Method for constructing DNA fingerprint map of pear varieties based on KASP primer combination and its application

Abstract

The present invention discloses a method for constructing a DNA fingerprint map of pear varieties based on a KASP primer combination and its application, belonging to the technical field of molecular marker development. Based on the whole-genome resequencing of 495 different pear germplasm resources from the Institute of Pomology, Chinese Academy of Agricultural Sciences, hundreds of thousands of SNP variant site data are obtained, and further analyzed using the KASP technology platform to identify and screen out 25 SNP markers and supporting KASP primer combinations. The present invention provides a complete set of SNP site detection primers for the research of pear varieties, which can be used for the development of pear variety SNP detection kits, the construction of pear variety DNA fingerprint maps, the genotyping of pear varieties, and the authenticity detection of pear varieties, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker development, and particularly relates to a method for constructing a DNA fingerprint map of pear varieties based on a KASP primer combination and its application. Background Art

[0002] China is the origin of pears and the differentiation center of oriental pears, and is one of the diversity centers of cultivated pears. The genetic resources of pear plants are rich and the cultivation history is long. Pear plants have the characteristic of self-incompatibility. There is no reproductive isolation between species of pear plants, and interspecific hybridization is common, with a complex genetic background. Identifying and utilizing pear germplasm resources can give full play to the quantity advantage of pear germplasm resources in China and lay a material foundation for pear variety improvement and breeding.

[0003] DNA molecular markers, with advantages such as short cycle, not affected by the environment, and enabling high-throughput detection, provide new technical means for pear variety identification, such as SSR molecular markers, KASP molecular markers, etc. However, due to factors such as limited SSR marker quantity and detection throughput, high detection cost, and time-consuming and laborious data reading, their application in identifying pear varieties is restricted. KASP molecular markers have the advantages of being more efficient, flexible, accurate, and low-cost, and are widely used at home and abroad. At present, the identification of pear varieties is only limited to the application in specific several varieties, and it cannot effectively distinguish many varieties, which leads to the situation of fake replacing real among common similar varieties in the market, and is also not conducive to the development and utilization of excellent germplasm resources. Therefore, it is very necessary to develop new molecular markers for pear variety identification. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for constructing a DNA fingerprint map of pear varieties based on a KASP primer combination and its application, so as to solve the problems existing in the above-mentioned prior art, realize the genotyping and authenticity identification of pear varieties, and construct a DNA fingerprint map of pear varieties.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides the application of a reagent for detecting the genotypes of 25 SNP loci in the pear genome in any one of the following:

[0007] (1) Application in preparing a kit for detecting the genotypes of pear SNP loci;

[0008] (2) Application in genotyping pear varieties;

[0009] (3) Application in constructing a DNA fingerprint map of pear varieties;

[0010] (4) Application in detecting true and false pear varieties;

[0011] The physical positions of the 25 SNP loci were determined based on the alignment of the whole genome sequence of pear. The gene accession number of the whole genome of pear on NCBI is GCA_007844245.1. The 25 SNP loci are SNP1 - SNP25:

[0012] SNP1 is located at position 16868304 on chromosome 1, and its base is C or T;

[0013] SNP2 is located at position 3483609 on chromosome 2, and its base is G or C;

[0014] SNP3 is located at position 26191031 on chromosome 2, and its base is A or G;

[0015] SNP4 is located at position 4991610 on chromosome 3, and its base is G or C;

[0016] SNP5 is located at position 17841568 on chromosome 4, and its base is A or G;

[0017] SNP6 is located at position 23947857 on chromosome 4, and its base is T or C;

[0018] SNP7 is located at position 3714662 on chromosome 5, and its base is T or C;

[0019] SNP8 is located at position 12064959 on chromosome 6, and its base is C or T;

[0020] SNP9 is located at position 18415632 on chromosome 6, and its base is A or T;

[0021] SNP10 is located at position 5417364 on chromosome 7, and its base is G or T;

[0022] SNP11 is located at position 6167915 on chromosome 8, and its base is G or A;

[0023] SNP12 is located at position 22160552 on chromosome 8, and its base is T or A;

[0024] SNP13 is located at position 508439 on chromosome 9, and its base is A or T;

[0025] SNP14 is located at position 8663266 on chromosome 9, and its base is C or T;

[0026] SNP15 is located at position 5690624 on chromosome 11, and its base is C or T;

[0027] SNP16 is located at position 16140350 on chromosome 11, and its base is T or C;

[0028] SNP17 is located at position 33330733 on chromosome 11, and its base is C or T;

[0029] SNP18 is located at position 3512965 on chromosome 12, and its base is C or A;

[0030] SNP19 is located at position 17378039 on chromosome 12, and its base is C or G;

[0031] SNP20 is located at position 21953151 on chromosome 13, and its base is A or C;

[0032] SNP21 is located at position 26179272 on chromosome 13, and its base is T or C;

[0033] SNP22 is located at position 2476344 on chromosome 14, and its base is A or G;

[0034] SNP23 is located at position 38652743 on chromosome 15, and its base is A or G;

[0035] SNP24 is located at position 1017338 on chromosome 17, and its base is A or G;

[0036] SNP25 is located at position 8230144 on chromosome 17, and its base is T or G.

[0037] The present invention also provides a KASP primer combination for detecting the 25 SNP sites. The KASP primers corresponding to each SNP site to be detected include two forward primers and one reverse primer;

[0038] The KASP primers for detecting SNP1 are two forward primers shown in SEQ ID NO.1-2 and a reverse primer shown in SEQ ID NO.3;

[0039] The KASP primers for detecting SNP2 are two forward primers shown in SEQ ID NO.4-5 and a reverse primer shown in SEQ ID NO.6;

[0040] The KASP primers for detecting SNP3 are two forward primers shown in SEQ ID NO.7-8 and a reverse primer shown in SEQ ID NO.9;

[0041] The KASP primers for detecting SNP4 are two forward primers shown in SEQ ID NO.10-11 and a reverse primer shown in SEQ ID NO.12;

[0042] The KASP primers for detecting SNP5 are two forward primers shown in SEQ ID NO.13-14 and a reverse primer shown in SEQ ID NO.15;

[0043] The KASP primers for detecting SNP6 are two forward primers shown in SEQ ID NO.16-17 and a reverse primer shown in SEQ ID NO.18;

[0044] The KASP primers for detecting SNP7 are two forward primers shown in SEQ ID NO.19-20 and a reverse primer shown in SEQ ID NO.21;

[0045] The KASP primers for detecting SNP8 are two forward primers shown in SEQ ID NO.22-23 and a reverse primer shown in SEQ ID NO.24;

[0046] The KASP primers for detecting SNP9 are two forward primers shown in SEQ ID NO.25-26 and a reverse primer shown in SEQ ID NO.27;

[0047] The KASP primers for detecting SNP10 are two forward primers shown in SEQ ID NO.28-29 and a reverse primer shown in SEQ ID NO.30;

[0048] The KASP primers for detecting SNP11 are two forward primers shown in SEQ ID NO.31-32 and a reverse primer shown in SEQ ID NO.33;

[0049] The KASP primers for detecting SNP12 are two forward primers shown in SEQ ID NO.34-35 and a reverse primer shown in SEQ ID NO.36;

[0050] The KASP primers for detecting SNP13 are two forward primers shown in SEQ ID NO.37-38 and a reverse primer shown in SEQ ID NO.39;

[0051] The KASP primers for detecting SNP14 are two forward primers shown in SEQ ID NO.40-41 and a reverse primer shown in SEQ ID NO.42;

[0052] The KASP primers for detecting SNP15 are two forward primers shown in SEQ ID NO.43-44 and a reverse primer shown in SEQ ID NO.45;

[0053] The KASP primers for detecting SNP16 are two forward primers shown in SEQ ID NO. 46-47 and a reverse primer shown in SEQ ID NO. 48;

[0054] The KASP primers for detecting SNP17 are two forward primers shown in SEQ ID NO. 49-50 and a reverse primer shown in SEQ ID NO. 51;

[0055] The KASP primers for detecting SNP18 are two forward primers shown in SEQ ID NO. 52-53 and a reverse primer shown in SEQ ID NO. 54;

[0056] The KASP primers for detecting SNP19 are two forward primers shown in SEQ ID NO. 55-56 and a reverse primer shown in SEQ ID NO. 57;

[0057] The KASP primers for detecting SNP20 are two forward primers shown in SEQ ID NO. 58-59 and a reverse primer shown in SEQ ID NO. 60;

[0058] The KASP primers for detecting SNP21 are two forward primers shown in SEQ ID NO. 61-62 and a reverse primer shown in SEQ ID NO. 63;

[0059] The KASP primers for detecting SNP22 are two forward primers shown in SEQ ID NO. 64-65 and a reverse primer shown in SEQ ID NO. 66;

[0060] The KASP primers for detecting SNP23 are two forward primers shown in SEQ ID NO. 67-68 and a reverse primer shown in SEQ ID NO. 69;

[0061] The KASP primers for detecting SNP24 are two forward primers shown in SEQ ID NO. 70-71 and a reverse primer shown in SEQ ID NO. 72;

[0062] The KASP primers for detecting SNP25 are two forward primers shown in SEQ ID NO. 73-74 and a reverse primer shown in SEQ ID NO. 75.

[0063] Optionally, different fluorescent groups are connected to the 5'-ends of the two forward primers, and the fluorescent groups include FAM fluorescent group and HEX fluorescent group.

[0064] The present invention also provides a kit containing the above KASP primer combination.

[0065] The present invention also provides the application of the KASP primer combination or the kit as described above in any one of the following:

[0066] (1) Application in genotyping pear varieties;

[0067] (2) Application in constructing DNA fingerprint maps of pear varieties;

[0068] (3) Application in detecting true and false pear varieties.

[0069] The present invention also provides a method for genotyping pear varieties, comprising the following steps:

[0070] Extracting genomic DNA of the pear variety to be tested;

[0071] Using the genomic DNA as a template, preparing a reaction system according to the 25 SNP loci, and respectively performing PCR amplification using the corresponding primers in the KASP primer combination as described above;

[0072] Detecting the fluorescence signal of the PCR amplification product, and obtaining the genotype of the pear variety to be tested at the corresponding SNP locus according to the value of the fluorescence signal.

[0073] The present invention also provides a method for constructing a DNA fingerprint map of pear varieties, comprising the following steps:

[0074] Extracting genomic DNA of the pear variety to be tested;

[0075] Using the genomic DNA as a template, preparing a reaction system according to the 25 SNP loci, and respectively performing PCR amplification using the corresponding primers in the KASP primer combination as described above;

[0076] Detecting the fluorescence signal of the PCR amplification product, and obtaining the genotype of the pear variety to be tested at the corresponding SNP locus according to the value of the fluorescence signal, thereby constructing a DNA fingerprint map of pear varieties.

[0077] The present invention discloses the following technical effects:

[0078] Based on the whole-genome resequencing of 269 different pear germplasm resources from the Institute of Pomology, Chinese Academy of Agricultural Sciences, the present invention obtains hundreds of thousands of SNP variation site data, and further analyzes and identifies 25 SNP markers and the supporting KASP primer combination by using the KASP technology platform. The present invention provides a complete set of SNP locus detection primers for pear variety research, which can be used for the development of pear variety SNP detection kits, the construction of DNA fingerprint maps of pear varieties, the genotyping of pear varieties, and the detection of true and false pear varieties, etc. Description of the Drawings

[0079] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required for the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0080] Figure 1 It is the identification efficiency of 25 pairs of KASP primers. Detailed implementation manners

[0081] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0082] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0083] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0084] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and embodiments are only exemplary.

[0085] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0086] Example 1

[0087] 1. Experimental materials

[0088] The present invention uses 269 different pear varieties for experiments, and the specific information is shown in Table 1.

[0089] Table 1 Names of 269 materials

[0090] Number Germplasm Name Species Number Germplasm Name Species 1 Serissa japonica White Pear 42 Suizhong Xiehuatian White Pear 2 Xijiangwu White Pear 43 Fenhongxiao White Pear 3 Sumu White Pear 44 Dashuihe White Pear 4 Ya Pear White Pear 45 You Pear White Pear 5 Bo Pear (Chang) White Pear 46 Yalao White Pear 6 Bo Pear (Suo) White Pear 47 Ruanzhiqing White Pear 7 Zhengzhou E Pear White Pear 48 Yingzhiqing White Pear 8 Yanbian Dashan White Pear 49 Shuixiang White Pear 9 E Pear White Pear 50 Xinglong Su White Pear 10 ESu White Pear 51 Shuihongxiao White Pear 11 Misu White Pear 52 Mili White Pear 12 Shiliuzui White Pear 53 Jinsutang White Pear 13 Bayuexue White Pear 54 Pingguoli White Pear 14 Haicheng Ci White Pear 55 Daaoao White Pear 15 Ban Jinsu No.1 (14) White Pear 56 Qiubai White Pear 16 Zaoli White Pear 57 Hongzhi Muyang White Pear 17 Hanyuan Bai White Pear 58 Youhongxiao White Pear 18 Anning Zao White Pear 59 Chili White Pear 19 Duanba Jitui Tong Jinchuanxue White Pear 60 Dahai Bai White Pear 20 Longdeng Zao White Pear 61 Lanzhou Dongguo White Pear 21 Shimen Shuidonggua White Pear 62 Duli Hong White Pear 22 Xinxiang White Pear 63 Liuban White Pear 23 Maogong White Pear 64 Fojianxi White Pear 24 Qingpi Fengmi White Pear 65 Xinglong Ma White Pear 25 Boshan Chi White Pear 66 Qingpi Cao White Pear 26 Sajin White Pear 67 Jinan Xiaobai White Pear 27 Wuzi Huang White Pear 68 Taihuang White Pear 28 Dangshan Su White Pear 69 Jingchuan White Pear 29 Pingding Cui White Pear 70 Youping White Pear 30 Yinbai White Pear 71 Bingtang White Pear 31 Shui Bai White Pear 72 Zisu White Pear 32 Chuizhi Yali White Pear 73 Yanbian Mingyue White Pear 33 Qingli (Siyang) White Pear 74 Huangxian Changba White Pear 34 Hongmachai White Pear 75 Qixia Daxiangshui White Pear 35 Ruanba White Pear 76 Haitang Su White Pear 36 Yuanba White Pear 77 Ehuang White Pear 37 Suizhong Matihuang White Pear 78 Xiaohua White Pear 38 Jinba Bai White Pear 79 Xiangchun White Pear 39 Jinhua White Pear 80 Xuehua White Pear 40 Hongmali White Pear 81 Qinglong Tian White Pear 41 Chazi Li White Pear 82 Xiuyan Ci White Pear 83 Baizhi Muyang White Pear 124 Chenjia Dama Sand Pear 84 Golden Pear White Pear 125 Hongpi Jidan Sand Pear 85 Summer Pear White Pear 126 Hongxiang Sand Pear 86 Jidan Guan White Pear 127 Huangpi Sand Pear 87 Jin Chuizi White Pear 128 Dechang Fengmi Sand Pear 88 Yangbai Xiao White Pear 129 Xichang Houshan Li Sand Pear 89 Yuanxiang White Pear 130 Yanyuan Youzhima Sand Pear 90 Fengxian Jitui White Pear 131 Honghuo Ba Sand Pear 91 Qiuqian White Pear 132 Qiubai (Sand Pear) Sand Pear 92 Dayan White Pear 133 Fuyuan Huang Sand Pear 93 Etou Li White Pear 134 Shuizhi Li Sand Pear 94 Qingpi Zhong Sand Pear 135 Huangpi Shui Yabian Sand Pear 95 Pucheng Xue Sand Pear 136 Kunming Ma Sand Pear 96 Guangxi Li Sand Pear 137 Bakesi Sand Pear 97 Guanyang Xue Sand Pear 138 Niutou Sand Pear 98 Hengxian Mi (Lv) Sand Pear 139 Shuai Li Sand Pear 99 Huishui Jingai Sand Pear 140 Flowering Crabapple Sand Pear 100 Xingyi Haizi Li Sand Pear 141 Weishan Hongxueli Sand Pear 101 Zunyi Xue Sand Pear 142 Lu Sha Sand Pear 102 Ban Jinsu (15th Area) Sand Pear 143 Desheng Xiang Sand Pear 103 Chi Xianfeng Sand Pear 144 Yanzhou Xue Sand Pear 104 Mapiao Sand Pear 145 Pugua Sand Pear 105 Zhimasu Sand Pear 146 Yandang Xue Sand Pear 106 Hongfen Sand Pear 147 Edan Sand Pear 107 Tian Chengzi Sand Pear 148 Shengxian Qiubai Sand Pear 108 Lotus Sand Pear 149 Glutinous Rice Sand Pear 109 Kuixing Maqiao Sand Pear 150 Yiwu Zili Sand Pear 110 Pingding Qing Sand Pear 151 Anhui Snow Pear Sand Pear 111 Xihua Maqiao Sand Pear 152 Hongtang Li 1 Sand Pear 112 Cupi Su Sand Pear 153 Hongtang Li 2 Sand Pear 113 Jiangwan White Pear Sand Pear 154 Xiaojin Sand Pear 114 Jiangwan Tangli Sand Pear 155 Xiangmian Sand Pear 115 Ergong Bai Sand Pear 156 Dazi Su Sand Pear 116 [[ID ​ 157 ​ ​ 117 ​ ​ 158 ​ ​ 118 ​ ​ 159 ​ ​ 119 ​ ​ 160 ​ ​ 120 ​ ​ 161 ​ ​ 121 ​ ​ 162 ​ ​ 122 ​ ​ 163 ​ ​ 123 ​ ​ 164 ​ ​ 165 ​ ​ 206 ​ ​ 166 ​ ​ 207 ​ ​ 167 ​ ​ 208 ​ ​ 168 ​ ​ 209 ​ ​ 169 ​ ​ 210 ​ ​ 170 ​ ​ 211 ​ ​ 171 ​ ​ 212 ​ ​ 172 ​ ​ 213 ​ ​ 173 ​ ​ 214 ​ Xinjiang Pear 174 Wild Pear 2 Ussurian Pear 215 Oil Dumpling Xinjiang Pear 175 Danan Fruit Ussurian Pear 216 Wuwei Pig Head Xinjiang Pear 176 Mountain Pear 24 Ussurian Pear 217 Wuwei Banana Xinjiang Pear 177 False Straight Handle Ussurian Pear 218 Yellow Acid Xinjiang Pear 178 Sharp Handle Ussurian Pear 219 Qili Amuti Xinjiang Pear 179 Flower Cover Ussurian Pear 220 Kuqa Amute Xinjiang Pear 180 Flour Pear Ussurian Pear 221 Korla Fragrance Xinjiang Pear 181 Red Bali Fragrance Ussurian Pear 222 Red Nahe Xinjiang Pear 182 Straight Handle Ussurian Pear 223 Serkepu Xinjiang Pear 183 Sweet Ussurian Pear Ussurian Pear 224 Yubilin Da European Pear 184 Flat Pear Ussurian Pear 225 David European Pear 185 Wood Pear Ussurian Pear 226 Vera European Pear 186 Full Garden Fragrance Ussurian Pear 227 Conference European Pear 187 Flour Acid Ussurian Pear 228 Angouleme European Pear 188 Bali Fragrance Ussurian Pear 229 Flein European Pear 189 White Flower Pot Ussurian Pear 230 Gut Louis European Pear 190 Big Perfume Ussurian Pear 231 Lucas European Pear 191 Yellow Mountain Pear Ussurian Pear 232 Madaomei European Pear 192 Autumn Seed Ussurian Pear 233 Trevor European Pear 193 Hot Ussurian Pear Ussurian Pear 234 Jenny A European Pear 194 Hot Pear Ussurian Pear 235 Gaolici European Pear 195 Southern Fruit Ussurian Pear 236 All Red Selected Variety 196 Guanhongxiao Ussurian Pear 237 Jinshuisu Selected Variety 197 Small Perfume Ussurian Pear 238 Annong No.1 Selected Variety 198 Five Spices Ussurian Pear 239 Fengxiang Pear Selected Variety 199 Green Mountain Pear Ussurian Pear 240 51-3-5 Selected Variety 200 Yanbian Xiehuatian Ussurian Pear 241 p8 Selected Variety 201 Wheat Pear Ussurian Pear 242 Early Fragrance No.1 Selected Variety 202 An Pear Ussurian Pear 243 Early Fragrance No.2 Selected Variety 203 Jingbai Ussurian Pear 244 Qiaoma Selected Variety 204 Sharp Handle King Ussurian Pear 245 Jinmi Pear Selected Variety 205 Duck Guang Ussurian Pear 246 Jinsu Pear Selected Variety 247 Sha 01 Selected Variety 259 Hongxiangsu Selected Variety 248 Winter Honey Selected Variety 260 Beifeng Selected Variety 249 Longyuan European Pear Selected Variety 261 Hanxiang Selected Variety 250 Qiuxiang Selected Variety 262 Huajin Selected Variety 251 Big Pear Selected Variety 263 Jinfeng Selected Variety 252 Apple Fragrance Selected Variety 264 Hongxiu No.2 Selected Variety 253 New Apple Pear Selected Variety 265 Big Cili Pear Selected Variety 254 P16 Selected Variety 266 Huangguan Selected Variety 255 Xiangchi Selected Variety 267 Zaosu Selected Variety 256 Southern Apple Superior Strain 2 Selected Variety 268 Ganli Early 8 Selected Variety 257 Longxiang Selected Variety 269 Qin Su Selected Variety 258 Wujuxiang Selected Variety

[0091] 2. Screening of candidate SNP loci

[0092] The re-sequencing data of 495 pear samples were aligned with the Pyrus betulifolia reference genome (GCA_007844245.1) to obtain a dataset containing all variant sites. There were 453,801 sites that met the requirements of dimorphism and had no other mutations within 100 bp before and after the polymorphic sites, 34,282 sites with maf > 0.4, and 27,274 sites with an average sequencing depth of 10; 11,015 sites with a sample missing rate of 0 (missing rate 0). Using Perl scripts (Winfield et al., 2020), optimal primer combinations were tested. Considering the failure rate of primer design, surplus SNP sites were selected to develop KASP markers, which altogether included 34 SNP sites in four best combinations. Nine pairs of primers with poor genotyping effects were discarded, and 25 pairs of primers with good genotyping results were used to detect the genotypes of 269 samples. The 269 samples were 269 pear germplasm resource materials from 5 major cultivated pear systems and breeding varieties, which came from the National Pear and Apple Germplasm Repository. Details are shown in Table 1. The genomic DNA of young leaves of pear samples was extracted using the TIANGEN Plant Genomic DNA Extraction Kit, and the DNA quality was detected by 1% agarose gel. The concentration was diluted to 40 ng·μL using a DS-11 (Denovix, USA) ultra-micro spectrophotometer -1 For backup use

[0093] 3. Design and synthesis of KASP primers

[0094] One pair of KASP primers consists of 2 forward competitive primers (recognizing different SNPs) and 1 common reverse primer. The primer sequences are shown in Table 2. The primer design parameters are as follows: (1) GC content < 60%; (2) annealing temperature Tm between 55 - 62 °C; (3) PCR amplification product < 120 bp. They were synthesized by Shanghai Sangon Biotech Co., Ltd. The 5' end of forward primer X was added with FAM, and the sequence was (SEQ ID NO.76): GAAGGTGACCAAGTTCATGCT. The 5' end of forward primer Y was added with HEX, and the sequence was (SEQ ID NO.77) GAAGGTCGGAGTCAACGGATT

[0095] Table 2 Primer sequences of core KASP markers

[0096] Serial Number Primer Name Physical Location Genotyping Result Coordinate Primer Sequence (5’-3’) 1 Chr1_168 Chr1_16868304 X:CY:T Reverse F1(SEQ ID NO.1): AGTATGATCCTTGAAATGGAGTTTAAATAG F2(SEQ ID NO.2): CAGTATGATCCTTGAAATGGAGTTTAAATAAR(SEQ ID NO.3): CTGTACTTCAGGAAAACTAATTGCAGGAA 2 Chr2_348 Chr2_3483609 X:GY:C Reverse F1(SEQ ID NO.4): CCCAAAACTATACACATCGATCTTTTC F2(SEQID NO.5): CCCAAAACTATACACATCGATCTTTTG R(SEQ IDNO.6): CGCCCACACAACGCGAGTGAAT 3 Chr2_261 Chr2_26191031 X:AY:G Reverse F1 (SEQ ID NO.7): CCCTGCTTTTTATATATACTTTCTAAAGAT 4 F2 (SEQ ID NO.8): CCCTGCTTTTTATATATACTTTCTAAAGAC R (SEQ ID NO.9): GTCCAAATTAAACCAATGACAACTCCTGAA Chr3_499 Chr3_4991610 5 X:GY:C Forward F1 (SEQ ID NO.10): GAGTTTTCAAGTAGATTAATGCCTAATAT F2 (SEQ ID NO.11): GAGTTTTCAAGTAGATTAATGCCTAATAT R (SEQ ID NO.12): GGATCTTCCTTTGACTTCAAACCACAATT 6 Chr4_178 Chr4_17841568 X:AY:G Forward F1 (SEQ ID NO.13): TTCTTGGCTTCTTCAACCAACCCA 7 F2 (SEQ ID NO.14): CTTGGCTTCTTCAACCAACCC R (SEQ ID NO.15): GCCTGGAATATGTTGGTTGAAGCGTA Chr4_239 Chr4_23947857 8 X:TY:C Reverse F1 (SEQ ID NO.16): GTCTATGAATTGTAAGCATCAATCTTGTA F2 (SEQ ID NO.17): GTCTATGAATTGTAAGCATCAATCTTGT R (SEQ ID NO.18): GTTGGGCTTAAACATGACCGAAAAGATTA 9 Chr5_371 Chr5_3714662 X:TY:C Reverse F1 (SEQ ID NO.19): ACATTCCTTCGAAGAATCGTTACAAGA F2 (SEQ ID NO.20): CATTCCTTCGAAGAATCGTTACAAG R (SEQ ID NO.21): GACACTCTAACATTCCTGCCACACAA Chr6_120 Chr6_12064959 X:CY:T Reverse F1 (SEQ ID NO.22): AGTCCCCGAGCAATGCCTATG F2 (SEQ ID NO.23): GAGTCCCCGAGCAATGCCTATA R (SEQ ID NO.24): GTTAAGTTGGACACGGCGCATGAAA Chr6_184 Chr6_18415632 X:AY:T Forward F1 (SEQ ID NO.25): CTTTTATCTGGATTCTCGTTGCTTTAA 10 F2 (SEQ ID NO.26): CTCTTTTATCTGGATTCTCGTTGCTTTAA R (SEQ ID NO.27): GTGCATCTCAGAAGCATCGTATACAAATA Chr7_541 Chr7_5417364 11 X:GY:T reverse F1 (SEQ ID NO.28): CGAATCTGTTCATGAAGTTCCAGTC F2 (SEQ ID NO.29): CCGAATCTGTTCATGAAGTTCCAGT R (SEQ ID NO.30): GTATGGTCGTGCGCATTGATATTTATGTT 12 Chr8_616 Chr8_6167915 X:GY:A reverse F1 (SEQ ID NO.31): AGGCCTTGTCATAAATAGAAGAGGC 13 F2 (SEQ ID NO.32): AAGGCCTTGTCATAAATAGAAGAGGT R (SEQ ID NO.33): GCGTAAAGCATTGATTTGTATGCTGAGTT Chr8_221 Chr8_22160552 14 X:TY:A reverse F1 (SEQ ID NO.34): GGAAACATGATAAACATAAAAACTGTATAAACA F2 (SEQ ID NO.35): GGAAACATGATAAACATAAAAACTGTATAAACA R (SEQ ID NO.36): GAGTTTCTTCAGCCTTGATGTCATCAAA 15 Chr9_508 Chr9_508439 X:AY:T forward F1 (SEQ ID NO.37): CCGTGAAGAATTCATCCAATAATTTATAAATTA F2 (SEQ ID NO.38): CCGTGAAGAATTCATCCAATAATTTATAAATTT R (SEQ ID NO.39): CGGCTCATAAACACAAAAGTATTGTACGAA Chr9_866 Chr9_8663266 X:CY:T forward F1 (SEQ ID NO.40): TTTAGGCCAAATTTTGTTGTTTACTATG F2 (SEQ ID NO.41): CTTTTTAGGCCAAATTTTGTTGTTTACTATG R (SEQ ID NO.42): CGCGTAAGTTAAAATGCCCTTAAACTGTT Chr11_569 Chr11_5690624 X:CY:T reverse F1 (SEQ ID NO.43): GAAACATAAAGAACTTTTTGAGAATGAACT 16 F2 (SEQ ID NO.44): CGAAACATAAAGAACTTTTTGAGAATGAACT AR (SEQ ID NO.45): CAGCTGGTAAGAAATTTAAACTTCGACCTA Chr11_161 Chr11_16140350 17 X:TY:C Forward F1 (SEQ ID NO.46): TTAAGCTACATAACAAACACAACATAACT F2 (SEQ ID NO.47): TAAGCTACATAACAAACACAACATAAC CR (SEQID NO.48): TACCTGCGCAATTACTCAATATAATTGGAT 18 Chr11_333 Chr11_33330733 X:CY:T Reverse F1 (SEQ ID NO.49): CACATATGTAATAACAGTCACCATCAG 19 F2 (SEQ ID NO.50): CTCACATATGTAATAACAGTCACCATCA AR (SEQID NO.51): TTCACGAGTCCTTGTGGAATACTTGATTT Chr12_351 Chr12_3512965 20 X:CY:A Forward F1 (SEQ ID NO.52): CGCAACACCTAATTACAAACCCC F2 (SEQ IDNO.53): AGCGCAACACCTAATTACAAACCCC AR (SEQ ID NO.54): TGGGTTTTAAGAATTGCTTAATACTCGCAA 21 Chr12_173 Chr12_17378039 X:CY:G Forward F1 (SEQ ID NO.55): TTCCGCCAAGCTGGAAATATCTTC F2 (SEQID NO.56): TCCGCCAAGCTGGAAATATCTTG AR (SEQ ID NO.57): AAGTAGAAAGACGCCACGCTGGAT Chr13_219 Chr13_21953151 X:AY:C Reverse F1 (SEQ ID NO.58): CAGTCAAAGCGTACGTTATCCGAATT F2 (SEQID NO.59): AGTCAAAGCGTACGTTATCCGAATG AR (SEQ IDNO.60): CGAGACATCGGAGGAAGATAGTTTAATTT Chr13_261 Chr13_26179272 X:TY:C Forward F1 (SEQ ID NO.61): GAGCTTGGCCTCACTTCCAACTF2 (SEQ ID NO.62): AGCTTGGCCTCACTTCCAACCR (SEQ ID NO.63): CCTCGGTATTTACTCCTTATCATGTATGAA 22 Chr14_247 Chr14_2476344 X:AY:G forward F1 (SEQ ID NO.64): ATATAAAATGAGTTATGGATCTTACTCCAF2 (SEQ ID NO.65): ATATAAAATGAGTTATGGATCTTACTCCGR (SEQ ID NO.66): CGAGTGAAAACTATAACCCCATCTGAAAA 23 Chr15_386 Chr15_38652743 X: AY:G forward F1 (SEQ ID NO.67): ATTATTTGAGAGCGGAGACTCGATATAF2 (SEQ ID NO.68): ATTTGAGAGCGGAGACTCGATATGR (SEQ ID NO.69): GTTCATACTTGTTTTTGGCGCACTTTCTT 24 Chr17_101 Chr17_1017338 X:AY:G reverse F1 (SEQ ID NO.70): GAGCTTTTGATGCTGACAAAAATATGAAAATF2 (SEQ ID NO.71): AGCTTTTGATGCTGACAAAAATATGAAAACR1 (SEQ ID NO.72): GGATGCATTGGTCATTTCTTAATATGGTTT 25 Chr17_823 Chr17_8230144 X:TY:G reverse F1 (SEQ ID NO.73): ATGGTTCAAGGGCAAAGGAAACGAF2 (SEQ ID NO.74): GGTTCAAGGGCAAAGGAAACGCR (SEQ ID NO.75): CCGACACTTGAGAATGATGATTATGCTT

[0097] To further clarify the characteristics of the above markers, the base type of each SNP marker site plus the downstream 100 bp sequence are shown in Table 3.

[0098] Table 3 100 bp sequences before and after the variant site

[0099] Locus Gene sequence (5'-3') Chr1_16868304 (SEQ ID NO.78) TTCCAAATTCAAAGATTAATCCGTCTGCTTAATTTTCCATCTCAAACAAAAATTGATCGCCTGCACACTGTACTTCAGGAAAACTAATTGCAGGAATGAA[C / T]TATTTAAACTCCATTTCAAGGATCATACTGTAGACTAATATGCTATAGCAGAAGTTGAATAAACATATTTCTCACTCGTTCAGAAAAACATGGGTACGAT Chr2_3483609 (SEQ ID NO.79) CTAAAGAAGCATGTGTATTTTCTTTCATTTCGCATTAGCAATCTGTAAACTGATTTTTCTTACCCTAAATGCAGAATACGCCCACACAACGCGAGTGAAT[G / C]AAAAGATCGATGTGTATAGTTTTGGGGTCATCCTTCTGGAGTTGACAACCGGCAGGGAAGCCAACCACGGCGATGAACACACTGCCCTCGCTGAATGGGC Chr2_26191031 (SEQ ID NO.80) CATATAATCATTCTCTTAAGAGTGAACAAAAATGACAACAGGAAACAGAAAATAAAGGTAGGACATCGAGTCCAAATTAAACCAATGACAACTCCTGAAA[A / G]TCTTTAGAAAGTATATATAAAAAGCAGGGAGAAGAATTAGATTTTATATGAAAGTTAATGAGAATTTGAAGTTTCAAAGAGACTCCAGTCAGTGGCATAT Chr3_4991610 (SEQ ID NO.81) AAAATGGTGGGTTTGGGGACACTCGGTTACCCTACTCCTACATTTGCGTGTAATGGTTGGCAAAAGTCTCAGAGTTTTCAAGTAGATTAATGCCTAATAT[G / C]AAATTGTGGTTTGAAGTCAAAGGAAGATCCCTATTCCCTATATCCAAGGCCAAGGGAAATGGGAAAGGAAACGGACTGAAAGATTTTTCAGTGTGATCAA Chr4_17841568 (SEQ ID NO.82) ATGCAATTCCATTGGCAAGGCTGCCATAAGTTGCCACATCAGGGTGAAACCCGTTATCTTTGATTCTCTGAATGATTTTCTTGGCTTCTTCAACCAACCC[A / G]AGTCTGCAGTACGCTTCAACCAACATATTCCAGGCAACCAAATCAGCCTTCACCCGAGGATCATTAAGCATCTCATCAAACACCTTGTTTGCCAGCTTCG Chr4_23947857 (SEQ ID NO.83) TGTGAAATGTGATTAACAACTAGCGCTTTTCGGCTTAGGGTGTCAAAGTTGGGCTTAAACATGACCGAAAAGATTATGGTTAAAAAAGAACATTGCAGAT[T / C]ACAAGATTGATGCTTACAATTCATAGACATTTGTAAGGTTTTTAAGATTCGATGGAACAATTCCCGTCAGAAAATTTCCATCAAGCCGACTGTTACAGAC Chr5_3714662 (SEQ ID NO.84) AGAGGATTAAACAATGGCTTCGCGAAATGACAACACTTCCCCTAACATTGCTGCCCCATAAAGTCATTATAACGACACTCTAACATTCCTGCCACACAAG[T / C]CTTGTAACGATTCTTCGAAGGAATGTATTGAAATGGGGCATAGCGAGCTTGAGTTCCATCGGGAGTTGCTGAAAATAAACCTGATATTCATACCTGCCTA Chr6_12064959 (SEQ ID NO.85) GCATTTGATTTCTATAGGTCTCACGGGCTCTAACCATAATTTATTTTGATGCAGTTGGAGAAGGATGCCAGTTAAGTTGGACACGGCGCATGAAAATTAT[C / T]ATAGGCATTGCTCGGGGACTCAAATATTTTCACTCGGAACTTGAGCCACCATTTACCATATCGGAGTTGAATTCTAGTGCTGTATATCTTACGGAACATT Chr6_18415632 (SEQ ID NO.86) GTCATCATTTTACAAGATGCACCATGTTTTTCTTTCTCTTTTTTGTAGTGTTATCATTTCATTTATCTTTAACTCTTTTATCTGGATTCTCGTTGCTTTA[A / T]TTTCATTGTTTTATTTGTATACGATGCTTCTGAGATGCACCTTGGTTTTATTAACAAAGCAGATGGCTAATTCACCATCAATCCATGGTAACATAGAAAG Chr7_5417364 (SEQ ID NO.87) CAGTGTTTGATAATAATTCCATATTCTGTGTTGGAGGGAATATCGTTCTGATGGGGCGAGGGTTTTCCGTATGGTCGTGCGCATTGATATTTATGTTGCT[G / T]ACTGGAACTTCATGAACAGATTCGGGAGCATGGATGTTGCGGAGGCTTTTGATAATGGTGGCAACGAACCTACCTTTGTTCCTGCAAGTGAGGCTTCCAT Chr8_22160552 (SEQ IDNO.88) GAGCTCATAGTTGGCCCTCTTGATTGGGTCTTTAAAAGTTAAAATTAGATAGACGATCAGTTCCGGTCGAGTTTCTTCAGCCTTGATGTCATCAAAATAA[T / A]GTTTATACAGTTTTTATGTTTATCATGTTTCCATATCTGAAATCAACTTATTGTACTGTAGAATTTGGATATAATGCTGCATAGGGGCTTGAGCGCCGAG Chr8_6167915 (SEQ ID NO.89) AGGGATTAACAAGCAATTAGTGTTTTTCTCAGGGTGTAAAAGGGTTTCGCGTAAAGCATTGATTTGTATGCTGAGTTGAAGATCTTTTTCTTGTTATGAA[G / A]CCTCTTCTATTTATGACAAGGCCTTAAGGATGAGCAAGTTTGCCGAGGTAAAGTCCTAATAAGATTGATCCTCCTCAACATTTCCCATAATACTCCAACC Chr9_508439 (SEQ ID NO.90) CTGGTATATTACGGTCCGTCATTCGTTAAACCGTTTTCCAATTCGAATAATCAAAGTTCAAATGGCTTCCGTGAAGAATTCATCCAATAATTTATAAATT[A / T]ATATTTTCAAAAAAGTTCGTACAATACTTTTGTGTTTATGAGCCGAAGTTTTAACACAAGAAAATCGAAGTATATATTTTATTCGATATAAACTCTTTTT Chr9_8663266 (SEQ ID NO.91) TTATTAAACAAGGAATTGTCCCGTGCATCGAGTGCAAATCACCGTTTTATTATGACATTAACATATAGTCTTTTTAGGCCAAATTTTGTTGTTTACTATG[C / T]TTCATCAACAGTTTAAGGGCATTTTAACTTACGCGGAAAGAAATTGGACGGAATGATAGTTATTCCCATTGTGGATTACTTCTTAATCTTATATATAAAG Chr11_5690624 (SEQ IDNO.92) ATGCCAGGTAGATCAGGATCAGCTAACAGATGCTGCTTTATGTATGAGCAGATCTATCAGCTGGTAAGAAATTTAAACTTCGACCTAATTTACCAAAAAC[C / T]AGTTCATTCTCAAAAAGTTCTTTATGTTTCGGCATTGTAAAAAATATGGGAAGCATTACAACTTACAAACAATTTTGTACGTTGTAACATATAATTTCCT Chr11_16140350 (SEQ IDNO.93) CAGAAGTCATAAGACCAAGGGGATAAATAACCTCAATATGCAGCACAAATTACAGCTTGCCTAAGACAGACTTTAAGCTACATAACAAACACAACATAAC[T / C]ATCCAATTATATTGAGTAATTGCGCAGGTATTGTTTGACATGTAAAAATGTGAATCACAATTGTGAATAGAAGCGAAGCACATCAACAATTGAAACAAAT Chr11_33330733 (SEQ IDNO.94) ACATAATTCTGTTCGTCCTCTTTGATGAGGTTTTGACTACCTTGTGCATCTTCTCTCATAGTAAGCATTCACGAGTCCTTGTGGAATACTTGATTTTTCA[C / T]TGATGGTGACTGTTATTACATATGTGAGGGCTTAAGATCATAGTATCATTCCAACAGGTCGCTTTTTATTTCTGTAAATGCCCGAAGTACTTTCAAGTTT Chr12_3512965 (SEQ ID NO.95) AGCAATGATATGAAAACAATGATTAAGCAATTACTTTTTCTTGTTGATACAAATGATATTGAGGATGAGACATTTGAGCGCAACACCTAATTACAAACCC[C / A]TTGCGAGTATTAAGCAATTCTTAAAACCCACAAGATAAATTGTACAAATAAAAGAATTCTACAAGGGATAAAATTAACTCAACATGGTCAACTTACATCA Chr12_17378039 (SEQ ID NO.96) ATCTTCAGACCCGCCAAGCGAGTCCTTGCGCTCACTGCTTTAGATTTCGTCCACCTCAGCATCCCCTCGGCCCCATTTTCCGCCAAGCTGGAAATATCTT[C / G]CATCCAGCGTGGCGTCTTTCTACTTCTACTGCATCTCCAGCCCTTGTTTTGTCCACGTCATCAATCGTTCAGCTCATCTCGGAGCTCATATGTTTCCCTC Chr13_21953151 (SEQ ID NO.97) GAAAAGCACGTAAATATCCACAATAGTTAGTACACAGTTCCGGAATTTCGGGAAAATGTCAAATTTTGCAACGAGACATCGGAGGAAGATAGTTTAATTT[A / C]ATTCGGATAACGTACGCTTTGACTGAGGGACTGAGTCGGATAGCTGAAGGAAGAAGAGAGCTTGCTTCTGCAAAAGTCCAGGTCGAAGCAGTTGTGATCG Chr13_26179272 (SEQ ID NO.98) TTCTGAACTTTTGCGTTTATTTAACTATTAACTTATTATGGTCTCTTCATAGCAATCGAAATGTAATATTCACATTAAGGAGCTTGGCCTCACTTCCAAC[T / C]TTCATACATGATAAGGAGTAAATACCGAGGAAAGAAAAATGAAGCAGTATGTCAAGGGTCCCCAAAAAGAATATAAACAAGTATTTTCTCAATAACCCCT Chr14_2476344 (SEQ ID NO.99) GTGAGAGGTTGTACGTTCGAATTTCATGACTAGCATGTTTGATCTCTTGCCTCTTAGTGTAGATATATCACTATATAAAATGAGTTATGGATCTTACTCC[A / G]GTTTTTCAGATGGGGTTATAGTTTTCACTCGAGGGATTTTGTTTTTCATTGATATGAGCCATATTGTGTACTGGAAAATTGAACACAAAACCTCGAGTAC Chr15_38652743 (SEQ ID NO.100) CACAAAATAAAAAATAAAAATAAACCAGCCTAGCTGTTCCTTAGAAAATTATATAATTAACATGCAAGCATTGCATTATTTGAGAGCGGAGACTCGATAT[A / G]GTTAATATATTAAAGAAAGTGCGCCAAAAACAAGTATGAACAGTAGGATTTCTTATACTCAGTCTCAACTTGGAGTGGGAACAGAATTATGCGTGTCAAT Chr17_1017338 (SEQ ID NO.101) TAATTCAAAGATACCTTTTTGGTTTTTCCCAACATTAATTCCATTGGATGCATTGGTCATTTCTTAATATGGTTTTTTACCTGCTAATTAATTAAAACTT[A / G]TTTTCATATTTTTGTCAGCATCAAAAGCTCATAAATCAGCCAACTTGAACTACTCGGCAAGTGCTAGCGACCACTTAACGATGCGTTTTTTTATCAGTAT Chr17_8230144 (SEQ ID NO.102) AGTTGAACCATGATGTGCTTGCTTCACTGTGTGTGTACAAATCTAGCTAGGTTTCTTCATGCCCCCACATGCCCGACACTTGAGAATGATGATTATGCTT[T / G]CGTTTCCTTTGCCCTTGAACCATGATGTGCTCGCTTCACTGTTTTGTCTTTGCCTTTACCTTGTCTTCCTTTTCCGTGGATTGCTTTACTTTTGTCTTGC

[0100] 4. PCR amplification and genotyping

[0101] KASP reaction system: KASP primer system was prepared, with 12µL of each specific primer (100µL), 30µL of universal primer (100µL), and 46µL of ddH2O. PCR was performed in a 96-well plate, with 10µL of reaction system in each well, and 5µL of concentration was 40 ng.μL -1 DNA, 5µL 2×Probe Mix, and 0.14µL Primer Mix.

[0102] The KASP genotyping instrument was a Bio-Rad CFX-96 fluorescence quantitative PCR instrument. The PCR cycle reaction program was as follows: 94°C pre-denaturation for 15 minutes; 94°C denaturation for 20 seconds, 61°C annealing for 60 seconds (0.6°C decrease per cycle, 10 cycles, reaching the final annealing temperature of 55°C); 94°C denaturation for 20 seconds, 55°C annealing for 60 seconds, 26 cycles. Read at 37°C for 1 minute. The amplification program for primers with low GC% content was: 94°C pre-denaturation for 15 minutes; 94°C denaturation for 20 seconds, 57°C annealing for 60 seconds, 36 cycles. Read at 37°C for 1 minute.

[0103] If after 36 cycles, the fluorescence signal is low and the clusters are dispersed, affecting data analysis, you can choose to add cycles, with the program of 94℃ denaturation for 20 seconds, 57℃ annealing for 60 seconds, and 3 cycles. If NTC is not amplified, up to 4 additional cycles can be performed.

[0104] 5. Data statistics and analysis

[0105] The SNPs were filtered and analyzed using vcftools and Excel software according to the KASP primer design rules, and the genotypes were read using the Bio-Rad CFX Manager3.1 software that comes with the fluorescence quantitative PCR instrument using the allele recognition mode.

[0106] 6. Identification efficiency of different primer combinations

[0107] To prove the identification ability of the KASP markers used in the fingerprint library provided in the present invention, in this example, the identification efficiency analysis was carried out on the genotype data of 269 germplasms detected by 25 KASP markers. Through the comparison of genotype data, it was found that 24 germplasm resources among the 269 germplasms were divided into 12 groups, and the corresponding genotypes were consistent. Among them, the genotypes of Qiuzǐ and Tianqiuzǐ (a bud mutation variety of Qiuzǐ), and Nanguo and Dananguo (a bud mutation variety of Nanguo) were consistent; the other 10 groups were respectively Zhengzhou E'li and Yinbai (suspected synonyms), Hongtangli and Hongtangli 2 (suspected synonyms), Shuixiang and Xiali (suspected synonyms), Mili and Jidanguan (suspected synonyms), Shimen Shuidonggua and Fujianxi (suspected synonyms), Fengxian Jituǐ and Puchengxue (suspected synonyms), Hanyuanbai and Jinbabei (suspected synonyms), Ban Jinsu and Kuixing Make (suspected synonyms), Sajin and Shengxian Qiubai (suspected synonyms), Fuyuan Huang and Huangpi Shuiya bian (suspected synonyms). After removing the germplasms with consistent genotypes, the observed heterozygosity values of the 25 pairs of primers were 0.3296 - 0.5556. Using the sorting function of the table, sorting by row in descending order according to the observed heterozygosity values of each primer, the order of the 25 pairs of primers from largest to smallest was Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, Chr11_333, Chr3_499, Chr11_569, Chr13_219, Chr17_101, Chr8_616, Chr6-184, Chr2_348, Chr7_541, Chr11_161, Chr14_247, Chr4_239, Chr6_120, Chr5_371, Chr12_351, Chr12_173, Chr15_386. After the arrangement, taking the first KASP primer locus as the keyword, sorting by column in ascending order; then displaying the data with unique genotypes. Three pairs of primers, Chr9_866, Chr8_221, and Chr2_261, obtained 5 unique genotypes; four pairs of primers, Chr9_866, Chr8_221, Chr2_261, and Chr1_168, obtained 20 unique genotypes; five primers, Chr9_866, Chr8_221, Chr2_261, Chr1_168, and Chr13_261, obtained 57 unique genotypes; six primers, Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, and Chr17_823, obtained 118 unique genotypes;168 unique genotypes were obtained with 7 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, and Chr9_5089; 203 unique genotypes were obtained with 8 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, and Chr4_178; 225 unique genotypes were obtained with 9 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, and Chr11_333; 236 unique genotypes were obtained with 10 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, Chr11_333, and Chr3_499; 239 unique genotypes were obtained with 11 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, Chr11_333, Chr3_499, and Chr11_569; 241 unique genotypes were obtained with 12 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, Chr11_333, Chr3_499, Chr11_569, and Chr13_219; 249 unique genotypes were obtained with 13 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, Chr11_333, Chr3_499, Chr11_569, Chr13_219, and Chr17_101; 253 unique genotypes were obtained with 14 primers, namely Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, Chr11_333, Chr3_499, Chr11_569, Chr13_219, Chr17_101, and Chr8_616;Chr9_866, Chr8_221, Chr2_261, Chr1_168, Chr13_261, Chr17_823, Chr9_508, Chr4_178, Chr11_333, Chr3_499, Chr11_569, Chr13_219, Chr17_101, Chr8_616, Chr6-184. A total of 257 unique genotypes were obtained with 15 primers. Finally, only 15 markers were needed to completely distinguish 257 germplasms, and the identification efficiency reached 95.54%. The identification efficiency is as follows: Figure 1 as shown

[0108] 7. Fingerprint map construction

[0109] According to the KASP genotyping results of 257 pear germplasm resources (see Tables 4 - 5), the SNP genotype information was converted into an Excel-readable format to construct a fingerprint map (for example, the wild type (consistent with the pear genome) was represented as 0, the mutant as 2, the heterozygous genotype as 1, and the missing site was recorded as "-").

[0110] Table 4

[0111] Number Germplasm resources A B C D E F G H I J K L M 1 Serissa foetida CT CC AG CC AG CC TT CT TT GT GA TA TT 2 Xijiangwu CT GC AG CC AG TC TT CC TT GT AA TA AT 3 Sumumu CT GC AG CC GG TC CC TT AT GT GA TA AT 4 Yali pear CC CC AG CC AA CC TC CT TT TT AA TA TT 5 Boli (Chang) CC GC GG CC AG CC TT TT TT GT AA AA AT 6 Boli (Suo) CT GC AA CC GG TT CC TT AT TT GA TT TT 7 Zhengzhou e'li CT GC AG GC GG CC CC CT TT TT GG TA AT 8 Yanbian Dashan TT GC GG CC GG CC TT CC AT TT AA TT AT 9 E'li CT GC AG GC AA TT TC CT TT TT AA TA AT 10 E'su CT GC AG GC AG TT TC CC TT TT AA AA AT 11 Misu CT GG AG GC GG CC CC TT TT GT GG TT AT 12 Shiliuzui CT GC AA GC GG TC CC CT AT TT AA TA AT 13 Bayuexue CT GC AG GC AG TC TC CT AT GT GA TA AT 14 Haichengci CC GG AG CC AG CC TC CC AT GT AA TA AT 15 Ban Jinsu No. 1 (14) CC GC AA GG AA TT TC CC AT GT AA TA AA 16 Zaoli CT CC GG CC AG TT TT CT TT GG GA TA AT 17 Hanyuanbai CT CC AG CC GG TC TC CC TT GT AA TT TT 18 Anningzao CC CC AG CC AG TC CC CT AT GT AA TT AT 19 Duanba Jitui CC CC AG CC AG TT TC CT TT GG AA TA TT 20 Longdengzao CT GC AG CC AA TC CC CT TT GT AA TA TT 21 Shimen Shuidonggua CC CC GG CC GG CC TC TT TT TT GA TA TT 22 Xinxiang CT GC GG GC GG TC CC CC TT GG AA TA AT 23 Maogong CT GC GG CC AG TC TC CT AT GT AA TA AT 24 Qingpi Fengmi CC CC GG GC AG CC TT ​ ​ ​ ​ ​ ​ 25 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 26 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 27 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ -- ​ AA 28 Dangshan Crisp CT GG AA GG GG TC TC CT AT TT AA TA AT 29 Flat top crisp CC GG AG CC AG CC TT TT TT GT AA AA AT 30 Water White CC GC GG CC AG CC TC TT TT GT AA AA AT 31 Weeping Pear CC CC AG CC AG CC TC CT TT TT AA TA TT 32 Green pear (Siyang) CT CC AG GC AA TT CC CC TT GT AA TT AT 33 Red Ma Cha CT CC AG GC AG TT TC CC TT TT AA TA TT 34 Soft handle CT GG GG CC GG CC CC CC TT GG GA AA AT 35 Round handle CC GC AG GC AG TT TC CC AT GT GA TT AT 36 Suizhong Horseshoe Yellow CC GC GG CC AA CC TT TT TT GT GA AA AT 37 Golden Flower CT GC AG GC AG TC CC CC TT GG AA TT AT 38 Red hemp pear CC GC GG CC AG CC TC TT AT GT GA AA TT 39 Chazi Pear CT CC GG CC AG TT TC CT TT GT GA TA TT 40 Xie Huatian from Suizhong CC CC AG CC AG CC CC TT TT TT GA TA TT 41 Pink Night CC CC AG CC AG CC CC TT TT TT GA TA TT 42 Large water core CT GC AG GC AG CC TT -- AT TT GA AA AT 43 Avocado CT CC AG GC GG CC CC TT TT GT GA TA AT 44 Duck Old CT CC AG GC AG CC TC TT TT TT AA -- TT 45 Soft Branch Green CT CC AG GC AG TT TC CC TT TT AA TA TT 46 Hard Branch Green CT GC AG GC AG TT TT CT TT GT GA TA AT 47 Water Fragrance CC GC AA CC AG CC CC TT TT GT AA TA TT 48 Xinglong Crisp CC CC GG CC AG TC TT TT TT GT AA TA TT 49 Water Red Night CC CC GG CC AA CC CC TT TT GG GA AA TT 50 Honey Pear CC CC GG CC AA CC CC TT TT GG GA AA TT 51 Golden candy CC CC GG GC AA CC CC TT TT TT AA TA AT 52 Apple Pear TT CC GG GC AG CC TT CC AA TT AA TA TT 53 big concave CC GC AG GC AA TT TC CC AT GT GA TT AT 54 Qiu Bai CC GC AG CC AA CC TC TT TT GT AA TA AT 55 Red Branch Mother Seedling CC GG GG CC AA CC TC TT AT GG GA AA AT 56 Oil red night CC GG AG CC AG CC TC TT TT GT GA TA TT 57 Chili CT CC AA GC AG TT TC TT TT TT AA TA AT 58 Large core white CT CC AG CC AG TC TC TT TT GG AA AA AT 59 Lanzhou Winter Fruit CT GC GG GC GG TC CC CT TT GT GA TA AA 60 Dulihong CT GC GG CC AA CC TC TT TT TT GA AA AT 61 Six petals CC GC AG CC AA CC TC TT TT GG AA AA AT 62 Xinglong Ma CC CC AG GC GG CC TC TT TT GT AA TT TT 63 Qingpi Rough CT GC AG GG AG TT TC CT TT TT AA TA TT 64 Jinan Xiaobai CC GC AA GC AA TC TC CT AT GG AA TA AT 65 Fetal Jaundice CC CC AG CC AG CC TC TT TT TT GA AA TT 66 Jingchuan CC GC AG GC AA TC TT CC AT GT AA TT AT 67 Oil Bottle CC GC AG GC GG CC TC CT TT TT AA AA AT 68 Rock Sugar CT CC AG GC GG TC CC TT TT TT GA TT AT 69 Purple Crisp CT GC AG GC AG TC TC CT TT TT AA TA AT 70 Yanbian Mingyue TT CC GG GG AG TC TT CC AT TT GG TA AT 71 County Magistrate Huang Ba CT GC AG GC AG TC TC CC AT GT GA TA AA 72 Qixia Big Perfume CC GG AG GC AG TC TC CC AT GT GA TA AA 73 Begonia Crisp CT CC AG GC AG TT TC CT TT TT GA TA AT 74 Goose Yellow CT CC AA GG GG TC TC CT TT TT AA TA AT 75 Little Flower CT GC AG GC AG TC TC CT AT GT AA AA AT 76 Toon CC GC GG CC GG TT TC CT TT GT GA TA AT 77 Snowflake CC GG AA GC AG TC CC CT TT GT AA TA AT 78 Qinglong Sweet CC CC GG CC AG CC TC TT TT GT AA TA TT 79 Xiuyan Ci TT GG AG GG AA TC TC CT AT TT GG TA AT 80 White Branch Mother Seedling CC GC GG CC AA CC TC TT TT GT AA TA AT 81 Golden Pear CC GC AA GG AA TT TC CC AT GT AA TA AA 82 Golden Hammer CT GG AG CC GG TC TT CC TT GT AA TA AT 83 Yangbai Small CC GC GG CC AG TC TC TT TT GT GA AA AT 84 Round Scent CT CC AG GC GG TT CC CT TT GG GA TT AA 85 Feng County Chicken Leg CT CC AG CC GG TC TC CT AT GT AA TA AT 86 Swing CC GC AA GC AG TC TC CC AT GG AA TA AT 87 Dayan CT CC ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 88 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 89 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 90 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 91 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 92 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 93 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 94 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 95 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 96 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ TT 97 Chi Xianfeng CC GC AA GC GG TT TT TT TT GT GG TT AT 98 Hemp Scoop TT CC AG CC AG CC TT CT TT TT GA TA TT 99 Sesame Crisp CT CC GG GC GG CC TC CC TT TT GA TT TT 100 Pink Powder TT GG AG GG GG TT TC CC AT TT AA TT AT 101 Sweet Orange CT GC AA GG AG TT TC CT TT GG AA TT TT 102 Lotus CT GC AG CC AG CC TT CC TT TT AA TA AT 103 Pingdingqing CT GC AA GC AG TC TC TT TT TT GA AA TT 104 Thin-flowered Hemp Shell CT GC AG CC GG CC TT CT TT TT GA AA AT 105 Coarse Skin Crisp CC GC AG GG GG TC TC TT TT GG GA TT TT 106 Jiangwan White Pear CT GC AA CC GG TC TC TT AT GT GG TA TT 107 Jiangwan Sugar Pear TT GC AG GC AA CC TC CC AT TT GA TA AA 108 Ninomiya White CC CC GG CC AA CC CC CC TT GT GA TA AT 109 Xiangnan CT CC AG GC AA TC TC CT AT GT GG TT AA 110 Brown Sugar Pear CC GC AG CC AG TC CC CT AT GG AA TA AT 111 Golden Pear CT GC GG GC AG TC CC CC AA TT GG TT AT 112 Large Hemp Pear CC CC GG CC GG TC TC CC AT GT AA TT AT 113 Fragrant Hemp CT GC GG GC AG TC TC CC TT GG AA TA TT 114 Long-handled Crisp CT CC AG CC GG CC TT TT TT GG GA TT TT 115 Sweet Potato Stick CC GC AA GC GG TT CC CC TT GG AA TT AT 116 Mulberry Bark Pear CT CC AG CC AA TC TC TT TT GG AA AA AT 117 Chen Family's Large Hemp CT GC AG GC AA TC TC CC TT GG AA TT AT 118 Red-Skinned Egg CT GC GG GC AA TC TT CT TT GG GA TA AT 119 Red Fragrance CT GC AG GC AG TC TC CT TT GG AA TA TT 120 Yellow Skin CT CC GG CC AA CC TC CC TT GT AA TA AT 121 Dechang Honey CT CC GG CC AA TT TC ​ ​ ​ ​ ​ ​ 122 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 123 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 124 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 125 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 126 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 127 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 128 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 129 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 130 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 131 ​ ​ ​ AG CC AA CC TC CT AT GT AA TA TT 132 Flower Red CT GC GG CC AG CC TT TT TT GT GG TT TT 133 Weishan Red Pear CT GC AA CC AG CC CC CT TT GG GA TT AT 134 Lusha CT CC AA GC GG CC TT TT TT GG GG TA TT 135 Deshengxiang CC GG GG CC GG CC CC CC TT GT GG TT AT 136 Yanzhou Snow CT GC AG CC AG TC CC CT TT GT GA TA TT 137 Pugua CC GC AA GG AA TC CC CC TT GT AA AA TT 138 Yandang Snow CT CC AA GC AA TT TC CC TT GG AA TT TT 139 Goose Egg CC CC AA GG AG TT TC CT AA TT GA TT TT 140 Glutinous Rice CC GC AG CC AG TC TC CT TT GT AA TT TT 141 Yiwu Pear CC CC AG GG AG CC TC TT TT TT AA TA TT 142 Anhui Pear TT GG AG GG AA TC TT CT AA GG GG TA TT 143 Brown Sugar Pear 1 CC GC AG CC AG CC TC TT TT GG AA TA AT 144 Xiaojin CC CC AG CC AA TC TC TT TT TT AA TA AT 145 Fragrant Noodles CT CC GG GC GG CC TC CC TT TT GA TT TT 146 Big Purple Crisp CT GC AG CC GG CC TC CC TT TT GA TT TT 147 Iron Sheet CT GC AA CC AG TC CC CT TT TT AA TT AT 148 Weining Rhubarb CC CC AA GC AG TC TT TT AT GT GA TA TT 149 Jeweled Pear TT GC AG GC GG TT CC TT TT GG GA AA AA 150 Mountain Pear 16 CC CC AG GC AG CC TC CT AT TT AA TA AA 151 Harbin Institute of Mountain Pear ② TT GG AG GG AG TT TT CC AA GT AA TA AA 152 Dwarf Mountain Pear TT GG GG GG AA TC TT CC AA GG AA AA AA 153 Ning'an Mountain Pear ② CT GG GG GG AG TC TT CC AA GT AA TA AA 154 Suiling Mountain Pear ④ TT GG AG GC AG TT TT CT AT GT AA TA AT 155 Sunwu Mountain Pear ③ CT GG GG GG GG TT TT CC AA TT [[ ​ ​ 156 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 157 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 158 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 159 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 160 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 161 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 162 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 163 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 164 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 165 Sorbus 24 CC GG GG GC GG CC TT CT AA GT AA TT TT 166 False straight handle CT GC AG GC AG TC TT CT AT GG AA TT AA 167 Sharp handle CT GG GG GC AA TC TT CC AA GT AA TT AT 168 Flower cover CC GC GG GC AG CC TT CT AT TT GA TA AT 169 Surface pear CT GC AG GC AA CC TT CT AT GT AA TA AT 170 Red Bali fragrance CT GG AA GG AA TC TT CT AA GT AA TA AT 171 Straight handle CT GC AG GC AG TC TT CT AT GG AA TT AA 172 Flat pear TT GG AG GC AG TC TT CT AT GT GA TT AT 173 Wood pear CT GC AA GC AG CC TT CT AA GG AA AA AA 174 Full garden fragrance CT GC AG GG AG TC TT CC AA GG AA TA TT 175 Surface sour CC GC GG GC AA TC TT CT AT TT GA TA AT 176 Bali fragrance CT GG AG GC AA TT TT CT AT GG AA TA AT 177 White flower pot CC GG AG GC AG CC TC CT AT GG AA TA AT 178 Big perfume CT GG AG GC AA TC TC CT AT GG AA AA AT 179 Huangshan pear CT GG AA GC AA CC TC CT AT TT AA TA AA 180 Autumn fruit CC CC AG CC AG CC TT CT AT GT AA AA AT 181 Hot autumn fruit TT GC AA CC AG TC TT TT AT GG GA TA AT 182 Hot pear CT GC AG GC AG CC TC CT AT TT AA AA AT 183 Southern fruit CT GC GG GG AG CC TT CT AA GT GA TA AT 184 Guanhongxiao CC GC GG GC AG CC CC CT AT TT AA AA AT 185 Small perfume CC GC AG GC AA CC TT CT AT TT GA TA AA 186 Five spices CC GC AG CC GG CC TC TT AT GT AA TA AT 187 Qingshan pear CC GC AG GC AG CC TT CT AA GT GA TT AA 188 Yanbian Xiehuatian CC GC AG GC AA CC TT CT AT TT GA TA AA 189 Mai pear CT GC AA GG GG CC TT CT AA [[ID=346 ​ ​ ​ 190 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 191 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 192 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 193 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 194 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 195 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 196 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 197 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 198 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 199 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ AA AA AA 200 Broom millet seedlings CC GG GG GC GG CC TT CT AT TT AA TA AT 201 Lanzhou long handle CT GC AA GC GG TC CC TT AT GT GA TT TT 202 Jute pear CT GC AG GG GG CC -- TT AA GG GA TT AT 203 Fried dumpling balls CT GG AA GG AG TC CC CT AA GT GG TT AT 204 Wuwei pig head CT GC AA GC GG TC CC TT AT GT GA TT AT 205 Wuwei banana CT CC AG GC AG TC CC TT AT GT AA TA AT 206 Yellow acid CT GC AA GC AG TT CC TT AT GT GG TT TT 207 Qiliamuti TT GG AA GG AG TC CC TT AA GT GG TT TT 208 Kuqa amut CT GC AG GC AA TT CC TT AT TT GA TA TT 209 Korla fragrance CT CC AG GC AG TT CC TT TT GT GA TA AT 210 Hongnahe TT GC AG GG AG TC CC TT AT GG GA TA AT 211 Selkepu TT GC GG GC GG TC CC TT AT GT AA AA AT 212 Yubilinda TT GG AG GG AG TC CC TT AA GG GG TA TT 213 David TT GG AA GG AA TT CC TT AA TT GG TA TT 214 Vera TT GG AG GG GG TC CC TT AA GT GG TA TT 215 Conferences TT GG AA GG AA TT CC TT AA TT GG AA TT 216 Angoulême TT GG -- GG AG TC TC TT AA GT GG TT TT 217 Feline TT GG AG GG AA TC CC TT AA TT GG TT TT 218 Gut Louis TT GG GG GG AA CC TC TT AA GT GG TA TT 219 Lucas TT GG AG GG AG TC TC TT AA GT GG TT TT 220 Madaomei TT GG AG GG AG TT CC TT AA GG GG AA TT 221 Trevor TT GG AG GG AA TT TT TT AA GG GG TT TT 222 Jenny A TT GG AG GG AA TT TC TT AA GT GG TT TT 223 Gaolici TT GC AG GC AG TC TC TT AA GG GA TA TT 224 Mantianhong TT GC AG GC AG CC CC CT [[ ​ ​ ​ ​ 225 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 226 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 227 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 228 51-3-5 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 229 p8 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 230 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 231 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 232 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 233 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 234 ​ CC CC AG GC AA TC TT CT AT TT AA TT AT 235 Sha 01 CT CC AG GC AG TT CC TT TT GT GA TA AT 236 Winter Honey CT GG AG GC AA CC TT CT AA GT GA AA AA 237 Longyuan Asian Pear CT GG AA GC AG TC CC CT AA TT GA TA TT 238 Qiuxiang CT GC AG GG AA TC TT CC AA GT AA TT AT 239 Large Pear TT GC GG CC GG CC TT CC AT TT AA TT AT 240 Pingxiang CT GC AG GG AG CC TT CC AT TT GG TT AT 241 New Ping Pear TT CC GG GG AG TC TT CC AT TT GA TA AT 242 P16 CT CC AG GC GG TC TC CT AT GT AA TA TT 243 Xiangchi CC CC GG CC AA CC CC CC AT GT GA TA AT 244 Southern Ping Pear Superior Strain 2 TT GC GG GC AG CC TT CC AA TT AA TA AT 245 Longxiang CC GG AA GC AG CC TC CT AT GT AA TA AT 246 Red Fragrant Crisp CT GC AA GC AG TT CC TT TT GG AA AA AT 247 Wujuxiang CT GC AA GC AG CC CC TT AT GT GA TA TT 248 Beifeng CT GG GG GG AA TC TT CT AA TT GA TA AA 249 Hanxiang TT GG AG GG AA TC TC CC AA GT AA AA TT 250 Huajin TT GG AG GG AA TT TT CT AA TT GG TA AT 251 Jinfeng CT CC AG GG AG TC TT CT AT TT AA TT AT 252 Red Show 2 CT GC GG CC AA CC CC CC AA GT AA AA TT 253 Daci Pear CT CC AG GC AG TC TT CT AT TT AA TT TT 254 Huangguan CT GC AA GG AG TC CC CC AT TT GA TA AA 255 Zaosu TT GC GG GG AA TC TT CC AT TT GA TT TT 256 Ganlizao 8 TT GG GG GC AA TC TC CC AT GT GA TA AT 257 Qin Su CC GG AA GG GG TC CC CT AT TT AA AA AA

[0112] Table 5

[0113] Number Germplasm Resources N O P Q R S T U V W X Y 1 Liuyuexue CT TT TC CT AA CG CC CC AG[[ ​ ​ ​ 2 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 3 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 4 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 5 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 6 ​ ​ -- ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 7 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 8 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 9 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 10 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 11 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 12 ​ ​ ​ ​ ​ ​ ​ AC CC AG AG AA TG 13 August Snow CT CT TC CT AA CC AC CC AG AA GG TT 14 Haicheng Cixi CT CT CC TT CA CC AC CC GG AG AA TG 15 Half Jin Crisp 1 (14) TT CC TC TT AA CC CC CC AG AA AA TG 16 Early Pear CT CC CC CC AA CG AC CC AG AA GG TG 17 Hanyuan White CT CC TC CT AA CG CC TC AA AG GG TT 18 Anning Early CT CC CC CT AA CG AA TC AG AA GG TT 19 Short Stem Chicken Leg CT CC CC CC AA GG AC TC AA AA GG TT 20 Dragon Lantern Early CT CT TC TT AA GG AC TC AA AA AG TG 21 Shimen Water Melon CT CC TT CT AA CG CC TC AA GG AA TG 22 Fragrant CT CC CC TT AA GG AC TC AA AA AG TT 23 Maogong CC CC CC CT AA CG AA TC AG AA GG TT 24 Green Peel Honey TT CT CC CT AA CG CC TC AA AA AG TG 25 Boshan Pond CC CT TC TT CA CG AA TC GG GG AG TG 26 Sajin CT CC TT CT AA CC CC TC GG AG GG TT 27 Seedless Yellow CT TT TT CT AA CC AC TT GG AG AA GG 28 Dangshan Crisp CC CC TC CC CA CC AA TC GG AA AG TG 29 Flat Top Crisp CT CT TT CT AA CC AC TC AG GG GG GG 30 Water White TT CC TC TT AA CC AC TC GG GG AG GG 31 Weeping Branch Duck Pear CT TT TC CT CA CG CC CC AA AG AG TG 32 Green Pear (Siyang) CC CC CC TT AA CC CC TT AG GG AA TT 33 Red Hemp Chai TT CT CC CC CA CC AC TC AA AG AA TG 34 Soft Stem TT TT TC CC CA CG AC CC AA AG AA TG 35 Round Stem CT CT CC TT AA CC CC TC AG AA AG TG 36 Suizhong Horseshoe Yellow CT CC TT CC AA CC AC TC GG GG AA TG 37 Golden Flower CT CT TC CT AA GG AA TT AA AG AG TG 38 Red Hemp Pear CT CC TT CT AA CC CC TT AG GG AG TG 39 [[ID=344 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 40 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 41 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 42 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 43 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 44 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 45 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 46 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 47 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 48 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 49 ​ ​ ​ ​ CT AA CC CC TT GG GG GG TG 50 Honey pear CT CT TC CC AA CC CC TT AG GG GG TT 51 Golden crispy candy CT CC TT CT AA CC AC TC GG GG GG TG 52 Apple pear CT CC TC CC CC GG CC TT AG AA AG TG 53 Big concave CT CT CC TT AA CC CC TC AG AA AG TG 54 Qiubai CT CT TC CT AA CC AC CC GG GG AG TG 55 Red branch mother seedling TT CT TT CT AA CC AC TC GG GG AG GG 56 Youhongxiao CT CC TT CC CA CC CC TC GG GG AG GG 57 Chili pear CT CT CC TT CA CC AA TC AG AA GG TT 58 Big kernel white TT CC TC CT AA CC CC TC GG GG AG TT 59 Lanzhou winter fruit CC CT CC CT AA CC CC TC AA AG AG TG 60 Duli red CT CC TC CC AA CC CC TC AG GG AG TT 61 Six petals CT CT TC CT CA CC AC TC AG GG GG TG 62 Xinglong hemp CT CT TC CC AA GG CC CC AA AG AG TG 63 Green peel rough CT CC TC CT AA CC AA CC GG AG AG TG 64 Jinan Xiaobai TT CT TC CT AA CC AC CC AA AG AA GG 65 Fetal jaundice CT CC TT CC CA CC AC TC AA GG AG TG 66 Jingchuan CT CC TC CT AA CG AC TC GG AA AG GG 67 Oil bottle TT CT TT TT AA CG CC CC AA GG GG GG 68 Rock sugar CC TT TC TT CA CG AC TC AA GG AA TG 69 Purple crisp CC CC TC CT AA CC AC CC GG AG AG TG 70 Yanbian Mingyue CC CC TC CT CC CG CC TC AG AG AG TT 71 Huang County Magistrate Ba TT CT TT CT CA CG CC CC AG AA AG TG 72 Qixia Big Perfume TT CT TC CT CA CG CC CC AG AA AA TG 73 Begonia crisp CT CT CC CT CA CC AC TC AG AG AG TT 74 Goose yellow TT CC CC CT AA CC AC TC GG AG AG TG 75 Xiaohua TT CC TC CT AA CC AC TC AA AG [[ID ​ 76 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 77 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 78 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 79 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 80 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 81 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 82 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 83 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 84 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 85 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 86 ​ ​ ​ ​ ​ ​ ​ ​ CC AG AA AA GG 87 Dayan CC CT CC CT AA CC AC TC AA AA AG TT 88 Goose Head Pear CT CC TC CC AA CC CC CC AA GG AG TG 89 Green Skin Bell TT CT TC TT AA CC AC TC AG GG GG TG 90 Guangxi Pear TT CC TC TT AA CG AC TC GG AG AG TG 91 Guanyang Snow CT TT CC TT AA CG AC CC AA AG GG TT 92 Hengxian Honey (Green) TT CT TC CC AA CG AC TT GG AG AA TT 93 Huishui Golden Cover CC CT CC CT CA CC CC CC GG GG GG TT 94 Xingyi Haizi Pear CC CT CC CT CA CC CC CC AG GG GG TT 95 Zunyi Snow CC CT CC CT CA CC CC CC AG GG GG TT 96 Half Jin Crisp (15) CT TT CC CT AA CG AC TC AG AG GG TT 97 Late Xianfeng CT CC TC TT AA CG CC TT AG AG AG TG 98 Hemp Ladle CT TT TC TT AA CG CC CC AG AA AG TG 99 Sesame Crisp TT CC TC CC AA CG CC CC AG AG AG TT 100 Pink Powder TT CT TC TT AA CC CC TT AG AA GG TT 101 Sweet Orange CT CC TC TT CA CC AA TT AA AA AA TG 102 Lotus CT TT CC TT AA CC AA TC AA AA AA TG 103 Flat Top Green CT CC CC TT AA CC AC TC AG AG GG TG 104 Thin Flower Hemp Shell CC TT TC CT AA CC AC CC AG AA GG TT 105 Coarse Skin Crisp CC CT TC TT CA CC AC TC AG AA AA GG 106 Jiangwan White Pear CT TT CC CC AA CC AC CC AG AA GG TT 107 Jiangwan Sugar Pear TT TT TT CT AA CC CC TT AG AA AG TG 108 Ninomiya White CT TT TC CT AA CG CC TC AA AA AA TG 109 Xiangnan TT TT TT CC AA CC CC TT AA AA AA TT 110 Brown Sugar Pear CT CT TC TT CA CC AC TC AA GG GG TT 111 Golden Pear CT TT TT CC AA CC CC TT AA AA AA TG 112 Big Hemp Pear CT CT CC CC AA CG CC CC AA AA AA TG 113 Fragrant Hemp [[ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 114 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 115 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 116 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 117 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 118 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 119 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 120 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 121 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 122 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 123 ​ ​ CT TC TT CA CG CC TC AG AG AG TG 124 Red torch TT CC TT CC CA CG AA TC GG AG AA TT 125 Qiubai (sand pear) CT TT TC CC CA CG CC TT AG AA AG TT 126 Fuyuan yellow CT CT TC CT AA CG CC TC AA AG AG TG 127 Shuizhi pear CT CT CC CT CC CC AC TC GG GG GG GG 128 Kunming hemp CC CT TC CT AA CG AC TT AG GG AA GG 129 Bacchus CT CT TC TT CA CC AC TC AG AA AG TG 130 Bull head CC CC CC TT AA CC CC TC AG AA GG GG 131 Shuai pear CT CC TT CC CA GG CC TC AG AG AG GG 132 Flower red CT CT TC CT CC CG AC TT GG AA AA GG 133 Weishan red snow pear CT CT TT CT CA CC CC TT GG GG AA GG 134 Lu sand CC CT TC TT CC CC CC TC AG GG AG GG 135 Desheng fragrance CT TT TT CT CA CC CC TC AA AG GG TG 136 Yanzhou snow TT TT TC TT AA CG AC TT AG GG AG TT 137 Pugua CC TT CC CT AA CC AC TC GG AA GG TT 138 Yandang snow TT CC TC CT AA CG AC TT AG AG GG TT 139 Goose egg CT CC TT TT CA CC CC TC AG AG AG TT 140 Glutinous rice TT CC TC TT AA CG AA TC AA AA AG TT 141 Yiwu pear CT CC TC TT AA CC AC TC AA AA AA TT 142 Anhui snow pear CC CC TT CT CA CG AC TT AG AA AA TG 143 Brown sugar pear 1 TT CC TC CC CA CG AC TT AA GG GG TG 144 Xiaojin CT TT TC CC AA CG AC TC GG GG AG TG 145 Xiangmian TT CC TC CC AA CG CC CC AG AG AG TG 146 Dazi su CT CT TT CC CA CC CC CC AG GG AA GG 147 Tin sheet CC CC CC CT AA CC AC TC AG AG AG TG 148 Weining rhubarb CT CC TC CT CA CG CC CC GG GG GG TG 149 Baozhu pear CC TT TT CC CA GG CC [[ID=34 ​ ​ ​ ​ 150 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 151 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 152 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 153 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 154 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 155 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 156 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 157 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 158 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 159 ​ ​ ​ ​ ​ ​ ​ AA CC GG GG GG GG 160 Goat milk fragrance CT TT CC CT CC GG AA TC AG GG GG TT 161 Golden pair hemp CT CC TC CC CC GG AC TC GG GG AA TT 162 Kuandian pear ① TT TT TT CT CC GG AA TC GG GG GG GG 163 Sand bumps CT CT TC CC CA CC AA CC GG GG AA TG 164 Wild pear 2 CC CT CC CT AA CC AC TC GG GG GG GG 165 Pear 24 CT CT TT CC CA CC AC TT AG GG GG TT 166 False straight handle TT CC TC CT CA CG AC CC GG GG -- GG 167 Sharp handle CC CT CC CC CA CG AC TT AG GG GG TG 168 Flower cover CC TT TT CC CA CG CC TC GG GG GG TG 169 Surface pear CC TT TT CC CA CG AA CC GG GG AG GG 170 Red Bali fragrance CT CT TC CT CC GG AA TC GG GG GG TG 171 Straight handle TT CC TC CT CA CG AC CC GG GG AG GG 172 Flat pear CC TT CC CC CA CG AA CC GG GG GG TT 173 Wood pear TT CT TC CC CC GG AA CC GG GG AG TT 174 Full garden fragrance CC TT TC CT CC GG AA TC AA GG GG TG 175 Surface sour CC TT TT CC CA CG CC TC GG GG GG TG 176 Bali fragrance CT CT CC CT CA CG AA CC AG GG AG TT 177 White flower pot CT CT TT CC AA CG AC TC AG GG AG TG 178 Big perfume CC CT TT CT CC CG AC TT AG GG AG GG 179 Yellow mountain pear TT TT TT CC CA GG AA CC GG GG GG TG 180 Autumn fruit CT TT CC CT AA CC AC TC GG GG AG GG 181 Hot autumn fruit CC CT CC CC CC GG AA CC GG AG AG TT 182 Hot pear TT CT TT CT CA GG AC TC AG GG AG TT 183 Southern fruit CT TT TC CC CC GG AA TC AG GG AG TG 184 Guanhongxiao CT CT TC TT AA CC CC TC GG GG AG TG 185 Small perfume TT TT CC CC CA GG AA TC GG AG AG TG 186 Five spices CT CT [[ID ​ ​ ​ ​ ​ ​ ​ ​ ​ 187 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 188 ​ ​ ​ ​ ​ ​ -- ​ ​ ​ ​ ​ ​ 189 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 190 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 191 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 192 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 193 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 194 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 195 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 196 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ TT 197 Zaomi CT CT TC CC CA CG AA CC AG GG AG TG 198 Xingcheng Xiehuatian CT CT CC CC AA CC AA CC GG GG AA TG 199 Qingtang TT CT CC CT CA CG AA CC GG GG AG TG 200 Broomcorn Seedlings CC CT TT CT CA CG AC TT AG GG AG GG 201 Lanzhou Changba CT CT TT TT CC GG AC TC AA AG AA TT 202 Huangmali CT CT TC CT CC GG AA CC AG AG AA TG 203 Youjiaotuan CT CT TT CC CC CG AA TC AG AA AA TT 204 Wuwei Zhutou CT CT TC TT CC GG AC TC AG AG AA TG 205 Wuwei Banana CC TT TC CT CA CG AC TC AA GG AG TG 206 Huangsuan TT CT TT TT CA CG AC CC GG AG AA TT 207 Qiliamuti TT TT TT CT CC GG AA TT GG AA AG TG 208 Kuqa Amute CT CT TC CT CA CG AC CC GG AG GG TG 209 Korla Fragrance CT CT TT CC CA CC AC TC GG GG AA GG 210 Hongnahe CT TT TT CT CA CG AC TT GG AG AA TG 211 Selkepu CC TT TT CT CA CG AA TC GG AG AA TG 212 Yubilinda CT CT TT CT CC GG AA TC AG AA AG TG 213 Davy TT CC TT CC CC GG AA TC AG AA AG TT 214 Vera CT TT TT CT CC GG AA CC AG AG AA TG 215 Conferences TT CT TT CT CC GG AA TT GG AA AA TT 216 Angouleme CT CT TT TT CC GG AA TT GG AA AA TT 217 Feline TT CT TT CT CC GG AA TC AG AA GG TT 218 Gutelouis CT CC TT CT CC GG AA TC GG AA AG TT 219 Lucas TT CT TT CT CC GG AA TC GG AA AG TT 220 Madome TT CC TT CC CC GG AA TC AG AA AA TT 221 Trevor CC CT CC TT CC GG AA TC GG AG GG TG 222 Jeanne TT CC TT TT CC GG AC TC AG AG AG TT 223 Gao Lici CT TT TC CT CA [[ID ​ ​ ​ ​ ​ ​ 224 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 225 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 226 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 227 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 228 51-3-5 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 229 p8 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 230 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 231 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 232 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 233 ​ ​ ​ ​ ​ ​ ​ ​ TC AG AG AG TG 234 Jinsu Pear CT CT TC TT CA CC CC CC AA AA AA GG 235 Sha 01 CC CT TT CC CA CC AC TC GG GG AG GG 236 Dongmi TT CT TC CC CA GG AC CC AG AG GG GG 237 Longyuan Asian Pear CC CT TT CT CC GG AA CC GG GG AG TG 238 Qiuxiang CT CC TC CC CC GG AA TT GG AG GG TG 239 Big Pear CT CC TC CC CA CG AC TT GG AG AG TG 240 Pingxiang TT CT CC CC CC GG AC TT GG AA GG TT 241 Xinping Pear CC CC TC CT CC CG CC TC AG AG AG TT 242 P16 CT CT TC CC CA CC AC TC GG GG AG TG 243 Xiangchi CT TT TC CT AA CG CC TC AA AA AG TG 244 Nanping Superior Strain 2 TT CT TT CC CC GG AC TC AG AG AG TG 245 Longxiang CT CT TT CT CC GG AC TC GG GG AG GG 246 Hongxiangsu TT CT TT CC CA CC AC CC AG GG AG GG 247 Wujiuxiang CT CT TT CC CA GG AC CC AG AA AG TG 248 Beifeng TT TT TT TT CA GG AC TC GG AG AA TT 249 Hanxiang CC TT TT CT CA CG AC TT AG AG AG TG 250 Huajin TT CT TT CT CA GG CC TT AA AA AG TG 251 Jinfeng CT CT CC CT CC CG AC TC AG AA AG TG 252 Hongxiu 2 CT CT TT CT CC CG AC TT AG AG AG GG 253 Dacili CT CC TC CT AA CG AA TC GG AG AG TG 254 Huangguan CT CT TC CT CA CC CC CC AA AA GG TG 255 Zaosu CT CC TT CC CC GG AC TT AG AA GG TT 256 Ganlizao 8 CT CT TC CC CA GG CC TT AG AA GG TT 257 Qinsu CT CC TC CC CA CC AC CC GG AA AG GG

[0114] Note: In the above Tables 4 - 7, A: Chr1_16868304; B: Chr2_3483609; C: Chr2_26191031; D: Chr3_4991610; E: Chr4_17841568; F: Chr4_23947857; G: Chr5_3714662; H: Chr6_12064959; I: Chr6_18415632; J: Chr7_5417364; K: Chr8_6167915; L: Chr8_22160552; M: Chr9_508439; N: Chr9_8663266; O: Chr11_5690624; P: Chr11_16140350; Q: Chr11_33330733; R: Chr12_3512965; S: Chr12_17378039; T: Chr13_21953151; U: Chr13_26179272; V: Chr14_2476344; W: Chr15_38652743; X: Chr17_1017338; Y: Chr17_8230144.

[0115] Using this set of markers to type 269 pear germplasm experimental materials, develop pear DNA fingerprint maps, and identify pear germplasm resources is beneficial to the preservation, collection and utilization of pear germplasm resources, as well as the protection and rights protection of cultivated varieties.

[0116] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a reagent for detecting the genotype of 25 SNP loci in the pear genome in any of the following: (1) Application in the preparation of a kit for constructing a DNA fingerprint of pear varieties or detecting authentic pear varieties; (2) Application in genotyping of pear germplasm resources; (3) Application in constructing DNA fingerprint of pear varieties; (4) Application in detecting authentic pear varieties; The physical positions of the 25 SNP sites are determined based on the alignment of the whole genome sequence of pear. The gene accession number of the whole genome of pear on NCBI is GCA_007844245.

1. The 25 SNP sites are SNP1-SNP25: SNP1 is located at position 16868304 on chromosome 1, and its base is C or T; SNP2 is located at position 3483609 on chromosome 2, and its base is G or C; SNP3 is located at position 26191031 on chromosome 2, and its base is A or G; SNP4 is located at position 4991610 on chromosome 3, and its base is G or C; SNP5 is located at position 17841568 on chromosome 4, and its base is A or G; SNP6 is located at position 23947857 on chromosome 4, and its base is T or C; SNP7 is located at position 3714662 on chromosome 5, and its base is T or C; SNP8 is located at position 12064959 on chromosome 6, and its base is C or T; SNP9 is located at position 18415632 on chromosome 6, and its base is either A or T; SNP10 is located at position 5417364 on chromosome 7, and its base is G or T; SNP11 is located at position 6167915 on chromosome 8, and its base is G or A; SNP12 is located at position 22160552 on chromosome 8, and its base is T or A; SNP13 is located at position 508439 on chromosome 9, and its base is A or T; SNP14 is located at position 8663266 on chromosome 9, and its base is C or T; SNP15 is located at position 5690624 on chromosome 11, and its base is C or T; SNP16 is located at position 16140350 on chromosome 11, and its base is T or C; SNP17 is located at position 33330733 on chromosome 11, and its base is C or T; SNP18 is located at position 3512965 on chromosome 12, and its base is C or A; SNP19 is located at position 17378039 on chromosome 12, and its base is C or G; SNP20 is located at position 21953151 on chromosome 13, and its base is A or C; SNP21 is located at position 26179272 on chromosome 13, and its base is T or C; SNP22 is located at position 2476344 on chromosome 14, and its base is A or G; SNP23 is located at position 38652743 on chromosome 15, and its base is either A or G; SNP24 is located at position 1017338 on chromosome 17, and its base is A or G; SNP25 is located at position 8230144 on chromosome 17, and its base is T or G.

2. A KASP primer combination for detecting the 25 SNP sites described in claim 1, characterized in that: The KASP primers corresponding to each SNP site to be tested include two forward primers and one reverse primer; The KASP primers used to detect SNP1 are two forward primers shown in SEQ ID NO.1-2 and a reverse primer shown in SEQ ID NO.3; The KASP primers used to detect SNP2 are two forward primers shown in SEQ ID NO.4-5 and a reverse primer shown in SEQ ID NO.6; The KASP primers used to detect SNP3 are two forward primers as shown in SEQ ID NO.7-8 and a reverse primer as shown in SEQ ID NO.9; The KASP primers used to detect SNP4 are two forward primers as shown in SEQ ID NO.10-11 and a reverse primer as shown in SEQ ID NO.12; The KASP primers used to detect SNP5 are two forward primers as shown in SEQ ID NO.13-14 and a reverse primer as shown in SEQ ID NO.15; The KASP primers used to detect SNP6 are two forward primers as shown in SEQ ID NO.16-17 and a reverse primer as shown in SEQ ID NO.18; The KASP primers used to detect SNP7 are two forward primers as shown in SEQ ID NO.19-20 and a reverse primer as shown in SEQ ID NO.21; The KASP primers used to detect SNP8 are two forward primers as shown in SEQ ID NO.22-23 and a reverse primer as shown in SEQ ID NO.24; The KASP primers used to detect SNP9 are two forward primers as shown in SEQ ID NO.25-26 and a reverse primer as shown in SEQ ID NO.27; The KASP primers used to detect SNP10 are two forward primers as shown in SEQ ID NO.28-29 and a reverse primer as shown in SEQ ID NO.30; The KASP primers used to detect SNP11 are two forward primers shown in SEQ ID NO.31-32 and a reverse primer shown in SEQ ID NO.33; The KASP primers used to detect SNP12 are two forward primers as shown in SEQ ID NO.34-35 and a reverse primer as shown in SEQ ID NO.36; The KASP primers used to detect SNP13 are two forward primers as shown in SEQ ID NO.37-38 and a reverse primer as shown in SEQ ID NO.39; The KASP primers used to detect SNP14 are two forward primers as shown in SEQ ID NO.40-41 and a reverse primer as shown in SEQ ID NO.42; The KASP primers used to detect SNP15 are two forward primers as shown in SEQ ID NO.43-44 and a reverse primer as shown in SEQ ID NO.45; The KASP primers used to detect SNP16 are two forward primers as shown in SEQ ID NO.46-47 and a reverse primer as shown in SEQ ID NO.48; The KASP primers used to detect SNP17 are two forward primers as shown in SEQ ID NO.49-50 and a reverse primer as shown in SEQ ID NO.51; The KASP primers used to detect SNP18 are two forward primers as shown in SEQ ID NO.52-53 and a reverse primer as shown in SEQ ID NO.54; The KASP primers used to detect SNP19 are two forward primers as shown in SEQ ID NO.55-56 and a reverse primer as shown in SEQ ID NO.57; The KASP primers used to detect SNP20 are two forward primers as shown in SEQ ID NO.58-59 and a reverse primer as shown in SEQ ID NO.60; The KASP primers used to detect SNP21 are two forward primers as shown in SEQ ID NO.61-62 and a reverse primer as shown in SEQ ID NO.63; The KASP primers used to detect SNP22 are two forward primers as shown in SEQ ID NO.64-65 and a reverse primer as shown in SEQ ID NO.66; The KASP primers used to detect SNP23 are two forward primers as shown in SEQ ID NO.67-68 and a reverse primer as shown in SEQ ID NO.69; The KASP primers used to detect SNP24 are two forward primers as shown in SEQ ID NO.70-71 and a reverse primer as shown in SEQ ID NO.72; The KASP primers used to detect SNP25 are two forward primers shown as SEQ ID NOs. 73-74 and a reverse primer shown as SEQ ID NO.

75.

3. The KASP primer combination according to claim 2, characterized in that The 5' ends of the two forward primers are connected to different fluorescent groups, including a FAM fluorescent group and a HEX fluorescent group.

4. A kit comprising the KASP primer combination according to claim 2 or 3.

5. Use of the KASP primer combination according to claim 2 or 3 or the kit according to claim 4 in any of the following: (1) Application in genotyping of pear varieties; (2) Application in constructing DNA fingerprint of pear varieties; (3) Application in detecting authentic pear varieties.

6. A method for genotyping pear varieties, characterized in that: The following steps are involved: Extracting genomic DNA of the pear varieties to be tested; Using the genomic DNA as a template, preparing a reaction system according to the 25 SNP sites described in claim 1, and using the corresponding primers in the KASP primer combination described in claim 2 or 3 to perform PCR amplification respectively; The fluorescence signal of the PCR amplification product is detected, and the genotype of the pear variety to be tested at the corresponding SNP site is obtained according to the value of the fluorescence signal.

7. A method for constructing a DNA fingerprint of pear varieties, characterized in that: The following steps are involved: Extracting genomic DNA of the pear varieties to be tested; Using the genomic DNA as a template, preparing a reaction system according to the 25 SNP sites described in claim 1, and using the corresponding primers in the KASP primer combination described in claim 2 or 3 to perform PCR amplification respectively; The fluorescence signal of the PCR amplification product is detected, and the genotype of the pear variety to be tested at the corresponding SNP site is obtained according to the value of the fluorescence signal, thereby constructing a DNA fingerprint of the pear variety.

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