provided in the example of the present application;
FIG. 29 is a schematic diagram showing the electrophoresis result of the primer pair 26 provided in the example of the present application;
FIG. 30 is a diagram illustrating the electrophoresis result of the primer pair 27 provided in the present embodiment;
FIG. 31 is a diagram showing the electrophoresis result of the primer pair 28 provided in the example of the present application;
FIG. 32 is a diagram showing the electrophoresis result of the primer pair 29 provided in the example of the present application;
FIG. 33 is a diagram showing the electrophoresis result of the primer pair 30 provided in the example of the present application;
FIG. 34 is a diagram illustrating the electrophoresis result of the primer pair 31 provided in the embodiment of the present application;
FIG. 35 is a diagram showing the electrophoresis result of the primer pair 32 provided in the example of the present application;
FIG. 36 is a diagram illustrating the electrophoresis result of the primer pair 33 provided in the present embodiment;
FIG. 37 is a diagram showing the electrophoresis result of the primer pair 34 provided in the example of the present application;
FIG. 38 is a diagram showing the electrophoresis result of the primer pair 35 provided in the present embodiment;
FIG. 39 is a diagram showing the electrophoresis result of the primer pair 36 provided in the example of the present application;
FIG. 40 is a diagram illustrating the electrophoresis result of the primer pair 37 provided in the present embodiment;
FIG. 41 is a schematic diagram showing the electrophoresis result of the primer pair 38 provided in the example of the present application;
FIG. 42 is a diagram showing the electrophoresis result of the primer pair 39 provided in the present embodiment;
FIG. 43 is a schematic diagram showing the electrophoresis result of the primer pair 40 provided in the example of the present application;
FIG. 44 is a diagram showing the electrophoresis result of the primer pair 41 provided in the example of the present application;
FIG. 45 is a schematic diagram showing the electrophoresis result of the primer pair 42 provided in the embodiment of the present application;
FIG. 46 is a diagram illustrating the electrophoresis result of the primer pair 43 provided in the embodiment of the present application;
FIG. 47 is a diagram illustrating electrophoresis results of primer pair 44 provided in an embodiment of the present application;
FIG. 48 is a diagram showing the electrophoresis result of the primer pair 45 provided in the example of the present application;
FIG. 49 is a schematic diagram showing the electrophoresis result of the primer pair 46 provided in the example of the present application;
FIG. 50 is a schematic diagram showing the electrophoresis result of the primer pair 47 provided in the example of the present application;
FIG. 51 is a schematic diagram showing the electrophoresis result of the primer pair 48 provided in the example of the present application;
FIG. 52 is a diagram showing the electrophoresis result of the primer pair 49 provided in the example of the present application;
FIG. 53 is a diagram illustrating the electrophoresis result of the primer pair 50 provided in the example of the present application;
FIG. 54 is a diagram showing the electrophoresis result of the primer pair 51 provided in the example of the present application;
FIG. 55 is a diagram showing the electrophoresis result of the primer pair 52 provided in the present embodiment;
FIG. 56 is a diagram showing the electrophoresis result of the primer pair 53 provided in the example of the present application;
FIG. 57 is a schematic diagram showing the electrophoresis result of the primer pair 54 provided in the example of the present application;
FIG. 58 is a diagram showing the results of electrophoresis of the primer pair 55 provided in the examples of the present application;
FIG. 59 is a diagram showing the results of electrophoresis of the primer pair 56 provided in the examples of the present application;
FIG. 60 is a diagram illustrating the electrophoresis result of the primer pair 57 provided in the present embodiment;
FIG. 61 is a schematic diagram showing the electrophoresis result of the primer pair 58 provided in the examples of the present application;
FIG. 62 is a diagram showing electrophoresis results of a primer pair 59 provided in an embodiment of the present application;
FIG. 63 is a schematic diagram showing the electrophoresis result of the primer pair 60 provided in the example of the present application;
FIG. 64 is a diagram showing the electrophoresis result of the primer pair 61 provided in the example of the present application;
FIG. 65 is a schematic diagram showing the electrophoresis result of the primer pair 62 provided in the example of the present application;
FIG. 66 is a diagram showing the electrophoresis result of the primer pair 63 provided in the example of the present application;
FIG. 67 is a schematic diagram showing the electrophoresis result of the primer pair 64 provided in the example of the present application;
FIG. 68 is a schematic diagram showing the electrophoresis result of the primer pair 65 provided in the example of the present application;
FIG. 69 is a diagram showing the electrophoresis result of the primer pair 66 provided in the example of the present application;
FIG. 70 is a diagram showing the electrophoresis result of the primer pair 67 provided in the present embodiment;
FIG. 71 is a schematic diagram showing the electrophoresis result of the primer pair 68 provided in the example of the present application;
FIG. 72 is a diagram illustrating electrophoresis results of a primer pair 69 provided in an embodiment of the present application;
FIG. 73 is a schematic diagram showing the electrophoresis result of the primer pair 70 provided in the example of the present application;
FIG. 74 is a diagram showing the electrophoresis result of a primer pair 71 provided in the present embodiment;
FIG. 75 is a diagram showing the results of electrophoresis of the primer pair 72 provided in the example of the present application;
FIG. 76 is a diagram showing the electrophoresis result of the primer pair 73 provided in the present embodiment;
FIG. 77 is a diagram illustrating the electrophoresis results of the primer pair 74 provided in the present embodiment;
FIG. 78 is a schematic diagram showing the electrophoresis result of the primer pair 75 provided in the examples of the present application;
FIG. 79 is a schematic diagram showing the electrophoresis result of the primer pair 76 provided in the example of the present application;
FIG. 80 is a diagram showing the results of electrophoresis of a primer pair 77 provided in the examples of the present application;
FIG. 81 is a schematic diagram showing the results of electrophoresis of a primer pair 78 provided in the examples of the present application;
FIG. 82 is a diagram showing the results of electrophoresis of a primer pair 79 provided in the examples of the present application;
FIG. 83 is a diagram illustrating electrophoresis results of a primer pair 80 provided in an embodiment of the present application;
FIG. 84 is a diagram showing the results of electrophoresis of a primer pair 81 provided in the example of the present application;
in the schematic diagram of the electrophoresis result, the genomes aligned sequentially from left to right are wufeng B and guanghui 308.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making creative efforts shall fall within the protection scope of the present application.
The inventive thinking of the application is that: wuyou 308 is an indica hybrid rice combination developed by Guangdong rice institute, has high quality and high yield, and parents (Wufeng A/B and Guanghui 308) of the Wuyou hybrid rice combination have multiple excellent traits and can be used for genetic basic research and excellent trait gene excavation; based on a deep sequencing technology, the invention uses genome sequences of Wufeng B and Guanghui 308 as templates, carries out primer design aiming at base insertion/deletion (In/Del) sequences thereof, develops a set of In/Del molecular markers containing 81 pairs of primers, has good polymorphism, and can be distinguished by only 3% agarose gel electrophoresis.
In one embodiment of the present application, there is provided a whole genome sequencing-based polymorphic molecular marker, wherein the molecular marker comprises 81 sets of primer pairs, the sequences of the primer pairs are shown as SEQ ID No.1 to SEQ ID No.162, and are specifically shown in table 1, and the molecular marker is prepared from the whole genome of indica maintainer hybrid rice and indica restorer hybrid rice.
TABLE 1 molecular marker sequence case Table
Wherein m, y and w represent degenerate bases, m = a/c, y = c/t and w = a/t.
In some alternative embodiments, the hybrid rice of the indica type maintainer line is indica rice variety wufeng B and the hybrid rice of the indica type restorer line is kukuohui 308.
In the application, specific genome sequences are defined by defining specific hybrid rice of the indica type maintainer line and hybrid rice of the indica type restorer line, and further guarantee is provided for specificity of molecular markers.
In one embodiment of the present application, as shown in fig. 1, there is provided a method for preparing a polymorphic molecular marker based on whole genome sequencing, the method for preparing the molecular marker, the method comprising:
s1, obtaining indica type maintainer line hybrid rice and indica type restorer line hybrid rice respectively;
s2, carrying out DNA library construction and sequencing on the hybrid rice of the indica type maintainer line and the hybrid rice of the indica type restorer line to obtain original data containing genomes of the hybrid rice of the indica type maintainer line and the hybrid rice of the indica type restorer line;
s3, performing quality control screening according to the original data to obtain effective data after quality control;
s4, comparing and assembling the effective data and the reference genome data to obtain a comparison file;
s5, respectively obtaining a consistency sequence of the hybrid rice of the indica type maintainer line and a consistency sequence of the hybrid rice of the indica type restorer line according to the comparison file;
s6, comparing the consistency sequence of the hybrid rice of the indica type maintainer line with the consistency sequence of the hybrid rice of the indica type restorer line to obtain an InDel locus;
s7, obtaining a plurality of pairs of designed primer sequences according to each InDel locus;
s8, searching and comparing the designed primer sequence in the indica type maintainer line hybrid rice genome and the indica type restorer line hybrid rice genome to obtain a labeled primer with high specificity;
s9, carrying out PCR amplification and electrophoresis detection on the marker primer by taking the genome of the hybrid rice of the indica type maintainer line and the genome of the hybrid rice of the indica type restorer line as templates to obtain an electrophoresis result picture;
s10, judging whether the labeled primer is a polymorphic molecular marker or not according to the electrophoresis result picture;
if so, the labeled primer is a polymorphic molecular marker, and the labeled primer is output as the polymorphic molecular marker;
if not, the labeled primer is not a polymorphic molecular marker, and primer design is carried out again;
wherein the sequencing can be performed by Illumina HiseqTMAnd (3) a sequencing system, wherein the comparison file is a bam file, and the software used for designing the primers is Primer Premier 5.
In some alternative embodiments, as shown in fig. 2 and fig. 3, the obtaining multiple pairs of designed primer sequences according to each InDel site specifically includes:
s71, designing primers according to the InDel sites to respectively obtain a plurality of pairs of primer sequences and corresponding product fragment lengths;
s72, judging whether the ratio of the difference lengths of the primer sequences to the lengths of the corresponding product fragments is more than or equal to 8 percent;
if so, outputting the designed primer sequence if the designed primer is the required designed primer sequence.
In the application, whether the primer sequence accords with the expected polymorphism can be accurately determined by judging the ratio of the primer sequence to the product fragment length, and then a reasonable diversity molecular marker can be obtained on the basis E which accords with the expected polymorphism.
In some alternative embodiments, the primer design comprises: and selecting a preset number of InDel sites with different lengths at each interval of a preset length.
Further, the preset length is 2M-10M, the preset number is 2-3, and the difference length is 15 bp-60 bp.
The positive effect of the preset length of 2-10M in the application is that in the range of the preset length, the designed molecular marker can cover the original data containing the indica maintainer line hybrid rice genome and the indica restorer line hybrid rice genome, and the diversity of the obtained molecular marker is further ensured; when the length is greater than the maximum value of the end point of the range, the adverse effect is that overlong length causes partial deletion of InDel sites, the diversity of molecular markers is influenced, and when the length is less than the minimum value of the end point of the range, the adverse effect is that overlong length causes excessive generation of InDel sites, so that a primer sequence with less functionality and difference is obtained, the saving principle is not met, and the detection process is complicated.
The preset number of 2-3 has the positive effects that in the number range, the number of the selected samples can be ensured to be enough, and sufficient samples are provided for the subsequent second selection; when the value of the number is larger than the maximum value of the end point of the range, the adverse effect is that excessive preset number will cause excessive InDel sites to be generated, and further, primer sequences with less functionality and difference are obtained, so that the saving principle is not met, and the detection process is complicated, and when the value of the number is smaller than the minimum value of the end point of the range, the adverse effect is that too few sample number will cause too few InDel sites to be obtained, so that the second selection is not facilitated.
The difference length of 15 bp-60 bp has the positive effects that in the length range, the number of InDel sites can be effectively controlled, the length of the primer is suitable, and the accuracy of molecular marking is ensured; when the length value is greater than the maximum value of the end point of the range, the accuracy of the molecular marker is reduced due to the overlong difference length, meanwhile, the InDel sites are too few due to the overlong difference length, which is not beneficial to the second selection, and when the length value is less than the minimum value of the end point of the range, the length of the molecular marker is too small, and meanwhile, the excessive InDel sites are generated, so that a primer sequence with less functionality and difference is obtained, which is not in accordance with the saving principle and leads to the complex detection process.
In some alternative embodiments, the electrophoretic detection comprises an agarose gel detection, wherein the agarose gel concentration of the electrophoretic detection is 2% to 4%.
In the application, the positive effect that the agarose gel concentration detected by electrophoresis is 2% -4% is that within the concentration range, the electrophoresis result can be accurately obtained; when the value of the concentration is larger than the maximum value of the end point of the range, the adverse effect is that excessive concentration causes raw material waste, the saving principle is not met, and when the value of the concentration is smaller than the minimum value of the end point of the range, the adverse effect is that the electrophoretic result is fuzzy due to too low concentration, the resolution is not facilitated, and the judgment process of the molecular marker is influenced.
Furthermore, the time for electrophoresis detection is 0.5 h-1.5 h.
In the application, the positive effect that the time for electrophoresis detection is 0.5 h-1.5 h is that in the time range, the amplified product can be ensured to be fully fixed and separated, so that an accurate result is obtained; when the value of the time is greater than the maximum value of the end point of the range, the result is adversely affected that the overall detection time is too long due to too long time, the result is accurately judged, and when the value of the time is less than the minimum value of the end point of the range, the result is adversely affected that the result is not completely fixed due to too short time, and the result accuracy is affected.
In one embodiment, the present application also provides the use of polymorphic molecular markers based on whole genome sequencing, comprising: the molecular marker is used for rice genetic map construction, QTL positioning, molecular marker assisted breeding and genetic diversity analysis.
Example 1
The polymorphism molecular marker based on whole genome sequencing comprises 81 groups of primer pairs, the sequences of the primer pairs are shown as SEQ ID NO. 1-SEQ ID NO.162, wherein the molecular marker is prepared from the whole genomes of indica rice variety Wufeng B and Guanghui 308.
As shown in fig. 1, 2 and 3, there is provided a method for preparing a polymorphic molecular marker based on whole genome sequencing, for preparing a molecular marker, comprising:
s1, obtaining indica type maintainer line hybrid rice and indica type restorer line hybrid rice respectively;
s2, carrying out DNA library construction and sequencing on the hybrid rice of the indica type maintainer line and the hybrid rice of the indica type restorer line to obtain original data containing the genome of the hybrid rice of the indica type maintainer line and the genome of the hybrid rice of the indica type restorer line;
s3, screening quality control according to the original data to obtain effective data after quality control;
s4, assembling the effective data and the reference genome data to obtain a comparison file;
s5, respectively obtaining a consistency sequence of the hybrid rice of the indica type maintainer line and a consistency sequence of the hybrid rice of the indica type restorer line according to the comparison file;
s6, comparing the consistency sequence of the hybrid rice of the indica type maintainer line with the consistency sequence of the hybrid rice of the indica type restorer line to obtain an InDel locus;
s71, designing primers according to the InDel locus to respectively obtain a plurality of pairs of primer sequences and corresponding product fragment lengths;
s72, judging whether the ratio of the difference length of the primer sequences to the length of the corresponding product fragment is more than or equal to 8 percent;
if so, designing the primer as a required designed primer sequence, and outputting the designed primer sequence;
if not, the primer sequence is not the required designed primer sequence, and then the primer design is carried out again;
s8, searching and comparing the designed primer sequences in the indica type maintainer line hybrid rice genome and the indica type restorer line hybrid rice genome to obtain a labeled primer with high specificity;
s9, carrying out PCR amplification and electrophoresis detection on the labeled primer by taking the genome of the indica maintainer line hybrid rice and the genome of the indica restorer line hybrid rice as templates to obtain electrophoresis result graphs shown in the figures 3 to 83;
s10, judging whether the labeled primer is a polymorphic molecular marker or not according to the electrophoresis result picture;
if yes, the labeled primer is a polymorphic molecular marker, and the labeled primer is output as the polymorphic molecular marker;
if not, the labeled primer is not the polymorphic molecular marker, and primer design is carried out again;
wherein the first sequencing may be by Illumina HiseqTMThe sequencing system is used for sequencing, the comparison file is a bam file, and the software used for Primer design is Primer Premier 5.
The first selection comprises: and selecting a preset number of InDel sites with different lengths at each interval of preset length.
The preset length is 2M-10M, the preset number is 2, and the difference length is 15 bp-60 bp.
The electrophoresis detection comprises agarose gel detection, and the agarose gel concentration of the electrophoresis detection is 3%.
The time of electrophoresis detection is 0.5 h-1.5 h.
The system used for PCR amplification is shown in Table 2, and the PCR amplification procedure is as follows: 3min at 94 ℃;94 ℃ 20s,55 ℃ 20s,72 ℃ 40s and 35 cycles; 5min at 72 ℃; 2min at 20 ℃.
TABLE 2 PCR amplification System
| Total volume of the system | 20μL |
| Genomic DNA | 0.5μg |
| 10×PCR buffer(2.0mM MgCl2) | 2.0μL |
| dNTPs(2.5mM) | 2.0μL |
| Forward primer (10. Mu.M) | 1.0μL |
| Reverse primer (10. Mu.M) | 1.0μL |
| Taq DNA polymerase (2.5 Units/. Mu.L) | 0.5μL |
| ddH2O | 11.5μL |
Example 2
Comparing example 2 with example 1, example 2 differs from example 1 in that:
the preset number is 2.
The agarose gel concentration detected by electrophoresis is 2%, and the time for detection by electrophoresis is 0.5h.
Example 3
Example 3 was compared with example 1, and example 3 differs from example 1 in that:
the preset number is 3.
The agarose gel concentration detected by electrophoresis is 4%, and the time for detection by electrophoresis is 1.5h.
Related experiments:
the molecular markers obtained in example 1 were collected and used to detect the whole genomes of indica rice varieties wufeng B and kuohui 308, respectively, and the results are shown in table 3;
test methods of the related experiments:
the difference rate: if the two sequences have InDel sites, the length of the InDel sites is 15 bp-60 bp, the ratio of the length of the insertion or deletion to the length of the corresponding amplification product is the difference rate, and when the difference rate is more than or equal to 8%, the difference can be shown in the electrophoresis detection, namely the polymorphism is shown.
TABLE 3
Specific analysis of table 3:
as can be seen from the data in table 3, the 81 molecular marker primer pairs provided in the examples of the present application have good polymorphisms in the genomic DNA of indica rice cultivar wufeng B and kuohui 308.
One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:
(1) According to the molecular marker provided by the embodiment of the application, the whole genome of the indica maintainer line hybrid rice and the indica restorer line hybrid rice is used as a template, and a plurality of groups of molecular markers are designed at intervals of 2-10M base specificity, so that the obtained molecular markers have high specificity and are uniformly distributed on 12 rice chromosomes.
(2) According to the method provided by the embodiment of the application, whether the primer sequence meets the expected specificity can be accurately determined by introducing the standard difference degree and the estimated standard ratio of the product fragment length, and the reasonable polymorphic molecular marker can be obtained on the basis of meeting the expected specificity.
(3) The method provided by the embodiment of the application can screen out the molecular marker with specificity from a plurality of groups of designed primer pairs through two-step screening, embodies the specificity of the molecular marker and simultaneously can ensure the polymorphism of the molecular marker.
(4) According to the application provided by the embodiment of the application, the obtained molecular marker is used for rice genetic map construction, QTL positioning, molecular marker assisted breeding and genetic diversity analysis, so that a proper molecular marker can be screened from multiple molecular markers according to different application scenes, and the universality of the molecular marker is embodied.
It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrases "comprising a," "8230," "8230," or "comprising" does not exclude the presence of additional like elements in a process, method, article, or apparatus that comprises the element.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Rice research institute of Guangdong province academy of agricultural sciences
<120> polymorphic molecular marker based on whole genome sequencing, preparation method and application
<160> 162
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agccaaagca atagaag 17
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaccaatcag agcccac 17
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
actccatccg tccttaag 18
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
acatactctt cccgtctc 18
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgtagattaa tgtggtcc 18
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gtactccatg tatgtttc 18
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctcggttgcc ctcactctc 19
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cccacctatt tagatccttg 20
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atcccacaag tagacatc 18
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aggaagggaa gaaggttg 18
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tggcactaac tttatcac 18
<210> 12
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aatccatctg cttctatc 18
<210> 13
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atctatgagc cagattac 18
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cattgtggga gaagtgtg 18
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agtgaagggt atttcttgta 20
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
actaaaccat tggctctg 18
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
actgtcctcc aatcaaaccg 20
<210> 18
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tacctccatc ctgacaac 18
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ctaaagacta tgccagtatc 20
<210> 20
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tcagcagtgt tcgctagg 18
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ttatttaggg tgaatttccg 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tcgaatggag gtttcagatc 20
<210> 23
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgtcacttac gagcgacg 18
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ggttctattg ctacgatg 18
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gtttggtaat ttgtactttg 20
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
actggagcag aacaacgtg 19
<210> 27
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tttaggacac ccgcaatg 18
<210> 28
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gcaggtagga acgagaag 18
<210> 29
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
atagtatagc ccgctgag 18
<210> 30
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
atcctggtgt ttggtgaa 18
<210> 31
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgcccgttca catccatc 18
<210> 32
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tcatcaccat tccaccaa 18
<210> 33
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
aacaatgtga cggaaaag 18
<210> 34
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
agacaagtaa tatgggac 18
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gcgaattagt ccaatgttat g 21
<210> 36
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tacatcgggc ctactgttg 19
<210> 37
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tacctctaag gctcaagac 19
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tcataagaac actgcgagtc 20
<210> 39
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tagcaacacg cgaactac 18
<210> 40
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tacgaaccag gaggaatg 18
<210> 41
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cagcgaaggt gaatagac 18
<210> 42
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gacaatgtga tgctacag 18
<210> 43
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tatctacttc atgccaagc 19
<210> 44
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tacctccatc ctgacaac 18
<210> 45
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
agtatggtgg actcgatg 18
<210> 46
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
cctctttgcc gatctcag 18
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
tgaggagatt gttgggctag 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
cgcgcgaaag gaaaggatat 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
acagtacaga attcgtgttg 20
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
atcagtcctg tccattatag 20
<210> 51
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ctcgtactgc tccacttg 18
<210> 52
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tttggtttct catcgtta 18
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
cgaaatgctc ttgagtcttg 20
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gggaacagtc agatttatgc 20
<210> 55
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gaacccgatm tgacaaat 18
<210> 56
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gtgtagcgtg tttagcag 18
<210> 57
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tctttggagc gtttgtag 18
<210> 58
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cgtcgtacat ttacgtgc 18
<210> 59
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
gcaaaatctt ctgacccc 18
<210> 60
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ggagtatcgt gcgtggtg 18
<210> 61
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
tggaatactt gctcctac 18
<210> 62
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gtgtcactca taccgaac 18
<210> 63
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
ttcggagctg gtgttattgg 20
<210> 64
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
acactacagg ctggatgg 18
<210> 65
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
tctagtcgca gtgtcttg 18
<210> 66
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
cggtttgttg ttgtgttg 18
<210> 67
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
cttaaatcgg gcaggaaatc 20
<210> 68
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
tattgattgg actcagcagc 20
<210> 69
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ccacatcata aagcacag 18
<210> 70
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
tactacagta ctacttcctc 20
<210> 71
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ttcagacttc agaggatacc 20
<210> 72
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
gagtgggacg aacacctg 18
<210> 73
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gagtgggacg aacacctg 18
<210> 74
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
ctgcctccct tcttattatc 20
<210> 75
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
gtgctacgag tatgactg 18
<210> 76
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
acaccaaggt aggatgag 18
<210> 77
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
ttgtgcctct gtaggtgttg 20
<210> 78
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
aagtgatgtc ttcatcattg 20
<210> 79
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ttgcccttct ttacgatatg 20
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
gtgctaaccc aaacttcatc 20
<210> 81
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
tttcagaaag akatttggag 20
<210> 82
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
atccttgcta gtgatggc 18
<210> 83
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
atggctttgg ctgatagc 18
<210> 84
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
attggggaga cgcatttg 18
<210> 85
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
atcctgccac tgttgaag 18
<210> 86
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gcagcgagaa agcgaaag 18
<210> 87
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
caaatggagc agaagatg 18
<210> 88
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
aagaggtagt tgtcgagg 18
<210> 89
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
aaatagmatc aaggaaaacc 20
<210> 90
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
atgcggayaa tgcatgaaag 20
<210> 91
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cacaactagt ctaaacttag 20
<210> 92
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gcattttgag ttatccggag 20
<210> 93
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
atcagcaacc gggaagaaag 20
<210> 94
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
gagcacaaat gagatacaag 20
<210> 95
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
aaggttggat gatgaatctg 20
<210> 96
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
aggggcggat aaaagagag 19
<210> 97
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
cggggaagca tcttaatatc 20
<210> 98
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tcagtttgga ctgtgattag 20
<210> 99
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
ccgagatgcc attaaacc 18
<210> 100
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
tttcagcctt gttcgtca 18
<210> 101
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
gtaataactt catctgcgtc 20
<210> 102
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
atcaatcact caaggaaaac 20
<210> 103
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
ttagtatcat ttcggtttgg 20
<210> 104
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
tgcactacct tctcctttcg 20
<210> 105
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
caccgtgtct cctctttg 18
<210> 106
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
caatcagatg tgatagggtg 20
<210> 107
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
tcgacccata cactcttg 18
<210> 108
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ggactcttgc tcccactc 18
<210> 109
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
aaagtaatac ggtgccaa 18
<210> 110
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
atagtatcca atcggtgagg 20
<210> 111
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
tgaaactcac ccgtcacc 18
<210> 112
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
ctcgcaacat cccactat 18
<210> 113
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
tctcgcacta tgctgctc 18
<210> 114
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
ctattacgtg atgtttag 18
<210> 115
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
tgctgcaacg cctaatgtg 19
<210> 116
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
ctgagctgag tggacga 17
<210> 117
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
acacggcttt aggtaggg 18
<210> 118
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
tttcgacgca cttgtttg 18
<210> 119
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
agataaggca tttcagtact 20
<210> 120
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
aatgtaagcc tctgtagcag 20
<210> 121
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
tcatggcagg aacgaatg 18
<210> 122
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
gttggtgatt gggaattg 18
<210> 123
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
cctaagcgtt ctatgatttg 20
<210> 124
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ttaggctctg tttatttcgc 20
<210> 125
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
acactccacc actcacttc 19
<210> 126
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
tgcagcatct gctaaaaac 19
<210> 127
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
gatccaaaat aaagcaac 18
<210> 128
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
cgcatagagt acaagaag 18
<210> 129
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
aagggtattg gtggatt 17
<210> 130
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
tgcacgtaag ctgttag 17
<210> 131
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
tagcacattg gcacatcg 18
<210> 132
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
ctccctccgt ctaggaac 18
<210> 133
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
gtcttgttgc tgctgatg 18
<210> 134
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
agcycaatcc cttccaaa 18
<210> 135
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
aatggcacga caaggctgg 19
<210> 136
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
ttggtgcgtg cgaataagg 19
<210> 137
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
tggtttacgg agcacaac 18
<210> 138
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
tgacgaagga aggtgaac 18
<210> 139
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
aactaaaacg gttgccag 18
<210> 140
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
gaagaccagc tcagtaac 18
<210> 141
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
tggtaggttg tgcggttg 18
<210> 142
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gagggataga ctcaaagaa 19
<210> 143
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
aacaatacca gggatagg 18
<210> 144
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
tgtaccgact tcttcgtc 18
<210> 145
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
cctaatgctg cttgcttg 18
<210> 146
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
ctcaaaagat tcgtctcg 18
<210> 147
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
gtgggattca tgttcatc 18
<210> 148
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
ccaagcaaag tatgtaag 18
<210> 149
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
ggtgagtgag aacgcata 18
<210> 150
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
gaagctgtgc caaaacag 18
<210> 151
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
agtggwtaga tttcggatac 20
<210> 152
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
ctcattagca tcacggttag 20
<210> 153
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
ttttcccgct cctgaatg 18
<210> 154
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
cttcgagggt tatacctg 18
<210> 155
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
tccacccagc actactcc 18
<210> 156
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
tagccaagtt gtcctacc 18
<210> 157
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
acaatcgacg tagttagg 18
<210> 158
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
tggaatcgga atcagtta 18
<210> 159
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
aaacccaatg ctacaaac 18
<210> 160
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
gagtatcatc aaaggcac 18
<210> 161
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
atgtcttcct caattccc 18
<210> 162
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
gcattgtatt accaactgc 19
Claims (2)
1. The polymorphic molecular marker based on whole genome sequencing is characterized by comprising 81 sets of primer pairs, wherein the sequences of the primer pairs are shown as SEQ ID No. 1-SEQ ID No.162, the molecular marker is prepared from whole genomes of indica maintainer line hybrid rice and indica restorer line hybrid rice, wherein the indica maintainer line hybrid rice is indica maintainer line Wufeng B, and the indica restorer line hybrid rice is Guanghui 308.
2. Use of a polymorphic molecular marker based on whole genome sequencing, wherein said use comprises: the molecular marker of claim 1 is used in rice genetic map construction, QTL positioning, molecular marker assisted breeding and genetic diversity analysis.
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| CN114622035B (en) * | 2022-04-29 | 2023-05-26 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato whole genome high-flux specific InDel molecular marker primer group and application thereof |
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| CN101323878A (en) * | 2008-05-08 | 2008-12-17 | 复旦大学 | Method for identifying indica rice and japonica rice by inserting or deleting molecular marker in rice DNA |
| CN103725768A (en) * | 2012-10-15 | 2014-04-16 | 丁筱玲 | Research method for rice genomics |
| CN108441574A (en) * | 2018-04-08 | 2018-08-24 | 中国科学院遗传与发育生物学研究所 | A kind of gene finely positioning method being sequenced for Plant Genome based on map based cloning principle and segregating population is inter-subspecies hybrid |
| CN110029187A (en) * | 2019-04-29 | 2019-07-19 | 华南农业大学 | A kind of application for marking the method for map based on competitive equipotential PCR building rice molecular and it being utilized to carry out breeding |
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| CN114350841A (en) * | 2022-01-13 | 2022-04-15 | 广东省农业科学院水稻研究所 | Polymorphic molecular marker based on whole genome sequencing, preparation method and application |
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- 2022-05-23 ZA ZA2022/05632A patent/ZA202205632B/en unknown
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| CN101323878A (en) * | 2008-05-08 | 2008-12-17 | 复旦大学 | Method for identifying indica rice and japonica rice by inserting or deleting molecular marker in rice DNA |
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| ZA202205632B (en) | 2022-09-28 |
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