CN112029890A - SNP (Single nucleotide polymorphism) site primer combination for identifying melon germplasm authenticity and application - Google Patents

SNP (Single nucleotide polymorphism) site primer combination for identifying melon germplasm authenticity and application Download PDF

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CN112029890A
CN112029890A CN202010938455.3A CN202010938455A CN112029890A CN 112029890 A CN112029890 A CN 112029890A CN 202010938455 A CN202010938455 A CN 202010938455A CN 112029890 A CN112029890 A CN 112029890A
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CN112029890B (en
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温常龙
杨静静
张建
赵泓
张晓飞
王超
罗江
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a core SNP (single nucleotide polymorphism) site and primer combination for identifying the authenticity of melon germplasm resources, a DNA (deoxyribonucleic acid) fingerprint database and database construction method based on the SNP site, an identification method for the authenticity of melon germplasm resources based on the SNP site and application. The SNP sites are selected from any 1 to 32 of the first to the third twelve SNP sites. The invention establishes a DNA fingerprint database for identifying the authenticity of melon germplasm resources based on high-throughput sequencing. The method provided by the invention can be used for identifying unknown melon germplasm resources and also can be used for identifying the authenticity of known melon germplasm resources. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

Description

SNP (Single nucleotide polymorphism) site primer combination for identifying melon germplasm authenticity and application
Technical Field
The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a core SNP (single nucleotide polymorphism) site and primer combination for identifying the authenticity of melon germplasm resources, a DNA (deoxyribonucleic acid) fingerprint database and database construction method based on the SNP site, an identification method for the authenticity of melon germplasm resources based on the SNP site and application.
Background
Germplasm resources are the foundation of breeding. The nineteenth article of the seed method states that germplasm resources refer to a basic material for breeding new varieties of plants, including propagation materials of cultivars and wild species of various plants, and genetic materials of various plants artificially created using the propagation materials. The germplasm resource is an important natural resource formed by natural evolution and artificial creation, accumulates extremely rich genetic variation, and is a material basis for breeding new varieties and developing agricultural production by human beings. The discovery and utilization of germplasm resources determine the key to the breakthrough development of modern crop breeding. Therefore, the identification and protection of germplasm resources are more important in the aspects of identifying authenticity and optimizing sources compared with specific varieties. However, at present, the identification and protection work of most germplasm resources is in a blank state.
Melons (Cucumis melo L.) are important economic crops in cucurbitaceae and are widely planted in China. In 2018, FAOSTAT publishes that the yield of the melons in China is 127 ten thousand tons, which accounts for 46.8 percent of the total yield of the melons in the world. Melons are divided into two subspecies, c.melo ssp. The difference of the fruit shape, the fruit flesh color, the sugar content, the fruit thickness and other properties is obvious. The melon germplasm resource carries a plurality of excellent characters, and is the basis of melon variety breeding. At the end of the 70 s in the 20 th century, China develops the collection and preservation work of melon germplasm resources, and at present, thousands of melon germplasm resources are preserved in a plurality of germplasm resource libraries, so that a good material library is provided for melon breeding and scientific research workers in China. The identification, evaluation and genetic diversity research of melon germplasm resources are the premise and the basis of melon genetic breeding and resource development and utilization. However, the germplasm resource library has not been subjected to germplasm resource identification work in the collection process. The germplasm resource library has multiple germplasm resources redundancy, and the cost of germplasm preservation is increased. The background of melon germplasm resources is unknown, and the deeper development and utilization of germplasm resources are also limited. The stealing of germplasm resources not only damages the intellectual property of breeders, but also causes the narrow genetic background among species. How to identify, evaluate and discover the utilization value of the biological.
The melon germplasm resource identification mainly comprises field identification and molecular marking methods. The traditional field planting identification period is long, is easily influenced by environmental factors, and is time-consuming and labor-consuming. Therefore, the field identification method is not feasible for the muskmelon germplasm resources with large quantity and serious homogenization. The DNA fingerprint identification technology represented by SSR and SNP molecular markers has the advantages of accuracy, reliability, simplicity, rapidness and easy automation, and is a recognized crop DNA fingerprint identification technology at home and abroad. The method can realize accurate and rapid identification of the melon resources by constructing the DNA fingerprint of the melon resource standard sample and a DNA fingerprint database management system. The SNP marker (which refers to the variation of a single nucleotide on the same site of a genome) is usually a double allele, presents the characteristics of non-same, is easy to judge, and has the advantages of simple data statistics, strong data compatibility, high throughput and the like compared with an SSR marker. Therefore, the identification of melon germplasm resources by using the SNP molecular markers is the most feasible method, and a method for identifying the authenticity of melon germplasm resources by using the SNP molecular markers is needed in scientific research and practice.
Disclosure of Invention
The invention provides a core SNP locus and a primer combination for identifying melon germplasm resources, a DNA fingerprint database and a database construction method based on the SNP locus, and an identification method and application of the melon germplasm resources based on the SNP locus.
The invention is realized by the following technical scheme:
identifying core SNP loci of melon germplasm resources authenticity, wherein the SNP loci are selected from any 1 to 32 of the following first SNP loci to third twelve SNP loci: a first SNP locus, wherein the first SNP locus is located at 1028867 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or T; a second SNP locus, wherein the second SNP locus is located at 15305628 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a third SNP locus, wherein the third SNP locus is located at 35346336 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A; a fourth SNP locus, wherein the fourth SNP locus is located at 1090543 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T; a fifth SNP locus, wherein the fifth SNP locus is located at 1709164 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a sixth SNP locus, wherein the sixth SNP locus is located at 13775704 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T; a seventh SNP locus, wherein the seventh SNP locus is located at 7930146 rd chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or A; an eighth SNP locus, wherein the eighth SNP locus is located at 18037611 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a ninth SNP locus, wherein the ninth SNP locus is located at 28288534 rd chromosome 3 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a tenth SNP locus, wherein the tenth SNP locus is located at 4189054 th chromosome 4 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or G; an eleventh SNP locus, wherein the eleventh SNP locus is located at 16536384 th chromosome 4 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T; a twelfth SNP locus, wherein the twelfth SNP locus is located at 14981598 th chromosome 5 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or C; a thirteenth SNP locus, wherein the thirteenth SNP locus is located at 28011864 th chromosome 5 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or C; a fourteenth SNP locus, wherein the fourteenth SNP locus is located at 8120366 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a fifteenth SNP locus, wherein the fifteenth SNP locus is located at 18900074 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A; a sixteenth SNP locus, wherein the sixteenth SNP locus is located at 19620053 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a seventeenth SNP site, wherein the seventeenth SNP site is located at 1104024 th chromosome of a7 th chromosome of a melon reference genome or a corresponding site on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the site is G or C; an eighteenth SNP locus, wherein the eighteenth SNP locus is located at 3254461 th chromosome of a reference genome of the melon, or a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is C or T; a nineteenth SNP locus, wherein the nineteenth SNP locus is located at 23111340 th chromosome 7 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A; a twentieth SNP locus, wherein the twentieth SNP locus is located at 4876476 th chromosome 8 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources thereof, and the nucleotide base of the locus is G or C; a twenty-first SNP locus, wherein the twenty-first SNP locus is located at 6383781 th chromosome 8 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T; a twenty-second SNP locus, wherein the twenty-second SNP locus is located at 32379197 th chromosome 8 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a twenty-third SNP locus, wherein the twenty-third SNP locus is located at 1849869 th chromosome of a melon reference genome 9 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or C; a twenty-fourth SNP locus, wherein the twenty-fourth SNP locus is located at 12154715 th chromosome of a melon reference genome 9 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A; a twenty-fifth SNP locus, wherein the twenty-fifth SNP locus is located at 20428406 th chromosome of a9 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T; a twenty-sixth SNP locus, wherein the twenty-sixth SNP locus is located at 9552072 th chromosome of 10 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T; a twenty-seventh SNP locus, wherein the twenty-seventh SNP locus is located at 20472868 th chromosome of the 10 th chromosome of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or T; a twenty-eighth SNP locus, wherein the twenty-eighth SNP locus is located at 7009967 th chromosome of the reference genome of the melon, or at a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is A or C; a twenty-ninth SNP locus, wherein the twenty-ninth SNP locus is located at 17070009 th chromosome of the 11 th chromosome of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or C; a thirtieth SNP locus, wherein the thirtieth SNP locus is located at 27232414 th chromosome of the melon reference genome 11 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or G; a thirty-first SNP locus, wherein the thirty-first SNP locus is located at 3459537 th chromosome of a 12 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or G; a thirty-second SNP locus, wherein the thirty-second SNP locus is located at 13742304 th chromosome of the reference genome of the melon or a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is G or A; wherein the melon reference genome is melon DHL92 reference genome V3.5.1.
In some embodiments, the sequence of the first SNP site and bases upstream and downstream thereof is SEQ ID NO: 97 or a genome fragment homologous between germplasm resources thereof, more preferably a fragment identical to the sequence shown in SEQ ID NO: 97, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequences of the second SNP locus and bases at the upstream and downstream of the second SNP locus are SEQ ID NO: 98 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 98, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the sequences of the third SNP locus and bases at the upstream and downstream are SEQ ID NO: 99 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 99 nucleotide sequences having greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the fourth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 100 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 100, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequences of the fifth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 101 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 101, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the sequences of the sixth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 102 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 102 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the seventh SNP locus and the sequences of bases on the seventh SNP locus and upstream and downstream thereof are SEQ ID NO: 103 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 103 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequences of the eighth SNP locus and bases at the upstream and downstream are SEQ ID NO: 104 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 104 greater than or equal to 95%, 96%, 97%, 98%, or 99%; the ninth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 105 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 105 nucleotide sequence identity greater than or equal to 95%, 96%, 97%, 98% or 99%; the tenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 106 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 106 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequence of the eleventh SNP site and bases on the eleventh SNP site is SEQ ID NO: 107 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 107 greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the twelfth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 108 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 108 greater than or equal to 95%, 96%, 97%, 98% or 99% identity; the thirteenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 109 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 109 by greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the fourteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 110 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 110 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the sequence of the fifteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 111 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 111 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the sixteenth SNP locus and bases on the sixteenth SNP locus is SEQ ID NO: 112 or an inter-germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 112 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the sequence of the seventeenth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 113 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 113 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequence of the eighteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 114 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 114 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the nineteenth SNP site and the sequences of bases on the nineteenth SNP site are SEQ ID NO: 115 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 115, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-second SNP site and the sequences of bases on the twenty-second SNP site are SEQ ID NO: 116 or a germplasm resource homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 116 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the twenty-first SNP locus and the sequences of bases on the twenty-first SNP locus and bases on the twenty-first SNP locus are SEQ ID NO: 117 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence set forth in SEQ ID NO: 117 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequence of the second twelve SNP locus and the base sequences of the second twelve SNP locus are SEQ ID NO: 118 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 118 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the twenty-third SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 119 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 119, or greater than 95%, 96%, 97%, 98%, or 99%; the sequence of the twenty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 120 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 120, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-fifth SNP locus and the sequences of bases on the twenty-fifth SNP locus and the upstream and downstream of the twenty-fifth SNP locus are SEQ ID NO: 121 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 121, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the twenty-sixth SNP locus and the sequences of bases on the twenty-sixth SNP locus and bases on the twenty-sixth SNP locus are SEQ ID NO: 122 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 122 is greater than or equal to 95%, 96%, 97%, 98% or 99%; the twenty-seventh SNP locus and the sequences of bases on the twenty-seventh SNP locus and the upstream and downstream of the twenty-seventh SNP locus are SEQ ID NO: 123 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 123 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequences of the twenty-eight SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 124 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 124, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-ninth SNP locus and the sequences of bases on the twenty-ninth SNP locus are SEQ ID NO: 125 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 125, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the thirty-third SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 126 or a resource-homologous genome fragment thereof, more preferably a fragment of a sequence shown in SEQ ID NO: 126, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the thirty-first SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 127 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 127, 95%, 96%, 97%, 98% or 99%; the sequences of the third twelve SNP loci and bases at the upper and lower ends of the third twelve SNP loci are SEQ ID NO: 128 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 128 is greater than or equal to 95%, 96%, 97%, 98% or 99%.
The core SNP primer set for identifying the authenticity of melon germplasm resources is used for amplifying the SNP sites respectively, and comprises the following components: a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site; a ninth SNP primer set for amplifying the ninth SNP site; a tenth SNP primer set for amplifying the tenth SNP site; an eleventh SNP primer set for amplifying the eleventh SNP site; a twelfth SNP primer set for amplifying the twelfth SNP site; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a fourteenth SNP primer set for amplifying the fourteenth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a sixteenth SNP primer set for amplifying the sixteenth SNP site; a seventeenth SNP primer set for amplifying the seventeenth SNP site; an eighteenth SNP primer set for amplifying the eighteenth SNP site; a nineteenth SNP primer set for amplifying the nineteenth SNP site; a twentieth SNP primer set for amplifying the twentieth SNP site; a twenty-first SNP primer set for amplifying the twenty-first SNP site; a second twelve SNP primer set for amplifying the second twelve SNP sites; a twenty-third SNP primer set for amplifying the twenty-third SNP site; a twenty-fourth SNP primer set for amplifying the twenty-fourth SNP site; a twenty-fifth SNP primer set for amplifying the twenty-fifth SNP site; a twenty-sixth SNP primer set for amplifying the twenty-sixth SNP site; a twenty-seventh SNP primer set for amplifying the twenty-seventh SNP site; a twenty-eighth SNP primer set for amplifying the twenty-eighth SNP site; a twenty-ninth SNP primer set for amplifying the twenty-ninth SNP site; a thirtieth SNP primer set for amplifying the thirtieth SNP site; a thirty-first SNP primer set for amplifying the thirty-first SNP site; a thirty-second SNP primer set for amplifying the thirty-second SNP site.
In some embodiments, the first SNP primer set, the specific portion of the first forward primer, the specific portion of the second forward primer, and the downstream primer are each identical to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second SNP primer set are respectively matched with the sequence shown in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the third SNP primer set are respectively matched with the sequences shown in SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the fifth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and in the sixth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and in the seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the eighth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the ninth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the tenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the eleventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 33, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and the twelfth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39, greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and in the fourteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the fifteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the sixteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and in the seventeenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the eighteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 52. SEQ ID NO: 53. SEQ ID NO: 54 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the nineteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 55. SEQ ID NO: 56. SEQ ID NO: 57 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the twentieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 58. SEQ ID NO: 59. SEQ ID NO: 60, preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; the twenty-first SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the sequence shown in SEQ ID NO: 61. SEQ ID NO: 62. SEQ ID NO: 63 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second twelve SNP primer set are respectively matched with the sequence shown in SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the twenty-third SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the twenty-fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the twenty-fifth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the primers shown in SEQ ID NO: 73. SEQ ID NO: 74. SEQ ID NO: 75 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the twenty-sixth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the primers shown in SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and in the twenty-seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 79. SEQ ID NO: 80. SEQ ID NO: 81 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second eighteen SNP primer set are respectively connected with the sequences shown in SEQ ID NO: 82. SEQ ID NO: 83. SEQ ID NO: 84 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the twenty-ninth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the thirtieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 88. SEQ ID NO: 89. SEQ ID NO: 90 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the thirty-first SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 91. SEQ ID NO: 92. SEQ ID NO: 93 greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the third twelve SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 94. SEQ ID NO: 95. SEQ ID NO: 96 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; preferably, the first upstream primer and the second upstream primer in each set of primers are linked to different fluorescent molecules, more preferably, the fluorescent molecules are selected from FAM, HEX.
A core SNP kit for identifying the authenticity of melon germplasm resources is prepared into a competitive allele specificity PCR reaction system; the reaction system comprises: the SNP primer sets preferably comprise a first upstream primer, a second upstream primer and a downstream primer of each primer set, wherein the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer in the SNP primer sets in the system is 2:2: 5.
A melon germplasm resource DNA fingerprint database based on core SNP markers, the DNA fingerprint database comprising: and the genotype of the SNP locus of the standard melon germplasm resource.
In some embodiments, the standard melon germplasm resources are selected from the following 441 melon germplasm resources:
PLANTERE JUMLED 76134, PERSIAN MEDIUM, CURTEOWPE PLANTER JUMBO, 76W-2-5, 76W-3, CATTA LOLLE, 76W-1, 76W-2, 76W-4, CNTALOU PE P.M.R450, CASABA GOLDEN QUOMI GUAUT, ZUO, mating, PF, mating, Japan, GOLDEN mating, Japan, POLIOORR F (RS 815), CATTOR HYBRID F, PLATE PE, PLATE, LONGANG mating, VR 6072, CA 456, CA M761232, (MUSKMELON) BURPEE' S BRIS, GURON mating, japan3, ェリザベス, HONEY ROCK, first generation OF Pachyrhizus communis, swan, MELAO HALE 'S BEST, bvrc5, Anemone MELO, CUCUCUMIS MELO L., IROQUOIS, ALASKA F1, サカタ mating プリンスメロン PF6, Japan4, サカタ mating ェ - ェ, ェ mating ェ - ェ, golden nine MELON, ェ - ェ, HYBRID SUMMET, JUMBO HALS BEST, type 1, type 2, BUEE' S AMBROSHIBRID F ェ, HERADES OF GOLO, SSIC HYBRD F ェ, BRA- ェ -ARO ESPEAL, bvV BO ェ, CV.HONEYOYW, POLIDOOR ェ, CLA MELO MELT, CALMOCHORON ェ, CALSTAVSTAVSTAVSTAVSBO, CAVSBO- ェ, CAVSBO-PHONZO-PHONYOR ェ, CAVSB ェ, WAVSBO-PHONZO ェ, CAVSB ェ, WAVSBO-PHONZO ェ, CAVSB ェ, WAVSB ェ, WAVSBO-PHONZO ェ, WAVSB ェ, WAVSBO-PHONZO-PHO, sunrise (original Sweet), snow char (Hami melon type), Jade Dew (Orien Sweet), farmer NO.2(Oriental Sweet, Silver Light (Oriental Sweet), Pride, Hearts of Gold, Delicous 51, Honey Rock, Roadside, Samson, Hales Best Jumbo, SUPER KET HYBRID, PLANTER ' S JUMBO VGR679, GUTO 45 QI L620, TOP MARK VGG 2663, PERLITA VGR, EDISTO VGL 415, JUMBO HALE ' S BEST 97849, FERRY-MORE SEEDS, HALE ' BEST JUMBO, MUSKON 6593-8, bvvrc 2, Sweet potato, snow melon, Japanese melon, Sweet potato, Japanese melon メロン, Sweet potato 3526, Sweet potato 3627, Sweet potato 3655, Sweet potato 3526, Sweet potato 3645, Sweet potato 3655, Sweet potato 35, Sweet potato 3655, Sweet potato 3526, Sweet potato 1, Sweet potato 35, Sweet potato 1, Sweet potato 3680, Sweet potato 1, Sweet potato 35, Sweet potato 1, Sweet potato 35, Sweet potato 357, Sweet potato, ハ - ベスト 5, ハ - ベスト 6, white muskmelon, Horey louge, cotton white, Taiwan sweet, CD ELDORATO YELLOW, CD CASTOR, Yanjiao sweet, Daphne potpourri, Huangdao, M26, M29, M285, ANANAS, CV SPECIAL HONEY DEW EYPE, Cucumis melo, OGEN, Japanese, Tiger skin Cucumis melo, Changlan persimmon 2, Ielizabeth, Galaoui, CHINESE MELON, ALIER F1, ANWAS D 'AMERIGUE, IDIAAN, TOJONA, SHIRAY, white dao, Huadao, green cucumber, red lantern, white sweet, black-peel MELON, bvrc8, Hongcheng MELON, AVGNON, manna, ZANDA (SANDA 03, ZA 03-03, ZA-03, ZA-03-12, SANIA-259, SAVOR F1, ANWAS D' AMORE E DEE, SAE DEE, SANIA, SANI-35, SANI-NI, 03-252-3-54, 03-252-3-90, 03-252-3-41, 03-252-3-69, 03-210-17-2,7, 03-204-1-1,3,5,6,7, 03-203-1-12,22,23,24,25, 03-208-1-15,16, 03-210-16-2, 03-209-1-1,11, 03-209-9-12,13,14,16,17,18,19, 03-210-3-9, 03-206-7,8,9,12, 03-204-2-18, 03-199-16-1, 03-199-18-11, 03-198-31-1, 03-205-2-9, 03-211-2, 03-210-13-1, 03-210-11-1, 03-199-14-1, 03-207-2-12, 03-195-1-4,5, 03-253-3,5.6,9,12,15, 03-252-1-12, 03-310-1-2,3, 03-337-2-20, 03-314-3-3, 03-308-2-5, 03-328-3, 03-345-9, 03-339-1-4,10,18, 03-325-2-2, 03-317-1-1, 03-299-1-5, 03-283-4-6, 03-296-2-8, 03-310-2-1,3,5,8, 03-210-14-4,6,9, 03-209-6-1, 03-199-17-24, 03-209-3-1,2,3,5,6,11, 03-203-1-9, 03-210-1-7, 03-210-2-9, 03-205-1-4,7, 03-199-12-18, 03-198-8-3, 03-198-7-11, 03-195-2-3, 03-210-11-19,41, 03-198-3-15, 03-210-11-17,24,28, 03-210-12-10, 03-210-2-19, 03-307-6-5, 03-293-1-6, 03-325-2-13, 03-252-2-17, 03-236-1,3,4,5,6,11, 03-295-1-7, 03-298-7-9,10, 03-291-1,3, 03-341-2,4,5,6,8, 03-283-1-4, 03-252-3-26, 03-339-2-4,6, 03-210-11-31, 03-196-2-10, 03-196-2-16, 03-196-3-20, 03-210-4-1, 03-210-8-2, 03-204-3-19,20,21,23, 03-210-9-1-2,3,6, 03-210-11-15,36,37, 03-210-10, 03-210-16-17, 03-210-16-13, 03-198-14-2, 03-210-11-2,25,29, 03-210-4-5, 03-210-7-1, 03-210-5-9, 03-210-6-2, 03-209-4-2, 03-198-10-10, 03-339-3-9,10, 03-320-2,5, 03-223-2-19, 03-287-1-1,10, 03-307-2-3, 03-306-1-8, 03-285-1-1, 03-318-3, 03-283-5-9, 03-289-2-5, 03-315-1,4, 03-310-3-3, bvrc15, 03-304-1-9, 03-286-3-1, 03-314-1-1, 03-319-3,4,5, 03-312-3-4,5, 03-314-2-1, 03-286-1-12,13, 03-342-2-9, 03-343-1-4, 03-346-2,3,4,5,6,7, 03-344-3,6, 03-297-8, 03-342-1-6,7, 03-312-4, 03-316-1, bvrc2, 04-562-1, 04-560-1, 04-555-7, 04-552-1, bvrc4, 04-551-6, 04-560-6, 04-545-7, 04-533-4, 04-1, 04-519-2, 04-539-1, 04-537-5, 04-521-12, 05-189-8, 04-528-1, 04-513-2, 04-511-1, 04-556-5, 04-558-1, 04-557-7, 04-556-1, 04-552-15, 04-557-1, 04-538-2, 04-528-12, 04-529, 04-517-1, 04-551-2, 04-563-1, 04-561-7, 04-554, 04-552-5, 04-532-2, 04-518-1, 04-523-1, 04-568-1, 04-566-9, 04-571-1, 04-571-13, 04-576-1, 04-566-1, 04-571-10, 04-565-2, 04-599-1, 04-570-6, 04-567-9, 04-578, 05-60-8, 05-56-3, 05-60-7, 05-65-4, 05-67-10, 05-63-1, 05-69-8, 05-67-7, 05-55-2, 05-69-6, 05-51-7, 05-41-6, 05-40-2, 05-46-15, 05-46-11, 05-48-5, 05-51-4, 05-50-8, 05-50-2, 05-50-9, 05-45-1, 05-38-6, 05-45-14, 05-38-9, 05-45-8, 05-93-4-1, 05-71-2, 05-77-4, 05-86-1, 05-70-1, 05-79-1, 05-70-4-2, 05-63-6, 05-66-2, 05-66-7, 05-51-9, 05-66-9, 05-67-1, 05-46-7, 05-44-1, 05-95-1-21,23,26, 05-95-3-1,3,4,6,11,12, 05-94-2-1,10,31,35, 05-93-1-22, 05-88-6, 05-76-2, 05-93-3-7, 05-93-4-26, 05-99-3-9,11,22,28,50,52,58,68,86, 05-99-12-2, 05-99-24-25,32,37, 05-99-14-8,9,11, 05-98-7-1,2,5, 05-97-1-5,9, 05-96-7,9,12,14,15,16,18, 05-99-7-12,13, 05-95-8-10,25,28,30,31, 05-99-19-34,36,37,42,44, 05-100-3-3,7,9,14, 05-171-2-10,16, 05-193-2, 05-136-20, 05-137-1-2, 05-133-3-1,2, 05-73-1, 05-80-1, May-83, 05-81-3, 05-77-8-3,5, 05-72-3, 05-87-2, 05-91-10, 05-77-1, 05-90-1,3,4, 05-273-1, 05-262-2, 05-271-1, 05-222-16, 05-276-2, 05-196-15, 05-270-1, 05-224-10, 05-269-2, 05-282, 05-344-1, 05-277-1, 05-170-2-1, 05-194-5, 05-185-2,3,5,8,11, 05-141-1-12, 05-146-13,14,15, 05-170-9-9,13, 05-137-2-14, 05-169-2-38, 05-191-8, 05-335-2, 05-298-5,10, 05-94-7-1,2,3,4,5,6,7,8, 05-348-2, 05-196-5, 05-268-1, 05-278-2, 05-223-10.
In some embodiments, the DNA fingerprint database is constructed by a method comprising: and (3) PCR reaction steps: carrying out competitive allele specific PCR amplification reaction on a standard melon germplasm resource by adopting the PCR reaction system to obtain a PCR reaction product; SNP locus genotype obtaining step: detecting the PCR reaction product to obtain the genotype of the SNP locus; and (4) directly sequencing.
A detection method for identifying the authenticity of melon germplasm resources comprises the following steps: the method comprises the following steps: detecting the genotype of the SNP locus of the melon to be detected; step two: and (3) judging the germplasm resources of the melon to be detected: if the number of the genotype of the melon to be detected based on the 32 SNP loci and the number of the difference loci of a certain specified germplasm resource in the standard melon germplasm resources in the database based on the genotype of the 32 SNP loci are 0-2, judging the melon to be detected and the specified germplasm resource as similar germplasm resources; if the number of the genotype of the melon to be detected based on the 32 SNP loci and the number of the difference loci of a certain specified germplasm resource in the standard melon germplasm resources in the database based on the genotype of the 32 SNP loci are more than 2, judging the melon to be detected and the specified germplasm resource as different melon germplasm resources; preferably, the result of the determination is obtained from a cluster analysis.
The SNP locus, or the SNP primer combination, or the SNP kit, or the DNA fingerprint database obtained by the construction method, or the detection method is applied to the following X1 or X2: x1: identifying whether the germplasm resources of the melon to be detected belong to one of the standard melon germplasm resources; x2: and identifying the specific germplasm resources of the melon to be detected as the standard melon germplasm resources. "C (B)
Compared with the prior art, the invention has the following beneficial effects:
1. the invention establishes the DNA fingerprint database for identifying the authenticity of the melon germplasm resources based on high-throughput sequencing, can be used for carrying out early identification on the melon germplasm resources in tissues or organs such as seeds, seedlings, leaves and the like, practically protects the rights and interests of breeders, and provides technical support for melon germplasm resource protection.
2. The method provided by the invention can be used for identifying unknown melon germplasm resources and also can be used for identifying the authenticity of known melon germplasm resources. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.
3. The invention utilizes 150 parts of core germplasm resource data in 1175 parts of melon germplasm resource sequencing published by public database NCBI to carry out big data mining of melon germplasm resource SNP, provides candidate sites for melon germplasm resource identification, develops specific primers of the melon germplasm resource SNP by adopting an allele competitive specific PCR method, carries out high-throughput, low-cost and automatic rapid detection, and finally obtains the melon germplasm resource SNP genotype.
4. The reported melon variety identification core locus is not suitable for melon germplasm resources due to different germplasm resources and variety genetic characteristics of melons. By taking more than 2 different SNP sites as a threshold value for identifying the germplasm resources, the 32 SNP sites in the patent with the application number of 201910079722.3 can only identify 76.4% of samples of 150 melon germplasm resources, and the Pearson correlation coefficient based on genetic distance with all SNPs is 0.54, which shows that the 32 SNPs can not effectively identify the melon germplasm resources and can not represent SNPs of all genes. Compared with the prior art for identifying the melon variety based on the SNP locus, the SNP locus for identifying the melon germplasm resources developed by the invention can identify 441 melon germplasm resources, and has strong identification capability; the Pearson correlation coefficient based on genetic distance with the whole genome SNP is high, and the whole genome SNP can be represented; the screening process is more complicated.
Drawings
FIG. 1: comparison of SNP site identification Capacity for different combinations in example 1. The threshold value in the invention is that the genotypes of at least 2 SNP loci between any two samples are different, and the two samples are considered as different melon germplasm resources. The curve of the circular marker is 32 SNPs in the invention, the curve of the triangular marker is 32 SNPs randomly selected, the curve of the square marker is 32 SNPs in patent application 201910079722.3, and the graph of FIG. 1 shows the difference of the identification capability of the 32 SNP sites of different groups on 150 melon germplasm resources.
Fig. 2 and 3: the SNP typing results of the 32 primer sets in part of the tested melon germplasm resources in example 2 are shown schematically.
FIG. 4: a cluster map of 441 test melon germplasm resources on the 32 SNP primer sets set forth in example 2 was created.
FIG. 5: identification capability curve chart of 441 tested melon germplasm with 32 SNP loci in example 2.
Detailed Description
The definition is as follows:
authenticity of melon germplasm resources: essentially refers to the real correspondence of melon germplasm resources and genetic backgrounds thereof; in actual work, whether a given germplasm resource has authenticity means whether the given germplasm resource conforms to a file record (such as a germplasm resource specification, a label and the like).
Germplasm resource homologous genome fragment: refers to non-repetitive (single copy) homologous genomic segments of the same segment of the same chromosome in different germplasm resources of a species, such as the melon, including but not limited to the 441 standard germplasm resources described herein. Non-duplication means that in a germplasm resource, the genome fragment exists only at one genome position, and can be homozygous or heterozygous for polyploidy; there are NO highly homologous genomic fragments elsewhere in the genome of one germplasm resource, but highly homologous genomic fragments are ubiquitous in different germplasm resources, e.g., SEQ ID NO: 97 show one of the homologous genomic fragments between melon germplasm resources.
Interspecies resource homologous genomic fragments for melon: the reference genome of the melon is a genome segment which is homologous with the reference genome sequence and is positioned in the same region of a chromosome in the melon of other germplasm resources except for the reference genome V3.5.1 sequence of the melon DHL 92. For example, with respect to a particular genomic fragment, including but not limited to, genomic fragments that share homology with the reference genomic sequence in the 441 standard germplasm resources of the invention. For example, in a particular case, a small mutation occurs within 20 or more bases upstream and downstream of one or some of the 32 SNPs characterized by the invention relative to the homologous sequence of the reference genome, the mutation is random and has no prevalence within a germplasm resource, whereas examining the SNP after ignoring the mutation, the SNP has prevalence within the germplasm resource.
Corresponding sites on the homologous genomic fragments between germplasm resources: refers to a species of germplasm resources (such as, but not limited to, one of the 441 standard germplasm resources described in this invention of melon) as the reference germplasm resources, which has a specific polymorphic genome segment, in which the base at a specific site (such as, the base at position 21 of the sequence shown in SEQ ID NO: 97) is highly polymorphic (such as, the sequence shown in SEQ ID NO: 97 is mainly G or T at position 21, and possibly other bases), between different germplasm resources, and a sequence downstream of the site (such as, the sequence shown in SEQ ID NO: 97 at positions 1-20 and 22-41) is highly conserved between different germplasm resources (this region may have small mutations, which are random and not diffused into the population, which mutations do not have a prevalence within one germplasm resource, ignoring the mutation and looking at the SNP, the SNP has a prevalence within the germplasm resources), the highly polymorphic base in the highly conserved genomic fragment, among the germplasm resource homologous genomic fragments of the species, is the corresponding site on the germplasm resource homologous genomic fragment.
The version number of the reference genome sequence of the melon DHL92 is V3.5.1 (download address: ftp:// curbitangenomics. org/pub/curbitat/genome/melon/v 3.5.1 /).
The method is based on 1175 muskmelon germplasm resources re-sequencing data published by NCBI (national biological information center), detects whole genome genetic variation, and screens perfect SNP which has high polymorphism, conserved flanking sequences of two wings and unique comparison of the flanking sequences to a reference genome. Perfect SNP has been applied to cucumber, pepper and eggplant, is proved to be superior to randomly screened SNP, and has high typing success rate and stable typing result. Based on a perfect SNP database, a set of germplasm resource identification algorithm is developed, and a group of SNP combinations with high Pearson correlation coefficient and strong germplasm resource identification capability are screened out. Meanwhile, the unit stores thousands of melon germplasms. Based on the method, the melon germplasm resource DNA fingerprint database is constructed, and the validity of the SNP loci of the melon germplasm identification is verified through a KASP platform. The development of the work can greatly solve the problem of melon germplasm redundancy; drawing the background of melon germplasm resources; greatly improves the breeding utilization efficiency and promotes the cultivation of new species.
In a first aspect, the present invention provides core SNP loci (core SNP loci are defined as a group of minimum SNP combinations capable of identifying target germplasm resources and representing genome-wide SNPs as much as possible; the core loci are distinguished from non-core loci by higher polymorphism of the core loci and capability of distinguishing target germplasm resources using minimum markers) for identifying the authenticity of melon germplasm resources, wherein the core SNP loci are selected from any 1 to 32 of the following first SNP loci to the third twelve SNP loci, as shown in Table 1:
a first SNP locus, wherein the first SNP locus is located at 1028867 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or T;
a second SNP locus, wherein the second SNP locus is located at 15305628 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a third SNP locus, wherein the third SNP locus is located at 35346336 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A;
a fourth SNP locus, wherein the fourth SNP locus is located at 1090543 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a fifth SNP locus, wherein the fifth SNP locus is located at 1709164 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a sixth SNP locus, wherein the sixth SNP locus is located at 13775704 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a seventh SNP locus, wherein the seventh SNP locus is located at 7930146 rd chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or A;
an eighth SNP locus, wherein the eighth SNP locus is located at 18037611 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a ninth SNP locus, wherein the ninth SNP locus is located at 28288534 rd chromosome 3 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a tenth SNP locus, wherein the tenth SNP locus is located at 4189054 th chromosome 4 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or G;
an eleventh SNP locus, wherein the eleventh SNP locus is located at 16536384 th chromosome 4 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twelfth SNP locus, wherein the twelfth SNP locus is located at 14981598 th chromosome 5 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or C;
a thirteenth SNP locus, wherein the thirteenth SNP locus is located at 28011864 th chromosome 5 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or C;
a fourteenth SNP locus, wherein the fourteenth SNP locus is located at 8120366 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a fifteenth SNP locus, wherein the fifteenth SNP locus is located at 18900074 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A;
a sixteenth SNP locus, wherein the sixteenth SNP locus is located at 19620053 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a seventeenth SNP site, wherein the seventeenth SNP site is located at 1104024 th chromosome of a7 th chromosome of a melon reference genome or a corresponding site on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the site is G or C;
an eighteenth SNP locus, wherein the eighteenth SNP locus is located at 3254461 th chromosome of a reference genome of the melon, or a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is C or T;
a nineteenth SNP locus, wherein the nineteenth SNP locus is located at 23111340 th chromosome 7 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A;
a twentieth SNP locus, wherein the twentieth SNP locus is located at 4876476 th chromosome 8 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources thereof, and the nucleotide base of the locus is G or C;
a twenty-first SNP locus, wherein the twenty-first SNP locus is located at 6383781 th chromosome 8 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twenty-second SNP locus, wherein the twenty-second SNP locus is located at 32379197 th chromosome 8 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a twenty-third SNP locus, wherein the twenty-third SNP locus is located at 1849869 th chromosome of a melon reference genome 9 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or C;
a twenty-fourth SNP locus, wherein the twenty-fourth SNP locus is located at 12154715 th chromosome of a melon reference genome 9 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a twenty-fifth SNP locus, wherein the twenty-fifth SNP locus is located at 20428406 th chromosome of a9 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twenty-sixth SNP locus, wherein the twenty-sixth SNP locus is located at 9552072 th chromosome of 10 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twenty-seventh SNP locus, wherein the twenty-seventh SNP locus is located at 20472868 th chromosome of the 10 th chromosome of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or T;
a twenty-eighth SNP locus, wherein the twenty-eighth SNP locus is located at 7009967 th chromosome of the reference genome of the melon, or at a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is A or C;
a twenty-ninth SNP locus, wherein the twenty-ninth SNP locus is located at 17070009 th chromosome of the 11 th chromosome of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or C;
a thirtieth SNP locus, wherein the thirtieth SNP locus is located at 27232414 th chromosome of the melon reference genome 11 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or G;
a thirty-first SNP locus, wherein the thirty-first SNP locus is located at 3459537 th chromosome of a 12 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or G;
a thirty-second SNP locus, wherein the thirty-second SNP locus is located at 13742304 th chromosome of the reference genome of the melon or a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is G or A;
wherein the melon reference genome is melon DHL92 reference genome V3.5.1.
In some embodiments, the sequence of the first SNP site and bases upstream and downstream thereof is SEQ ID NO: 97 or a genome fragment homologous between germplasm resources thereof, more preferably a fragment identical to the sequence shown in SEQ ID NO: 97, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequences of the second SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 98 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 98, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the sequences of the third SNP locus and bases at the upstream and downstream are SEQ ID NO: 99 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 99 nucleotide sequences having greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the fourth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 100 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 100, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the fifth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 101 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 101, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the sequences of the sixth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 102 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 102 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the seventh SNP locus and the sequences of bases on the seventh SNP locus and upstream and downstream thereof are SEQ ID NO: 103 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 103 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequences of the eighth SNP locus and bases at the upstream and downstream are SEQ ID NO: 104 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 104 greater than or equal to 95%, 96%, 97%, 98%, or 99%; the ninth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 105 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 105 nucleotide sequence identity greater than or equal to 95%, 96%, 97%, 98% or 99%; the tenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 106 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 106 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequence of the eleventh SNP site and bases on the eleventh SNP site is SEQ ID NO: 107 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 107 greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the twelfth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 108 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 108 greater than or equal to 95%, 96%, 97%, 98% or 99% identity; the thirteenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 109 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 109 by greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the fourteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 110 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 110 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the sequence of the fifteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 111 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 111 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the sixteenth SNP locus and bases on the sixteenth SNP locus is SEQ ID NO: 112 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 112 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the sequence of the seventeenth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 113 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 113 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequence of the eighteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 114 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 114 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the nineteenth SNP site and the sequences of bases on the nineteenth SNP site are SEQ ID NO: 115 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 115, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-second SNP site and the sequences of bases on the twenty-second SNP site are SEQ ID NO: 116 or a germplasm resource homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 116 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the twenty-first SNP locus and the sequences of bases on the twenty-first SNP locus and bases on the twenty-first SNP locus are SEQ ID NO: 117 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence set forth in SEQ ID NO: 117 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequence of the second twelve SNP locus and the base sequences of the second twelve SNP locus are SEQ ID NO: 118 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 118 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the twenty-third SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 119 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 119, or greater than 95%, 96%, 97%, 98%, or 99%; the sequence of the twenty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 120 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 120, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-fifth SNP locus and the sequences of bases on the twenty-fifth SNP locus and the upstream and downstream of the twenty-fifth SNP locus are SEQ ID NO: 121 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 121, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the twenty-sixth SNP locus and the sequences of bases on the twenty-sixth SNP locus and bases on the twenty-sixth SNP locus are SEQ ID NO: 122 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 122 is greater than or equal to 95%, 96%, 97%, 98% or 99%; the twenty-seventh SNP locus and the sequences of bases on the twenty-seventh SNP locus and the upstream and downstream of the twenty-seventh SNP locus are SEQ ID NO: 123 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 123 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequences of the twenty-eight SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 124 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 124, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-ninth SNP locus and the sequences of bases on the twenty-ninth SNP locus are SEQ ID NO: 125 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 125, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the thirty-third SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 126 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 126, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the thirty-first SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 127 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 127, 95%, 96%, 97%, 98% or 99%; the sequences of the third twelve SNP loci and bases at the upper and lower ends of the third twelve SNP loci are SEQ ID NO: 128 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 128 is greater than or equal to 95%, 96%, 97%, 98% or 99%.
In a second aspect, the present invention provides a combination of core SNP primer sets for identifying the authenticity of melon germplasm resources, the core SNP primer sets comprising: a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site; a ninth SNP primer set for amplifying the ninth SNP site; a tenth SNP primer set for amplifying the tenth SNP site; an eleventh SNP primer set for amplifying the eleventh SNP site; a twelfth SNP primer set for amplifying the twelfth SNP site; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a fourteenth SNP primer set for amplifying the fourteenth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a sixteenth SNP primer set for amplifying the sixteenth SNP site; a seventeenth SNP primer set for amplifying the seventeenth SNP site; an eighteenth SNP primer set for amplifying the eighteenth SNP site; a nineteenth SNP primer set for amplifying the nineteenth SNP site; a twentieth SNP primer set for amplifying the twentieth SNP site; a twenty-first SNP primer set for amplifying the twenty-first SNP site; a second twelve SNP primer set for amplifying the second twelve SNP sites; a twenty-third SNP primer set for amplifying the twenty-third SNP site; a twenty-fourth SNP primer set for amplifying the twenty-fourth SNP site; a twenty-fifth SNP primer set for amplifying the twenty-fifth SNP site; a twenty-sixth SNP primer set for amplifying the twenty-sixth SNP site; a twenty-seventh SNP primer set for amplifying the twenty-seventh SNP site; a second eighteen SNP primer set for amplifying the second eighteen SNP site; a twenty-ninth SNP primer set for amplifying the twenty-ninth SNP site; a thirtieth SNP primer set for amplifying the thirtieth SNP site; a thirty-first SNP primer set for amplifying the thirty-first SNP site; a thirty-second SNP primer set for amplifying the thirty-second SNP site.
In some embodiments, the first SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) of the first SNP primer set, the downstream primer (R) of the first SNP primer set, are identical to SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the second SNP primer set, which includes a specific portion of the first upstream primer (F1) of the second SNP primer set, a specific portion of the second upstream primer (F2) of the second SNP primer set, and a downstream primer (R) of the second SNP primer set, are identical to SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the third SNP primer set, which includes a specific portion of the first upstream primer (F1) of the third SNP primer set, a specific portion of the second upstream primer (F2) of the third SNP primer set, and a downstream primer (R) of the third SNP primer set, are identical to SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fourth SNP primer set, are linked to SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fifth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fifth SNP primer set, are linked to SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the sixth SNP primer set, including the specific portion of the first upstream primer (F1) of the sixth SNP primer set, the specific portion of the second upstream primer (F2) of the sixth SNP primer set, and the downstream primer (R) of the sixth SNP primer set, are linked to SEQ ID NOs: 16. SEQ ID NO: 17. SEQ ID NO: 18, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the seventh SNP primer set, including the specific portion of the first upstream primer (F1) of the seventh SNP primer set, the specific portion of the second upstream primer (F2) of the seventh SNP primer set, and the downstream primer (R) of the seventh SNP primer set, are linked to SEQ ID NOs: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighth SNP primer set, including the specific portion of the first upstream primer (F1) of the eighth SNP primer set, the specific portion of the second upstream primer (F2) of the eighth SNP primer set, and the downstream primer (R) of the eighth SNP primer set, are linked to SEQ ID NOs: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the ninth SNP primer set, including the specific portion of the first upstream primer (F1) of the ninth SNP primer set, the specific portion of the second upstream primer (F2) of the ninth SNP primer set, and the downstream primer (R) of the ninth SNP primer set, are linked to SEQ ID NOs: 25. SEQ ID NO: 26. SEQ ID NO: 27 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the tenth SNP primer set, including the specific portion of the first upstream primer (F1) of the tenth SNP primer set, the specific portion of the second upstream primer (F2) of the tenth SNP primer set, and the downstream primer (R) of the tenth SNP primer set, are linked to SEQ ID NOs: 28. SEQ ID NO: 29. SEQ ID NO: 30 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eleventh SNP primer set, including the specific portion of the first upstream primer (F1) of the eleventh SNP primer set, the specific portion of the second upstream primer (F2) of the eleventh SNP primer set, and the downstream primer (R) of the eleventh SNP primer set, are linked to SEQ ID NOs: 31. SEQ ID NO: 32. SEQ ID NO: 33, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the twelfth SNP primer set, which includes a specific portion of the first upstream primer (F1) of the twelfth SNP primer set, a specific portion of the second upstream primer (F2) of the twelfth SNP primer set, and a downstream primer (R) of the twelfth SNP primer set, is identical to the sequence set shown in SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the thirteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the thirteenth SNP primer set, the specific portion of the second upstream primer (F2) of the thirteenth SNP primer set, the downstream primer (R) of the thirteenth SNP primer set, is identical to SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39, greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the fourteenth SNP primer set, which includes a specific portion of the first upstream primer (F1) of the fourteenth SNP primer set, a specific portion of the second upstream primer (F2) of the fourteenth SNP primer set, and a downstream primer (R) of the fourteenth SNP primer set, is identical to SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fifteenth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fifteenth SNP primer set, are linked to SEQ ID NOs: 43. SEQ ID NO: 44. SEQ ID NO: 45 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the sixteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the sixteenth SNP primer set, the specific portion of the second upstream primer (F2) of the sixteenth SNP primer set, and the downstream primer (R) of the sixteenth SNP primer set, are linked to SEQ ID NOs: 46. SEQ ID NO: 47. SEQ ID NO: 48, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the seventeenth SNP primer set, including the specific portion of the first upstream primer (F1) of the seventeenth SNP primer set, the specific portion of the second upstream primer (F2) of the seventeenth SNP primer set, and the downstream primer (R) of the seventeenth SNP primer set, are linked to SEQ ID NOs: 49. SEQ ID NO: 50. SEQ ID NO: 51, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the eighteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the eighteenth SNP primer set, the specific portion of the second upstream primer (F2) of the eighteenth SNP primer set, and the downstream primer (R) of the eighteenth SNP primer set, are linked to SEQ ID NOs: 52. SEQ ID NO: 53. SEQ ID NO: 54 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the nineteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the nineteenth SNP primer set, the specific portion of the second upstream primer (F2) of the nineteenth SNP primer set, and the downstream primer (R) of the nineteenth SNP primer set, are identical to SEQ ID NOs: 55. SEQ ID NO: 56. SEQ ID NO: 57 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twentieth SNP primer set, including the specific portion of the first upstream primer (F1) of the twentieth SNP primer set, the specific portion of the second upstream primer (F2) of the twentieth SNP primer set, and the downstream primer (R) of the twentieth SNP primer set, are linked to SEQ ID NOs: 58. SEQ ID NO: 59. SEQ ID NO: 60, preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; the twenty-first SNP primer set including a specific portion of the first upstream primer (F1) of the twenty-first SNP primer set, a specific portion of the second upstream primer (F2) of the twenty-first SNP primer set, a downstream primer (R) of the twenty-first SNP primer set, and a sequence listing corresponding to SEQ ID NOs: 61. SEQ ID NO: 62. SEQ ID NO: 63 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the second twelve SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) of the second twelve SNP primer set, the downstream primer (R) of the second twelve SNP primer set, and the sequence numbers of SEQ ID NOs: 64. SEQ ID NO: 65. SEQ ID NO: 66 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the thirteenth SNP primer set including a specific portion of the first upstream primer (F1) of the thirteenth SNP primer set, a specific portion of the second upstream primer (F2) of the thirteenth SNP primer set, and a downstream primer (R) of the thirteenth SNP primer set, are identical to SEQ ID NOs: 67. SEQ ID NO: 68. SEQ ID NO: 69 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twenty-fourth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the twenty-fourth SNP primer set, are identical to SEQ ID NOs: 70. SEQ ID NO: 71. SEQ ID NO: 72 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twenty-fifth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the twenty-fifth SNP primer set, are identical to SEQ ID NOs: 73. SEQ ID NO: 74. SEQ ID NO: 75 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twenty-sixth SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) and the downstream primer (R) of the twenty-sixth SNP primer set, respectively, have the same sequence as SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the twenty-seventh SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the twenty-seventh SNP primer set, are identical to SEQ ID NOs: 79. SEQ ID NO: 80. SEQ ID NO: 81 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the second eighteen SNP primer set, including the specific portion of the first upstream primer (F1) of the second eighteen SNP primer set, the specific portion of the second upstream primer (F2) of the twenty-eighth SNP primer set, the downstream primer (R) of the second eighteen SNP primer set, are identical to SEQ ID NOs: 82. SEQ ID NO: 83. SEQ ID NO: 84 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twenty-ninth SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) and the downstream primer (R) of the twenty-ninth SNP primer set, respectively, have the same sequence as SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the thirtieth SNP primer set comprises a specific part of the first upstream primer (F1) of the thirtieth SNP primer set, a specific part of the second upstream primer (F2) of the thirtieth SNP primer set and a downstream primer (R) of the thirtieth SNP primer set, which are respectively connected with the sequence numbers of SEQ ID NOs: 88. SEQ ID NO: 89. SEQ ID NO: 90 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the thirty-first SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the thirty-first SNP primer set, are identical to SEQ ID NOs: 91. SEQ ID NO: 92. SEQ ID NO: 93 greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the third twelve SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) and the downstream primer (R) of the third twelve SNP primer set, respectively, are identical to SEQ ID NOs: 94. SEQ ID NO: 95. SEQ ID NO: 96 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; preferably, one primer of each of said primer pairs is linked to a fluorescent molecule, more preferably said fluorescent molecule is selected from FAM, HEX.
In a preferred embodiment, the SNP primer combination is selected from one or more of the primer sets 01 to 32; the DNA sequence information of the primer groups 01-32 is shown in a sequence table SEQ ID: 1-96, see table 2.
In the primer group, the 5' end of the upstream primer can be provided with a fluorescent label sequence for fluorescent PCR detection, the first upstream primer and the second upstream primer in each group of primers are connected with different fluorescent molecules, and more preferably, the fluorescent molecules are selected from FAM and HEX;
for example, the fluorescence signal of FAM fluorescent tag sequence is blue, and the fluorescence signal of HEX fluorescent tag sequence is red.
In a third aspect, the present invention provides a core SNP kit for identifying the authenticity of melon germplasm resources, the SNP reagents being formulated as a competitive allele-specific PCR reaction system, preferably comprising:
in the SNP primer sets, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer of each primer set in the system is 2:2: 5;
reagents, consumables and instruments in the reaction system were provided by LGC company, including reagent amounts, usage and the whole experimental procedure were performed according to the LGC company's operating manual KASP user guide and manual (www.lgcgenomics.com), KASPar reaction was performed in 384 well plate (Part No. KBS-0750-001) or 96 well plate (Part No. KBS-0751-001), and the reaction system was 3. mu.l or 1. mu.l, as shown in the following table.
Table: KASP reaction system of 384-well plate or 96-well plate
Figure RE-GDA0002754435980000271
The preparation method of the kit comprises the step of packaging each primer in any one primer group separately.
In a fourth aspect, the invention provides the above mentioned DNA fingerprint database for melon germplasm resources based on core SNP markers, where the above mentioned DNA fingerprint database includes: and (3) the genotype of the core SNP locus of the standard melon germplasm resource.
The standard melon germplasm resources comprise 441 melon germplasm resources selected from the following:
PLANTERE JUMLED 76134, PERSIAN MEDIUM, CURTEOWPE PLANTER JUMBO, 76W-2-5, 76W-3, CATTA LOLLE, 76W-1, 76W-2, 76W-4, CNTALOU PE P.M.R450, CASABA GOLDEN QUOMI GUAUT, ZUO, mating, PF, mating, Japan, GOLDEN mating, Japan, POLIOORR F (RS 815), CATTOR HYBRID F, PLATE PE, PLATE, LONGANG mating, VR 6072, CA 456, CA M761232, (MUSKMELON) BURPEE' S BRIS, GURON mating, japan3, ェリザベス, HONEY ROCK, first generation OF Pachyrhizus communis, swan, MELAO HALE 'S BEST, bvrc5, Anemone MELO, CUCUCUMIS MELO L., IROQUOIS, ALASKA F1, サカタ mating プリンスメロン PF6, Japan4, サカタ mating ェ - ェ, ェ mating ェ - ェ, golden nine MELON, ェ - ェ, HYBRID SUMMET, JUMBO HALS BEST, type 1, type 2, BUEE' S AMBROSHIBRID F ェ, HERADES OF GOLO, SSIC HYBRD F ェ, BRA- ェ -ARO ESPEAL, bvV BO ェ, CV.HONEYOYW, POLIDOOR ェ, CLA MELO MELT, CALMOCHORON ェ, CALSTAVSTAVSTAVSTAVSBO, CAVSBO- ェ, CAVSBO-PHONZO-PHONYOR ェ, CAVSB ェ, WAVSBO-PHONZO ェ, CAVSB ェ, WAVSBO-PHONZO ェ, CAVSB ェ, WAVSB ェ, WAVSBO-PHONZO ェ, WAVSB ェ, WAVSBO-PHONZO-PHO, sunrise (original Sweet), snow char (Hami melon type), Jade Dew (Orien Sweet), farmer NO.2(Oriental Sweet, Silver Light (Oriental Sweet), Pride, Hearts of Gold, Delicous 51, Honey Rock, Roadside, Samson, Hales Best Jumbo, SUPER KET HYBRID, PLANTER ' S JUMBO VGR679, GUTO 45 QI L620, TOP MARK VGG 2663, PERLITA VGR, EDISTO VGL 415, JUMBO HALE ' S BEST 97849, FERRY-MORE SEEDS, HALE ' BEST JUMBO, MUSKON 6593-8, bvvrc 2, Sweet potato, snow melon, Japanese melon, Sweet potato, Japanese melon メロン, Sweet potato 3526, Sweet potato 3627, Sweet potato 3655, Sweet potato 3526, Sweet potato 3645, Sweet potato 3655, Sweet potato 35, Sweet potato 3655, Sweet potato 3526, Sweet potato 1, Sweet potato 35, Sweet potato 1, Sweet potato 3680, Sweet potato 1, Sweet potato 35, Sweet potato 1, Sweet potato 35, Sweet potato 357, Sweet potato, ハ - ベスト 5, ハ - ベスト 6, white muskmelon, Horey louge, cotton white, Taiwan sweet, CD ELDORATO YELLOW, CD CASTOR, Yanjiao sweet, Daphne potpourri, Huangdao, M26, M29, M285, ANANAS, CV SPECIAL HONEY DEW EYPE, Cucumis melo, OGEN, Japanese, Tiger skin Cucumis melo, Changlan persimmon 2, Ielizabeth, Galaoui, CHINESE MELON, ALIER F1, ANWAS D 'AMERIGUE, IDIAAN, TOJONA, SHIRAY, white dao, Huadao, green cucumber, red lantern, white sweet, black-peel MELON, bvrc8, Hongcheng MELON, AVGNON, manna, ZANDA (SANDA 03, ZA 03-03, ZA-03, ZA-03-12, SANIA-259, SAVOR F1, ANWAS D' AMORE E DEE, SAE DEE, SANIA, SANI-35, SANI-NI, 03-252-3-54, 03-252-3-90, 03-252-3-41, 03-252-3-69, 03-210-17-2,7, 03-204-1-1,3,5,6,7, 03-203-1-12,22,23,24,25, 03-208-1-15,16, 03-210-16-2, 03-209-1-1,11, 03-209-9-12,13,14,16,17,18,19, 03-210-3-9, 03-206-7,8,9,12, 03-204-2-18, 03-199-16-1, 03-199-18-11, 03-198-31-1, 03-205-2-9, 03-211-2, 03-210-13-1, 03-210-11-1, 03-199-14-1, 03-207-2-12, 03-195-1-4,5, 03-253-3,5.6,9,12,15, 03-252-1-12, 03-310-1-2,3, 03-337-2-20, 03-314-3-3, 03-308-2-5, 03-328-3, 03-345-9, 03-339-1-4,10,18, 03-325-2-2, 03-317-1-1, 03-299-1-5, 03-283-4-6, 03-296-2-8, 03-310-2-1,3,5,8, 03-210-14-4,6,9, 03-209-6-1, 03-199-17-24, 03-209-3-1,2,3,5,6,11, 03-203-1-9, 03-210-1-7, 03-210-2-9, 03-205-1-4,7, 03-199-12-18, 03-198-8-3, 03-198-7-11, 03-195-2-3, 03-210-11-19,41, 03-198-3-15, 03-210-11-17,24,28, 03-210-12-10, 03-210-2-19, 03-307-6-5, 03-293-1-6, 03-325-2-13, 03-252-2-17, 03-236-1,3,4,5,6,11, 03-295-1-7, 03-298-7-9,10, 03-291-1,3, 03-341-2,4,5,6,8, 03-283-1-4, 03-252-3-26, 03-339-2-4,6, 03-210-11-31, 03-196-2-10, 03-196-2-16, 03-196-3-20, 03-210-4-1, 03-210-8-2, 03-204-3-19,20,21,23, 03-210-9-1-2,3,6, 03-210-11-15,36,37, 03-210-10, 03-210-16-17, 03-210-16-13, 03-198-14-2, 03-210-11-2,25,29, 03-210-4-5, 03-210-7-1, 03-210-5-9, 03-210-6-2, 03-209-4-2, 03-198-10-10, 03-339-3-9,10, 03-320-2,5, 03-223-2-19, 03-287-1-1,10, 03-307-2-3, 03-306-1-8, 03-285-1-1, 03-318-3, 03-283-5-9, 03-289-2-5, 03-315-1,4, 03-310-3-3, bvrc15, 03-304-1-9, 03-286-3-1, 03-314-1-1, 03-319-3,4,5, 03-312-3-4,5, 03-314-2-1, 03-286-1-12,13, 03-342-2-9, 03-343-1-4, 03-346-2,3,4,5,6,7, 03-344-3,6, 03-297-8, 03-342-1-6,7, 03-312-4, 03-316-1, bvrc2, 04-562-1, 04-560-1, 04-555-7, 04-552-1, bvrc4, 04-551-6, 04-560-6, 04-545-7, 04-533-4, 04-1, 04-519-2, 04-539-1, 04-537-5, 04-521-12, 05-189-8, 04-528-1, 04-513-2, 04-511-1, 04-556-5, 04-558-1, 04-557-7, 04-556-1, 04-552-15, 04-557-1, 04-538-2, 04-528-12, 04-529, 04-517-1, 04-551-2, 04-563-1, 04-561-7, 04-554, 04-552-5, 04-532-2, 04-518-1, 04-523-1, 04-568-1, 04-566-9, 04-571-1, 04-571-13, 04-576-1, 04-566-1, 04-571-10, 04-565-2, 04-599-1, 04-570-6, 04-567-9, 04-578, 05-60-8, 05-56-3, 05-60-7, 05-65-4, 05-67-10, 05-63-1, 05-69-8, 05-67-7, 05-55-2, 05-69-6, 05-51-7, 05-41-6, 05-40-2, 05-46-15, 05-46-11, 05-48-5, 05-51-4, 05-50-8, 05-50-2, 05-50-9, 05-45-1, 05-38-6, 05-45-14, 05-38-9, 05-45-8, 05-93-4-1, 05-71-2, 05-77-4, 05-86-1, 05-70-1, 05-79-1, 05-70-4-2, 05-63-6, 05-66-2, 05-66-7, 05-51-9, 05-66-9, 05-67-1, 05-46-7, 05-44-1, 05-95-1-21,23,26, 05-95-3-1,3,4,6,11,12, 05-94-2-1,10,31,35, 05-93-1-22, 05-88-6, 05-76-2, 05-93-3-7, 05-93-4-26, 05-99-3-9,11,22,28,50,52,58,68,86, 05-99-12-2, 05-99-24-25,32,37, 05-99-14-8,9,11, 05-98-7-1,2,5, 05-97-1-5,9, 05-96-7,9,12,14,15,16,18, 05-99-7-12,13, 05-95-8-10,25,28,30,31, 05-99-19-34,36,37,42,44, 05-100-3-3,7,9,14, 05-171-2-10,16, 05-193-2, 05-136-20, 05-137-1-2, 05-133-3-1,2, 05-73-1, 05-80-1, May-83, 05-81-3, 05-77-8-3,5, 05-72-3, 05-87-2, 05-91-10, 05-77-1, 05-90-1,3,4, 05-273-1, 05-262-2, 05-271-1, 05-222-16, 05-276-2, 05-196-15, 05-270-1, 05-224-10, 05-269-2, 05-282, 05-344-1, 05-277-1, 05-170-2-1, 05-194-5, 05-185-2,3,5,8,11, 05-141-1-12, 05-146-13,14,15, 05-170-9-9,13, 05-137-2-14, 05-169-2-38, 05-191-8, 05-335-2, 05-298-5,10, 05-94-7-1,2,3,4,5,6,7,8, 05-348-2, 05-196-5, 05-268-1, 05-278-2, 05-223-10.
In a fifth aspect, the invention provides a method for constructing the melon germplasm resource DNA fingerprint database, which comprises the following steps:
s1: KASP reaction: carrying out competitive allele specificity PCR amplification reaction on the standard melon germplasm resources by adopting the reaction system to obtain a PCR reaction product;
the PCR reaction program is: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles.
S2: SNP locus genotype acquisition: and detecting the PCR reaction product to obtain the genotype of the SNP locus.
The above detection method may be selected from: for fluorescent signal detection, direct sequencing and restriction enzyme digestion are carried out.
In a sixth aspect, the invention provides an authenticity detection method for identifying melon germplasm resources, which comprises the following steps:
s1, detecting the genotype of the SNP locus of the melon to be detected:
respectively taking genome DNA of the muskmelon to be detected as a template, and respectively adopting the primer group in the SNP primer combination to carry out competitive allele specificity PCR amplification reaction to obtain PCR amplification products;
s2, judging the germplasm resources of the melon to be tested: comparing the genotype of the SNP locus of the melon to be detected with the DNA fingerprint database, obtaining a result through cluster analysis and judging, wherein the judgment standard is as follows:
if the number of the ectopic difference points between the melon germplasm resource to be detected and a standard melon germplasm resource (to-be-detected melon germplasm resource) is more than 2, judging the melon germplasm resource to be detected and the standard melon germplasm resource as different melon germplasm resources; the greater the number of differential sites, the more distant the genetic relationship.
If the number of different sites between the melon germplasm resource to be tested and a standard melon germplasm resource (to-be-tested melon germplasm resource) is 0-2, the melon germplasm resource to be tested and the standard melon germplasm resource are judged to be similar melon germplasm resources.
In a seventh aspect, the invention provides the above SNP sites, SNP primer combinations, SNP kits, DNA fingerprint database detection methods, for use in X1 or X2:
x1: identifying whether the germplasm resources of the melon to be detected belong to one of the standard melon germplasm resources;
x2: identifying the specific germplasm resources of the melon to be detected as standard melon germplasm resources;
both X1 and X2 belong to the application of identifying the authenticity of melon germplasm resources, wherein the authenticity of melon germplasm resources is identified.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1
Acquisition of site and primer combination for identifying authenticity of melon germplasm resources
Discovery of 32 core SNP sites
The method screens 150 core germplasm resources by group structure and main component analysis based on the resequencing data of 1175 melon germplasm resources. And (3) deeply excavating to obtain 32 SNP sites by using the 150 core germplasm resources. The 150 melon germplasm resources comprise two subspecies, 6 varieties. The method comprises the main germplasm resource types in the Chinese market, has great differences in multiple characters such as fruit shape, flesh color, peel color and the like, and has high genetic diversity.
The core SNP site screening criteria are as follows: (1) the SNP sites with MAF <0.05 and deletion rate >0.3 in the re-sequencing SNP dataset were removed to obtain 1124208 SNPs. (2) In the result (1), 483745 SNPs were obtained by eliminating the SNP sites having other mutations within 30bp of each of the right and left wings. (3) The sequence composed of 30bp above and below the SNP site is aligned with the reference genome blast, and if the sequence can be aligned to the reference genome at more than two positions (including two positions), the SNP site is removed, and 549435 SNPs are obtained. (4) Extracting 60bp of each SNP flanking sequence to design a KASPar SNP primer, and removing SNP sites failed in primer design to obtain 484273 SNPs; among these SNP sites, 32 randomly selected SNP sites in FIG. 1 were randomly selected by random selection using a random function. (5) In order to screen a representative set of SNPs, the Pearson correlation coefficient between the genetic distance calculated from the candidate SNP site and the genetic distance calculated from 484273 SNPs is required to be more than 0.90; meanwhile, at least 2 SNP genotypes are different between any two germplasm resources. And (3) programming by using java to meet the condition, and finally screening out 32 SNP sites for identifying melon germplasm resources.
The basic information of the 32 SNP sites is detailed in Table 1. Wherein the position of the SNP locus on the chromosome is determined based on the comparison of the reference genome sequence of the melon DHL92, and the version number of the reference genome sequence of the melon DHL92 is V3.5.1 (the download address is: ftp:// curbitangenetics. org/pub/curbitat/genome/melon/v3.5.1 /).
TABLE 1.32 basic information of SNP sites
Figure RE-GDA0002754435980000321
Figure RE-GDA0002754435980000331
Figure RE-GDA0002754435980000341
Figure RE-GDA0002754435980000351
Second, 32 SNPs to 150 muskmelon germplasm resource efficiency evaluation
In FIG. 1, with more than 2 SNP sites as the threshold for identifying germplasm resources, the circular marker curve represents the identification capability of the 32 SNPs in the invention on 150 melon germplasm resources, and the result shows that the discrimination rate of the 32 SNPs in 150 melon germplasm resources is 100%; the triangular marker curve represents the identification capability of 32 SNPs randomly selected from the 484273 SNP loci obtained in the step (4) of "discovery of one and 32 core SNP loci" in example 1 on 150 melon germplasm resources, and the discrimination rate is 81.0%; the square marker curve represents the capability of identifying 150 melon germplasm resources by 32 SNPs in the patent (application No. CN201910079722.3), and the discrimination rate was 76.4%.
Thirdly, obtaining of primer combination for identifying melon germplasm resource authenticity
And (3) developing a primer combination which can represent the whole genome SNP and is suitable for identifying melon germplasm resources by using an allele competitive specific PCR (KASP) method according to the 32 SNP loci discovered in the step one.
The SNP primer set consists of 32 primer sets, and the name of each primer set is shown in Table 2. Each primer group consists of 3 primer sequences, comprises a first upstream primer, a second upstream primer and a downstream primer, and is used for amplifying one SNP site. The nucleotide sequences of the individual primers in the 32 primer sets are shown in Table 2. In columns 2-4 of Table 2, the fluorescent tag sequence for labeling FAM is single underlinedGAAGGTGACCAAGTTCATGCTDouble-underlined is a fluorescent tag sequence for labeling HEXGAAGGTCGGAGTCAACGGATTThe sequence of the specific part is not underlined.
Table 2: statistical table of SNP primer nucleic acid sequences of 32 SNP sites
Figure RE-GDA0002754435980000352
Figure RE-GDA0002754435980000361
Figure RE-GDA0002754435980000371
Figure RE-GDA0002754435980000381
Figure RE-GDA0002754435980000391
Example 2
This example is a validation test of the SNP primer combination developed in example 1, and is also based on the construction of the melon germplasm resource DNA fingerprint database with the 32 core SNP markers.
The 441 tested melon germplasm resources in this example are all common excellent germplasm resources or germplasm resources introduced abroad, and are all stored in the vegetable center germplasm resource library of agriculture and forestry academy of sciences in Beijing. The details are shown in Table 3 below.
Table 3: sources of planting resources
Figure RE-GDA0002754435980000401
Figure RE-GDA0002754435980000411
Figure RE-GDA0002754435980000421
Figure RE-GDA0002754435980000431
Figure RE-GDA0002754435980000441
Figure RE-GDA0002754435980000451
Figure RE-GDA0002754435980000461
Figure RE-GDA0002754435980000471
Figure RE-GDA0002754435980000481
1. Obtaining the genome DNA of the tested melon germplasm resources:
and respectively extracting the genome DNA of 441 leaves (30 seeds of each germplasm resource grow out of true leaves and equal amount of leaves are picked and mixed) of the test melon germplasm resources by a Cetyl Trimethyl Ammonium Bromide (CTAB) method to obtain the genome DNA of the test melon germplasm resources and the genome DNA of the test melon germplasm resources to be tested.
The CTAB method is specifically operated as follows: 1) equivalently picking and mixing the tender leaves of the germplasm resource material, and dehydrating for 24 hours by using a freeze dryer (CoolSafe 55-4); 2) drying the leaves, grinding the leaves with a high-throughput grinder (Geno/Grind6875), and putting 200mg of dry leaf powder into 200ul of centrifuge tube; 3) adding 800 μ L CTAB extractive solution (2% CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0, 20mM EDTA pH8.0, 1% PVP-40 and 0.2% beta-mercaptoethanol), shaking the extractive solution and dry leaf powder thoroughly, placing in a 65 deg.C water bath, and shaking once every 10 min; 4) after 30min an equal volume of chloroform/isoamyl alcohol (v: v ═ 24: 1) shaking again and standing for 20 min; 5) placing in a centrifuge, centrifuging at 10000rpm/min for 10min, carefully sucking 500ul of supernatant, transferring to a new centrifuge tube, adding pre-cooled 400ul of isopropanol, slightly inverting, mixing, and placing in a refrigerator at-20 deg.C for 30 min; 6) centrifuging the frozen centrifuge tube at 4 deg.C for 10min at 12,000r/min, carefully removing supernatant, adding 70% ethanol solution, and carefully sucking for 2 times; 7) drying under natural condition, adding 100 μ L ddH2Dissolving DNA in the O to finally obtain the genome DNA of the melon germplasm resource to be tested; 8) the concentration of the DNA after the dissolution is carried outAnd (4) detecting, and placing in a refrigerator at 4 ℃ for standby.
The quality and concentration of the stored genome DNA both need to meet the PCR requirement, and the standard of the standard is as follows: agarose electrophoresis showed that the DNA band was single and not dispersed significantly; detecting that the ratio of A260 to A280 is about 1.8 and the ratio of A260 to A230 is more than 1.8 by using an ultraviolet spectrophotometer Nanodrop2000 (Thermo); the concentration of DNA ranged from 30-50 ng/. mu.L.
2. Obtaining a PCR amplification product: and (3) taking the genomic DNA of 441 tested melon germplasm resources as templates, and respectively adopting 32 primer groups to perform competitive allele-specific PCR amplification. In each PCR reaction system, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer is 2:2: 5.
Reagents, consumables and instruments in the reaction system were provided by LGC company, including reagent amounts, usage and the whole experimental procedure were performed according to the LGC company's operating manual KASP user guide and manual (www.lgcgenomics.com), KASPar reaction was performed in 384 well plates (Part No. KBS-0750-001) or 96 well plates (Part No. KBS-0751-001), and the reaction system was 3. mu.l or 10. mu.l, as shown in Table 4 below.
Table 4: KASP reaction system of 384-well plate or 96-well plate
Figure RE-GDA0002754435980000491
Kits supplied by LGC company or otherwise having AS-PCR detection capability
The reaction procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min, and continuous amplification for 26 cycles; final extension: 10min at 72 ℃. The resulting amplification product was stored at 4 ℃ before electrophoresis.
3. And (3) fluorescent signal detection: after the step 2 is completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, the fluorescence value is read through FAM and HEX light beam scanning of a microplate reader (the reading value is observed when the FAM fluorescent label sequence is at 485nm of exciting light and 520nm of emitted light, the reading value is observed when the HEX fluorescent label sequence is at 528nm of exciting light and 560nm of emitted light), and the genotype of 441 tested melon germplasm resources based on each SNP site is judged according to the color of the fluorescence signal.
The specific judgment principle is as follows:
if a certain SNP locus of a certain test melon germplasm resource shows a blue fluorescent signal, the genotype of the test melon germplasm resource based on the SNP locus is homozygote of a complementary base of the 1 st base at the 3' tail end of a first upstream primer for amplifying the SNP locus;
if the test melon germplasm resource shows a red fluorescent signal based on a certain SNP locus, the genotype of the test melon germplasm resource based on the SNP locus is homozygous for the complementary base of the 1 st base at the 3' end of the second upstream primer for amplifying the SNP locus;
if a certain SNP locus of a certain test melon germplasm resource shows a green fluorescent signal, the genotype of the test melon germplasm resource based on the SNP locus is a heterozygote type, one base is a complementary base of the 1 st base at the 3 'tail end of a first upstream primer for amplifying the SNP locus, and the other base is a complementary base of the 1 st base at the 3' tail end of a second upstream primer for amplifying the SNP locus.
The genotype of each germplasm resource in each of the 441 test melon germplasm resources at each of the 32 SNP sites constitutes a DNA fingerprint database of the melon germplasm resources based on the 32 core SNP markers, and the database can be used for identifying whether or not a certain unknown melon germplasm resource belongs to the 441 test germplasm resources, or specifically belongs to one of the 441 test melon germplasm resources.
If the fluorescence signal is weak after the PCR amplification is finished and affects data analysis, cycles (denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min and 5 cycles) can be added until the result is satisfactory.
As shown in fig. 2 and 3, the fluorescence signal of PCR amplification products of each SNP site at 441 melon varieties (441 samples) tested clearly appeared in 3 forms: 1) blue, aggregating in the sample near the X-axis, genotype is the allele that joins the HEX fluorescent tag sequence; 2) red, aggregating samples near the Y-axis, genotype is the allele that connects FAM fluorescent tag sequences; 3) green, X-axis and Y-axis samples, genotype is a heterozygote of the two alleles. Due to poor DNA quality or too low a concentration, the amplification product was not clearly typed, and samples with few or no fluorescent signals or no discrimination were shown to be pink. Therefore, the amplification effect of each primer is good, and the genotypes of 441 tested melon germplasm resources can be obviously distinguished.
4. Cluster analysis
And carrying out cluster analysis on the 441 tested melon germplasm resources by using MEGA7 software. The cluster map is shown in fig. 4. The result shows that the 32 primer groups can completely distinguish 441 tested melon germplasm resources in the table. Therefore, the SNP primer combination developed in the embodiment 1 can be applied to construction of DNA fingerprint databases of melon germplasm resources and identification of variety authenticity.
5. Evaluation of resource efficiency of 441 melon germplasm resources by 32 SNPs
The melon germplasm resource authenticity identification can reduce the workload by adopting a sequential analysis mode. The invention compares the relation between the number of SNP markers (namely the number of primer sets) and the distinguishing rate of 441 melon germplasm resource regions to be tested. In fig. 5, 441 melon germplasm could be completely identified by 32 SNPs.
Example 3
The embodiment is a method for detecting whether a to-be-detected melon germplasm resource belongs to the germplasm resources of 441 to-be-detected melon germplasm resources, the to-be-detected melon germplasm resource is unknown, and whether the to-be-detected melon germplasm resource is one of the 441 germplasm resources needs to be obtained through the detection method of the embodiment. The actual melon germplasm resources to be tested in the embodiment are PI 618838, and do not belong to the 441 melon germplasm resources; and under the condition that the melon germplasm resources to be tested are known, verifying whether the melon resources to be tested belong to one of the 441 melon resources by using the method disclosed by the invention. If the verification result is in accordance with the actual result, the method of the invention can be used for detecting whether the unknown melon germplasm resource is one of the 441. The detection method of the embodiment comprises the following steps:
1. obtaining of genome DNA of muskmelon germplasm resource to be detected
The leaves of the melon germplasm resources to be tested are taken from the test base of the vegetable research center of the academy of agriculture and forestry, Beijing.
According to the method of the step 1 in the embodiment 2, the 'leaf of the melon germplasm resource to be tested' is replaced by the 'leaf of the melon germplasm resource to be tested', and other steps are not changed, so that the genome DNA of the melon germplasm resource to be tested is obtained.
2. Configuration of SNP primer and PCR reaction system
Replacing the 'genomic DNA of the melon germplasm resource to be tested' with the 'genomic DNA of the melon germplasm resource to be tested' according to the method in the step 2 in the embodiment 2, and carrying out competitive allele specific PCR reaction without changing other steps to obtain a PCR product of the melon germplasm resource to be tested.
3. Fluorescence signal detection
Taking a PCR product of the melon germplasm resource to be detected, judging the genotype of the melon germplasm resource to be detected based on each site in the 32 SNP sites according to the color of the fluorescent signal according to the method of the step 2 in the embodiment 2, wherein the specific judgment principle is as described in the step 3 in the embodiment 2, and replacing the 'melon germplasm resource to be detected' with the 'melon germplasm resource to be detected'.
4. Specific germplasm resource judgment of melon germplasm resources to be detected
Comparing the genotypes of the 32 SNP loci of the melon germplasm resources to be detected with the melon germplasm resource DNA fingerprint database consisting of 91 test melon germplasm resources in the embodiment 2, counting the number of different loci of the melon germplasm resources to be detected and each test melon germplasm resource, and then judging as follows:
if the number of the ectopic difference points between the melon germplasm resource to be detected and a standard melon germplasm resource (to-be-detected melon germplasm resource) is more than 2, judging the melon germplasm resource to be detected and the standard melon germplasm resource as different melon germplasm resources; the greater the number of differential sites, the more distant the genetic relationship.
If the number of different sites between the melon germplasm resource to be tested and a standard melon germplasm resource (to-be-tested melon germplasm resource) is 0-2, the melon germplasm resource to be tested and the standard melon germplasm resource are judged to be similar melon germplasm resources.
The result shows that the number of the different sites of the melon germplasm resource to be detected and the number of the different sites of the melon germplasm resources to be detected on the 32 SNP sites are more than 4, so that the melon germplasm resource to be detected does not belong to any one of the 441 melon germplasm resources to be detected, namely the melon germplasm resource to be detected is not one of the 441 melon germplasm resources to be detected.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> agriculture and forestry academy of sciences of Beijing City
<120> SNP locus primer combination for identifying melon germplasm resource authenticity and application
<130> C1CNCN180989
<141> 2020-09-07
<160> 128
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 1
acacacacac tattagcata aaatatcaaa g 31
<210> 2
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 2
aaacacacac actattagca taaaatatca aat 33
<210> 3
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 3
cgtttttatg atatctgatt cctttttctt gaaa 34
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 4
ttagatggta tcaaaacaaa tggttcaaag 30
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 5
tgttattcaa atgggcttag ttgaaaatca agtaagaata t 41
<210> 6
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 6
cttgaaccat ttgttttgat accatcaagt ttt 33
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 7
ccaaattttg gctcgccgga ca 22
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 8
ccaaattttg gctcgccgga ct 22
<210> 9
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 9
agatgcggaa agaaaaattc ccagaatatt a 31
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 10
cggttttctt ttcttttttt caaataggac 30
<210> 11
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 11
gcggttttct tttctttttt tcaaatagga t 31
<210> 12
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 12
cgtaacattt gtttttttgt ttaactattt ttggat 36
<210> 13
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 13
tctggtgtta ttcaaatggg cttagt 26
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 14
ctggtgttat tcaaatgggc ttagc 25
<210> 15
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 15
cccaatctcc ttacaaagca aaacaaaac 29
<210> 16
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 16
aatcggatgc cattgtctaa aaccttt 27
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 17
tcggatgcca ttgtctaaaa ccttg 25
<210> 18
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 18
gcatgttttt ggggttcaca aaaggttta 29
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 19
aaggaaattc tgtcagtcaa atgttagata 30
<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 20
ggaaattctg tcagtcaaat gttagatg 28
<210> 21
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 21
gaaaagttct acagaagtat ctttggtaat ttat 34
<210> 22
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 22
gtgttttggg gtattagtgt agtcaaaaa 29
<210> 23
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 23
gttttggggt attagtgtag tcaaaag 27
<210> 24
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 24
tggacatgga aaaatttctt gattggatca a 31
<210> 25
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 25
gactacgata gaagagatat gtctctaa 28
<210> 26
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 26
actacgatag aagagatatg tctctag 27
<210> 27
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 27
acacagcaaa cattttacat agtgcattgt a 31
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 28
acaattcatc ccaccgccac c 21
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 29
gacaattcat cccaccgcca ct 22
<210> 30
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 30
gatcagggtt tacgatgacg agacata 27
<210> 31
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 31
ttctcattag tcatttggta ttgcaagc 28
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 32
atttctcatt agtcatttgg tattgcaagt 30
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 33
acgaagaagt acgctgtcag tcgat 25
<210> 34
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 34
atgcattatt gttcatggaa ttcgcac 27
<210> 35
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 35
gatgcattat tgttcatgga attcgcat 28
<210> 36
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 36
tcttcagtga attatctgaa tgtgcaaata aata 34
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 37
gtggtcagtt aatcctgtca ccg 23
<210> 38
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 38
ttgtggtcag ttaatcctgt cacca 25
<210> 39
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 39
acatagagtt aacatttgtg tttaagtttg gtta 34
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 40
gtgtctcctc tgttgaagag gc 22
<210> 41
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 41
agtgtctcct ctgttgaaga ggt 23
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 42
cgggaacact gctttgcacc cta 23
<210> 43
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 43
gttacagctt agagtctaga acaaattc 28
<210> 44
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 44
gttacagctt agagtctaga acaaatta 28
<210> 45
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 45
ctaaaattac tctccttacg tcagtttttc ttt 33
<210> 46
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 46
gcgtaagcct cgaaagaaac cg 22
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 47
aagcgtaagc ctcgaaagaa acca 24
<210> 48
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 48
attgtcaggt tgccttctca ccaaataa 28
<210> 49
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 49
tctttctttc gttcagcgtc ttcg 24
<210> 50
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 50
tctttctttc gttcagcgtc ttcc 24
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 51
atcggagatg gtggcgggag aa 22
<210> 52
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 52
atgaacaaca tacttttatt tgaagacaca ata 33
<210> 53
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 53
gaacaacata cttttatttg aagacacaat c 31
<210> 54
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 54
gttcctatat acaggatatc atttcactat gaa 33
<210> 55
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 55
ttgcacctta gtttgaagaa aatggag 27
<210> 56
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 56
ttttgcacct tagtttgaag aaaatggaa 29
<210> 57
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 57
ctccacacat atagagagta tgatgcaa 28
<210> 58
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 58
ataggcctct aaattttata gggtaactta 30
<210> 59
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 59
aggcctctaa attttatagg gtaacttg 28
<210> 60
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 60
tttggacaca aattcaaaca cgaaatacct a 31
<210> 61
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 61
ttgaggtgtg acgtctagaa atacaa 26
<210> 62
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 62
ttgaggtgtg acgtctagaa atacat 26
<210> 63
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 63
catcctgatt attttattgt taatgaaata tgtgaa 36
<210> 64
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 64
aatttatatg tgataagttt ctagagggat c 31
<210> 65
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 65
ataatttata tgtgataagt ttctagaggg att 33
<210> 66
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 66
attggtctat ggagagtcaa attcaaaatt ataa 34
<210> 67
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 67
agaaaaaaac gcacaacgca aattctag 28
<210> 68
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 68
aaagaaaaaa acgcacaacg caaattctaa 30
<210> 69
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 69
tgactccttt tcttttcctc tttcttttta tata 34
<210> 70
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 70
ttcctatctg ttaaaaaaat tacacgatcg 30
<210> 71
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 71
ttcctatctg ttaaaaaaat tacacgatcc 30
<210> 72
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 72
tcacgcacac gagtaatctt cttctaaa 28
<210> 73
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 73
tgaatctatg aaaggggtaa caaaaaatc 29
<210> 74
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 74
ctgaatctat gaaaggggta acaaaaaatt 30
<210> 75
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 75
ctgagagccc tgttagaaaa attggaat 28
<210> 76
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 76
ttcaaagaaa gatataatcc atcataaaat gtta 34
<210> 77
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 77
ttcaaagaaa gatataatcc atcataaaat gttt 34
<210> 78
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 78
ttttatcggg gagaggaata tgtttaatgt a 31
<210> 79
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 79
gaggcaatcc aaatgcaagc ttac 24
<210> 80
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 80
gaggcaatcc aaatgcaagc ttag 24
<210> 81
<211> 35
<212> DNA
<213> Artificial Sequence
<400> 81
aatcaaagtc aaagaaatca ttttgttata agctt 35
<210> 82
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 82
cacaccagaa gaataaaatg acgtattc 28
<210> 83
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 83
atcacaccag aagaataaaa tgacgtattt 30
<210> 84
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 84
atgccaaggc aaccatcttc ccaat 25
<210> 85
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 85
aaatcatatg tcttctttcg atcttgaatg 30
<210> 86
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 86
gaaatcatat gtcttctttc gatcttgaat a 31
<210> 87
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 87
ttcttattca actattttag cttaggaaag agat 34
<210> 88
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 88
gtgtcattta tatgtatcgc actaagc 27
<210> 89
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 89
gtgtcattta tatgtatcgc actaagg 27
<210> 90
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 90
gaatcgagta gcgataattt ctaacaatca aat 33
<210> 91
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 91
gtctcaacat ctttgatccg aaaaaag 27
<210> 92
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 92
ttgtctcaac atctttgatc cgaaaaaaa 29
<210> 93
<211> 35
<212> DNA
<213> Artificial Sequence
<400> 93
cccttgatat gaaaattcta aaaaatttgg aatta 35
<210> 94
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 94
taatcataat agagatcagg gaggtaac 28
<210> 95
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 95
attaatcata atagagatca gggaggtaat 30
<210> 96
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 96
ttgaggtttg taagttggaa tagggctt 28
<210> 97
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 97
tattagcata aaatatcaaa ggggtttcaa gaaaaaggaa t 41
<210> 98
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 98
atcaaaacaa atggttcaaa gaaaacttga tggtatcaaa a 41
<210> 99
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 99
aaaaattccc agaatattac tgtccggcga gccaaaattt g 41
<210> 100
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 100
tttctttttt tcaaatagga ctatttcata acaaaatcca a 41
<210> 101
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 101
gaaaattgtg agggagaagg gaattattta aattctgggt t 41
<210> 102
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 102
tggggttcac aaaaggttta aaaggtttta gacaatggca t 41
<210> 103
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 103
ctgtcagtca aatgttagat acgataaatt accaaagata c 41
<210> 104
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 104
gatcaagaat gtagaaaatt tttttgacta cactaatacc c 41
<210> 105
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 105
atagaagaga tatgtctcta atacaatgca ctatgtaaaa t 41
<210> 106
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 106
cgagacatag tggcggagga ggtggcggtg ggatgaattg t 41
<210> 107
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 107
tagtcatttg gtattgcaag catcgactga cagcgtactt c 41
<210> 108
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 108
tgaatgtgca aataaatagt gtgcgaattc catgaacaat a 41
<210> 109
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 109
tgtgtttaag tttggttacg cggtgacagg attaactgac c 41
<210> 110
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 110
cactgctttg caccctacgc gcctcttcaa cagaggagac a 41
<210> 111
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 111
cttagagtct agaacaaatt cagaaagaaa aactgacgta a 41
<210> 112
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 112
cgtaagcctc gaaagaaacc gttatttggt gagaaggcaa c 41
<210> 113
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 113
cggagatggt ggcgggagaa cgaagacgct gaacgaaaga a 41
<210> 114
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 114
cttttatttg aagacacaat attcatagtg aaatgatatc c 41
<210> 115
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 115
atatagagag tatgatgcaa ctccattttc ttcaaactaa g 41
<210> 116
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 116
attcaaacac gaaataccta taagttaccc tataaaattt a 41
<210> 117
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 117
ttaatgaaat atgtgaatga ttgtatttct agacgtcaca c 41
<210> 118
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 118
aattataatt tctctggctt gatccctcta gaaacttatc a 41
<210> 119
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 119
aacgcacaac gcaaattcta gttaacttat ataaaaagaa a 41
<210> 120
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 120
gttaaaaaaa ttacacgatc gtttagaaga agattactcg t 41
<210> 121
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 121
tgaaaggggt aacaaaaaat cgattccaat ttttctaaca g 41
<210> 122
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 122
tgtacatttc tagatttttc taacatttta tgatggatta t 41
<210> 123
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 123
ttataagctt acattttgct gtaagcttgc atttggattg c 41
<210> 124
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 124
gaagaataaa atgacgtatt cgattgggaa gatggttgcc t 41
<210> 125
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 125
gtcttctttc gatcttgaat gatctctttc ctaagctaaa a 41
<210> 126
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 126
ataatttcta acaatcaaat gcttagtgcg atacatataa a 41
<210> 127
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 127
acatctttga tccgaaaaaa gattctaatt ccaaattttt t 41
<210> 128
<211> 41
<212> DNA
<213> Cucumis melo L.
<400> 128
aatagagatc agggaggtaa cagaagccct attccaactt a 41

Claims (10)

1. Identifying core SNP loci of melon germplasm resources authenticity, wherein the SNP loci are selected from any 1 to 32 of the following first SNP loci to third twelve SNP loci:
a first SNP locus, wherein the first SNP locus is located at 1028867 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or T;
a second SNP locus, wherein the second SNP locus is located at 15305628 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a third SNP locus, wherein the third SNP locus is located at 35346336 th chromosome 1 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A;
a fourth SNP locus, wherein the fourth SNP locus is located at 1090543 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a fifth SNP locus, wherein the fifth SNP locus is located at 1709164 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a sixth SNP locus, wherein the sixth SNP locus is located at 13775704 th chromosome 2 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a seventh SNP locus, wherein the seventh SNP locus is located at 7930146 rd chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or A;
an eighth SNP locus, wherein the eighth SNP locus is located at 18037611 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a ninth SNP locus, wherein the ninth SNP locus is located at 28288534 rd chromosome 3 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a tenth SNP locus, wherein the tenth SNP locus is located at 4189054 th chromosome 4 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or G;
an eleventh SNP locus, wherein the eleventh SNP locus is located at 16536384 th chromosome 4 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twelfth SNP locus, wherein the twelfth SNP locus is located at 14981598 th chromosome 5 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or C;
a thirteenth SNP locus, wherein the thirteenth SNP locus is located at 28011864 th chromosome 5 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or C;
a fourteenth SNP locus, wherein the fourteenth SNP locus is located at 8120366 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a fifteenth SNP locus, wherein the fifteenth SNP locus is located at 18900074 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A;
a sixteenth SNP locus, wherein the sixteenth SNP locus is located at 19620053 th chromosome 6 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a seventeenth SNP site, wherein the seventeenth SNP site is located at 1104024 th chromosome of a7 th chromosome of a melon reference genome or a corresponding site on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the site is G or C;
an eighteenth SNP locus, wherein the eighteenth SNP locus is located at 3254461 th chromosome of a reference genome of the melon, or a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is C or T;
a nineteenth SNP locus, wherein the nineteenth SNP locus is located at 23111340 th chromosome 7 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or A;
a twentieth SNP locus, wherein the twentieth SNP locus is located at 4876476 th chromosome 8 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources thereof, and the nucleotide base of the locus is G or C;
a twenty-first SNP locus, wherein the twenty-first SNP locus is located at 6383781 th chromosome 8 of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twenty-second SNP locus, wherein the twenty-second SNP locus is located at 32379197 th chromosome 8 of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a twenty-third SNP locus, wherein the twenty-third SNP locus is located at 1849869 th chromosome of a melon reference genome 9 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or C;
a twenty-fourth SNP locus, wherein the twenty-fourth SNP locus is located at 12154715 th chromosome of a melon reference genome 9 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or A;
a twenty-fifth SNP locus, wherein the twenty-fifth SNP locus is located at 20428406 th chromosome of a9 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twenty-sixth SNP locus, wherein the twenty-sixth SNP locus is located at 9552072 th chromosome of 10 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is C or T;
a twenty-seventh SNP locus, wherein the twenty-seventh SNP locus is located at 20472868 th chromosome of the 10 th chromosome of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is G or T;
a twenty-eighth SNP locus, wherein the twenty-eighth SNP locus is located at 7009967 th chromosome of the reference genome of the melon, or at a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is A or C;
a twenty-ninth SNP locus, wherein the twenty-ninth SNP locus is located at 17070009 th chromosome of the 11 th chromosome of the melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is T or C;
a thirtieth SNP locus, wherein the thirtieth SNP locus is located at 27232414 th chromosome of the melon reference genome 11 or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or G;
a thirty-first SNP locus, wherein the thirty-first SNP locus is located at 3459537 th chromosome of a 12 th chromosome of a melon reference genome or a corresponding locus on a homologous genome fragment among germplasm resources of the melon reference genome, and the nucleotide base of the locus is A or G;
a thirty-second SNP locus, wherein the thirty-second SNP locus is located at 13742304 th chromosome of the reference genome of the melon or a corresponding locus on a homologous genome fragment among germplasm resources of the melon, and the nucleotide base of the locus is G or A;
wherein the melon reference genome is melon DHL92 reference genome V3.5.1.
2. The SNP site according to claim 1, wherein:
the sequences of the first SNP locus and bases at the upstream and downstream are SEQ ID NO: 97 or a genome fragment homologous between germplasm resources thereof, more preferably a fragment identical to the sequence shown in SEQ ID NO: 97, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequences of the second SNP locus and bases at the upstream and downstream of the second SNP locus are SEQ ID NO: 98 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 98, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity;
the sequences of the third SNP locus and bases at the upstream and downstream are SEQ ID NO: 99 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 99 nucleotide sequences having greater than or equal to 95%, 96%, 97%, 98%, or 99% identity;
the fourth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 100 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 100, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the fifth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 101 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 101, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity;
the sequences of the sixth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 102 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 102 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the seventh SNP locus and the sequences of bases on the seventh SNP locus and upstream and downstream thereof are SEQ ID NO: 103 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 103 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequences of the eighth SNP locus and bases at the upstream and downstream are SEQ ID NO: 104 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 104 greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the ninth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 105 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 105 nucleotide sequence identity greater than or equal to 95%, 96%, 97%, 98% or 99%;
the tenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 106 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 106 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequence of the eleventh SNP site and bases on the eleventh SNP site is SEQ ID NO: 107 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 107 greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the twelfth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 108 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 108 greater than or equal to 95%, 96%, 97%, 98% or 99% identity;
the thirteenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 109 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 109 by greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the fourteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 110 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 110 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequence of the fifteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 111 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 111 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the sixteenth SNP locus and bases on the sixteenth SNP locus is SEQ ID NO: 112 or an inter-germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 112 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequence of the seventeenth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 113 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 113 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequence of the eighteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 114 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 114 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the nineteenth SNP site and the sequences of bases on the nineteenth SNP site are SEQ ID NO: 115 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 115, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the twenty-second SNP site and the sequences of bases on the twenty-second SNP site are SEQ ID NO: 116 or a germplasm resource homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 116 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the twenty-first SNP locus and the sequences of bases on the twenty-first SNP locus and bases on the twenty-first SNP locus are SEQ ID NO: 117 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence set forth in SEQ ID NO: 117 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequence of the second twelve SNP locus and the base sequences of the second twelve SNP locus are SEQ ID NO: 118 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 118 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the twenty-third SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 119 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 119, or greater than 95%, 96%, 97%, 98%, or 99%;
the sequence of the twenty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 120 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 120, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the twenty-fifth SNP locus and the sequences of bases on the twenty-fifth SNP locus and the upstream and downstream of the twenty-fifth SNP locus are SEQ ID NO: 121 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 121, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity;
the twenty-sixth SNP locus and the sequences of bases on the twenty-sixth SNP locus and bases on the twenty-sixth SNP locus are SEQ ID NO: 122 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 122 is greater than or equal to 95%, 96%, 97%, 98% or 99%;
the twenty-seventh SNP locus and the sequences of bases on the twenty-seventh SNP locus and the upstream and downstream of the twenty-seventh SNP locus are SEQ ID NO: 123 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 123 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequences of the twenty-eight SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 124 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 124, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the twenty-ninth SNP locus and the sequences of bases on the twenty-ninth SNP locus are SEQ ID NO: 125 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 125, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the thirty-third SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 126 or a resource-homologous genome fragment thereof, more preferably a fragment of a sequence shown in SEQ ID NO: 126, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the thirty-first SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 127 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 127, 95%, 96%, 97%, 98% or 99%;
the sequences of the third twelve SNP loci and bases at the upper and lower ends of the third twelve SNP loci are SEQ ID NO: 128 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 128 is greater than or equal to 95%, 96%, 97%, 98% or 99%.
3. A core SNP primer set for identifying the authenticity of melon germplasm resources, the SNP primer set being used for respectively amplifying the SNP sites as claimed in claim 1, and the SNP primer set comprising:
a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site; a ninth SNP primer set for amplifying the ninth SNP site; a tenth SNP primer set for amplifying the tenth SNP site; an eleventh SNP primer set for amplifying the eleventh SNP site; a twelfth SNP primer set for amplifying the twelfth SNP site; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a fourteenth SNP primer set for amplifying the fourteenth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a sixteenth SNP primer set for amplifying the sixteenth SNP site; a seventeenth SNP primer set for amplifying the seventeenth SNP site; an eighteenth SNP primer set for amplifying the eighteenth SNP site; a nineteenth SNP primer set for amplifying the nineteenth SNP site; a twentieth SNP primer set for amplifying the twentieth SNP site; a twenty-first SNP primer set for amplifying the twenty-first SNP site; a second twelve SNP primer set for amplifying the second twelve SNP sites; a twenty-third SNP primer set for amplifying the twenty-third SNP site; a twenty-fourth SNP primer set for amplifying the twenty-fourth SNP site; a twenty-fifth SNP primer set for amplifying the twenty-fifth SNP site; a twenty-sixth SNP primer set for amplifying the twenty-sixth SNP site; a twenty-seventh SNP primer set for amplifying the twenty-seventh SNP site; a second eighteen SNP primer set for amplifying the second eighteen SNP site; a twenty-ninth SNP primer set for amplifying the twenty-ninth SNP site; a thirtieth SNP primer set for amplifying the thirtieth SNP site; a thirty-first SNP primer set for amplifying the thirty-first SNP site; a thirty-second SNP primer set for amplifying the thirty-second SNP site.
4. The SNP primer set according to claim 3, wherein:
the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the first SNP primer set are respectively matched with the sequence shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second SNP primer set are respectively matched with the sequence shown in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the third SNP primer set are respectively matched with the sequences shown in SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and the fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the fifth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
and in the sixth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
and in the seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the eighth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the ninth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the tenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and the eleventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 33, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
and the twelfth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39, greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
and in the fourteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the fifteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the sixteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
and in the seventeenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the eighteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 52. SEQ ID NO: 53. SEQ ID NO: 54 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the nineteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 55. SEQ ID NO: 56. SEQ ID NO: 57 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and the twentieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 58. SEQ ID NO: 59. SEQ ID NO: 60, preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity;
the twenty-first SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the sequence shown in SEQ ID NO: 61. SEQ ID NO: 62. SEQ ID NO: 63 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second twelve SNP primer set are respectively matched with the sequence shown in SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the twenty-third SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the twenty-fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the twenty-fifth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the primers shown in SEQ ID NO: 73. SEQ ID NO: 74. SEQ ID NO: 75 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the twenty-sixth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the primers shown in SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
and in the twenty-seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 79. SEQ ID NO: 80. SEQ ID NO: 81 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second eighteen SNP primer set are respectively connected with the sequences shown in SEQ ID NO: 82. SEQ ID NO: 83. SEQ ID NO: 84 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the twenty-ninth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the thirtieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 88. SEQ ID NO: 89. SEQ ID NO: 90 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the thirty-first SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 91. SEQ ID NO: 92. SEQ ID NO: 93 greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
and in the third twelve SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 94. SEQ ID NO: 95. SEQ ID NO: 96 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
preferably, the first upstream primer and the second upstream primer in each set of primers are linked to different fluorescent molecules, more preferably, the fluorescent molecules are selected from FAM, HEX.
5. The core SNP kit for identifying the authenticity of melon germplasm resources is characterized in that: the SNP kit is prepared into a competitive allele specificity PCR reaction system; the reaction system comprises:
the SNP primer set according to claim 3 or 4,
preferably, in the SNP primer sets, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer of each primer set in the system is 2:2: 5.
6. A melon germplasm resource DNA fingerprint database based on core SNP markers is characterized in that: the DNA fingerprint database includes: the genotype of the SNP site of claim 1 of a standard melon germplasm resource.
7. The DNA fingerprint database of claim 6, wherein: the standard melon germplasm resources are selected from the following 441 melon germplasm resources:
PLANTERE JUMLED 76134, PERSIAN MEDIUM, CURTEOWPE PLANTER JUMBO, 76W-2-5, 76W-3, CATTA LOLLE, 76W-1, 76W-2, 76W-4, CNTALOU PE P.M.R450, CASABA GOLDEN QUOMI GUAUT, ZUO, mating, PF, mating, Japan, GOLDEN mating, Japan, POLIOORR F (RS 815), CATTOR HYBRID F, PLATE PE, PLATE, LONGANG mating, VR 6072, CA 456, CA M761232, (MUSKMELON) BURPEE' S BRIS, GURON mating, japan3, ェリザベス, HONEY ROCK, first generation OF Pachyrhizus, swan, Melao HALE 'S BEST, bvrc5, Anemone MELO, CUCUCUMIS MELO L., IROQUOIS, ALASKA F1, サカタ mating プリンスメロン PF6, Japan4, サカタ mating ェ - ヮ ン メロン, サカタ mating アム - アム, golden MELON, アム - アム, HYBRID SUMMET, JUMBO HALS BEST, TYPE 1, TYPE 2, BUEE' S AMBROSHIBRID F アム, HEARTS OF GOLO, SSIC HYBRD F アム, BRA- アム -ARO ESPEAL, bvV HONEYOYW, POLOR F アム, CLAOSMELT OF 1 MELT, CARE MELT 72, CARE-PALOCARO ESPEAL, BREAR アム, CORVSB アム, PLANSHAVSB アム, CORVSWAVSWAVSWAVSWAVSWANSHASSON アム, WAVSWAVSWAVSWAVSWAVSWAVSWANSHASSON アム, WAVSWAVSWAVSWAVSWAVSWAVSWAVSWANSHASSON アム, WAVSWAVSON アム, WAVSHONESSOBOSSWE アム, WAVSTAVSTAVSTAVSTAVSTAVSTAVSTAVSTAVSON アム, CAVSTAVSTAVSTAVSK アム, CAVSBO 36SUSCOBRONTE アム, CALSOBSER アム, WAVSOBSER 36SU, snow charm (Hami melon type), Jade Dew (OrienSweet), Farmers NO.2(Oriental Sweet, Silver Light (Oriental Sweet), Pride, Hearts of Gold, Delcious 51, Honey Rock, Roadside, Samson, Hales Best Jumbo, SUPER MARKET HYBRID, PLANTER ' S JUMBO VGR679, GUTO 45MSL620, TOP MARK VGG 2663, PERLITA VGR 771, EDISTO VGL 415, JUMHALE ' S BEST 97849, FERY-MORSE SEEDS, HALE ' S JUMBO, SKMELON 6593-8, Kazu Berc 2, Branch melon, snow melon, Japanese cucumber, Bingkuai melon, Bikuai Kazuki Ke, Japanese melon, Japan Sweet potato メロン, Japanese melon 3527, Japanese Sweet potato 3528, Japanese Sweet potato 3680, Japan Sweet potato 357, Japan Sweet potato 3645, Japan Sweet potato 365631, Japan Sweet potato 3611, Japan Sweet potato 3680, Japan Sweet potato 357, Japan Sweet potato 3645, Japan Sweet potato 3611, Sweet potato yellow Sweet potato 3645, Sweet potato yellow Sweet potato 3611, Japan Sweet potato yellow Sweet potato 3526, Japan Sweet potato yellow Sweet potato, Sweet potato yellow Sweet potato, Sweet potato yellow Sweet potato (Japan Sweet potato yellow Sweet potato), Sweet potato yellow Sweet potato, ハ - ベスト 5, ハ - ベスト 6, white muskmelon, Horey louge, cotton white, Taiwan sweet, CD ELDORATO YELLOW, CD CASTOR, Yanjiao sweet, Daphne potpourri, Huangdao, M26, M29, M285, ANANAS, CV SPECIAL HONEY DEW EYPE, Cucumis melo, OGEN, Japanese, Tiger skin Cucumis melo, Changlan persimmon 2, Ielizabeth, Galaoui, CHINESE MELON, ALIER F1, ANWASD' AMERIGUE, IDIAAN, TOJONA, SHIRY, Baidao, Huadao, Qingkuo, Red lantern, Baishami, Black-Miyao, bvao 8, Hongcheng, AVGNON, Mannhan, Manna (NDA), SEMAN 03-03, ZA 03-03, ZA-03, SAIpomoea-3-252-3-12, SANI-259, 03-252-3-90, 03-252-3-41, 03-252-3-69, 03-210-17-2,7, 03-204-1-1,3,5,6,7, 03-203-1-12,22,23,24,25, 03-208-1-15,16, 03-210-16-2, 03-209-1-1,11, 03-209-9-12,13,14,16,17,18,19, 03-210-3-9, 03-206-7,8,9,12, 03-204-2-18, 03-199-16-1, 03-199-18-11, 03-198-31-1, 03-205-2-9, 03-211-2, 03-210-13-1, 03-210-11-1, 03-199-14-1, 03-207-2-12, 03-195-1-4,5, 03-253-3,5.6,9,12,15, 03-252-1-12, 03-310-1-2,3, 03-337-2-20, 03-314-3-3, 03-308-2-5, 03-328-3, 03-345-9, 03-339-1-4,10,18, 03-325-2-2, 03-317-1-1, 03-299-1-5, 03-283-4-6, 03-296-2-8, 03-310-2-1,3,5,8, 03-210-14-4,6,9, 03-209-6-1, 03-199-17-24, 03-209-3-1,2,3,5,6,11, 03-203-1-9, 03-210-1-7, 03-210-2-9, 03-205-1-4,7, 03-199-12-18, 03-198-8-3, 03-198-7-11, 03-195-2-3, 03-210-11-19,41, 03-198-3-15, 03-210-11-17,24,28, 03-210-12-10, 03-210-2-19, 03-307-6-5, 03-293-1-6, 03-325-2-13, 03-252-2-17, 03-236-1,3,4,5,6,11, 03-295-1-7, 03-298-7-9,10, 03-291-1,3, 03-341-2,4,5,6,8, 03-283-1-4, 03-252-3-26, 03-339-2-4,6, 03-210-11-31, 03-196-2-10, 03-196-2-16, 03-196-3-20, 03-210-4-1, 03-210-8-2, 03-204-3-19,20,21,23, 03-210-9-1-2,3,6, 03-210-11-15,36,37, 03-210-10, 03-210-16-17, 03-210-16-13, 03-198-14-2, 03-210-11-2,25,29, 03-210-4-5, 03-210-7-1, 03-210-5-9, 03-210-6-2, 03-209-4-2, 03-198-10-10, 03-339-3-9,10, 03-320-2,5, 03-223-2-19, 03-287-1-1,10, 03-307-2-3, 03-306-1-8, 03-285-1-1, 03-318-3, 03-283-5-9, 03-289-2-5, 03-315-1,4, 03-310-3-3, bvrc15, 03-304-1-9, 03-286-3-1, 03-314-1, 03-319-3,4,5, 03-312-3-4,5, 03-314-2-1, 03-286-1-12,13, 03-342-2-9, 03-343-1-4, 03-346-2,3,4,5,6,7, 03-344-3,6, 03-297-8, 03-342-1-6,7, 03-312-4, 03-316-1, bvrc2, 04-562-1, 04-560-1, 04-555-7, 04-552-1, bvrc4, 04-551-6, 04-560-6, 04-545-7, 04-533-4, 04-544-1, 04-519-2, 04-539-1, 04-537-5, 04-521-12, 05-189-8, 04-528-1, 04-513-2, 04-511-1, 04-556-5, 04-558-1, 04-557-7, 04-556-1, 04-552-15, 04-557-1, 04-538-2, 04-528-12, 04-529, 04-517-1, 04-551-2, 04-563-1, 04-561-7, 04-554, 04-552-5, 04-532-2, 04-518-1, 04-523-1, 04-568-1, 04-566-9, 04-571-1, 04-571-13, 04-576-1, 04-566-1, 04-571-10, 04-565-2, 04-599-1, 04-570-6, 04-567-9, 04-578, 05-60-8, 05-56-3, 05-60-7, 05-65-4, 05-67-10, 05-63-1, 05-69-8, 05-67-7, 05-55-2, 05-69-6, 05-51-7, 05-41-6, 05-40-2, 05-46-15, 05-46-11, 05-48-5, 05-51-4, 05-50-8, 05-50-2, 05-50-9, 05-45-1, 05-38-6, 05-45-14, 05-38-9, 05-45-8, 05-93-4-1, 05-71-2, 05-77-4, 05-86-1, 05-70-1, 05-79-1, 05-70-4-2, 05-63-6, 05-66-2, 05-66-7, 05-51-9, 05-66-9, 05-67-1, 05-46-7, 05-44-1, 05-95-1-21,23,26, 05-95-3-1,3,4,6,11,12, 05-94-2-1,10,31,35, 05-93-1-22, 05-88-6, 05-76-2, 05-93-3-7, 05-93-4-26, 05-99-3-9,11,22,28,50,52,58,68,86, 05-99-12-2, 05-99-24-25,32,37, 05-99-14-8,9,11, 05-98-7-1,2,5, 05-97-1-5,9, 05-96-7,9,12,14,15,16,18, 05-99-7-12,13, 05-95-8-10,25,28,30,31, 05-99-19-34,36,37,42,44, 05-100-3-3,7,9,14,05-171-2-10, 16, 05-193-2, 05-136-20, 05-137-1-2, 05-133-3-1,2, 05-73-1, 05-80-1, May-83, 05-81-3, 05-77-8-3,5, 05-72-3, 05-87-2, 05-91-10, 05-77-1, 05-90-1,3,4, 05-273-1, 05-262-2, 05-271-1, 05-222-16, 05-276-2, 05-196-15, 05-270-1, 05-224-10, 05-269-2, 05-282, 05-344-1, 05-277-1, 05-170-2-1, 05-194-5, 05-185-2,3,5,8,11, 05-141-1-12, 05-146-13,14,15, 05-170-9-9,13, 05-137-2-14, 05-169-2-38, 05-191-8, 05-335-2, 05-298-5,10, 05-94-7-1,2,3,4,5,6,7,8, 05-348-2, 05-196-5, 05-268-1, 05-278-2, 05-223-10.
8. The method of constructing a DNA fingerprint database according to claim 6, wherein: the construction method comprises the following steps:
and (3) PCR reaction steps: carrying out competitive allele specific PCR amplification reaction on a standard melon germplasm resource by adopting the PCR reaction system as claimed in claim 5 to obtain a PCR reaction product;
SNP locus genotype obtaining step: detecting the PCR reaction product to obtain the genotype of the SNP locus;
preferably, the detection is fluorescence signal detection or direct sequencing.
9. A detection method for identifying the authenticity of melon germplasm resources is characterized by comprising the following steps: the detection method comprises the following steps:
the method comprises the following steps: detecting the genotype of the SNP locus of the melon to be detected according to claim 1;
step two: and (3) judging the germplasm resources of the melon to be detected:
if the number of the genotype of the melon to be tested based on the 32 SNP sites and the number of the different sites of a specified germplasm resource in the standard melon germplasm resources in the database of claim 6 or 7 based on the genotype of the 32 SNP sites are 0-2, judging the melon to be tested as a similar germplasm resource with the specified germplasm resource;
if the number of the genotype of the melon to be tested based on the 32 SNP sites and the number of the different sites of the genotype of a specified germplasm resource in the standard melon germplasm resources in the database according to claim 6 or 7 based on the 32 SNP sites is more than 2, judging the melon to be tested and the specified germplasm resource as different melon germplasm resources;
preferably, the result of the determination is obtained from a cluster analysis.
10. The SNP site according to claim 1 or 2, or the SNP primer combination according to claim 3 or 4, or the SNP kit according to claim 5, or the DNA fingerprint database according to claim 6 or 7, or the DNA fingerprint database obtained by the construction method according to claim 8, or the detection method according to claim 9, wherein the SNP site is used in the following X1 or X2:
x1: identifying whether the germplasm resources of the melon to be detected belong to one of the standard melon germplasm resources;
x2: and identifying the specific germplasm resources of the melon to be detected as the standard melon germplasm resources.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112481408A (en) * 2020-12-16 2021-03-12 武汉市农业科学院 MNP core primer combination for molecular identification of eggplant DNA varieties and application thereof
NL2031136B1 (en) * 2022-03-02 2023-09-08 Inst Vegetables & Flowers Caas Set of kompetitive allele specific pcr (kasp)-based core single nucleotide polymorphism (snp) markers for eggplant and use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109666756A (en) * 2019-01-28 2019-04-23 北京市农林科学院 A kind of method for identifying melon variety authenticity and its combination of dedicated SNP primer
CN110331226A (en) * 2019-06-28 2019-10-15 北京市农林科学院 A kind of method for identifying melon variety authenticity and its combination of dedicated SSR primer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109666756A (en) * 2019-01-28 2019-04-23 北京市农林科学院 A kind of method for identifying melon variety authenticity and its combination of dedicated SNP primer
CN110331226A (en) * 2019-06-28 2019-10-15 北京市农林科学院 A kind of method for identifying melon variety authenticity and its combination of dedicated SSR primer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JOSÉ BLANCA等: "Transcriptome sequencing for SNP discovery across Cucumis melo", 《BMC GENOMICS》 *
SAMER OMARI等: "Landraces of snake melon, an ancient Middle Eastern crop, reveal extensive morphological and DNA diversity for potential genetic improvement", 《BMC GENETICS》 *
王富强等: "SNP分子标记在作物品种鉴定中的应用和展望", 《植物遗传资源学报》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112481408A (en) * 2020-12-16 2021-03-12 武汉市农业科学院 MNP core primer combination for molecular identification of eggplant DNA varieties and application thereof
CN112481408B (en) * 2020-12-16 2021-06-11 武汉市农业科学院 MNP core primer combination for molecular identification of eggplant DNA varieties and application thereof
NL2031136B1 (en) * 2022-03-02 2023-09-08 Inst Vegetables & Flowers Caas Set of kompetitive allele specific pcr (kasp)-based core single nucleotide polymorphism (snp) markers for eggplant and use thereof

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