CN114410815A - Method for constructing Xinjiang upland cotton variety fingerprint spectrum - Google Patents
Method for constructing Xinjiang upland cotton variety fingerprint spectrum Download PDFInfo
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Abstract
The invention provides a method for constructing a fingerprint of Xinjiang upland cotton variety, and relates to the technical field of crop molecular identification. The method screens the Xinjiang examined cotton upland cotton varieties by SSR markers based on capillary electrophoresis, can meet the requirements of integration and automation of data among different laboratories, makes up the limitation of SSR molecular marker technology, saves time, is beneficial to large sample processing, is convenient for application in variety authenticity and purity detection, and is simple, convenient, rapid and high in accuracy when performing variety DNA fingerprint detection. In the embodiment of the invention, SSR molecular fingerprints are respectively constructed for 2 series Xinjiang cotton upland cotton varieties based on capillary electrophoresis, and the cotton varieties can be identified because the target spectrum band read by the capillary electrophoresis amplification product can be accurate to 1 bp.
Description
Technical Field
The invention belongs to the technical field of crop molecular identification, and particularly relates to a method for constructing a fingerprint of a Xinjiang upland cotton variety.
Background
Due to the development of molecular biology technology, the establishment of the DNA fingerprint of the crop variety by using molecular marker technology has the advantages of rapidness, simplicity and accuracy in detection, and becomes an important technical tool for variety discrimination and seed purity detection. For example, the SSR created in 1991 has great advantages in DNA fingerprint identification compared with RFLP, RAPD and AFLP, has high polymorphism, covers the whole genome and is large in quantity, has become a molecular marker selected by DUS test in the International plant variety rights protection organization, and has been widely used for mainly examining and establishing fingerprint spectrum databases of crops such as rice, wheat, soybean and corn.
However, when the PCR amplification product is applied to cotton, especially in the technology of cotton DNA fingerprint map construction, the separation and specific detection of the PCR amplification product are the most critical and difficult items. At present, in the construction process of cotton DNA fingerprint, the separation of PCR amplification products mainly adopts a polyacrylamide gel electrophoresis method, which wastes time and labor, can not detect the size of a specific target band, and is difficult to satisfy the identification and comparison of large-scale primers and multiple batches of cotton varieties.
Disclosure of Invention
In view of the above, the invention aims to provide a method for constructing fingerprint spectra of Xinjiang upland cotton varieties, which screens SSR markers on the cotton varieties based on capillary electrophoresis, meets the requirements of integration and automation of data among different laboratories, makes up the limitation of SSR molecular marker technology, and fills the defects of polyacrylamide gel electrophoresis; and can be used to big sample processing, and the processing precision is high.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for constructing Xinjiang upland cotton variety fingerprint comprises the following steps: screening core primers from known cotton SSR primers positioned on chromosomes, carrying out PCR amplification on Xinjiang examined upland cotton varieties by using the core primers, and carrying out full-automatic capillary electrophoresis on amplification products to obtain molecular fingerprint.
Preferably, the screening of the core primer includes screening for polymorphic markers and screening for genetic diversity of the markers.
Preferably, the screening for genetic diversity of the marker comprises screening using the information content of the site polymorphism.
Preferably, the core primers include 73 pairs, which are respectively: HAU3193, NAU3736b, MUSS422a, NBRI _ HQ526730, NBRI _ HQ527566, MON _ CGR5007, MON _ SHIN-1481, MON _ SHIN-1584, NAU3419c, HAU0875, HAU2588, NBRI _ HQ527820, CK988221-PR-ss, NAU7182, HAU3371, Gh107, BNL2865, MON _ CGR5732, HAU195 b, MON _ DPL0024, HAU 256, MON _ DPL0375c, DPL0238, MON _ DPL0811b, MON _ SHIN-1585, HAU-44113, NAU6734, MON _ SHIN-1494b, NAL 034831, NAU5323, MON-DPU 0815, MON-40211, MON-40251, MON-40233, NAU-364749, NAU-363946, NAU-36397346, NADPU-363048, NANO-NO-3, NANO-NO-.
Preferably, the Xinjiang approved upland cotton varieties comprise Xinluzao series cotton varieties and Xinluzhong series cotton varieties.
Preferably, the PCR amplification system is 10 μ l, and comprises: 2.50. mu.l of DNA template, 0.16. mu.l of forward primer, 0.16. mu.l of reverse primer, 0.25. mu.l of dNTP, 1.00. mu.l of Taqbuffer, MgCl20.80. mu.l Taq enzyme 0.16. mu.l and the balance ddH2O。
Preferably, the procedure of PCR amplification comprises: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 50sec, annealing at 58 ℃ for 45sec, extension at 72 ℃ for 1min, 35 cycles; extension at 72 ℃ for 10 min.
Preferably, the distance distribution of the core primers on the chromosome is 0-238.658 cM, and the size of the polymorphism band amplified by the PCR is 75-400 bp.
Preferably, according to the relative position of the amplified product after PCR amplification on an electrophoresis gel, different polymorphisms generated by each pair of primers are marked, the polymorphism information content of the loci is calculated, and according to the difference of the genotypes of each pair of primers in different materials, a DNA molecular fingerprint is constructed.
The invention also provides the application of the Xinjiang upland cotton variety fingerprint spectrum constructed by the construction method in variety identification.
Has the advantages that: the invention provides a construction method of Xinjiang upland cotton variety fingerprint, which screens the Xinjiang approved cotton variety by SSR markers based on capillary electrophoresis, can realize the requirements of integration and automation of data among different laboratories, makes up the limitation of SSR molecular marker technology, and fills the defect of polyacrylamide gel electrophoresis; the capillary electrophoresis does not need to carry out sample genotype analysis through complex processes such as dyeing, developing and the like, so that the time is saved, the large sample processing is facilitated, and the application in variety authenticity and purity detection is facilitated; the capillary electrophoresis detection method compares the position of a target peak of a variety to be detected with an internal standard of the same capillary lane, and directly reads the size of a specific target band of target DNA by a fluorescent line to be accurate to 1 bp. In the embodiment of the invention, SSR molecular fingerprint spectrums are respectively constructed for 2 series Xinjiang cotton upland cotton varieties based on capillary electrophoresis, and the target spectrum band read by a capillary electrophoresis amplification product can be accurate to 1bp, so that the variety can be identified from other varieties when a certain primer has a unique specific spectrum band, and the method is simple, convenient, quick and high in accuracy when the DNA fingerprint detection of the variety is carried out.
Drawings
FIG. 1 is a DNA fingerprint of capillary electrophoresis of Xinluo early cotton;
FIG. 2 is DNA fingerprint of capillary electrophoresis of Xinluzhong cotton.
Detailed Description
The invention provides a method for constructing Xinjiang upland cotton variety fingerprint, which comprises the following steps: screening core primers from known cotton SSR primers positioned on chromosomes, carrying out PCR amplification on Xinjiang examined upland cotton varieties by using the core primers, and carrying out full-automatic capillary electrophoresis on amplification products to obtain molecular fingerprint.
The known cotton SSR primers positioned on the chromosome comprise primer markers (https:// www.cottongen.org/data/download) which are constructed by the university of agriculture in Huazhong and positioned on a genetic linkage map containing 5152 markers on the chromosome. The invention preferably screens the primers, including screening of polymorphic markers and screening of marker genetic diversity, wherein the screening of the polymorphic markers preferably comprises the steps of preliminarily screening 436 pairs of polymorphic markers from upland cotton on the basis of capillary electrophoresis and reading of polymorphic bands of more than 100bp for 8 selected core cotton varieties (see table 1); then, by selecting marking primers 225 pairs which have high Polymorphism and good stability, are positioned on 26 chromosomes of cotton and are uniformly distributed, carrying out PCR amplification on the Xinjiang upland cotton variety by utilizing the screened 225 pairs of primers, detecting various polymorphic genotype sites, and by calculating PIC (polymorphic information content of sites) values and data analysis among markers, screening the core SSR primers 73 pairs which have high Polymorphism, good effectiveness, large marked allelic variation and high polymorphic index and are beneficial to variety fingerprint construction, the method comprises the following steps: HAU3193, NAU3736b, MUSS422a, NBRI _ HQ526730, NBRI _ HQ527566, MON _ CGR5007, MON _ SHIN-1481, MON _ SHIN-1584, NAU3419c, HAU0875, HAU2588, NBRI _ HQ527820, CK988221-PR-ss, NAU7182, HAU3371, Gh107, BNL2865, MON _ CGR5732, HAU195 b, MON _ DPL0024, HAU 256, MON _ DPL0375c, DPL0238, MON _ DPL0811b, MON _ SHIN-1585, HAU-44113, NAU6734, MON _ SHIN-1494b, NAL 034831, NAU5323, MON-DPU 0815, MON-40211, MON-40251, MON-40233, NAU-364749, NAU-363946, NAU-36397346, NADPU-363048, NANO-NO-3, NANO-NO-. The present invention preferably calculates the PIC value using the software PowerMaker V3.25.
The Xinjiang approved upland cotton variety preferably comprises Xinluzao series cotton varieties and Xinluzhong series cotton varieties. In the embodiment of the invention, fingerprint spectrums are preferably constructed for 70 parts of new Luzao variety and 80 parts of new Luzao variety respectively.
Table 18 germplasm representing genetic diversity of Xinjiang upland cotton variety
TABLE 2 SSR marker information for Xinjiang cotton variety screening
TABLE 373 primer polymorphism information for SSR markers in 240 Xinjiang cotton varieties
TABLE 4 New Luzao Cotton variety information
TABLE 5 New cotton variety information in New land
The PCR amplification system of the present invention is preferably 10. mu.l, and preferably comprises: 2.50. mu.l of DNA template (10 ng/. mu.l), 0.16. mu.l of forward primer (25. mu.M), 0.16. mu.l of reverse primer (25. mu.M), 0.25. mu.l of dNTP (10mM), 1.00. mu.l of Taq buffer (10 ×), MgCl20.80. mu.l, Taq enzyme (5U/. mu.l) 0.16. mu.l and ddH as the remainder2And O. The PCR amplification process of the present invention preferably comprises: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 50sec, annealing at 58 ℃ for 45sec, extension at 72 ℃ for 1min, 35 cycles; extension at 72 ℃ for 10 min. The extraction method of the DNA template is not specially limited, and the extraction is preferably carried out by referring to a silent method (screening of SSR core primers of hybrid cotton varieties and authenticity and purity identification [ M)]University of agriculture in china, 2011, 45-50.). The distance distribution of the core primers on the chromosome is determined to be 0-238.658 cM, and the size of the polymorphism band amplified by the PCR is 75-400 bp.
The invention preferably carries out full-automatic capillary electrophoresis on the amplification product on an apparatus Fragment Analyzer (FA) and Zero Agarose Gel (ZAG) for 45min, then preferably marks different polymorphisms generated by each pair of primers according to the relative position of the amplification product after PCR amplification on an electrophoresis Gel, calculates the polymorphism information content of the loci, and constructs a DNA molecular fingerprint spectrum according to the difference of genotypes of each pair of primers in different materials.
After the DNA molecular fingerprint is obtained, the reliability and the practicability of the constructed molecular fingerprint are preferably verified. When the reliability verification is carried out, the method preferably utilizes the confidence probability of calculating the SSR molecular fingerprint, repeats SSR fingerprint core SSR primers to scan varieties, and selects 3 random district test materials to detect the authenticity and the purity based on the constructed primary database of the molecular fingerprint. In order to test the repeatability of 2 SSR molecular fingerprints constructed based on capillary electrophoresis, the invention preferably screens and analyzes the difference and polymorphism of each series of cotton varieties again according to SSR core primers used by the constructed SSR molecular fingerprints. In order to test the repeatability of the 4 SSR molecular fingerprints constructed based on capillary electrophoresis, the invention screens and analyzes the difference and polymorphism of each series of cotton varieties again according to SSR core primers used by the constructed SSR molecular fingerprints. The repeatability experiment shows that the SSR molecular fingerprint constructed based on capillary electrophoresis has high reliability and is beneficial to subsequent variety identification and purity identification.
In the embodiment of the invention, fingerprint analysis is carried out on 70 new Luzao varieties of 73 pairs of SSR core primers. Wherein 18 parts of the Xinluzao variety has specific primers: xinluzao29, Xinluzao8, Xinluzao47 and Xinluzao58 have 3, 2 and 2 pairs of specific primers respectively, and the other 12 varieties (Xinluzao1, Xinluzao9, Xinluzao22, Xinluzao24, Xinluzao57, Xinluzao59, Xinluzao61, Xinluzao63, Xinluzao64, Xinluzao65, Xinluzao83 and Xinluzao84) have a pair of specific primers. 52 Xinluzao cotton varieties in the New Luzao are distinguished by adopting a combined primer consisting of 42 pairs of primers, 45 cotton varieties can be distinguished by only combining 2 pairs of primers, and 7 cotton varieties (Xinluzao3, Xinluzao15, Xinluzao17, Xinluzao18, Xinluzao18, Xinluzao39 and Xinluzao54) need 3 pairs of primer combinations to be distinguished from other varieties. Thus, the fingerprints of 52 pairs of primers (BNL2449, BNL2535b, BNL3031, BNL3043, CCRI596 28, CK988221-PR-ss, DPL0238, Gh330, HAU0875, HAU0938, HAU1496, HAU1952b, HAU1968a, HAU2481, HAU2770, HAU2846, HAU3071a, HAU3193, HAU4483, HAU4748, HAU-SNP113, MGHES31, MON _ CGR5447, MON _ CGR5732, MON _ CGR6012, MON _ DPL0024, MON _ DPL03 c, MON _ DPL0502, MON _ DPL 050406, MON _ CGR 483 4832, MON _ NAU 363946, MON 5246, MON 334135, MON _ NAU 365246, MON 3246, MON 3352, NAU 364135, NANO 3, NAU 364135, NAU3, NAHQ 3, NANO. NAQ 3, NANO. 2, NANO. A3, NANO. 2, NAU 48, NAU3, NANO. A3, NAU 36693, NAU3, NANO. A. The 52 pairs of primers are distributed on 23 chromosomes, the distance between the primers on the chromosomes is 0-238.658 cM, and the size of the amplified polymorphism bands is 75-400 bp.
In the embodiment of the invention, when the fingerprint of a series of cotton varieties in new lands is constructed, fingerprint analysis is carried out on 80 new-land varieties by using 73 pairs of core SSR primers, 15 new-land cotton varieties have specific primers, wherein the Xinluzhong66 has 15 pairs of specific primers, the Xinluzhong41 has 6 pairs of specific primers, the Xinluzhong67, the Xinluzhong74, the Xinluzhong76 and the Xinluzhong79 have 2 pairs of specific primers, and 9 new-land cotton varieties (Xinluzhong5, the Xinluzhong8, the Xinluzhong15, the Xinluzhong37, the Xinluzhong50, the Xinluzhong55, the Xinluzhong62, the Xinluzhong78 and the Xinluzhong86) have only 1 specific primer. 15 of 80 neoland cotton varieties can be distinguished by using a specific primer at one time, and the rest 65 neoland cotton varieties need a combined primer to realize the distinction of the varieties from other varieties. 65 new cotton varieties in the new land are distinguished by adopting a combined primer pair consisting of 41 pairs of primers, wherein the Xinluzhong16, the Xinluzhong45 and the Xinluzhong49 can be distinguished by 3 pairs of primer combinations, and the rest 62 new cotton varieties in the new land can be distinguished by only 2 pairs of primer combinations. Thus, the unique primers of 15 parts of new terrestrial cultivars and 65 parts of combined primers of new terrestrial cultivars can be combined, and the detailed maps of the unique primers (BNL2449, BNL2865, BNL3031, BNL3043, CCRI303, CCRI596a, DPL0238, Gh330, HAU0875, HAU0938, HAU1952b, HAU2588, HAU2846, HAU3071a, HAU3193, HAU4022, HAU4483, HAU-SNP085, MGHES31, MON _ CGR5007, MON _ CGR5447, MON _ CGR5732, MON _ CGR6012, MON _ CGR6812, MON _ DPL0010, MON _ DPL0024, MON _ DPL 03383875, MON _ DPL0502, MON _ DPL 6011, MON _ CGR6812, MON _ DPL0010, MON _ DPL0024, MON _ DPL03 c, MON _ DPL 05052402, MON _ NAHQ 3246, MON _ NAU 3252, MON-NAU-37083, MON-NAU-3776, MON-NAU-NAH-3, MON-NAU-NAH-H3, and their RNA-H3, and their DNA sequences can be selected as well as the minimum 51. 51 pairs of primers are distributed on 21 chromosomes, the distance of the primers on the chromosomes is distributed between 0 and 238.658cM, and the size of the amplified polymorphism bands is between 75 and 400 bp.
The invention also provides the application of the Xinjiang upland cotton variety fingerprint spectrum constructed by the construction method in variety identification.
Based on the fingerprint spectrum, the present Xinjiang upland cotton variety can be identified, for example, in the embodiment, specific primer identification or combined primer identification is carried out. Wherein the specific primer identification comprises: marker HAU1952b polymorphic bands amplified in 70 new early-in-the-land varieties: 319bp, 305bp, 254bp and 244bp, the Xinluzao47, the Xinluzao59 and the Xinluzao77 can be identified at one time in the 249bp fragment, and the characteristic fingerprint information of the 0-1 reading band is respectively 1000, 0000 and 1110; the identification of the combined primer comprises the following steps: in the new-land series of cotton varieties, Xinluzhong87 and Xinluzhong41 have 2 polymorphic bands of 254bp and 278bp in a primer MON _ SHIN-1585, Xinluzhong87 has no specificity at 220bp amplified by a primer HAU3193, and Xinluzhong41 has specificity at 220bp amplified by a primer HAU3193, and the Xinluzhong87 can be successfully identified by using the 2 pairs of combined primers.
TABLE 6 construction of specific primer information Table for 70 New Oryza sativa variety
TABLE 7 construction of a specific primer information Table for 80 New Gossypium hirsutum varieties
The method for constructing fingerprint of Xinjiang upland cotton variety provided by the invention is described in detail with reference to the following examples, but they should not be construed as limiting the scope of the invention.
Example 1
1. Performing total DNA extraction of cotton by referring to a silent method;
2. detecting an SSR amplification product;
SSR-PCR reaction System (10. mu.l): 2.50. mu.l of DNA template (10 ng/. mu.l), 0.16. mu.l of forward primer (25. mu.M), 0.16. mu.l of reverse primer (25. mu.M), 0.25. mu.l of dNTP (10mM), 1.00. mu.l of Taq buffer (10 ×), MgCl20.80. mu.l, Taq enzyme (5U/. mu.l) 0.16. mu.l and ddH as the remainder2O。
PCR amplification procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 50sec, annealing at 58 ℃ for 45sec, extension at 72 ℃ for 1min, 35 cycles; extending at 72 deg.C for 10min, and storing at 4 deg.C for 10min
3. The amplification products were subjected to full-automatic capillary electrophoresis on a Fragment Analyzer (FA) and Zero Agarose Gel (ZAG) instrument for 45 minutes.
4. And (3) data statistics: and marking the different polymorphisms generated by each pair of primers according to the relative positions of the PCR amplification products on the electrophoresis gel.
5. The content of site Polymorphism Information (PIC) was calculated using the software PowerMaker V3.25.
6. According to the genotype difference of each pair of primers in different materials, a DNA molecular fingerprint is constructed, and according to the construction of Wuyusheng and the like (Wuyusheng, Yanghong, Zheng-3 corn hybrids and a parent SSR fingerprint [ J]The formula 1/2 for calculating the probability of appearance of fingerprint of the crop variety 2003(04) 496-500.)n(2 is the presence or absence of the same migration site, n is the number of alleles, 2nThe number of all possible varieties involved in detecting n alleles) the confidence probability of the SSR fingerprint constructed in this study is calculated.
(1) Constructing a fingerprint spectrum of a new Luzao series cotton variety;
through the analysis of the genetic diversity of the markers, 73 pairs of SSR core primers with high polymorphism, good effectiveness, large allelic variation and high polymorphism index are determined to carry out fingerprint analysis on 70 new-land-early varieties. Wherein 18 parts of the Xinluzao variety has specific primers: xinluzao29, Xinluzao8, Xinluzao47 and Xinluzao58 have 3, 2 and 2 pairs of specific primers respectively, and the other 12 varieties (Xinluzao1, Xinluzao9, Xinluzao22, Xinluzao24, Xinluzao57, Xinluzao59, Xinluzao61, Xinluzao63, Xinluzao64, Xinluzao65, Xinluzao83 and Xinluzao84) have a pair of specific primers.
And secondly, 52 Xinluzao cotton varieties are distinguished by adopting a combined primer consisting of 42 pairs of primers, 45 cotton varieties can be distinguished by only combining 2 pairs of primers, and 7 cotton varieties (Xinluzao3, Xinluzao15, Xinluzao17, Xinluzao18, Xinluzao18, Xinluzao39 and Xinluzao54) can be distinguished from other varieties by combining 3 pairs of primers.
Therefore, by combining the characteristic primers of 18 new Luzao varieties and the combined primers of 52 new Luzao varieties, at least 52 pairs of primers can be selected to construct detailed molecular fingerprint maps of 70 new Luzao varieties. The 52 pairs of primers are distributed on 23 chromosomes, the distance between the primers on the chromosomes is 0-238.658 cM, and the size of the amplified polymorphism bands is 75-400 bp.
(2) Constructing a series of cotton variety fingerprints in New land;
fingerprinting 80 new-land cultivars with 73 pairs of core SSR primers, 15 new-land cotton cultivars with specific primers, where Xinluzhong66 had 15 pairs of specific primers, Xinluzhong41 had 6 pairs of specific primers, Xinluzhong67, Xinluzhong74, Xinluzhong76, and Xinluzhong79 had 2 pairs of specific primers, and 9 new-land cotton cultivars (Xinluzhong5, Xinluzhong8, Xinluzhong15, Xinluzhong37, Xinluzhong50, Xinluzhong55, Xinluzhong62, Xinluzhong78, Xinluzhong86) had only 1 specific primer.
15 of 80 neoland cotton varieties can be distinguished by using a specific primer at one time, and the rest 65 neoland cotton varieties need a combined primer to realize the distinction of the varieties from other varieties. 65 new cotton varieties in the new land are distinguished by adopting a combined primer pair consisting of 41 pairs of primers, wherein the Xinluzhong16, the Xinluzhong45 and the Xinluzhong49 can be distinguished by 3 pairs of primer combinations, and the rest 62 new cotton varieties in the new land can be distinguished by only 2 pairs of primer combinations.
Therefore, by combining the characteristic primers of 15 parts of new terrestrial species and the combined primers of 65 parts of new terrestrial species, at least 51 pairs of primers can be selected to construct a detailed molecular fingerprint of 80 parts of new terrestrial species. 51 pairs of primers are distributed on 21 chromosomes, the distance of the primers on the chromosomes is distributed between 0 and 238.658cM, and the size of the amplified polymorphism bands is between 75 and 400 bp.
Example 2
(1) Identification of specific primers
Marker HAU1952b polymorphic bands amplified in 70 new early-in-the-land varieties: 319bp, 305bp, 254bp and 244bp, and at 249bp, Xinluzao47, Xinluzao59 and Xinluzao77 can be identified at one time, and the fingerprint information is 1000, 0000 and 1110 according to the characteristics of 0-1 reading bands.
(2) Identification with composite primers
In the new-land series of cotton varieties, Xinluzhong87 and Xinluzhong41 have 2 polymorphic bands of 254bp and 278bp in a primer MON _ SHIN-1585, Xinluzhong87 has no specificity at 220bp amplified by a primer HAU3193, and Xinluzhong41 has specificity at 220bp amplified by a primer HAU3193, and the Xinluzhong87 can be successfully identified by using the 2 pairs of combined primers.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for constructing Xinjiang upland cotton variety fingerprint spectrum is characterized by comprising the following steps: screening core primers from known cotton SSR primers positioned on chromosomes, carrying out PCR amplification on Xinjiang examined upland cotton varieties by using the core primers, and carrying out full-automatic capillary electrophoresis on amplification products to obtain molecular fingerprint.
2. The method of claim 1, wherein the screening of the core primer comprises screening for polymorphic markers and screening for genetic diversity of the markers.
3. The method of claim 2, wherein the screening for genetic diversity of the marker comprises screening using the information content of site polymorphisms.
4. The method of claim 1, wherein the core primers comprise 73 pairs of primers: HAU3193, NAU3736b, MUSS422a, NBRI _ HQ526730, NBRI _ HQ527566, MON _ CGR5007, MON _ SHIN-1481, MON _ SHIN-1584, NAU3419c, HAU0875, HAU2588, NBRI _ HQ527820, CK988221-PR-ss, NAU7182, HAU3371, Gh107, BNL2865, MON _ CGR5732, HAU195 b, MON _ DPL0024, HAU 256, MON _ DPL0375c, DPL0238, MON _ DPL0811b, MON _ SHIN-1585, HAU-44113, NAU6734, MON _ SHIN-1494b, NAL 034831, NAU5323, MON-DPU 0815, MON-40211, MON-40251, MON-40233, NAU-364749, NAU-363946, NAU-36397346, NADPU-363048, NANO-NO-3, NANO-NO-.
5. The method of claim 1, wherein the Xinjiang approved upland cotton varieties comprise Xinluohao series cotton varieties and Xinluohao series cotton varieties.
6. The construction method according to claim 1, wherein the PCR amplification system is 10 μ l, and comprises: 2.50. mu.l of DNA template, 0.16. mu.l of forward primer, 0.16. mu.l of reverse primer, 0.25. mu.l of dNTP, 1.00. mu.l of Taqbuffer, MgCl20.80. mu.l Taq enzyme 0.16. mu.l and the balanceddH2O。
7. The method of construction according to claim 1 or 6, wherein the PCR amplification procedure comprises: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 50sec, annealing at 58 ℃ for 45sec, extension at 72 ℃ for 1min, 35 cycles; extension at 72 ℃ for 10 min.
8. The construction method of claim 1, wherein the distance distribution of the core primers on the chromosome is 0-238.658 cM, and the size of the polymorphism band amplified by the PCR is 75-400 bp.
9. The construction method according to claim 8, characterized in that different polymorphisms generated by each pair of primers are marked according to the relative positions of the amplification products after PCR amplification on an electrophoresis gel, the content of site polymorphism information is calculated, and a DNA molecular fingerprint is constructed according to the difference of genotypes of each pair of primers in different materials.
10. The application of the Xinjiang upland cotton variety fingerprint spectrum constructed by the construction method of any one of claims 1-9 in variety identification.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776274A (en) * | 2012-03-22 | 2012-11-14 | 艾先涛 | Method for identifying cotton variety |
| CN105734124A (en) * | 2016-02-23 | 2016-07-06 | 新疆农垦科学院 | Cotton blastogenesis identification method based on SSR markers and capillary electrophoresis |
| CN107312827A (en) * | 2017-03-22 | 2017-11-03 | 新疆农业科学院经济作物研究所 | A kind of SSR molecular marker method for identifying non-Bt cotton variety authentication |
| WO2018085971A1 (en) * | 2016-11-08 | 2018-05-17 | 南京农业大学 | Cotton whole genome snp chip and application thereof |
| CN111041126A (en) * | 2020-01-16 | 2020-04-21 | 石河子大学 | Polymorphic molecular marker for identifying series cotton varieties in new land and application thereof |
-
2021
- 2021-12-31 CN CN202111669260.4A patent/CN114410815A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776274A (en) * | 2012-03-22 | 2012-11-14 | 艾先涛 | Method for identifying cotton variety |
| CN105734124A (en) * | 2016-02-23 | 2016-07-06 | 新疆农垦科学院 | Cotton blastogenesis identification method based on SSR markers and capillary electrophoresis |
| WO2018085971A1 (en) * | 2016-11-08 | 2018-05-17 | 南京农业大学 | Cotton whole genome snp chip and application thereof |
| CN107312827A (en) * | 2017-03-22 | 2017-11-03 | 新疆农业科学院经济作物研究所 | A kind of SSR molecular marker method for identifying non-Bt cotton variety authentication |
| CN111041126A (en) * | 2020-01-16 | 2020-04-21 | 石河子大学 | Polymorphic molecular marker for identifying series cotton varieties in new land and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| ARUNITA RAKSHIT等: "Evaluation of SSR Markers for the Assessment of Genetic Diversity and Fingerprinting of Gossypium hirsutum Accessions", JOURNAL OF PLANT BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 19, no. 2, 31 December 2010 (2010-12-31), pages 153 - 160 * |
| 王欣怡等: "基于SSR标记新疆陆地棉的DNA指纹图谱构建及遗传多样性分析", 棉花学报, vol. 30, no. 04, 15 July 2018 (2018-07-15), pages 308 - 315 * |
| 秦利等: "新疆陆地棉SSR标记指纹图谱构建和杂种纯度鉴定研究", 新疆农业科学, vol. 42, no. 06, 30 December 2005 (2005-12-30), pages 399 - 401 * |
| 聂新辉等: "新陆早棉花品种DNA指纹图谱的构建及遗传多样性分析", 作物学报, vol. 40, no. 12, 31 December 2014 (2014-12-31), pages 2104 - 2117 * |
| 雷杰杰等: "新疆陆地棉经济性状优异等位基因位点的遗传解析", 棉花学报, vol. 32, no. 3, 8 June 2020 (2020-06-08), pages 186 * |
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