MNP molecular markers, detection primer combinations, kits and applications for identification of Brasenia schrenkiana varieties

Abstract

The present invention discloses MNP molecular markers, detection primer combinations, kits and applications for identification of water shield varieties. The present invention provides MNP molecular marker sites for identification of water shield varieties, which are genomic regions with multiple nucleotide polymorphisms in water shield populations screened on a simplified water shield genome, specifically including marker sites of MNP-1 to MNP-100 on the simplified water shield genome; the present invention further provides a multiplex PCR primer pair combination for detecting the MNP molecular marker site, which is composed of the nucleotide sequence shown in SEQ ID No.1-200; the present invention applies the MNP molecular marker site or the multiplex PCR primer combination for detecting the MNP molecular marker site to aspects including authenticity identification of water shield varieties, identification of substantially derived varieties of water shield, construction of a DNA fingerprint database of water shield varieties, genetic diversity analysis of water shield germplasm resources or molecular breeding of water shield.

Description

Technical Field

[0001] The invention relates to molecular marker sites for identifying water shield varieties, in particular to MNP molecular markers for identifying water shield varieties and a detection PCR primer combination, a kit and applications thereof, and belongs to the field of molecular identification of water shield varieties. Background Art

[0002] Brasenia schreberi is a perennial aquatic plant of the Brasenia genus of the Brasenaceae family. It is a relict and rare species of the Tertiary flora and one of the world's famous rare aquatic vegetables. Brasenia schreberi is widely distributed in the world, and is distributed in Africa, East Asia, Australia, North America and Central America, and is concentrated in the subtropical areas south of 40° north latitude. In China, Brasenia schreberi has both cultivated varieties and wild populations. Wild Brasenia schreberi is distributed in Yunnan, Hunan, Jiangxi, Fujian and Heilongjiang in China, and is a national Class II key protected wild plant in China. The main production areas of cultivated Brasenia schreberi are Leibo County, Sichuan Province, Lichuan City, Hubei Province, Shizhu County, Chongqing City, Taihu Lake area, Jiangsu Province, West Lake, Hangzhou, Zhejiang Province, etc. China has a history of more than 3,000 years of eating Brasenia schreberi. .

[0003] Brasenia schrenkiana has long been regarded as a precious food. Its main edible part is the young and tender leaves that are rich in colloid and not unfolded. It is rich in polysaccharides, proteins, vitamins and trace elements, and has high medicinal value. With the increasing market demand and high economic value, it is common to see inferior products and mixed varieties in the market, which damages the rights and interests of consumers and is not conducive to the sustainable development of the Brasenia schrenkiana industry. At present, the identification of Brasenia schrenkiana varieties is mainly based on the morphological characteristics of the plants, such as flower color, the color of the front and back of the leaves, etc., but sometimes it is difficult to distinguish them using these phenotypic characteristics. For example, the flowers of the West Lake Brasenia schrenkiana variety and the Lichuan Brasenia schrenkiana variety are both red, and although there are some differences in the colors of the front and back of the leaves, the differences are not obvious.

[0004] Traditional field planting observation and identification can no longer meet the needs of large sample, accurate and rapid identification. DNA fingerprint identification technology has become an important means of identifying water shield because it is not affected by environmental and human factors and has the characteristics of rapidity and accuracy. Although a few research papers have published the application of ISSR markers in genetic analysis and molecular identity cards of water shield, the number of marker sites is relatively small. For example, the published paper "Construction of molecular identity cards of water shield varieties in Lichuan, Central China and Suzhou and Hangzhou, East China based on ISSR maps" only studied the effect of 6 ISSR sites on the differentiation of 6 water shield regional groups in Suzhou and Hangzhou and Lichuan. Although the effect of variety differentiation can be achieved, it is impossible to identify substantial derived varieties due to the small number of markers.

[0005] Therefore, the development of new highly polymorphic molecular markers and their detection technology for the identification of water shield varieties has become a technical problem that needs to be solved urgently. Summary of the invention

[0006] One of the purposes of the present invention is to provide a MNP molecular marker site for identification of Brasenia schrenkiana varieties;

[0007] The second object of the present invention is to provide a multiplex PCR primer combination for detecting the MNP molecular marker site;

[0008] The third object of the present invention is to provide a multiplex PCR kit for detecting the MNP molecular marker sites.

[0009] The fourth object of the present invention is to apply the MNP molecular marker site, the multiplex PCR primer combination for detecting the MNP molecular marker site, or the multiplex PCR kit for detecting the MNP molecular marker site to aspects including authenticity identification of water shield varieties, identification of substantially derived varieties, or genetic analysis of water shield varieties.

[0010] The above object of the present invention is achieved through the following technical solutions:

[0011] In order to achieve the above object, the present invention adopts the following technical solution:

[0012] The present invention first provides an MNP molecular marker site for identifying water shield varieties. The MNP marker site is a genomic region with multiple nucleotide polymorphisms in a water shield population obtained by screening on a simplified water shield genome. The MNP molecular marker site includes marker sites MNP-1 to MNP-100 on the simplified water shield genome.

[0013] Another aspect of the present invention provides a multiplex PCR primer combination for detecting the MNP molecular marker site, wherein the PCR detection primer for each MNP molecular marker site consists of a forward primer and a reverse primer, and the detection PCR primer pairs for detecting the molecular marker sites of MNP-1 to MNP-100 are the PCR primer pairs shown in (1) to (100) below:

[0014] (1) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 1 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 2;

[0015] (2) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 4;

[0016] (3) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 5 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 6;

[0017] (4) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 7 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 8;

[0018] (5) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 9 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 10;

[0019] (6) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 11 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 12;

[0020] (7) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 13 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 14;

[0021] (8) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 15 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 16;

[0022] (9) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 17 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 18;

[0023] (10) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 19 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 20;

[0024] (11) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 21 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 22;

[0025] (12) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 23 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 24;

[0026] (13) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 25 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 26;

[0027] (14) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 27 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 28;

[0028] (15) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 29 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 30;

[0029] (16) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 31 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 32;

[0030] (17) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 33 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 34;

[0031] (18) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 35 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 36;

[0032] (19) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 37 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 38;

[0033] (20) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 39 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 40;

[0034] (21) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 41 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 42;

[0035] (22) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 43 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 44;

[0036] (23) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 45 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 46;

[0037] (24) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 47 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 48;

[0038] (25) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 49 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 50;

[0039] (26) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 51 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 52;

[0040] (27) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 53 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 54;

[0041] (28) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 55 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 56;

[0042] (29) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 57 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 58;

[0043] (30) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 59 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 60;

[0044] (31) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No.61 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.62;

[0045] (32) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 63 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 64;

[0046] (33) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 65 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 66;

[0047] (34) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 67 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 68;

[0048] (35) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 69 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 70;

[0049] (36) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 71 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 72;

[0050] (37) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 73 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 74;

[0051] (38) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 75 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 76;

[0052] (39) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 77 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 78;

[0053] (40) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 79 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 80;

[0054] (41) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 81 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 82;

[0055] (42) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 83 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 84;

[0056] (43) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 85 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 86;

[0057] (44) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 87 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 88;

[0058] (45) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 89 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 90;

[0059] (46) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 91 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 92;

[0060] (47) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 93 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 94;

[0061] (48) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 95 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 96;

[0062] (49) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 97 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 98;

[0063] (50) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 99 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 100;

[0064] (51) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 101 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 102;

[0065] (52) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 103 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 104;

[0066] (53) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 105 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 106;

[0067] (54) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 107 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 108;

[0068] (55) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 109 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 110;

[0069] (56) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 111 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 112;

[0070] (57) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 113 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 114;

[0071] (58) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 115 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 116;

[0072] (59) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 117 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 118;

[0073] (60) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 119 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 120;

[0074] (61) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 121 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 122;

[0075] (62) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 123 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 124;

[0076] (63) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 125 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 126;

[0077] (64) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 127 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 128;

[0078] (65) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 129 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 130;

[0079] (66) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 131 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 132;

[0080] (67) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 133 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 134;

[0081] (68) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 135 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 136;

[0082] (69) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 137 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 138;

[0083] (70) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 139 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 140;

[0084] (71) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 141 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 142;

[0085] (72) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 143 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 144;

[0086] (73) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 145 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 146;

[0087] (74) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 147 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 148;

[0088] (75) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 149 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 150;

[0089] (76) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 151 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 152;

[0090] (77) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 153 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 154;

[0091] (78) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 155 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 156;

[0092] (79) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 157 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 158;

[0093] (80) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 159 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 160;

[0094] (81) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 161 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 162;

[0095] (82) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 163 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 164;

[0096] (83) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 165 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 166;

[0097] (84) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 167 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 168;

[0098] (85) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 169 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 170;

[0099] (86) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 171 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 172;

[0100] (87) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 173 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 174;

[0101] (88) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 175 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 176;

[0102] (89) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 177 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 178;

[0103] (90) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 179 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 180;

[0104] (91) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 181 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 182;

[0105] (92) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 183 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 184;

[0106] (93) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 185 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 186;

[0107] (94) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 187 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 188;

[0108] (95) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 189 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 190;

[0109] (96) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 191 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 192;

[0110] (97) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 193 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 194;

[0111] (98) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 195 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 196;

[0112] (99) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 197 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 198;

[0113] (100) A PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 199 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 200.

[0114] Another aspect of the present invention is to provide a multiplex PCR detection kit for detecting the MNP marker site, the multiplex PCR detection kit comprising: a multiplex PCR premix, multiplex PCR detection primers; wherein the multiplex PCR detection primers consist of the nucleotide sequences shown in SEQ ID No.1-SEQ ID No.200.

[0115] Another aspect of the present invention is to apply the MNP molecular marker site or the multiplex PCR primer combination for detecting the MNP molecular marker site to aspects including authenticity identification of water shield varieties, identification of substantially derived varieties of water shield, construction of a DNA fingerprint database of water shield varieties, genetic diversity analysis of water shield germplasm resources or molecular breeding of water shield.

[0116] A preferred specific embodiment of the present invention provides a method for constructing a DNA fingerprint database of water shield varieties using the multiple PCR primer combination of the MNP molecular marker site, comprising: (1) using the MNP marker primer combination to perform multiple amplification on the DNA of multiple water shield germplasms to be detected; (2) sequencing, data analysis and comparison of the amplified products to obtain genotype site data; and (3) importing the genotype site data into Excel 2016 software, recording the genotypes of 100 sites of each sample to construct an MNP fingerprint map database.

[0117] In a preferred specific embodiment of the present invention, the present invention provides a method for identifying whether a water shield sample to be tested is a known control water shield variety by using the multiplex PCR primer combination of the MNP molecular marker site, comprising:

[0118] (1) Extract genomic DNA from the tested water shield sample and the control water shield variety, perform DNA quality detection and MNP marker identification, and obtain MNP site genotype fingerprint data; (2) Compare the MNP site genotype data of the two to determine whether the tested water shield sample and the control water shield variety are the same variety.

[0119] A preferred specific embodiment of the present invention provides a method for grouping water shield resources using the multiplex PCR primer combination of the MNP molecular marker site, comprising: (1) using the MNP core marker primer combination to construct water shield standard DNA fingerprint information; (2) using Power-Marker v3.25 software to perform cluster analysis on 100 site genotype fingerprint data of the water shield to be detected, using the Euclidean genetic distance algorithm UPGMA, and using MEGA7 software to draw a cluster dendrogram, and combining the water shield standard DNA fingerprint information to group the water shield resources.

[0120] The present invention provides an MNP marker site, a primer combination and a kit for identifying water shield varieties and applications thereof. The 100 MNP sites of water shield provided and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products can be sequenced in combination with a second-generation sequencing platform, so that the detection of water shield varieties has the characteristics of high throughput, high discrimination, high accuracy, etc. Experiments have proved that the 100 MNP marker sites, the primer combination and the kit provided by the present invention can not only meet the needs of authenticity identification and substantial derivation identification of water shield varieties, but also realize the free comparison of variety data in a DNA fingerprint database; therefore, the 500 MNP marker sites and the primer combination provided by the present invention can be applied to authenticity identification, substantial derivation identification, database construction, genetic diversity analysis of germplasm resources and other related applications of water shield varieties, provide strong technical support for molecular breeding, intellectual property protection, market supervision and other aspects of water shield varieties, and promote the healthy development of the industry.

[0121] The present invention firstly provides an MNP marker site, a primer composition and a kit for identifying water shield varieties and their applications, which can not only identify the authenticity of water shield varieties and substantially derived varieties, but also perform genetic analysis on water shield varieties, and has the effects of strong discrimination, high throughput and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 The horizontal axis represents the name of the water shield variety, and the vertical axis represents the number of MNP marker sites.

[0123] Figure 2 The figure shows the difference distribution of MNP marker sites among 43 pairs of water shield samples. The vertical axis represents the logarithm of pairwise comparisons, and the horizontal axis represents the percentage distribution of difference sites among pairwise comparisons among varieties.

[0124] Figure 3 Cluster analysis diagram of 43 water shield varieties based on genotype data of MNP loci. DETAILED DESCRIPTION

[0125] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0126] Example 1 Screening of MNP core marker primer combinations for molecular identification of Brasenia schrenkiana DNA varieties

[0127] The whole genome sequencing data of Brasenia schrenkiana with project number PRJNA698426 downloaded from NCBI was assembled to obtain a preliminary reference sequence named SRR13594045.sra. Simplified genome sequencing was performed on 35 Brasenia schrenkiana germplasm resources collected by our unit, and a total of 12.9Gb of sequencing data was obtained. The data was compared with the reference sequence SRR13594045.sra, and samples with low alignment rates were excluded. 516 MNP marker sites were screened from 56623 high-coverage SNP sites, and finally 100 MNP marker amplification primers with high discrimination in this batch of RAD data were synthesized. The detailed information of the primers is shown in Table 1.

[0128] Table 1 Sequence information of 100 pairs of MNP marker amplification primers

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] Example 2 Application of MNP core marker primer combination for molecular identification of Brasenia schrenkiana DNA varieties to construct Brasenia schrenkiana standard DNA fingerprint data

[0137] 1. DNA extraction and quality assessment

[0138] The genomic DNA of all Brasenia species used to construct the Brasenia fingerprint database was extracted using the conventional CTAB method. After RNAse treatment, the quality of the DNA was tested using agarose gel and UV spectrophotometer. If the DNA band is single, there is no obvious degradation, the A260 / 280 detected by the UV spectrophotometer is between 1.8-2.0, and the A260 / 230 is between 1.8-2.0, there is no obvious light absorption at 270nm, and the DNA concentration is greater than 50ng / ul, then the genomic DNA meets the relevant quality requirements and subsequent experiments can be carried out.

[0139] 2. MNP labeling detection

[0140] The 100 pairs of Brasenia schrenkiana MNP-labeled primer combinations screened in Example 1 were used to perform multiplex amplification, second-generation high-throughput sequencing and data analysis on 43 Brasenia schrenkiana germplasms provided by the Vegetable Research Institute of Wuhan Academy of Agricultural Sciences.

[0141] Based on the MNP data of 43 Brasenia schrenkiana germplasms, an average of 82 MNP markers were detected for each variety ( Figure 1 ), the number of detected markers far exceeds the number of existing SSR markers.

[0142] 3. Genotype fingerprint data

[0143] The aligned genotype site data were imported into Excel 2016 software, and the genotypes of 100 sites of each sample were recorded as the final MNP fingerprint data.

[0144] Example 3 Application of MNP core marker primer combination for molecular identification of Brasenia schrenkiana DNA varieties to detect the authenticity of Brasenia schrenkiana varieties 1. Determination of Brasenia schrenkiana MNP primer marker variety discrimination and determination criteria for Brasenia schrenkiana variety authenticity detection results

[0145] The 43 water shield samples in Example 2 were compared in pairs and the differences in their MNP marker sites were analyzed. A total of 903 pairs of comparison results were obtained. The average number of MNP marker differences between any varieties was 82. The difference in genetic distance of the 903 pairs of samples was between 2.4% and 53.7%, with an average difference of 32.1%. Using the developed marker detection method, all water shield varieties can be distinguished ( Figure 2 ), indicating that the Brasenia schrenkiana MNP marker provided by the present invention has high polymorphism and strong variety discrimination. Therefore, the 100 loci can be fully applied to the authenticity identification of Brasenia schrenkiana varieties.

[0146] 2. Identify whether the tested Brasenia schrenkiana sample is a known control Brasenia schrenkiana variety

[0147] The tested water shield sample (code A) and the control water shield variety Lichuan water shield (code B); the tested water shield sample (code C) and the control water shield variety Taihu water shield (code D); extract the genomic DNA of the above two groups of test samples, perform DNA quality detection and MNP marker identification, and obtain genotype fingerprint data. The specific method is shown in Example 2.

[0148] The MNP site genotype data obtained from sample A and sample B are compared, and the MNP site genotype data obtained from sample C and sample D are compared.

[0149] The results are as follows:

[0150] (1) The number of comparison sites between sample A (the tested Brasenia schrenkiana sample) and sample B (the known Brasenia schrenkiana variety Lichuan) was 83, the number of difference sites was 1, and the percentage of difference sites was 1.2%;

[0151] (2) The number of comparison sites between sample C (the tested Brasenia schrenkiana sample) and sample D (the known Brasenia schrenkiana variety Taihu) was 84, the number of difference sites was 35, and the percentage of difference sites was 41.6%.

[0152] According to the test results, the sample A to be tested was identified as the same variety or a similar variety as the Lichuan water shield variety; while the sample C to be tested was not the same variety as the Taihu water shield variety.

[0153] Example 4: Using the standard DNA fingerprint information of Brasenia schrenkiana constructed by the MNP core marker primer combination to classify Brasenia schrenkiana resources

[0154] The 100 MNP primers provided by the present invention can be used to construct a fingerprint map of Brasenia schrenkiana and analyze the Brasenia schrenkiana germplasm resources. The specific method includes: applying the standard DNA fingerprint information of Brasenia schrenkiana constructed by the MNP core marker primer combination in Example 2, using the 100 site genotype fingerprint data of 43 Brasenia schrenkiana in Example 3 using Power-Marker v3.25 software, selecting the Euclidean genetic distance algorithm UPGMA for cluster analysis, and using MEGA7 software to draw a cluster dendrogram. The 43 Brasenia schrenkiana germplasm resources are divided into different groups. The results are as follows: Figure 3 shown.

Claims

1. A multiplex PCR primer pair combination for identifying Brasenia schrenkiana varieties or constructing a Brasenia schrenkiana MNP fingerprint database, wherein the multiplex PCR primer pair consists of a forward primer and a reverse primer; characterized in that: The multiplex PCR primer pair combination consists of the PCR primer pairs shown in (1)-(100) below: (1) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 1 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 2; (2) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 4; (3) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 5 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 6; (4) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 7 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 8; (5) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 9 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 10; (6) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 11 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 12; (7) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 13 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 14; (8) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 15 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 16; (9) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 17 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 18; (10) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 19 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 20; (11) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 21 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 22; (12) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 23 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 24; (13) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 25 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 26; (14) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 27 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 28; (15) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 29 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 30; (16) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 31 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 32; (17) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 33 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 34; (18) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 35 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 36; (19) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 37 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 38; (20) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 39 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 40; (21) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 41 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 42; (22) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 43 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 44; (23) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 45 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 46; (24) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 47 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 48; (25) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 49 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 50; (26) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 51 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 52; (27) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 53 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 54; (28) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 55 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 56; (29) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 57 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 58; (30) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 59 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 60; (31) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No.61 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.62; (32) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 63 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 64; (33) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No.65 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.66; (34) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No.67 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.68; (35) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No.69 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.70; (36) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 71 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 72; (37) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 73 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 74; (38) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 75 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 76; (39) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 77 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 78; (40) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 79 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 80; (41) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 81 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 82; (42) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 83 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 84; (43) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 85 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 86; (44) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 87 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 88; (45) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No.89 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.90; (46) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No.91 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.92; (47) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 93 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 94; (48) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 95 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 96; (49) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 97 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 98; (50) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 99 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 100; (51) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 101 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 102; (52) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 103 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 104; (53) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 105 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 106; (54) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 107 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 108; (55) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 109 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 110; (56) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 111 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 112; (57) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 113 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 114; (58) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 115 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 116; (59) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 117 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 118; (60) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 119 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 120; (61) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 121 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 122; (62) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 123 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 124; (63) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 125 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 126; (64) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 127 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 128; (65) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 129 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 130; (66) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 131 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 132; (67) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 133 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 134; (68) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 135 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 136; (69) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 137 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 138; (70) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 139 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 140; (71) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 141 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 142; (72) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 143 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 144; (73) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 145 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 146; (74) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 147 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 148; (75) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 149 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 150; (76) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 151 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 152; (77) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 153 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 154; (78) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 155 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 156; (79) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 157 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 158; (80) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 159 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 160; (81) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 161 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 162; (82) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 163 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 164; (83) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 165 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 166; (84) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 167 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 168; (85) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 169 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 170; (86) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 171 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 172; (87) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 173 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 174; (88) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 175 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 176; (89) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 177 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 178; (90) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 179 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 180; (91) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 181 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 182; (92) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 183 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 184; (93) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 185 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 186; (94) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 187 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 188; (95) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 189 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 190; (96) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 191 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 192; (97) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 193 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 194; (98) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 195 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 196; (99) a PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 197 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 198; (100) A PCR primer pair consisting of a forward primer having a nucleotide sequence as shown in SEQ ID No. 199 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.

200.

2. A multiplex PCR detection kit for identifying Brasenia schrenkiana varieties or constructing a Brasenia schrenkiana MNP fingerprint database, the multiplex PCR detection kit comprising: Multiplex PCR premix, multiplex PCR detection primers; characterized in that the multiplex PCR detection primers consist of the nucleotide sequences shown in SEQ ID No.1-SEQ ID No.

200.

3. Use of the multiplex PCR primer pair combination according to claim 1 in authenticity identification of Brasenia schrenkiana varieties.

4. Use of the multiplex PCR primer pair combination according to claim 1 in identifying substantially derived varieties of Brasenia schrenkiana.

5. Use of the multiplex PCR primer pair combination according to claim 1 in constructing a DNA fingerprint database of Brasenia schrenkiana varieties.

6. Use of the multiplex PCR primer pair combination of claim 1 in genetic diversity analysis of Brasenia schrenkiana germplasm resources or Brasenia schrenkiana molecular breeding.

7. The use according to claim 3 or 4, characterized in that: include: (1) Extract genomic DNA from the tested water shield sample and the control water shield variety, perform DNA quality detection and MNP marker identification, and obtain MNP site genotype fingerprint data; (2) Compare the MNP site genotype data of the two to determine whether the tested water shield sample and the control water shield variety are the same variety.

8. The use according to claim 5, characterized in that: include: (1) using the multiple PCR primers to perform multiple amplification on the DNA of the multiple Brasenia schrenkiana germplasms to be tested; (2) The amplified products were sequenced, analyzed and compared to obtain the genotype site data; (3) The genotype site data were imported into Excel 2016 software, and the genotypes of 100 sites of each sample were recorded to construct the MNP fingerprint database.

9. The use according to claim 6, characterized in that: include: (1) The multiplex PCR primer pair combination was used to construct the standard DNA fingerprint information of Brasenia schrenkiana; (2) The 100-site genotype fingerprint data of the Brasenia schrenkiana to be tested were clustered using Power-Marker v3.25 software, the Euclidean genetic distance algorithm UPGMA was used for cluster analysis, and the cluster dendrogram was drawn using MEGA7 software. Combined with the standard DNA fingerprint information of Brasenia schrenkiana, the Brasenia schrenkiana resources were classified into groups.

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