CN108384886B - Detection primer group and kit for alfalfa bean SSR marker, application of detection primer group and kit and screening method for alfalfa bean self-compatibility genotype - Google Patents
Detection primer group and kit for alfalfa bean SSR marker, application of detection primer group and kit and screening method for alfalfa bean self-compatibility genotype Download PDFInfo
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Abstract
The invention provides a detection primer group and a kit for an SSR (simple sequence repeat) marker of Melissitus ruthenicus, application of the detection primer group and the kit containing the primer group, and a screening method for self-compatibility genotype of Melissitus ruthenicus, and relates to the technical field of biology. In the method for screening the alfalfa bean self-compatible genotype by using the primer group, the female parent of the self-compatible genotype can be found by comparing the female parent genotype with the progeny genotype to obtain the selfing rate of the female parent, so that the technical problem that the method for accurately and quickly screening the alfalfa bean self-compatible genotype is lacked in the prior art is solved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection primer group and a kit for SSR (simple sequence repeat) markers of alfalfa beans, application of the detection primer group and the kit, and a screening method for self-compatibility genotypes of alfalfa beans.
Background
Melissitus ruthenicus is a perennial legume, and is distributed in northern areas of China. It is cold-resistant, drought-resistant and strong in adaptability, and is an important rare gene resource. The research, the transformation and the utilization of the Melissitus ruthenicus seeds have practical production significance for improving the production capacity of grassland and promoting the development of animal husbandry.
Melissitus ruthenicus is an perennial cross-pollinated plant. At present, the breeding of the variety of Melissitus ruthenicus seeds is usually carried out by a recurrent breeding method. Compared with recurrent selective breeding, the crossbreeding method has the advantages of rapid aggregation of target characters, short breeding period and the like. In the breeding of Melissitus ruthenicus, the inbred line and cytoplasmic male sterile line are lacked, and the improvement of target characters by the crossbreeding method is limited.
With the development of biotechnology, molecular markers are widely applied to molecular breeding and genetic analysis, but no method for screening the female parent of a single plant of Melissitus ruthenicus with an compatible self-bred genotype by using a molecular breeding method exists at present.
Therefore, there is a need for a method for effectively screening alfalfa bean self-compatible genotypes using molecular breeding.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a detection primer group for alfalfa bean SSR markers, which alleviates the problem that a primer group capable of screening alfalfa bean self-compatibility genotype molecular markers through amplification is lacked in the prior art; the second object of the present invention is to provide a kit comprising the above-mentioned detection primer set; the technical problem that a kit capable of detecting the self-compatible genotype of the alfalfa bean is lacked in the prior art is solved; the third purpose of the invention is to provide the application of the detection primer group and the kit in screening the self-compatible genotype of the alfalfa bean, which can be widely applied to screening the self-compatible genotype of the alfalfa bean, self-bred progeny and hybrid progeny; the fourth purpose of the invention is to provide a method for screening self-compatible genotype of Melissitus ruthenicus seeds, which alleviates the problem of the prior art that a method capable of effectively screening self-compatible genotype of Melissitus ruthenicus seeds is lacked.
In order to solve the technical problems, the invention adopts the following technical scheme:
a detection primer set for Melissitus ruthenicus SSR markers, which comprises at least two primer pairs of the following primer pairs:
primer pair MrE-SSR 190: comprises MrE-SSR190-F and MrE-SSR190-R, wherein MrE-SSR190-F has a sequence shown as SEQ ID NO.1, and MrE-SSR190-R has a sequence shown as SEQ ID NO. 2;
primer pair MrE-SSR-578: comprises MrE-SSR-578-F and MrE-SSR-578-R, wherein MrE-SSR-578-F has a sequence shown as SEQ ID NO.3, and MrE-SSR-578-R has a sequence shown as SEQ ID NO. 4;
primer pair MrE-SSR 137: comprises MrE-SSR137-F and MrE-SSR137-R, wherein MrE-SSR137-F has a sequence shown as SEQ ID NO.5, and MrE-SSR137-R has a sequence shown as SEQ ID NO. 6;
primer pair MrE-SSR 492: comprises MrE-SSR492-F and MrE-SSR492-R, wherein MrE-SSR492-F has a sequence shown as SEQ ID NO.7, MrE-SSR492-R has a sequence shown as SEQ ID NO. 8;
primer pair MrE-SSR-669: comprises MrE-SSR-669-F and MrE-SSR-669-R, wherein MrE-SSR-669-F has a sequence shown as SEQ ID NO.9, and MrE-SSR-669-R has a sequence shown as SEQ ID NO. 10;
primer pair MrE-SSR 200: comprises MrE-SSR200-F and MrE-SSR200-R, wherein MrE-SSR200-F has a sequence shown as SEQ ID NO.11, and MrE-SSR200-R has a sequence shown as SEQ ID NO. 12;
primer pair MrE-SSR-534: comprises MrE-SSR-534-F and MrE-SSR-534-R, wherein MrE-SSR-534-F has a sequence shown as SEQ ID NO.13, and MrE-SSR-534-R has a sequence shown as SEQ ID NO. 14;
primer pair MrE-SSR-650: comprises MrE-SSR-650-F and MrE-SSR-650-R, wherein MrE-SSR-650-F has a sequence shown in SEQ ID NO.15, and MrE-SSR-650-R has a sequence shown in SEQ ID NO. 16;
primer pair MrE-SSR-676: comprises MrE-SSR-676-F and MrE-SSR-676-R, wherein MrE-SSR-676-F has a sequence shown in SEQ ID NO.17, and MrE-SSR-676-R has a sequence shown in SEQ ID NO. 18;
primer pair MrE-SSR 143: comprises MrE-SSR143-F and MrE-SSR143-R, wherein MrE-SSR143-F has a sequence shown as SEQ ID NO.19, MrE-SSR143-R has a sequence shown as SEQ ID NO. 20;
primer pair MrE-SSR 140: comprises MrE-SSR140-F and MrE-SSR140-R, wherein MrE-SSR140-F has a sequence shown as SEQ ID NO.21, MrE-SSR140-R has a sequence shown as SEQ ID NO. 22;
primer pair MrE-SSR-614: comprises MrE-SSR-614-F and MrE-SSR-614-R, wherein MrE-SSR-614-F has a sequence shown as SEQ ID NO.23, and MrE-SSR-614-R has a sequence shown as SEQ ID NO. 24;
primer pair MrE-SSR 256: comprises MrE-SSR256-F and MrE-SSR256-R, wherein MrE-SSR256-F has a sequence shown as SEQ ID NO.25, and MrE-SSR256-R has a sequence shown as SEQ ID NO. 26;
primer pair MrE-SSR 101: comprises MrE-SSR101-F and MrE-SSR101-R, wherein MrE-SSR101-F has a sequence shown as SEQ ID NO.27, and MrE-SSR101-R has a sequence shown as SEQ ID NO. 28;
primer pair MrE-SSR 539: comprises MrE-SSR539-F and MrE-SSR539-R, wherein MrE-SSR539-F has the sequence shown in SEQ ID NO.29, MrE-SSR539-R has the sequence shown in SEQ ID NO. 30;
primer pair MrE-SSR 727: comprises MrE-SSR727-F and MrE-SSR727-R, wherein MrE-SSR727-F has a sequence shown as SEQ ID NO.31, and MrE-SSR727-R has a sequence shown as SEQ ID NO. 32.
A kit for SSR marker detection of Melissitus ruthenicus seeds comprises the detection primer group.
An application of the detection primer group or the kit in screening alfalfa bean self-compatible genotypes.
A screening method of self-compatible genotype of Melissitus ruthenicus seeds, the screening method comprising amplifying DNA of a female parent of a Melissitus ruthenicus seed single plant and progeny of the female parent of the Melissitus ruthenicus seed single plant, screening the self-compatible genotype by comparing a band type of an amplified product after electrophoresis to obtain a distribution of types of the progeny of the female parent of the Melissitus ruthenicus seed single plant;
the amplification is to amplify the DNA of the alfalfa plant female parent and the progeny of the alfalfa plant female parent by using the detection primer set or the kit.
Further, the number of the filial generation of the single plant of Melissitus ruthenicus seeds is 25-30.
Further, the types of progeny include hybrid progeny and selfed progeny.
Further, in the amplification products of at least any six groups of primer pairs, the band type of the bands of the self-bred filial generation is completely identical to that of the bands of the female parent; or the like, or, alternatively,
the bands of the female parent comprise two bands, one of the bands of the selfed progeny being the same as one of the bands of the female parent.
Further, in the amplification products of at least any two sets of primer pairs, one of the bands of the filial generation is the same as one of the bands of the female parent, and the other band is different from any one of the bands of the female parent.
Further, the amplification comprises multiplex PCR amplification, and the reaction of the multiplex PCR amplification at least comprises two groups of primer pairs;
preferably, primer pair MrE-SSR190 and primer pair MrE-SSR-578 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR137 and primer pair MrE-SSR492 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR-669 and primer pair MrE-SSR200 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR-534 and primer pair MrE-SSR-650 are subjected to multiplex PCR amplification in the same PCR system;
preferably, the primer pair MrE-SSR-676 and the primer pair MrE-SSR143 are subjected to multiple PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR140 and primer pair MrE-SSR-614 perform multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR256 and primer pair MrE-SSR101 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR-539 and primer pair MrE-SSR-727 are subjected to multiplex PCR amplification within the same PCR system.
Further, the electrophoresis is 5% -10% polyacrylamide gel electrophoresis, and preferably 6% polyacrylamide gel electrophoresis.
Compared with the prior art, the invention has the following beneficial effects:
the SSR marker detection primer group for Melissitus ruthenicus seeds provided by the invention can specifically amplify SSR markers of Melissitus ruthenicus seeds respectively, and the SSR markers of the filial generation of the female parent and the female parent can be used for comparing the difference and identity of SSR marker bands of filial generation and the female parent respectively by using the detection primer group, so that the filial generation is judged to be filial generation or self-bred filial generation. The kit containing the detection primer group provided by the invention has the advantages of convenience and rapidness in use, and the SSR markers of the sample can be detected by amplifying the DNA of the sample through PCR by using the detection primer group contained in the kit. The detection primer group and the kit containing the detection primer group provided by the invention are wide in application, and can be used for screening the alfalfa bean female parent with self-compatible genotype and also can be used for screening the self-bred progeny or hybrid progeny of the alfalfa bean.
The invention provides a molecular marking method for screening self-compatible genotypes of Melissitus ruthenicus seeds. The method comprises the steps of amplifying DNA of a single plant female parent of Melissitus ruthenicus and progeny of the single plant female parent of Melissitus ruthenicus by using the detection primer group or the kit, obtaining distribution of types of the progeny of the single plant female parent of Melissitus ruthenicus by comparing band types of amplified products after electrophoresis, and screening the self-compatible genotype. Through screening of a large amount of Melissitus ruthenicus seeds, a genotype with high selfing rate can be discovered. The method provided by the invention is used for screening the self-compatible genotype of the alfalfa bean, can improve the screening accuracy and efficiency of the self-compatible genotype and lays a foundation for cultivating the alfalfa bean inbred line.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an electrophoretogram of DNA amplification products of female and progeny of Melissitus ruthenicus seeds provided in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings and embodiments, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a detection primer group for Melissitus ruthenicus SSR markers, which comprises at least two groups of primer pairs in the following table:
SSR (simple sequence repeat), also called microsatellite DNA (Microsolalite), is a series repeat sequence of dozens of nucleotides with 2-5 nucleotides as repeat units, widely distributed on different loci of the whole eukaryotic genome, and the number of the repeat units of each locus may not be completely the same, thus forming polymorphism, namely SSR molecular marker. Since most of the two ends of the SSR marker are relatively conserved single copy sequences, PCR amplification can be carried out by designing specific primers, and the SSR marker usually shows codominance.
The detection primer group for the alfalfa bean SSR marker provided by the invention can specifically amplify the SSR marker of alfalfa bean, and the SSR markers of the female parent and the progeny of the female parent can be respectively amplified by using the detection primer group to compare the similarities and differences of the progeny and the SSR markers of the female parent, so that the progeny is judged to be filial generations or self-bred progeny.
The invention also provides a kit for detecting the SSR marker of Melissitus ruthenicus seeds, which comprises the detection primer group. The kit containing the detection primer group provided by the invention has the advantages of convenience and rapidness in use, and the SSR markers of the sample can be detected by amplifying the DNA of the sample through PCR by using the detection primer group contained in the kit. In some alternative embodiments, the kit comprises a lyophilized powder of the detection primer set, or is dissolved in a buffer acceptable in any molecular biological assay; in some alternative embodiments, the kit comprises pre-mixed sets of primer pairs for use in multiplex PCR; optionally, some reagents acceptable in molecular biology experiments, such as but not limited to PCR buffer, dNTP, magnesium ions, or fluorescent dye for Q-PCR, can be included in the kit.
The invention also provides application of the detection primer group and the kit in screening of self-compatible genotypes of Melissitus ruthenicus seeds. Can be used for screening the alfalfa bean female parent with self-compatible genotype and can also be used for screening the self-bred progeny or hybrid progeny of the alfalfa bean.
The invention also provides a screening method of the self-compatibility genotype of the Melissitus ruthenicus seeds, which comprises the steps of amplifying DNA of a female parent of the Melissitus ruthenicus seeds and progeny of the female parent of the Melissitus ruthenicus seeds, obtaining distribution of types of the progeny of the female parent of the Melissitus ruthenicus seeds by comparing band types of amplified products after electrophoresis, and screening the self-compatibility genotype;
wherein the DNA of the female parent of the single plant of Melissitus ruthenicus and the DNA of the progeny of the female parent of Melissitus ruthenicus are amplified by using the detection primer set or the kit.
The method provided by the invention is used for screening the self-compatible genotype of the alfalfa bean, can improve the screening accuracy and efficiency of the self-compatible genotype and lays a foundation for cultivating the alfalfa bean inbred line.
In some alternative embodiments of the invention, the number of progeny that amplify the maternal plant of Melissitus ruthenicus is 25-30. When the screening method is implemented, 25-30 progeny of the single plant female parent can be selected for detection of each single plant female parent, and too small amount of progeny sample can reduce the screening accuracy and too large amount of progeny sample can increase the screening workload, so that the number of progeny is preferably 25-30.
In some alternative embodiments of the invention, the types of progeny include hybrid progeny and self-bred progeny. Alternatively, when the screening method is implemented, all self-bred progeny of a single plant of Melissitus ruthenicus can be selected, and whether the single plant of Melissitus ruthenicus is a self-compatible genotype or not can be judged according to the number ratio of the self-bred progeny in all the progeny; or selecting all filial generations of the female parent of the Melissitus ruthenicus seeds, and judging whether the Melissitus ruthenicus seeds female parent is of the self-compatible genotype according to the quantity ratio of the filial generations in all the filial generations.
In some alternative embodiments of the present invention, the selfing rate may be used to determine whether the single plant of alfalfa bean mother is of the self-compatible genotype, optionally, the selfing rate is calculated by formula (a) or formula (b):
formula (a): the selfing rate is the number of selfed filial generation/total number of filial generation multiplied by 100%;
formula (b): the selfing rate is (total number of filial generation-number of filial generation of hybrid)/total number of filial generation x 100%.
In the actual screening process, the self-compatibility genotype self-crossing rate range is divided according to the breeding purpose and the actual production requirement.
The detection primer group is located in the conservation regions at two ends of the SSR marker, and the repeating units in the SSR marker are variable regions, so that when one group of primer groups is used for amplifying the same SSR marker of the female parent and the filial generation thereof, the length of the amplified product is different due to different numbers of the repeating units in the variable regions, and the filial generation can be judged to be self-bred filial generation or hybrid filial generation by comparing the difference of the amplified products of the same SSR marker in the female parent and the filial generation.
In an alternative embodiment, in the amplification product of at least any six sets of primer pairs, the band pattern of the bands of the selfed progeny is identical to the band pattern of the bands of the female parent, or the bands of the female parent comprises two bands, one band of the bands of the selfed progeny being identical to one band of the bands of the female parent.
In an alternative embodiment, in the amplification products of at least any two primer pairs, one of the bands of the progeny of the cross is identical to one of the bands of the female parent, and the other band is not identical to any band of the female parent.
In some alternative embodiments of the invention, the amplification is amplified using a multiplex PCR method, wherein the reaction of the multiplex PCR amplification comprises at least two sets of primer pairs. By using multiple PCR, the screening workload can be reduced and the screening efficiency can be improved.
Preferably, primer pair MrE-SSR190 and primer pair MrE-SSR-578 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR137 and primer pair MrE-SSR492 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR-669 and primer pair MrE-SSR200 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR-534 and primer pair MrE-SSR-650 are subjected to multiplex PCR amplification in the same PCR system;
preferably, the primer pair MrE-SSR-676 and the primer pair MrE-SSR143 are subjected to multiple PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR140 and primer pair MrE-SSR-614 perform multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR256 and primer pair MrE-SSR101 are subjected to multiplex PCR amplification in the same PCR system;
preferably, primer pair MrE-SSR-539 and primer pair MrE-SSR-727 are subjected to multiplex PCR amplification within the same PCR system.
The reaction efficiency of PCR can be improved by optimizing the multiple PCR because of no selection, thereby improving the efficiency of the whole screening method.
In some alternative embodiments, the amplification products may be separated using high concentration agarose gel electrophoresis or polyacrylamide gel electrophoresis, preferably 5% to 10% polyacrylamide gel electrophoresis, preferably 6% polyacrylamide gel electrophoresis. The gel concentration is optimized to enable the band after electrophoresis to be clearer, and the contrast and the screening of the female parent and the filial generation are facilitated.
The following examples are provided to further illustrate the advantageous effects of the present invention.
Example 1
And (3) carrying out selfing rate detection on the single plant female parent of the alfalfa bean, the number M1 and the progeny (P1-P25) of the single plant female parent of the alfalfa bean by utilizing the SSR marker detection primer group. And performing multiplex PCR to amplify the DNA of the female parent and the progeny of the Melissitus ruthenicus seeds by using 12 sets of primer pairs.
Wherein, primer pairs MrE-SSR190, primer pairs MrE-SSR-578, primer pairs MrE-SSR137 and MrE-SSR492, primer pairs MrE-SSR-669 and MrE-SSR200, primer pairs MrE-SSR-534 and MrE-SSR-650, primer pairs MrE-SSR-676 and MrE-SSR143, and primer pairs MrE-SSR140 and MrE-SSR-614 are respectively subjected to multiplex PCR amplification in the same PCR system.
The test method is as follows:
DNA extraction: taking 20-50 individual parent and progeny of Melissitus ruthenicus, extracting DNA with a novel plant genome extraction kit (DP320, Tiangen Biochemical technology (Beijing)) and detecting the quality and concentration of DNA, wherein the qualified DNA sample is diluted to 10ng/μ l.
And (3) PCR reaction system: mu.l of PCR Supermix (AS112-3, Beijing Quanjin Biotechnology Co., Ltd.), 0.4. mu.l of each primer, 1. mu.l of template DNA, and 2.4. mu.l of ddH2O。
PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension 72 ℃ for 10 min.
Electrophoresis and color development: 6% polyacrylamide gel electrophoresis, silver staining and color development, and photographing.
The electrophoresis results of primer pair MrE-SSR190 and primer pair MrE-SSR-578 are shown in FIG. 1: in the figure, M represents a female parent, P represents a progeny, and the progeny of the cross is marked with an asterisk.
The selfing rate was calculated from the selfing rate, i.e., the number of selfed progeny/total number of progeny × 100%, and the results are shown in table 1, where √ denotes that the site represents a hybridization trait, and the selfing rate of the individual plant mother M2 of alfalfa bean is 28%, which is the maternal plant of alfalfa bean of incompatible selfing genotype.
TABLE 1 type distribution of progeny M1 for single plant of Melissitus ruthenicus
Primer pairs 1-16 in the above table are MrE-SSR190, MrE-SSR-578, MrE-SSR137, MrE-SSR492, MrE-SSR-669, MrE-SSR200, MrE-SSR-534, MrE-SSR-650, MrE-SSR-676, MrE-SSR143, MrE-SSR140 and MrE-SSR-614 in sequence.
Example 2
And (3) carrying out selfing rate detection on the single plant female parent of the alfalfa bean, the number M2 and the progeny (P1-P30) of the single plant female parent of the alfalfa bean by utilizing the SSR marker detection primer group. And performing multiplex PCR to amplify the DNA of the female parent and the progeny of the Melissitus ruthenicus seeds by using 16 primer pairs.
Wherein, primer pair MrE-SSR190 and primer pair MrE-SSR-578, primer pair MrE-SSR137 and primer pair MrE-SSR492, primer pair MrE-SSR-669 and primer pair MrE-SSR200, primer pair MrE-SSR-534 and primer pair MrE-SSR-650, primer pair MrE-SSR-676 and primer pair MrE-SSR143, primer pair MrE-SSR140 and primer pair MrE-SSR-614, primer pair MrE-SSR256 and primer pair MrE-SSR101, primer pair MrE-SSR-539 and primer pair MrE-SSR-727 are respectively carried out multiple PCR amplification in the same PCR system.
The test method was the same as in example 2, and the results are shown in Table 2, wherein V represents that the individual plant M2 of Melissitus ruthenicus seeds exhibited a hybridization trait at the site, the selfing rate of the individual plant M2 of Melissitus ruthenicus seeds was 73%, and the individual plant M2 of Melissitus ruthenicus seeds was a parent of Melissitus ruthenicus seeds of compatible selfing genotype.
TABLE 2 type distribution of progeny M2 for single plant of Melissitus ruthenicus
In the above table, primer pairs 1-16 are MrE-SSR190, MrE-SSR-578, MrE-SSR137, MrE-SSR492, MrE-SSR-669, MrE-SSR200, MrE-SSR-534, MrE-SSR-650, MrE-SSR-676, MrE-SSR143, MrE-SSR140, MrE-SSR-614, MrE-SSR256, MrE-SSR101, MrE-SSR-539 and MrE-SSR727 in sequence.
Example 3
Randomly selecting 10 Melissitus ruthenicus single plant female parents (M3-M12), randomly selecting 25 progeny of each Melissitus ruthenicus single plant female parent, respectively using 16 sets of primer pairs provided by the invention, screening a self-compatible genotype according to the test method provided by the embodiment 3, taking the Melissitus ruthenicus single plant female parent with the selfing rate of more than or equal to 50% as the self-compatible genotype single plant female parent, wherein the female parents M7, M10, M11 and M12 are the Melissitus ruthenicus single plant female parents of the self-compatible genotype, and the test results are shown in the following table.
TABLE 3 screening results of the female parent of self-compatible genotype Melissitus ruthenicus
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> institute of grassland of Chinese academy of agricultural sciences
<120> detection primer group and kit for Melissitus ruthenicus SSR marker, application of detection primer group and kit and application of SSR marker of Melissitus ruthenicus
Screening method
<160> 32
<170> PatentIn version 3.5
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agcagctgca atttctcgat 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
cttggtaagc catctggagc 20
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence
<400> 5
cgagcccatg aatttagatg a 21
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
tgtgtttttg gggaatgatg 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
gtttgtttgg tgcggaaact 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
tgcatccatc aacttcaacc 20
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence
<400> 9
tcgagacaaa aatgtttacc ca 22
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
tgatgcgtga gaggatgaag 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
taaaggtcct ggatgggatg 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence
<400> 12
gctccataag ctggcaattc 20
<210> 13
<211> 26
<212> DNA
<213> Artificial sequence
<400> 13
gcctctcttt tattgcatta tctttc 26
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
aatgcatgtc gttgttgctc 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence
<400> 15
aaacctcacc tccttcccat 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<400> 16
gcaagattgg aggcatgttt 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence
<400> 17
aagggttgac gttgatggag 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<400> 18
gcctcaaagg gaattgatga 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence
<400> 19
aaaaacccaa acccctcaag 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence
<400> 20
ctgctgagcc tgtgtctgag 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence
<400> 21
ctccctccag ttgcattagc 20
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence
<400> 22
tcatcaacag caaaaccagc 20
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence
<400> 23
gaaacgaagt gggatggaga 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence
<400> 24
ctttcccaca agtacagcgg 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence
<400> 25
cgttgacgag aatggaatga 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence
<400> 26
atcagggaga ttggagcaga 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence
<400> 27
cttactaatg ccacgccgtt 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence
<400> 28
ctgccacagt accaggacaa 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence
<400> 29
gatccttgtt cctccgatca 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence
<400> 30
gaaggagaga ggggatttgg 20
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence
<400> 31
cctcttcgat ccaaaaccaa 20
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence
<400> 32
tccaaaccta ttcccattcg 20
Claims (15)
1. A detection primer group for selfing-compatible genotype SSR markers of Melissitus ruthenicus seeds is characterized by consisting of the following primer pairs:
primer pair MrE-SSR 190: comprises MrE-SSR190-F and MrE-SSR190-R, wherein MrE-SSR190-F is shown as SEQ ID NO.1, MrE-SSR190-R is shown as SEQ ID NO. 2;
primer pair MrE-SSR-578: comprises MrE-SSR-578-F and MrE-SSR-578-R, wherein MrE-SSR-578-F is shown as SEQ ID NO.3, and MrE-SSR-578-R is shown as SEQ ID NO. 4;
primer pair MrE-SSR 137: comprises MrE-SSR137-F and MrE-SSR137-R, wherein MrE-SSR137-F is shown as SEQ ID NO.5, MrE-SSR137-R is shown as SEQ ID NO. 6;
primer pair MrE-SSR 492: comprises MrE-SSR492-F and MrE-SSR492-R, wherein MrE-SSR492-F is shown as SEQ ID NO.7, MrE-SSR492-R is shown as SEQ ID NO. 8;
primer pair MrE-SSR-669: comprises MrE-SSR-669-F and MrE-SSR-669-R, wherein MrE-SSR-669-F is shown as SEQ ID NO.9, and MrE-SSR-669-R is shown as SEQ ID NO. 10;
primer pair MrE-SSR 200: comprises MrE-SSR200-F and MrE-SSR200-R, wherein MrE-SSR200-F is shown as SEQ ID NO.11, MrE-SSR200-R is shown as SEQ ID NO. 12;
primer pair MrE-SSR-534: comprises MrE-SSR-534-F and MrE-SSR-534-R, wherein MrE-SSR-534-F is shown as SEQ ID NO.13, MrE-SSR-534-R is shown as SEQ ID NO. 14;
primer pair MrE-SSR-650: comprises MrE-SSR-650-F and MrE-SSR-650-R, wherein MrE-SSR-650-F is a sequence shown in SEQ ID NO.15, and MrE-SSR-650-R is a sequence shown in SEQ ID NO. 16;
primer pair MrE-SSR-676: comprises MrE-SSR-676-F and MrE-SSR-676-R, wherein MrE-SSR-676-F is shown as SEQ ID NO.17, MrE-SSR-676-R is shown as SEQ ID NO. 18;
primer pair MrE-SSR 143: comprises MrE-SSR143-F and MrE-SSR143-R, wherein MrE-SSR143-F is shown as SEQ ID NO.19, MrE-SSR143-R is shown as SEQ ID NO. 20;
primer pair MrE-SSR 140: comprises MrE-SSR140-F and MrE-SSR140-R, wherein MrE-SSR140-F is shown as SEQ ID NO.21, MrE-SSR140-R is shown as SEQ ID NO. 22;
primer pair MrE-SSR-614: comprises MrE-SSR-614-F and MrE-SSR-614-R, wherein MrE-SSR-614-F is shown as SEQ ID NO.23, MrE-SSR-614-R is shown as SEQ ID NO. 24;
primer pair MrE-SSR 256: comprises MrE-SSR256-F and MrE-SSR256-R, wherein MrE-SSR256-F is shown as SEQ ID NO.25, MrE-SSR256-R is shown as SEQ ID NO. 26;
primer pair MrE-SSR 101: comprises MrE-SSR101-F and MrE-SSR101-R, wherein MrE-SSR101-F is shown as SEQ ID NO.27, MrE-SSR101-R is shown as SEQ ID NO. 28;
primer pair MrE-SSR 539: comprises MrE-SSR539-F and MrE-SSR539-R, wherein MrE-SSR539-F is the sequence shown in SEQ ID NO.29, MrE-SSR539-R is the sequence shown in SEQ ID NO. 30;
primer pair MrE-SSR 727: comprises MrE-SSR727-F and MrE-SSR727-R, wherein MrE-SSR727-F is a sequence shown as SEQ ID NO.31, and MrE-SSR727-R is a sequence shown as SEQ ID NO. 32.
2. A kit for screening Melissitus ruthenicus self-compatible genotype, comprising the detection primer set of claim 1.
3. Use of the detection primer set of claim 1 or the kit of claim 2, for screening Melissitus ruthenicus self-compatible genotype.
4. A screening method for self-compatible genotype of Melissitus ruthenicus seeds is characterized in that the screening method comprises the steps of amplifying DNA of a mother plant of Melissitus ruthenicus seeds and progeny of the mother plant of Melissitus ruthenicus seeds, obtaining distribution of types of the progeny of the mother plant of Melissitus ruthenicus seeds by comparing band types of amplified products after electrophoresis, and screening the self-compatible genotype;
the amplifying is amplifying DNA of the maternal plant of the Melissitus ruthenicus plant and progeny of the maternal plant of the Melissitus ruthenicus plant using the set of detection primers of claim 1 or the kit of claim 2;
the number of the filial generations for amplifying the single plant female parent of Melissitus ruthenicus is 25-30;
the types of the filial generation comprise hybrid filial generation and self-bred filial generation;
the selfing rate = the number of selfed progeny/total number of progeny × 100%;
taking the single plant female parent of the alfalfa bean with the selfing rate of more than or equal to 50 percent as the self-compatible genotype single plant female parent.
5. The screening method of claim 4, wherein the amplification comprises multiplex PCR amplification, the reaction of which comprises at least two sets of primer pairs.
6. The screening method of claim 5, wherein primer pair MrE-SSR190 and primer pair MrE-SSR-578 are amplified by multiplex PCR in the same PCR system.
7. The screening method of claim 5, wherein primer pair MrE-SSR137 and primer pair MrE-SSR492 are amplified by multiplex PCR in the same PCR system.
8. The screening method according to claim 5, wherein primer pair MrE-SSR-669 and primer pair MrE-SSR200 are subjected to multiplex PCR amplification in the same PCR system.
9. The screening method of claim 5, wherein primer pair MrE-SSR-534 and primer pair MrE-SSR-650 are amplified by multiplex PCR in the same PCR system.
10. The screening method of claim 5, wherein primer pair MrE-SSR-676 and primer pair MrE-SSR143 are amplified by multiplex PCR in the same PCR system.
11. The screening method of claim 5, wherein primer pair MrE-SSR140 and primer pair MrE-SSR-614 are subjected to multiplex PCR amplification in the same PCR system.
12. The screening method according to claim 5, wherein primer pair MrE-SSR256 and primer pair MrE-SSR101 are subjected to multiplex PCR amplification in the same PCR system.
13. The screening method of claim 5, wherein primer pair MrE-SSR-539 and primer pair MrE-SSR-727 are subjected to multiplex PCR amplification in the same PCR system.
14. The screening method according to claim 4, wherein the electrophoresis is 5% to 10% polyacrylamide gel electrophoresis.
15. The screening method according to claim 14, wherein the electrophoresis is 6% polyacrylamide gel electrophoresis.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106521024A (en) * | 2017-01-15 | 2017-03-22 | 兰州大学 | Medicago-truncatula-gaertn microRNA-SSR molecular marker primer and application to alfalfa variety identification |
| CN106676176A (en) * | 2017-01-18 | 2017-05-17 | 天津农学院 | Method for performing SSR analysis on tetraploid alfalfa by utilizing multiple PCR |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106521024A (en) * | 2017-01-15 | 2017-03-22 | 兰州大学 | Medicago-truncatula-gaertn microRNA-SSR molecular marker primer and application to alfalfa variety identification |
| CN106676176A (en) * | 2017-01-18 | 2017-05-17 | 天津农学院 | Method for performing SSR analysis on tetraploid alfalfa by utilizing multiple PCR |
Non-Patent Citations (2)
| Title |
|---|
| Analysis of genetic diversity of Ruthenia Medic (Medicago ruthenica (L.) Trautv.) in Inner Mongolia using ISSR and SSR markers;Hong-yan Li等;《Genet Resour Crop Evol 》;20130213;第60卷(第5期);第1687-1694页 * |
| 扁蓿豆24个居群遗传多样性的SSR研究;赵敏等;《西北植物学报》;20121231;第32卷(第12期);第2405-2411页 * |
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