Donkey 40K liquid chip based on targeted sequencing and application thereof

Abstract

The present invention discloses a donkey liquid chip based on targeted sequencing and comprising 1000 SNP loci located on a donkey reference genome. Optionally the chip comprises a capture probe desig

Description

The present invention relates to the technical field of gene detection, and particularly relates to a donkey 40k liquid chip based on a genotyping by targeted sequencing (GBTS) technology and an application thereof in donkey genotyping. Background Single nucleotide polymorphism (SNP), with the characteristics of large number, wide distribution, easy rapid mass screening and easy genotyping, is the third-generation genetic marker after the first-generation restriction fragment length polymorphism marker and the second-generation microsatellite marker (i.e., short tandem repeat marker), and has important biological significance. At present, the SNP marker has become an important tool for identification of biotic population, analysis of genetic structure, positioning of functional genes and selection of genomes. With the development of the high-throughput SNP genotyping technology, the method based on whole genome sequencing or reduced-representation genome sequencing has become the mainstream of the SNP genotyping technology. For species with reference genome sequences, researchers generally design SNP chips based on the known sequences, and hybridize genomic DNA fragments interrupted randomly with oligonucleotide probes on the chips to obtain the genotype of the corresponding SNP locus. Currently, no mature commercial SNP chips can be used on donkeys. Therefore, a genotyping technology is needed to reduce the production cost of high-throughput SNP chips and customize SNP markers according to actual population conditions, so as to achieve rapid, economical, applicable and convenient genotyping. GBTS is a technology that achieves deep resequencing only for target loci by reducing the library abundance, which can significantly reduce the cost of genotyping. The technology has not been applied in donkeys yet. However, the reduced-representation genome sequencing typing technology has the problems of uneven marker density and poor polymorphism, which is not favorable for the application of the high-throughput SNP genotyping technology in basic researches and molecular breeding of donkeys. Summary The purpose of the present invention is to provide a donkey 40K liquid chip based on targeted sequencing and an application thereof. To achieve the above purpose, the present invention adopts the following technical solution: A donkey 40K liquid chip based on targeted sequencing, which is composed of a probe mixture and a hybridization capture reagent which are individually packaged, wherein the probe mixture comprises a capture probe designed and synthesized according to each SNP locus (genotyping object) screened, and the density of the donkey 40K liquid chip can be reduced to a low density of IK (the specific positions of 1000 SNP loci on the donkey reference genome of the EquAsil.O version are shown in Table II below) or expanded to a high density of 200K by flexible adjustment. Preferably, the genotyping object of the donkey 40K liquid chip also comprises other SNP loci located on the donkey reference genome of the EquAsil.O version and matched by a capture probe designed and synthesized according to each SNP locus screened (the capture probes corresponding to the above 1000 SNP loci can match the following SNP loci located on the donkey reference genome of the EquAsil.O version: chrl5:23191566, chr 15:26258385, chrl 5:24168300, etc.). Preferably, each SNP locus screened is determined by comparing the whole genome sequencing result of the donkey with the donkey reference genome of the EquAsil.O version and based on the following SNP locus screening principles: the SNP loci are evenly distributed on each chromosome of the donkey reference genome and have the heterozygosis rate less than 15% and good polymorphism, wherein the index of good polymorphism is MAF greater than 0.35 in 18 Chinese donkey breeds, 8 foreign donkey breeds and wild donkey clusters (see Table I below). Preferably, the design principles of the capture probe include: the length of the probe is 100-120 nt, the GC content of the probe is 30%-80%, and the selected areas do not contain a GAP area. Preferably, two nucleotide sequences overlapping each other by 60%-70% and covering the SNP loci are designed according to the screened SNP loci, and then two pieces of DNA with the length of 100-120 nt and with biotin groups modified at 5' ends are synthesized according to the designed nucleotide sequences, i.e., the capture probe corresponding to the SNP locus. Preferably, the capture probes corresponding to the screened SNP loci are mixed at an equal amount of substance, and a mixture of EDTA and Tris-HCl is used for a constant volume, i.e., the probe mixture. A donkey genotyping method based on targeted sequencing, comprising the following steps: SI Preparing a donkey liquid chip based on targeted sequencing, wherein the density of the chip can be adjusted flexibly, and can be reduced to a low density of IK or expanded to a high density of 200K based on a donkey 40K liquid chip; S2 constructing a donkey DNA high-throughput sequencing library by using the genomic DNA of a donkey to be tested; S3 Capturing a DNA fragment containing the corresponding SNP loci from the donkey DNA high-throughput sequencing library constructed in S2 by using the donkey liquid chip prepared in SI; S4 Amplifying and purifying the DNA fragment obtained in S3, carrying out high-throughput sequencing of the product, and comparing the sequencing result and the donkey reference genome of the EquAsil.O version, so as to acquire the genotyping data of the donkey to be tested. Preferably, a method of constructing a donkey DNA high-throughput sequencing library in S2 specifically comprises the following steps: fragmenting the genomic DNA of the donkey to be tested by ultrasonic interruption or enzyme cutting, obtaining a donkey DNA high-throughput sequencing library by carrying out end-filling and poly(A) tailing and connecting a sequencing adapter, and measuring the concentration of the donkey DNA high-throughput sequencing library using Qubit. Preferably, the capturing operation in S3 specifically comprises the following steps: attaching the target donkey genomic DNA fragment to magnetic beads covered with streptavidin by capture probes contained in the probe mixture, and removing DNA fragments in non-target areas by elution. Preferably, the operation of acquiring the genotyping data of the donkey to be tested in S4 3 specifically comprises the following steps: processing high-throughput sequencing data by BWA and GATK software to obtain the genotype of the corresponding SNP locus of the donkey to be tested. An application of the donkey 40K liquid chip based on targeted sequencing in molecular breeding of donkeys. Preferably, an SNP locus chrl5:25408816, chrl5:23191566, chrl5:26258385 or chrl5:24168300 in the genotyping object of the chip is an SNP marker significantly related to the coat color trait of the donkey. The present invention has the following beneficial effects: The present invention effectively solves the problem of shortage of donkey high-throughput SNP chips due to uneven marker density and poor polymorphism and provides an efficient genome typing approach for genetic diversity analysis, line identification and marker assisted breeding researches and applications of donkeys based on the research and development of donkey high-throughput SNP chips (such as a 40K liquid chip based on targeted sequencing genotyping technology) and considering the distribution of the captured SNP loci in the whole genome (donkey reference genome) and the need of flexible application of chips in probe design. Further, the present invention improves the polymorphism level of genomic loci for the design of capture probes by analyzing the genomic sample data of donkey breeds (18 Chinese donkey breeds and 8 foreign donkey breeds) from different regions and wild donkey clusters (MAF is required to be greater than 0.35), and provides an important reference for exploring candidate SNP markers. Further, the present invention designs two single-strand DNA probes with the sequences overlapping each other by 60%-70% for each SNP locus (the GC content is controlled to 30%-80%), which can effectively improve the stability of hybridization for the genome fragment where each SNP is located, so as to improve the efficiency of fragment capture. Meanwhile, this probe design also fully considers the genomic specificity of the locus flanking sequence, ensures the locus-specific capture rate and reduces the sequencing cost, which is conducive to the application in large-scale SNP typing of donkey-specific markers. Further, the probe designed by the present invention (the selected areas do not contain a GAP area) has high tolerance to the variation of the locus flanking sequence, and still can stably 4 capture target fragments under the condition that the variation of the flanking sequence is not more than 10%. In this way, the sequence information of part of upstream and downstream areas can be acquired in addition to the target SNP information, which provides more information support for molecular breeding of donkeys at the same cost. Description of Drawings Fig. 1 is a schematic diagram of designing probes according to SNP loci. Fig. 2 shows a distribution diagram of the number of SNP loci on different chromosomes in a donkey 40K liquid chip. Fig. 3 is a spacing diagram of adjacent SNP loci of a donkey 40K liquid chip. Fig. 4 is a PCA diagram of distinguishing 9 donkey breeds using a donkey 40K liquid chip. Fig. 5 is a Manhattan plot of genome-wide association study of coat color trait of Dezhou donkeys using a donkey 40K liquid chip. Detailed Description The present invention will be further described in detail below in combination with the drawings and the embodiments. The embodiments are only used for explaining the present invention, not used for limiting the protection scope of the present invention. (I) Donkey 40K liquid chip based on targeted sequencing The present invention aims to develop a completely new donkey high-throughput SNP chip, a donkey 40K liquid chip based on targeted sequencing, so as to promote the GBTS technology to basic researches and molecular breeding applications of donkeys on a large scale. The whole genome sequencing results of donkeys (165 Chinese domestic donkeys, 7 Kyrgyz donkeys, 17 Kenyan donkeys, 6 Nigerian donkeys, 8 Egyptian donkeys, 10 Ethiopian donkeys, 18 Spanish donkeys, 8 Iranian donkeys, 8 Australian donkeys and 8 wild donkeys are selected at the chip development stage, wherein blood samples and genomic DNA are collected from some breeds of Chinese domestic donkeys; see Table I) are compared with the donkey reference genome of the EquAsil.O version to screen SNP loci, and the principles of screening SNP loci are as follows: (1) the SNP loci are evenly distributed on each chromosome and have the heterozygosis rate less than 15% and good polymorphism, wherein the index of good polymorphism is MAF greater than 0.35 in 18 Chinese donkey breeds, 8 foreign donkey breeds and wild donkey clusters; and (2) the coverage area of the screened SNP loci also meets the set probe design principles (the length of the probe is 100-120 nt, the GC content of the probe is 30%-80%, and the selected areas do not contain a GAP area). Two nucleotide sequences (as shown in Fig. 1) overlapping by 60%-70% and covering the SNP locus are designed according 5 to each SNP locus screened, then probes are respectively synthesized according to the designed nucleotide sequences, and two synthesized DNA single strands with the length of 100-120 nt and with biotin groups modified at 5' ends are probes used for capturing the genome fragment where the SNP locus is located. The probe design of the donkey high-throughput SNP chip based on the targeted sequencing 10 genotyping technology is carried out respectively for a total of 45894 SNP loci (target loci) screened. Experiments show that the probe sequence designed according to the screened SNP loci can ensure the accuracy and sensitivity of the formed donkey 40K liquid chip. Meanwhile, Fig. 2 shows that the SNP loci involved in the donkey 40K liquid chip cover each chromosome; and Fig. 3 shows that the SNP loci involved in the donkey 40K liquid chip have reasonable 15 spacing (about 40 kb on average, and the spacing of the SNP loci on each chromosome is roughly the same). Table I. Sources of Sample Genome Data of Chinese and Foreign Donkey Breeds and Wild Donkey Clusters Breed Quantity Blood Sample Collecting Position Sampling Time Chinese Tibetan donkey 31 Changdu, Tibet 2022.5 Yunnan donkey 7 Yunnan 2022.4 Huaibei donkey 10 Browse - BioProject - CNCB-NGDC Xinjiang donkey 16 PRJNA675210 Turpan donkey 5 PRJNA767165 Guola donkey (Hetian Qing donkey) 10 Xinjiang 2022.4 Biyang donkey 10 Biyang, Henan 2022.4 Dezhou donkey 7 Browse - BioProject - CNCB-NGDC Gunsha donkey 7 Qinghai 2022.1 Guanzhong donkey 10 Browse - BioProject - CNCB-NGDC Jiami donkey 11 Jiaxian, Shamixi 2022.1 Mongolian donkey 2 Inner Mongolia 2022.5 Qinghai donkey 7 Qinghai 2022.1 Qingyang donkey 9 Qingyang, Gansu 2022.1 Liangzhou donkey 10 Hexi Corridor, Gansu 2022.1 Taihang donkey 5 Taihang Mountain Area, Hebei 2022.1 Guangling donkey 4 Browse - BioProject - CNCB-NGDC Kulun donkey 4 PRJNA767165 Foreign Kyrgyzstan donkey 7 PRJCA001131 Kenyan donkey 17 ENA Browser (ebi.ac.uk) Nigerian donkey 6 PRJEB24845 Egyptian donkey W 8 PRJNA767165 Ethiopian donkey I s 10 ENA Browser (ebi.ac.uk) Spanish donkey M 18 ENA Browser (ebi.ac.uk) Iranian donkey '7 8 ENA Browser (ebi.ac.uk) Australian donkey 8 ENA Browser (ebi.ac.uk) Wild donkey 7 Browse - BioSamplc - CNCB-NGDC Note: [1] Kyrgyz; [2] Kenya; [3] Nigeria; [4] Egypt; [5] Ethiopia; [6] Spain; [7] Iran; [8] Australia. Compared with the mainstream solid chips developed based on the Illumina platform and the Affymetrix platform in the market, the donkey 40K liquid chip has the following advantages 5 in addition to retaining the advantages of high detection accuracy and low price: (1) the intellectual property right is fully independent; (2) the density of the developed SNP chip can be adjusted flexibly, and can be reduced to a low density of IK (see Table II) or expanded to a high density of 200K based on a mainstream 40K liquid chip; (3) loci can be added at any time without sample size limitation; and (4) the detection cost is lower than that of the current solid chip. 10 Table II. Position and Mutation Information of 1000 SNP Loci Serial No. Position Mutation Type Serial No. Position Mutation Type Serial No. Position Mutation Type 001 1:2821410 A / G 002 1:3304319 A / G 003 1:4256987 A / C 004 1:8214369 T / G 005 1:8393144 G / A 006 1:13630087 C / T 007 1:15867974 C / A 008 1:16136849 C / T 009 1:17961449 T / C 010 1:19721474 G / A 011 1:22775063 C / A 012 1:24850106 C / T 013 1:29042828 A / G 014 1:29192879 T / C 015 1:31319101 T / G 016 1:35173280 C / A 017 1:35346427 C / G 018 1:40449588 A / C 019 1:41568543 T / G 020 1:43987039 G / A 021 1:47994232 G / C 022 1:48751758 G / A 023 1:54086898 C / A 024 1:54598045 G / C 025 1:64038936 A / G 026 1:67600674 T / C 027 1:75400728 C / T 028 1:77335867 C / T 029 1:78144679 T / C 030 1:78195979 C / T 031 1:81089296 T / G 032 1:83159091 T / A 033 1:83219464 T / G 034 1:90104401 G / A 035 1:95365178 T / C 036 1:95602803 C / A 037 1:96195246 A / G 038 1:102440145 Cl 039 1:105468784 C / T 040 1:107273988 G / T 041 1:109719942 G / A 042 1:112995579 G / C 043 1:113964133 T / A 044 1:116254348 A / G 045 1:117602185 C / T 046 2:1639522 T / C 047 2:1848659 C / A 048 2:2696207 C / G 049 2:4239982 A / G 050 2:8311555 G / C 051 2:8639778 A / C 052 2:12177221 A / G 053 2:15546207 A / T 054 2:15728803 C / A 055 2:16177892 G / C 056 2:32026630 A / G 057 2:33503191 T / C 058 2:42889719 G / A 059 2:43053138 C / G 060 2:48023098 C / G 061 2:48754806 T / C 062 2:49875521 T / G 063 2:53335179 G / A 064 2:55143447 T / C 065 2:57830937 T / G 066 2:62303018 G / A 067 2:64763399 T / G 068 2:68595061 G / T 069 2:73412009 G / C 070 2:76942241 A / G 071 2:78942961 G / T 072 2:81522730 N / . 073 2:82737198 C / T 074 2:84398012 1 / Cr 075 2:86048052 C / T 076 2:89547306 C / T 077 2:92428466 G / A 078 2:92927618 G / A 079 2:97626211 G / A 080 2:97869924 Cl 081 2:98259609 C / A 082 2:99828310 G / A 083 2:101145345 G / A 084 2:106077189 G / A 085 2:108574845 T / C 086 2:114251214 A / G 087 2:115485969 C / G 088 2:117362744 T / C 089 2:120099845 T / C 090 2:120368713 C / T 091 2:121524849 A / G 092 2:121895215 A / C 093 2:126737281 A / C 094 2:130468580 A / T 095 2:131253316 A / G 096 2:133236372 G / A 097 2:133248366 C / T 098 2:133444539 C / A 099 2:133469440 C / G 100 2:134479165 G / A 101 2:138534908 A / G 102 2:141893451 G / A 103 2:148211278 C / A 104 2:153628354 G / A 105 2:153797691 A / G 106 2:163995187 A / G 107 2:166636497 T / C 108 2:168627380 C / T 109 2:169989455 C / T 110 2:176103389 T / C 111 2:182040263 A / C 112 2:186534463 C / T 113 2:186595553 C / T 114 2:186616841 A / C 115 2:196779759 T / C 116 2:199646742 A / G 117 2:201291959 A / G 118 2:205436490 T / A 119 2:212360202 C / T 120 2:214606447 G / A 121 2:219091249 G / A 122 2:219613583 C / A 123 2:227654782 T / G 124 2:228782219 A / G 125 2:229583873 G / C 126 2:230465587 T / G 127 2:231037577 A / G 128 2:236237903 G / C 129 3:10059822 T / C 130 3:11355858 C / T 131 3:15783408 T / C 132 3:16410219 T / A 133 3:19141132 T / C 134 3:21594300 C / G 135 3:23843883 T / G 136 3:27344134 C / G 137 3:30921247 A / G 138 3:31964340 C / T 139 3:32588658 G / A 140 3:34121987 A / C 141 3:40662693 C / T 142 3:42693564 C / A 143 3:48931851 A / G 144 3:51487432 A / C 145 3:54941284 A / G 146 3:63629492 A / G 147 3:64914756 A / G 148 3:64930904 T / C 149 3:66203228 G / A 150 3:68975958 A / C 151 3:69362543 N / . 152 3:74947630 C / A 153 3:81179088 T / G 154 3:81828392 C / T 155 3:84781075 A / G 156 3:86785687 AG 157 3:89642177 C / A 158 3:91953513 C / G 159 3:92867681 C / G 160 3:93850030 T / C 161 3:99169138 A / G 162 3:100892305 A / T 163 3:104106305 A / G 164 3:109729435 G / A 165 3:121900552 A / G 166 3:126132760 G / C 167 3:127967567 T / C 168 3:130149418 A / G 169 3:130265967 C / T 170 3:130307501 G / T 171 3:130345258 C / T 172 3:130528902 G / T 173 3:130880810 A / G 174 3:132673599 A / G 175 3:132896897 A / G 176 3:138892308 A / G 177 3:139506743 C / T 178 3:139623569 T / C 179 3:141118818 C / T 180 3:143789879 T / C 181 3:145540346 A / C 182 3:147369860 A / G 183 3:149213221 C / A 184 3:156486273 A / T 185 3:156709295 A / G 186 3:156815559 G / C 187 3:162795797 G / A 188 3:162925633 C / G 189 3:164046868 A / G 190 3:166917707 C / T 191 3:167454130 G / A 192 3:167689274 G / A 193 3:169144872 A / G 194 3:169484762 C / T 195 3:170202903 T / C 196 3:170528565 C / A 197 3:174708468 C / G 198 3:176220418 C / A 199 3:177169254 A / T 200 3:178924663 A / C 201 3:179400342 G / A 202 4:3653964 C / T 203 4:6002611 C / T 204 4:6248861 G / A 205 4:6269014 A / G 206 4:6824685 G / A 207 4:11895637 A / G 208 4:13428280 G / C 209 4:18354897 C / T 210 4:18574182 C / A 211 4:20803496 A / G 212 4:21897104 A / C 213 4:22275147 G / A 214 4:23299297 T / G 215 4:27688890 C / T 216 4:32877869 C / A 217 4:34843111 A / G 218 4:35524372 A / G 219 4:35920799 A / G 220 4:37181072 G / A 221 4:39474777 A / G 222 4:43696416 A / C 223 4:44808377 T / C 224 4:45963117 A / G 225 4:48109887 A / G 226 4:49460518 G / A 227 4:49851364 A / T 228 4:50833213 C / A 229 4:50942198 T / C 230 4:51301060 T / G 231 4:51869927 G / A 232 4:54452703 C / G 233 4:54495976 T / C 234 4:55994354 A / C 235 4:56312621 C / G 236 4:61164444 A / G 237 4:63237246 T / C 238 4:63338095 T / C 239 4:63430239 G / A 240 4:73765898 T / C 241 4:76693646 G / T 242 4:79461179 G / A 243 4:83885031 T / C 244 4:88064950 A / G 245 5:2445985 A / T 246 5:4333203 T / C 247 5:6949114 G / C 248 5:12232852 T / C 249 5:13614954 T / C 250 5:14053251 G / A 251 5:15144241 G / A 252 5:18217006 C / T 253 5:19603617 C / T 254 5:20641617 T / C 255 5:20747358 T / C 256 5:23041535 T / C 257 5:23385081 T / C 258 5:36303775 G / T 259 5:38577053 A / C 260 5:44450111 C / T 261 5:46770220 A / G 262 5:50277984 A / G 263 5:52308053 T / C 264 5:57640315 A / G 265 5:58863669 C / T 266 5:63545689 A / C 267 5:68018477 C / T 268 5:68065715 l / (j 269 5:68596990 C / A 270 5:77709544 G / A 271 5:79029691 T / C 272 5:81599403 T / C 273 5:81679867 T / C 274 5:81805271 T / C 275 5:82024113 T / C 276 5:82344375 T / C 277 5:83290222 G / A 278 5:83412571 C / T 279 5:83659024 A / C 280 5:83962906 T / C 281 5:84128487 C / T 282 5:86831655 A / G 283 5:87172903 A / G 284 5:88038576 C / T 285 5:91761730 AG 286 5:94240419 G / A 287 5:94432835 C / A 288 5:95064213 T / C 289 5:100843912 G / A 290 5:102055508 T / G 291 5:102144434 G / T 292 5:102189367 T / C 293 5:102345386 G / A 294 5:102876259 G / C 295 5:109977105 G / A 296 5:111240164 A / T 297 6:5595455 G / A 298 6:7484067 A / G 299 6:9927297 C / G 300 6:21715192 G / A 301 6:22607368 G / A 302 6:25183820 C / T 303 6:25532380 C / T 304 6:26033662 T / C 305 6:30592864 A / G 306 6:30924327 T / C 307 6:31842306 T / C 308 6:34449996 A / G 309 6:36687651 T / G 310 6:37312133 A / G 311 6:37740386 G / A 312 6:40351358 G / A 313 6:40737423 A / G 314 6:45195252 T / C 315 6:46375784 T / Z'' l / (j 316 6:51486412 C / T 317 6:55315435 T / G 318 6:56396975 C / T 319 6:60933976 G / T 320 6:62716905 A / G 321 6:64526946 T / C 322 6:65998298 T / A 323 6:67510785 G / T 324 6:68459803 T / G 325 6:69969352 T / C 326 6:72354044 G / T 327 6:73644786 T / C 328 6:75401581 A / G 329 6:77176691 A / G 330 6:79225237 C / T 331 6:84715635 C / T 332 6:84932740 C / T 333 6:85908274 T / G 334 6:88718396 T / G 335 7:559587 C / T 336 7:2318455 C / A 337 7:2399133 C / T 338 7:9194016 G / A 339 7:14156217 G / C 340 7:20235127 G / A 341 7:30710350 C / T 342 7:31426147 T / C 343 7:34572363 T / C 344 7:35193243 G / A 345 7:36220063 A / G 346 7:47025476 C / A 347 7:47735132 A / T 348 7:49538698 T / G 349 7:51213703 T / A 350 7:52312049 A / C 351 7:60507045 C G 352 7:65049909 C / T 353 7:65336848 A / G 354 7:67245951 G / A 355 7:68290351 T / C 356 7:68538895 C / T 357 7:70709421 T / Z'' l / (j 358 7:72617422 C / G 359 7:85999552 A / C 360 7:86301750 T / C 361 7:89731832 G / A 362 7:91771282 G / C 363 7:92961427 T / C 364 7:94519044 G / C 365 7:96238980 A / G 366 7:100396698 T / C 367 7:104068276 G / A 368 7:107182961 G / A 369 7:110243736 A / G 370 7:110789681 A / G 371 7:111695881 C / A 372 7:113589503 T / C 373 7:117486751 G / A 374 7:120532274 C / T 375 7:122323353 T / G 376 8:2204542 C / A 377 8:4441528 C / T 378 8:5952611 G / A 379 8:6297425 T / C 380 8:6917049 T / C 381 8:9988841 C / T 382 8:10952390 A / C 383 8:12119662 C / A 384 8:12376842 C / G 385 8:13021236 A / G 386 8:14271834 G / A 387 8:15178083 T / C 388 8:18838190 A / G 389 8:20941221 C / T 390 8:21634485 G / T 391 8:22161517 T / C 392 8:25669382 T / C 393 8:25813782 C / T 394 8:31810891 A / T 395 8:31966290 C / T 396 8:32190241 T / C 397 8:32897200 C / T 398 8:33525693 G / A 399 8:41145275 A / C 400 8:41811745 A / G 401 8:42675861 G / C 402 8:49309645 G / A 403 8:49465255 A / G 404 8:50948912 T / A 405 8:56649735 T / C 406 8:56889792 T / C 407 8:57558388 A / C 408 8:58093946 G / A 409 8:59751525 G / T 410 8:62885789 G / A 411 8:67723492 C / T 412 8:69068233 G / A 413 8:71308710 C / T 414 8:71570691 G / A 415 8:73476243 G / A 416 8:74305675 G / A 417 8:74566570 A / C 418 8:76860994 C / T 419 8:84312050 C / T 420 8:84787011 A / C 421 8:88652081 T / G 422 8:91078378 C / T 423 8:96077168 T / G 424 9:15185871 G / C 425 9:16485531 IC 426 9:22526184 A / G 427 9:23538812 T / C 428 9:25494508 A / G 429 9:27962541 T / C 430 9:28246503 T / C 431 9:30066839 C / G 432 9:31793297 G / A 433 9:34534394 G / A 434 9:37749325 N / . 435 9:38053176 A / G 436 9:38055392 C / G 437 9:38813270 T / C 438 9:39441949 G / A 439 9:39795508 T / C 440 9:41218907 T / C 441 9:42816250 A / G 442 9:47329546 C / A 443 9:56178854 T / C 444 9:57564565 C / T 445 9:60285098 C / A 446 9:62413316 T / C 447 9:62788164 G / A 448 10:501590 G / A 449 10:1967485 C / A 450 10:3054106 T / C 451 10:7797602 G / A 452 10:9111659 A / G 453 10:10692361 C / T 454 10:14987267 A / G 455 10:17892599 C / T 456 10:18497860 G / A 457 10:20406972 T / C 458 10:23259334 C / A 459 10:23802265 T / C 460 10:24321909 A / T 461 10:25449801 T / C 462 10:26076414 A / C 463 10:26319509 N / . 464 10:27049951 G / C 465 10:27601202 C / T 466 10:28427917 G / A 467 10:34067973 Cl 468 10:38841082 A / T 469 10:41096175 G / C 470 10:43333554 A / T 471 10:45930241 C / T 472 10:52748219 A / G 473 10:53486886 G / A 474 10:54809742 T / A 475 10:56037674 G / A 476 10:57946372 T / C 477 10:62111107 T / C 478 10:63772670 A / G 479 10:65009596 G / A 480 10:66302419 A / G 481 10:66561268 C / T 482 10:69729141 T / C 483 10:70129762 A / G 484 10:71435926 T / A 485 10:75186923 G / C 486 10:79121808 A / G 487 10:80956704 A / G 488 10:83347368 T / C 489 10:85054189 C / T 490 10:87943825 G / T 491 11:1608860 C / T 492 11:3135701 C / T 493 11:5516300 A / C 494 11:7901494 G / C 495 11:8829287 T / C 496 11:9838649 G / C 497 11:10205394 G / A 498 11:14623780 G / A 499 11:15130619 A / G 500 11:16501843 T / C 501 11:17413536 C / T 502 11:20331918 C / T 503 11:21339192 T / C 504 11:26421211 A / G 505 11:28526023 T / G 506 11:29751453 T / C 507 11:31659044 T / A 508 11:37301201 T / C 509 11:39630736 G / A 510 11:41609139 T / C 511 11:43631504 T / C 512 11:47938099 T / C 513 11:48124614 G / C 514 11:50515439 G / C 515 11:51053452 C / T 516 11:51174695 C / T 517 11:51220969 A / G 518 11:51946761 G / C 519 11:54620229 G / A 520 11:61053496 T / C 521 11:62077306 A / C 522 11:71945501 T / G 523 11:76467241 G / A 524 11:85394050 T / C 525 12:3058123 C / T 526 12:3748492 G / A 527 12:9768907 A / G 528 12:9965523 C / T 529 12:13762495 C / A 530 12:15217226 IC 531 12:23388972 T / C 532 12:24521328 G / A 533 12:28113129 A / G 534 12:28591983 A / G 535 12:33031304 G / A 536 12:35273984 A / G 537 12:36010557 A / T 538 12:39101062 C / T 539 12:39567199 A / C 540 12:40060233 CT 541 12:42897837 G / A 542 12:43677883 G / A 543 12:44463207 G / T 544 12:47162341 A / G 545 12:47625982 T / C 546 12:48374842 G / A 547 12:49006527 G / A 548 12:52714208 A / G 549 12:53133484 C / A 550 12:55075301 T / C 551 12:55775640 A / G 552 12:55865055 T / C 553 12:56534771 G / A 554 12:56554141 IC 555 12:60689518 A / C 556 12:61651013 A / G 557 12:69882933 C / T 558 12:70478193 C / T 559 12:73374955 A / C 560 12:81323134 G / A 561 12:83153060 A / G 562 12:89365894 A / G 563 12:91845453 A / C 564 12:93274173 C / A 565 12:93738705 G / A 566 12:93944591 C / A 567 12:94799128 T / C 568 12:95229835 G / A 569 12:97415996 G / A 570 12:99165142 C / G 571 12:99543819 C / A 572 12:101433824 G / A 573 12:103507645 A / G 574 12:103810973 T / C 575 12:105504707 C / A 576 13:4563768 G / T 577 13:4884657 G / A 578 13:8632006 C / G 579 13:22096703 A / T 580 13:36469964 T / C 581 13:38218039 G / A 582 13:43136454 T / A 583 13:43613004 C / A 584 13:43624023 A / G 585 13:44557631 C / G 586 13:44804002 G / T 587 13:55248457 C / G 588 13:56221963 T / C 589 13:56594417 G / A 590 13:57098334 G / C 591 13:57255793 C / T 592 13:62258737 G / A 593 13:62738028 T / C 594 14:496416 G / C 595 14:1776501 T / C 596 14:2482151 IC 597 14:3109667 C / T 598 14:4882139 A / G 599 14:11370708 T / C 600 14:13916867 C / T 601 14:16034688 C / A 602 14:18167998 G / A 603 14:20165579 A / G 604 14:20726665 C / T 605 14:22074017 A / G 606 14:32972186 C / G 607 14:34520480 T / C 608 14:35110036 G / A 609 14:37557425 T / C 610 14:39933904 G / A 611 14:43915281 T / C 612 15:2246407 A / C 613 15:5906146 T / C 614 15:7416771 A / C 615 15:9096693 G / A 616 15:9392633 C / T 617 15:11387306 A / G 618 15:11870421 G / A 619 15:15837009 T / G 620 15:19495494 G / A 621 15:25408816 C / T 622 15:30643226 A / G 623 15:31992444 A / T 624 15:33443407 T / C 625 15:34559221 G / A 626 15:35541780 G / A 627 15:36065115 T / C 628 15:37870431 A / G 629 15:42354042 A / G 630 15:43536919 C / A 631 15:44315621 A / G 632 15:44413416 T / C 633 15:45889655 G / T 634 15:47673743 A / C 635 15:48286212 A / C 636 16:499263 A / G 637 16:3723679 T / C 638 16:6887230 C / T 639 16:7694581 T / C 640 16:7900661 G / C 641 16:8778140 T / G 642 16:10266338 C / T 643 16:10318086 C / T 644 16:12951849 G / A 645 16:13249028 A / C 646 16:13616110 A / G 647 16:15595003 C / T 648 16:16174612 A / G 649 16:17093159 G / A 650 16:19099047 G / A 651 16:24913554 T / C 652 16:32772441 G / C 653 16:35502412 T / C 654 16:37314520 G / T 655 16:39720328 A / G 656 16:40447923 C / T 657 16:41866488 A / G 658 16:44752407 C / G 659 16:45225818 IC 660 16:47674505 G / T 661 16:48072521 C / G 662 16:48263025 T / C 663 16:49561912 C / G 664 16:49974351 G / C 665 17:4147675 A / G 666 17:4468825 T / C 667 17:5811859 C / T 668 17:10285809 A / G 669 17:11773265 C / A 670 17:12017498 C / G 671 17:12255767 C / T 672 17:18921554 G / A 673 17:27880117 G / A 674 17:28436811 G / C 675 17:29286412 A / C 676 17:30504863 C / A 677 17:32163790 U71 678 17:33236962 A / C 679 17:33451338 C / T 680 17:33959025 C / T 681 17:34209687 G / C 682 17:34996026 A / G 683 17:35707041 Cl 684 17:36041512 C / T 685 17:40517576 A / G 686 17:42852297 C / T 687 17:43003291 G / A 688 17:44732926 G / A 689 17:45445540 C / T 690 17:45589844 G / A 691 17:46333362 C / A 692 18:2388633 T / G 693 18:3403977 C / T 694 18:3929143 G / A 695 18:8448312 C / T 696 18:8637951 C G 697 18:8969962 N / . 698 18:13256995 G / A 699 18:13677773 A / G 700 18:18723573 A / T 701 18:19695906 G / A 702 18:23296279 C / A 703 18:24293007 T / C 704 18:24655619 A / G 705 18:26308843 G / A 706 18:27719882 T / C 707 18:31027266 A / G 708 19:270335 T / A 709 19:2114429 C / A 710 19:2688730 G / A 711 19:3822831 C / T 712 19:4823160 G / C 713 19:4939074 C / T 714 19:6586515 G / A 715 19:7465436 C / T 716 19:7885892 G / T 717 19:8912993 A / G 718 19:13280204 G / C 719 19:15710560 G / T 720 19:15880188 A / C 721 19:20311234 C / T 722 19:20578931 C / T 723 19:21038538 C / T 724 19:26327677 T / C 725 19:26388739 AC 726 19:26979613 A / C 727 20:6339166 C / T 728 20:18789874 A / G 729 20:19028435 G / A 730 20:20544783 A / G 731 20:20587311 A / G 732 20:22094136 C / A 733 20:23981183 C / G 734 20:24190090 T / C 735 20:24255893 G / C 736 20:27972431 T / C 737 20:29400468 C / T 738 20:30812292 C G 739 20:31308804 / “1 / r-p 1171 740 20:33307955 C / T 741 20:34956051 G / A 742 20:35580553 A / C 743 20:35759881 T / C 744 20:37923934 T / C 745 20:39150517 C / G 746 20:39908284 G / A 747 20:46609310 G / A 748 20:49146900 G / A 749 20:56420906 C / T 750 20:58613269 A / G 751 20:61815866 G / C 752 20:65670420 C / T 753 20:67529154 T / C 754 20:68081138 T / C 755 20:77758727 T / C 756 20:78620487 C / T 757 20:79165013 C / T 758 20:79438296 A / C 759 20:89153730 C / G 760 20:89488213 T / G 761 20:89682416 C / T 762 20:90101085 G / C 763 20:91827736 A / G 764 20:91919844 C / G 765 20:93316651 G / A 766 20:94721217 T / G 767 20:98095802 C / T 768 20:99824776 C / A 769 21:3061812 A / G 770 21:5596362 C / T 771 21:10006507 T / C 772 21:11649291 T / C 773 21:11697961 G / A 774 21:12577280 T / C 775 21:14025462 G / A 776 21:14910709 A / G 777 21:15855493 A / G 778 21:18279939 C / T 779 21:18589104 A / G 780 21:19165597 A / T 781 21:19192497 C / T 782 21:19415640 G / A 783 21:21694365 G / A 784 21:22132508 A / G 785 21:23219115 T / C 786 21:26831360 G / A 787 21:30859844 G / A 788 21:31776920 Cl 789 21:33203955 T / C 790 21:36750047 G / C 791 21:38672493 A / G 792 21:39472741 T / C 793 21:41787491 T / C 794 21:43657013 A / G 795 21:45919522 C / G 796 21:52259783 A / C 797 21:68062875 G / A 798 21:68435856 C / A 799 21:70356379 A / G 800 21:76993977 T / C 801 21:80178714 C / T 802 21:81152642 T / C 803 21:81863949 G / A 804 21:82213523 G / T 805 21:82714747 T / C 806 21:84194566 G / C 807 21:84319127 G / A 808 21:85458891 C / T 809 21:86013145 T / G 810 21:86720773 C / T 811 21:92073932 G / A 812 21:94331915 A / G 813 21:98155322 C / T 814 22:2293346 G / T 815 22:2795330 G / A 816 22:5566092 G / C 817 22:6691812 T / C 818 22:10278062 A / C 819 22:12828416 T / C 820 22:22284782 A / G 821 22:22663170 A / G 822 22:23235076 C / G 823 22:23854673 G / T 824 22:25615574 T / A 825 22:28656358 C T 826 22:31095926 G / A 827 22:31475818 G / A 828 22:32919826 C / G 829 22:35009865 G / A 830 22:37345539 A / G 831 23:2690505 A / G 832 23:7424233 T / C 833 23:7480437 T / A 834 23:13115937 G / T 835 23:17079265 T / C 836 23:17594967 T / C 837 23:22106645 T / C 838 23:22735071 T / G 839 23:23473586 T / G 840 23:25265734 T / C 841 23:25614597 C / T 842 23:26904772 G / A 843 23:30330134 A / G 844 23:30606759 G / A 845 23:33858483 C / A 846 23:36925287 A / C 847 23:37266882 A / G 848 23:41423630 1 / Cr 849 23:41507322 G / A 850 23:43103465 C / G 851 23:43453190 T / G 852 23:44971381 A / G 853 24:6548872 C / A 854 24:7236806 Cl 855 24:11375375 C / T 856 24:16738616 A / C 857 24:19944593 G / A 858 24:21779446 A / G 859 24:22920106 A / T 860 24:23108642 C / T 861 24:26121521 T / C 862 24:26398180 A / G 863 24:28900111 T / C 864 24:36171465 A / C 865 24:42097099 T / A 866 24:43435423 C / T 867 25:655338 C A 868 25:3861620 1 / Cr 869 25:6487076 T / C 870 25:12398415 G / T 871 25:13386116 G / A 872 25:15011422 A / G 873 25:16031288 G / A 874 25:17910131 C / T 875 25:24136111 T / C 876 25:25957634 A / G 877 25:27659606 A / G 878 25:32508383 G / A 879 25:33043288 A / G 880 25:34716556 A / G 881 25:35536203 A / G 882 25:35815157 C / T 883 25:39089459 A / C 884 25:41396569 C / G 885 25:42022761 T / C 886 25:42435008 C / T 887 25:43275682 C / A 888 25:43568723 C / G 889 26:543339 G / A 890 26:987762 T / C 891 26:1532299 C / T 892 26:2596041 T / C 893 26:2762116 C / G 894 26:3273611 T / G 895 26:4048102 G / A 896 26:4192497 A / G 897 26:6962380 C / T 898 26:9127939 C / T 899 26:9778216 T / C 900 26:10080357 G / A 901 26:11031917 G / C 902 26:13594405 C / T 903 26:16603632 T / C 904 26:17484721 T / A 905 26:18631616 C / A 906 26:19505902 C / T 907 26:23000168 G / C 908 26:23018587 T / C 909 26:26144816 G / A 910 27:2493363 T / C 911 27:5747383 A / G 912 27:6316822 G / A 913 27:7334613 C / T 914 27:8024389 A / C 915 27:10219720 G / C 916 27:11924865 T / C 917 27:14111391 G / C 918 27:14742472 A / G 919 27:21940923 A / G 920 27:27168242 G / A 921 27:28964361 C / G 922 28:4819906 G / A 923 28:6789996 C / A 924 28:9198597 A / T 925 28:14634869 T / A 926 28:21872936 A / G 927 28:22336033 CT 928 28:22791960 T / C 929 28:22963896 C / T 930 28:23799071 C / T 931 28:25101440 G / C 932 28:26274706 T / C 933 28:29194191 C / T 934 28:29327411 G / A 935 28:30262767 T / C 936 28:32112512 C / T 937 28:35525801 A / G 938 28:36907777 T / C 939 28:41029449 C / T 940 28:49935930 T / G 941 28:52033004 G / C 942 28:57907245 G / A 943 28:58812955 T / C 944 29:645525 A / G 945 29:3436373 A / T 946 29:6355613 T / C 947 29:6877652 T / C 948 29:8297899 C / G 949 29:8766933 A / G 950 29:9886954 A / G 951 29:14735581 T / C 952 29:17740514 G / C 953 29:22535355 T / A 954 29:23309465 T / C 955 29:28401101 A / G 956 29:29684014 G / A 957 29:30972968 C / T 958 29:31409121 G / T 959 29:31600907 G / C 960 29:35489791 C / T 961 30:1307858 G / A 962 30:4967300 A / G 963 30:5252281 G / A 964 30:20270917 A / G 965 30:21059240 G / A 966 30:24064333 A / G 967 30:25327725 T / C 968 30:25461446 C / G 969 30:26258301 C / T 970 30:27286231 A / C 971 30:28692644 C / G 972 X:2345264 G / T 973 X:6372012 T / G 974 X: 17070409 G / A 975 X:31726773 T / G 976 X: 32142109 C / T 977 X:32707803 G / A 978 X:34385517 C / T 979 X:38962535 G / T 980 X:46079163 C / G 981 X:50308814 T / C 982 X: 57737117 A / G 983 X:66139797 G / A 984 X:67449901 A / C 985 X: 68229501 C / T 986 X:68620566 T / C 987 X:68765677 T / C 988 X:69951482 A / G 989 X:73801205 G / A 990 X:74229727 G / A 991 X: 78248795 A / G 992 X:85950525 G / A 993 X:92003559 G / T 994 X: 92491285 G / A 995 X: 96618977 G / A 996 X:97282210 G / A 997 X: 98984945 T / C 998 X:99438944 T / C 999 X: 106641828 T / C 1000 Y:4995971 A / C (II) Donkey genotyping method using donkey 40K liquid chip based on targeted sequencing SI. Preparing a donkey 40K liquid chip based on targeted sequencing The donkey 40K liquid chip is composed of a probe mixture and a hybridization capture 5 reagent which are individually packaged. Probe mixture: two DNA single strands with biotin groups at 5' ends, which are designed and synthesized according to each SNP locus screened, are mixed at an equal molar amount, and a mixture of EDTA and Tris-HCl is used for a constant volume (the concentration of each capture probe is 3 pmol / mL). 10 Hybridization capture reagent: a GenoBaits DNA seq Library Prep kit purchased from MolBreeding Biotech Ltd., specifically comprising: GenoBaits Block I, GenoBaits Block II for ILM / MGI, GenoBaits 2 / Hyb Buffer V3.3, GenoBaits Hyb Buffer Enhancer V3.3, GenoBaits 1 Ox Wash Buffer I, GenoBaits 10x Wash Buffer II, GenoBaits 10* Wash Buffer III and GenoBaits 10* Stringent Wash Buffer which are individually packaged. 15 S2 Constructing a donkey DNA high-throughput sequencing library by using the genomic DNA of a donkey to be tested 2.1 Extracting the genomic DNA of a donkey to be tested A TIANamp Genomic DNA Kit (blood / cell / tissue genomic DNA extraction kit) is used for extraction, and the specific method is as follows: (1) Treating materials: directly using 200 pL of fresh or frozen blood or blood with anticoagulant, and adding buffer GA to make up if less than 200 pL. (2) Adding 20 pL of ProteinaseK solution, and mixing well. (3) Adding 200 pL of buffer GB, mixing well by turning upside down, standing at 70°C for 10 min, and after the solution becomes clear, centrifuging the solution shortly to remove water drops on the inner wall of the tube cover. (4) Adding 200 pL of absolute ethyl alcohol, mixing well by full oscillation for 15 sec, flocculent precipitate may appear at this time, and centrifuging the solution shortly to remove water drops on the inner wall of the tube cover. (5) Adding the solution and flocculent precipitate obtained in the above step to an adsorption column CB3 (putting the adsorption column into a collecting tube), centrifuging at 12000 rpm (13400*g) for 30 sec, discarding the waste solution, and putting the adsorption column CB3 back into the collecting tube. (6) Adding 500 pL of buffer GD to the adsorption column CB3 (adding absolute ethyl alcohol before use), centrifuging at 12000 rpm (13400*g) for 30 sec, discarding the waste solution, and putting the adsorption column CB3 into the collecting tube. (7) Adding 600 pL of buffer PW to the adsorption column CB3 (adding absolute ethyl alcohol before use), centrifuging at 12000 rpm (13400*g) for 30 sec, discarding the waste solution, and putting the adsorption column CB3 into the collecting tube. (8) Repeating step 7 once. (9) Putting the adsorption column CB3 back into the collecting tube, centrifuging at 12000 rpm (13400*g) for 2 min, and discarding the waste solution. Placing the adsorption column CB3 at room temperature for several minutes to dry the adsorbed material thoroughly to remove the residual buffer. (10) Transferring the adsorption column CB3 to a clean centrifuging tube, dripping 50-200 pL of wash buffer to the middle part of an adsorption film, standing at room temperature for 2-5 16 min, centrifuging at 12000 rpm (13400*g) for 2 min, and collecting the solution into the centrifuging tube. (11) Measuring the length of the donkey genomic DNA extracted from the blood sample. 2.2 Constructing a donkey DNA high-throughput sequencing library (1) Preparing a reaction system with a total amount of 20uL in a PCR tube: 200 ng of donkey genomic DNA obtained in step 1, 4 pL of GenoBaits End Repair Buffer (MolBreeding Biotech Ltd.), 3.1 pL of GenoBaits End Repair Enzyme (MolBreeding Biotech Ltd.), and the balance of ultrapure water. (2) Gently mixing the reaction system well, and centrifuging shortly to collect the reaction liquid to the bottom of the tube. (3) Placing the PCR tube in a PCR instrument, carrying out reaction according to the conditions in Table 1, and heating the cover at 82°C: Table 1. Reaction Conditions of PCR Instrument Temperature Time 37°C 20min 72°C 20min 4°C Storage (4) Adding the following components (with a total amount of 20 pL) in the PCR tube: 2 pL of GenoBaits ULtra DNA Ligase (MolBreeding Biotech Ltd ), 8 pL of GenoBaits ULtra DNA Ligase Buffer (MolBreeding Biotech Ltd.), 2 pL of GenoBaits Adapter for MGI (MolBreeding Biotech Ltd.), and the balance of ultrapure water. (5) Gently shaking the PCR tube to mix the added components well, and centrifuging shortly to collect the liquid to the bottom of the tube. (6) Placing the PCR tube in the PCR instrument, carrying out reaction according to the conditions in Table 2, and canceling the operation of heating the cover: Table 2. Reaction Conditions of PCR Instrument Temperature Time 22°C 60min 4°C Storage (7) Adding 48 pL of DNA purification magnetic beads that have been in equilibrium at room temperature for more than 30 min in the PCR tube, shaking and mixing well, standing for 5 min, and centrifuging shortly to collect the liquid to the bottom of the tube. (8) Placing the PCR tube on a magnetic frame for at least 3 min until the solution becomes clear, and removing supernatant. (9) Keeping the PCR tube in the magnetic frame, and adding 100 pL of 80% ethanol; and incubating at room temperature for 30 sec, and removing supernatant. (10) Keeping the PCR tube in the magnetic frame, removing the cover and drying in the air for 5 min until the ethanol evaporates. (11) Removing the PCR tube from the magnetic frame, and drying the magnetic beads therein. (12) Preparing a reaction system with a total amount of 20 pL: 10 pL of GenoBaits PCR Master Mix (MolBreeding Biotech Ltd ), 1 pL of 15 Barcode(10pm)-MGI, 5 pL of 17 Barcode(2pm)-MGI and 4 pL of ultrapure water. (13) Adding the reaction system in step 12 in the PCR tube in step 11, resuspending the dried magnetic beads, and centrifuging shortly to collect the liquid to the bottom of the tube. (14) Placing the PCR tube in the PCR instrument, and carrying out reaction according to the conditions in Table 3: Table 3. Reaction Conditions of PCR Instrument Temperature Time Number of Cycles 98°C 2min None 98°C 30s 6-8 50°C 30s 72°C 40s 72°C 4min None The number of cycles in Table 3 is adjusted according to the DNA initial mass, as shown 5 in Table 4: Table 4. Reference for Number of Cycles of PCR Amplification Initial Mass Number of Cycles Ing-lOng 8 lOng-lOOng 6-8 100 ng and above 6 (15) Adding 20 pL of DNA purification magnetic beads that have been in equilibrium at room temperature for more than 30 min in the PCR tube, shaking and mixing well, standing for 10 5 min, and centrifuging shortly to collect the liquid to the bottom of the tube. (16) Placing the PCR tube on the magnetic frame for at least 3 min until the solution becomes clear, and removing supernatant. (17) Keeping the PCR tube in the magnetic frame, and adding 100 pL of 80% ethanol; and incubating at room temperature for 30 sec, and removing supernatant. 15 (18) Keeping the PCR tube in the magnetic frame, removing the cover and drying in the air for 10 min. (19) Removing the PCR tube from the magnetic frame, adding 35 pL of Tris HC1, shaking and mixing well, standing for 5 min, and centrifuging shortly to collect the liquid to the bottom of the tube. (20) Placing the PCR tube on the magnetic frame, and after the solution becomes clear (about 3 min), moving supernatant to a new tube to complete construction of a donkey DNA high-throughput sequencing library. Mass detection of library: 1 uL of prepared library sample is taken and quantified with Qubit, and the DNA total mass of the sequencing library is required to be more than 500 ng. 3 pL of prepared library sample is taken and subjected to 1% agarose gel electrophoresis experiment, and the range of DNA fragments of the electrophoresis result is 300-500 bp, which can ensure the effect of subsequent capture experiments. The donkey DNA high-throughput sequencing library is stored at 4°C for a short time or at -20°C for a long time. S3. Mixing the probe mixture of the donkey 40K liquid chip prepared in SI with the donkey DNA high-throughput sequencing library constructed in S2, so as to capture DNA fragments of target loci contained in the donkey DNA high-throughput sequencing library (1) Keeping the streptavidin magnetic beads in equilibrium at room temperature for more than 30 min, and placing the magnetic beads in a 0.2 mL PCR tube. (2) Adding 500 ng of donkey DNA high-throughput sequencing library, 5 pL of GenoBaits Block I, 2 pL of GenoBaits Block II for 1LM / MGI and probe mixture (containing 300 ng of probe) in the PCR tube to form a mixed solution containing the donkey DNA high-throughput sequencing library and capture probes. (3) Centrifuging the PCR tube at 12000 rpm for 1 min. (4) Checking the dissolution of the GenoBaits hybridization reagent; and if crystals are found, heating to 65°C until the crystals are dissolved completely. (5) Adding 8 pL of GenoBaits 2xHyb Buffer V3.3, 2 pL of GenoBaits Hyb Buffer Enhancer V3.3 and 6 pL of NucLease-Free Water in the PCR tube in step 3. (6) Absorbing the mixture in the PCR tube back and forth or stirring and mixing well, standing at room temperature for 5 min, absorbing the mixture back and forth or stirring and mixing well again, gently centrifuging, and transferring all the mixture in the PCR tube to a 0.2 mL EP tube. (7) Placing the EP tube in the PCR instrument, and keeping the PCR instrument in a warm bath at 95°C for 10 min (the cover heating temperature is 105°C). (8) Transferring the EP tube to another PCR instrument with the cover heated to 75°C, 20 keeping the PCR instrument in a warm bath at 65°C for 1 h, and binding DNA (DNA fragments where the target loci are located in the donkey DNA high-throughput sequencing library) with the streptavidin magnetic beads through the corresponding capture probes. (9) Adding 100 pL of GenoBaits 1 xWash Buffer I preheated at 65°C in the EP tube, shaking and stirring for 5 s, and centrifuging for 5 s, and then placing the EP tube on the magnetic frame, and after the magnetic beads are completely separated from the solution, removing supernatant (containing unbound DNA). (10) Adding 150 pL of GenoBaits 1 * Stringent Wash Buffer preheated (65°C) in the EP tube, and slowly absorbing the mixture back and forth for 10 times (avoiding bubbles); standing for 2 min after the samples are mixed well; and then placing the EP tube on the magnetic frame, and after the magnetic beads are completely separated from the solution, removing supernatant. (11) Adding 150 pL of GenoBaits 1 x Wash Buffer I (of room temperature) to the EP tube, and shaking for 2 min; and then placing the EP tube on the magnetic frame, and after the magnetic beads are completely separated from the solution, removing supernatant. (12) Adding 150 pL of GenoBaits 1 x Wash Buffer II (of room temperature) to the EP tube, and shaking for 1 min; and then placing the EP tube on the magnetic frame, and after the magnetic beads are completely separated from the solution, removing supernatant. (13) Adding 150 pL of GenoBaits lx Wash Buffer III of room temperature to the EP tube, and shaking for 30 sec; and then placing the EP tube on the magnetic frame, and after the magnetic beads are completely separated from the solution, removing supernatant. (14) Removing the EP tube from the magnetic frame, adding 20 pL of NucLease-Free Water, absorbing the mixture back and forth for 20 times to resuspend magnetic beads, and using 10 pL of resuspension for subsequent PCR amplification and purification, and reserving the remaining 10 pL for backup. The eluents used in the above steps, which require 65°C preheating, are placed in the PCR instrument in advance, and the remaining eluents are stored at room temperature. The eluents shall be prepared in advance (see Table 5): Table 5. Preparation of Eluent lOxBuffer Water Total GenoBaits 1 xWash Buffer I 28 pL 252 pL 280 pL GenoBaits 1 Wash Buffer II 18 pL 162 pL 180 pL GenoBaits 1 xWash Buffer III 18 pL 162 pL 180 pL GenoBaits 1x Stringent Wash Buffer 40 pL 360 pL 400 pL S4. Amplifying and purifying the DNA fragments obtaining in S3, carrying out high-5 throughput sequencing of the product, and comparing the sequencing result and the donkey reference genome, so as to acquire the genotyping data of the donkey to be tested. (1) Preparing a PCR reagent with a total amount of 30 pL in a 0.2 mL PCR tube: 15 pL of GenoBaits PCR Master Mix (MolBreeding Biotech Ltd ), 1.2 pL of GenoBaits Primer Mix for MGI (MolBreeding Biotech Ltd.), 10 pL of magnetic beads adsorbing captured DNA and 3.8 pL 10 of ultrapure water. (2) Stirring shortly, centrifuging and ensuring that the magnetic beads are in the solution. (3) Placing the PCR tube in the PCR instrument, with the cover heating temperature of 105°C, and performing PCR amplification according to procedures, see Table 6: Table 6. Reaction Conditions of PCR Instrument Temperature Time Number of Cycles 98°C 45 s (Predegeneration) None 98°C 15s 12 50°C 30s 72°C 30s 72°C 1 min (Extension) None 4°C Storage (4) Adding 45 pL (1.5* volume) of DNA purification magnetic beads that have been in equilibrium at room temperature for more than 30 min to the PCR reaction product, shaking and mixing well, avoiding bubbles, standing for 5 min, and centrifuging shortly to collect the liquid to the bottom of the tube; and placing the PCR tube on the magnetic frame for at least 3 min until the solution becomes clear, and removing supernatant. (5) Keeping the PCR tube in the magnetic frame, adding 100 pL of 80% ethanol, incubating at room temperature for 30 sec, and removing supernatant; and keeping the PCR tube in the magnetic frame, removing the cover and drying in the air for 10 min. (6) Removing the PCR tube from the magnetic frame, adding 35 uL of Tris HO, shaking and mixing well, standing for 5 min, and centrifuging shortly to collect the liquid to the bottom of the tube; and placing the PCR tube on the magnetic frame, and after the solution becomes clear (about 3 min), moving supernatant to a new tube, and storing at -20°C. The purified PCR fragments can be stored at -20°C for one week. (7) Measuring the captured DNA library using Qubits FLuorometer and Qubit dsDNA HS Assay Kit, and measuring the average fragment length of the DNA library captured on a digital electrophoresis system; and measuring the concentration of the captured DNA Library using the KAPA Library Quantification Kit. (8) Sequencing the captured DNA library using a sequenator, and processing the sequencing data through BWA and GATK software to obtain the genotype of the corresponding SNP locus of a donkey to be tested. (Ill) Genotyping quality evaluation of donkey 40K liquid chip based on targeted sequencing 1. Detection rate The SNP detection rate is an important index to measure the chip quality, which is generally measured by loci of autosome and X chromosome. The sample test shows that the average SNP detection rate of the donkey 40K liquid chip is 98.5%-99.1%, which exceeds the standard detection rate of 95%, indicating that the genotype detection quality of the donkey 40K liquid chip is good. 2. Analysis of genetic structure For the whole genome sequencing result of sample donkeys (except wild donkeys) in Table 23 I, genotype annotation is made with reference to the SNP loci involved in the donkey 40K liquid chip, and then principal component analysis (PCA) is conducted. With reference to Fig. 4, the donkey 40K liquid chip can clearly distinguish Chinese domestic donkey breeds from 8 foreign donkey breeds. The result also shows that the donkey 40K liquid chip can provide SNP markers for genetic structure division. (IV) Breeding application A total of 210 Dezhou donkeys are genotyped using the donkey 40K liquid chip, including 98 Sanfen donkeys and 112 Wutou donkeys. Significant molecular markers of coat color trait are screened using genome-wide association analysis, and after data quality control, 205 individuals and 44661 SNP loci are retained. The significance thresholds are defined as 0.05 / N (genomewide significance level) and 1 / N (chromosome significance level), wherein N is the number of SNP loci used. The result is shown as Fig. 5, and a total of 4 SNP loci reach the genome-wide significance level: chrl5:25408816 (P value = 1.67 x 10'34), chrl5:23191566 (P value = 3.19 x 10-9), chrl5:26258385 (P value = 9.94 x 10’8) and chrl 5:24168300 (P value = 1.05 x 10^’), wherein the most significant SNP locus (chrl5:25408816) is the SNP locus corresponding to 621 in Table II, and the genotype distribution of the locus in the Sanfen cluster is: CC: 3 donkeys, CT: 22 donkeys, and TT: 72 donkeys; and the genotype distribution of the locus in the Wutou cluster is: CC: 82 donkeys, CT: 25 donkeys, and TT: 1 donkey. It can be seen that the Sanfen cluster and the Wutou cluster can be quickly established by respectively selecting individuals with TT and CC genotypes. As annotated, the candidate gene affecting coat color trait is the ASIP (agouti signaling protein) gene. To sum up, the donkey 40K liquid chip developed by the present invention provides SNP variation information containing more than 200K for typing detection results, and can supplement SNP loci at any time. The new probes designed according to the new loci only need to be added into the existing probe mixture, and can also remove SNP loci that do not need genotyping. Meanwhile, different from the solid chip technology which has requirements for the sample size (one chip can usually detect 12,24,48 or even 96 samples), the donkey 40K liquid chip developed by the present invention has no requirement for the sample size, the whole experiment is performed in the PCR tube, and the reaction system can be added as needed, so one sample can also be detected, and the detection number of samples is flexible, which makes up for the 24 technical defects of solid chips and has more popularization value in genetic structure analysis and molecular breeding application of donkeys. In addition, the hybridization capture time of the present invention is only 1 h (step 8 in S3) by screening and optimizing the capture hybridization system. Compared with the overnight hybridization capture process of more than 16 h, the present 5 invention significantly shortens the time of genotype acquisition, so that the whole process of library construction and capture can be completed in one day, saving time for genotype detection.

Claims

1. A donkey liquid chip based on targeted sequencing, wherein the genotyping object of the chip comprises 1000 SNP loci located on a donkey reference genome, and the positions of the5   1000 SNP loci on the donkey reference genome are shown as follows:Serial No. Position Mutation Type Serial No. Position Mutation Type Serial No. Position Mutation Type 001 1:2821410 A / G 002 1:3304319 A / G 003 1:4256987 A / C 004 1:8214369 T / G 005 1:8393144 G / A 006 1:13630087 C / T 007 1:15867974 C / A 008 1:16136849 C / T 009 1:17961449 T / C 010 1:19721474 G / A 011 1:22775063 C / A 012 1:24850106 C / T 013 1:29042828 A / G 014 1:29192879 T / C 015 1:31319101 T / G 016 1:35173280 C / A 017 1:35346427 C / G 018 1:40449588 A / C 019 1:41568543 T / G 020 1:43987039 G / A 021 1:47994232 G / C 022 1:48751758 G / A 023 1:54086898 C / A 024 1:54598045 G / C 025 1:64038936 A / G 026 1:67600674 T / C 027 1:75400728 C / T 028 1:77335867 C / T 029 1:78144679 T / C 030 1:78195979 C / T 031 1:81089296 T / Z^ 1 / Cj 032 1:83159091 T / A 033 1:83219464 T / G 034 1:90104401 G / A 035 1:95365178 T / C 036 1:95602803 C / A 037 1:96195246 A / G 038 1:102440145 C / T 039 1:105468784 C / T 040 1:107273988 G / T 041 1:109719942 G / A 042 1:112995579 G / C 043 1:113964133 T / A 044 1:116254348 A / G 045 1:117602185 C / T 046 2:1639522 T / C 047 2:1848659 C / A 048 2:2696207 C / G 049 2:4239982 A / G 050 2:8311555 G / C 051 2:8639778 AC 052 2:12177221 A / G 053 2:15546207 A / T 054 2:15728803 C / A 055 2:16177892 G / C 056 2:32026630 A / G 057 2:33503191 T / C 058 2:42889719 G / A 059 2:43053138 C / G 060 2:48023098 C / G 061 2:48754806 T / C 062 2:49875521 T / G 063 2:53335179 G / A 064 2:55143447 T / C 065 2:57830937 T / G 066 2:62303018 G / A 067 2:64763399 T / G 068 2:68595061 G / T 069 2:73412009 G / C 070 2:76942241 A / G 071 2:78942961 G / T 072 2:81522730 N / . 073 2:82737198 C / T 074 2:84398012 T / G 075 2:86048052 C / T 076 2:89547306 C / T 077 2:92428466 G / A 078 2:92927618 G / A 079 2:97626211 G / A 080 2:97869924 C / T 081 2:98259609 C / A 082 2:99828310 G / A 083 2:101145345 G / A 084 2:106077189 G / A 085 2:108574845 T / C 086 2:114251214 A / G 087 2:115485969 C / G 088 2:117362744 T / C 089 2:120099845 T / C 090 2:120368713 C / T 091 2:121524849 AG 092 2:121895215 A / C 093 2:126737281 A / C 094 2:130468580 A / T 095 2:131253316 A / G 096 2:133236372 G / A 097 2:133248366 C / T 098 2:133444539 C / A 099 2:133469440 C / G 100 2:134479165 G / A 101 2:138534908 A / G 102 2:141893451 G / A103 2:148211278 C / A 104 2:153628354 G / A 105 2:153797691 A / G 106 2:163995187 A / G 107 2:166636497 T / C 108 2:168627380 C / T 109 2:169989455 C / T 110 2:176103389 T / C 111 2:182040263 A / C 112 2:186534463 C / T 113 2:186595553 C / T 114 2:186616841 A / C 115 2:196779759 T / C 116 2:199646742 A / G 117 2:201291959 A / G 118 2:205436490 T / A 119 2:212360202 C / T 120 2:214606447 G / A 121 2:219091249 G / A 122 2:219613583 C / A 123 2:227654782 T / Z'' l / (j 124 2:228782219 A / G 125 2:229583873 G / C 126 2:230465587 T / G 127 2:231037577 A / G 128 2:236237903 G / C 129 3:10059822 T / C 130 3:11355858 C / T 131 3:15783408 T / C 132 3:16410219 T / A 133 3:19141132 T / C 134 3:21594300 C / G 135 3:23843883 T / G 136 3:27344134 C / G 137 3:30921247 A / G 138 3:31964340 C / T 139 3:32588658 G / A 140 3:34121987 A / C 141 3:40662693 C / T 142 3:42693564 C / A 143 3:48931851 A / G 144 3:51487432 A / C 145 3:54941284 A / G 146 3:63629492 A / G 147 3:64914756 A / G 148 3:64930904 T / C 149 3:66203228 G / A 150 3:68975958 A / C 151 3:69362543 N / . 152 3:74947630 C / A 153 3:81179088 T / G 154 3:81828392 C / T 155 3:84781075 A / G 156 3:86785687 A / G 157 3:89642177 C / A 158 3:91953513 C / G 159 3:92867681 C / G 160 3:93850030 T / C 161 3:99169138 A / G 162 3:100892305 A / T 163 3:104106305 A / G 164 3:109729435 G / A 165 3:121900552 A / G 166 3:126132760 G / C 167 3:127967567 T / C 168 3:130149418 A / G 169 3:130265967 C / T 170 3:130307501 G / T 171 3:130345258 C / T 172 3:130528902 G / T 173 3:130880810 A / G 174 3:132673599 A / G 175 3:132896897 A / G 176 3:138892308 A / G 177 3:139506743 C / T 178 3:139623569 T / C 179 3:141118818 C / T 180 3:143789879 T / C 181 3:145540346 A / C 182 3:147369860 A / G 183 3:149213221 C / A 184 3:156486273 A / T 185 3:156709295 A / G 186 3:156815559 G / C 187 3:162795797 G / A 188 3:162925633 C / G 189 3:164046868 A / G 190 3:166917707 C / T 191 3:167454130 G / A 192 3:167689274 G / A 193 3:169144872 A / G 194 3:169484762 C / T 195 3:170202903 T / C 196 3:170528565 C / A 197 3:174708468 C / G 198 3:176220418 C / A 199 3:177169254 A / T 200 3:178924663 A / C 201 3:179400342 G / A 202 4:3653964 C / T 203 4:6002611 C / T 204 4:6248861 G / A 205 4:6269014 A / G 206 4:6824685 G / A 207 4:11895637 A / G 208 4:13428280 G / C 209 4:18354897 Cl 210 4:18574182 C / A 211 4:20803496 A / G 212 4:21897104 A / C 213 4:22275147 G / A 214 4:23299297 T / G 215 4:27688890 C / T 216 4:32877869 C / A 217 4:34843111 A / G 218 4:35524372 A / G 219 4:35920799 A / G 220 4:37181072 G / A 221 4:39474777 A / G 222 4:43696416 AC 223 4:44808377 T / C 224 4:45963117 A / G 225 4:48109887 A / G 226 4:49460518 G / A 227 4:49851364 A / T 228 4:50833213 C / A 229 4:50942198 T / C 230 4:51301060 T / G 231 4:51869927 G / A232 4:54452703 C / G 233 4:54495976 IC 234 4:55994354 A / C 235 4:56312621 C / G 236 4:61164444 A / G 237 4:63237246 T / C 238 4:63338095 T / C 239 4:63430239 G / A 240 4:73765898 T / C 241 4:76693646 G / T 242 4:79461179 G / A 243 4:83885031 T / C 244 4:88064950 A / G 245 5:2445985 A / T 246 5:4333203 T / C 247 5:6949114 G / C 248 5:12232852 T / C 249 5:13614954 T / C 250 5:14053251 G / A 251 5:15144241 G / A 252 5:18217006 C / T 253 5:19603617 C / T 254 5:20641617 T / C 255 5:20747358 T / C 256 5:23041535 T / C 257 5:23385081 T / C 258 5:36303775 G / T 259 5:38577053 A / C 260 5:44450111 C / T 261 5:46770220 A / G 262 5:50277984 A / G 263 5:52308053 T / C 264 5:57640315 A / G 265 5:58863669 C / T 266 5:63545689 A / C 267 5:68018477 C / T 268 5:68065715 T / G 269 5:68596990 C / A 270 5:77709544 G / A 271 5:79029691 T / C 272 5:81599403 T / C 273 5:81679867 T / C 274 5:81805271 T / C 275 5:82024113 IC 276 5:82344375 T / C 277 5:83290222 G / A 278 5:83412571 C / T 279 5:83659024 A / C 280 5:83962906 T / C 281 5:84128487 C / T 282 5:86831655 A / G 283 5:87172903 A / G 284 5:88038576 C / T 285 5:91761730 A / G 286 5:94240419 G / A 287 5:94432835 C / A 288 5:95064213 T / C 289 5:100843912 G / A 290 5:102055508 T / G 291 5:102144434 G / T 292 5:102189367 T / C 293 5:102345386 G / A 294 5:102876259 G / C 295 5:109977105 G / A 296 5:111240164 A / T 297 6:5595455 G / A 298 6:7484067 A / G 299 6:9927297 C / G 300 6:21715192 G / A 301 6:22607368 G / A 302 6:25183820 C / T 303 6:25532380 C / T 304 6:26033662 T / C 305 6:30592864 A / G 306 6:30924327 T / C 307 6:31842306 T / C 308 6:34449996 A / G 309 6:36687651 T / G 310 6:37312133 A / G 311 6:37740386 G / A 312 6:40351358 G / A 313 6:40737423 A / G 314 6:45195252 T / C 315 6:46375784 T / G 316 6:51486412 C / T 317 6:55315435 T / G 318 6:56396975 C / T 319 6:60933976 G / T 320 6:62716905 A / G 321 6:64526946 T / C 322 6:65998298 T / A 323 6:67510785 G / T 324 6:68459803 T / G 325 6:69969352 T / C 326 6:72354044 G / T 327 6:73644786 T / C 328 6:75401581 A / G 329 6:77176691 A / G 330 6:79225237 C / T 331 6:84715635 C / T 332 6:84932740 C / T 333 6:85908274 T / G 334 6:88718396 T / G 335 7:559587 C / T 336 7:2318455 C / A 337 7:2399133 C / T 338 7:9194016 G / A 339 7:14156217 G / C 340 7:20235127 G / A 341 7:30710350 C / T 342 7:31426147 T / C 343 7:34572363 T / C 344 7:35193243 G / A 345 7:36220063 A / G 346 7:47025476 C / A 347 7:47735132 A / T 348 7:49538698 T / G 349 7:51213703 T / A 350 7:52312049 A / C 351 7:60507045 C / G 352 7:65049909 C / T 353 7:65336848 A / G 354 7:67245951 G / A 355 7:68290351 T / C 356 7:68538895 C / T 357 7:70709421 T / G 358 7:72617422 C / G 359 7:85999552 A / C 360 7:86301750 T / C361 7:89731832 G / A 362 7:91771282 G / C 363 7:92961427 T / C 364 7:94519044 G / C 365 7:96238980 A / G 366 7:100396698 T / C 367 7:104068276 G / A 368 7:107182961 G / A 369 7:110243736 A / G 370 7:110789681 A / G 371 7:111695881 C / A 372 7:113589503 T / C 373 7:117486751 G / A 374 7:120532274 C / T 375 7:122323353 T / G 376 8:2204542 C / A 377 8:4441528 C / T 378 8:5952611 G / A 379 8:6297425 T / C 380 8:6917049 T / C 381 8:9988841 C / T 382 8:10952390 A / C 383 8:12119662 C / A 384 8:12376842 C / G 385 8:13021236 A / G 386 8:14271834 G / A 387 8:15178083 T / C 388 8:18838190 A / G 389 8:20941221 C / T 390 8:21634485 G / T 391 8:22161517 T / C 392 8:25669382 T / C 393 8:25813782 C / T 394 8:31810891 A / T 395 8:31966290 C / T 396 8:32190241 T / C 397 8:32897200 C / T 398 8:33525693 G / A 399 8:41145275 A / C 400 8:41811745 A / G 401 8:42675861 G / C 402 8:49309645 G / A 403 8:49465255 A / G 404 8:50948912 T / A 405 8:56649735 T / C 406 8:56889792 T / C 407 8:57558388 A / C 408 8:58093946 G / A 409 8:59751525 G / T 410 8:62885789 G / A 411 8:67723492 C / T 412 8:69068233 G / A 413 8:71308710 C / T 414 8:71570691 G / A 415 8:73476243 G / A 416 8:74305675 G / A 417 8:74566570 A / C 418 8:76860994 C / T 419 8:84312050 C / T 420 8:84787011 A / C 421 8:88652081 T / G 422 8:91078378 C / T 423 8:96077168 T / Z'' l / (j 424 9:15185871 G / C 425 9:16485531 T / C 426 9:22526184 A / G 427 9:23538812 T / C 428 9:25494508 A / G 429 9:27962541 T / C 430 9:28246503 T / C 431 9:30066839 C / G 432 9:31793297 G / A 433 9:34534394 G / A 434 9:37749325 N / . 435 9:38053176 A / G 436 9:38055392 C / G 437 9:38813270 T / C 438 9:39441949 G / A 439 9:39795508 T / C 440 9:41218907 T / C 441 9:42816250 A / G 442 9:47329546 C / A 443 9:56178854 T / C 444 9:57564565 C / T 445 9:60285098 C / A 446 9:62413316 T / C 447 9:62788164 G / A 448 10:501590 G / A 449 10:1967485 C / A 450 10:3054106 T / C 451 10:7797602 G / A 452 10:9111659 A / G 453 10:10692361 C / T 454 10:14987267 A / G 455 10:17892599 C / T 456 10:18497860 G / A 457 10:20406972 T / C 458 10:23259334 C / A 459 10:23802265 T / C 460 10:24321909 A / T 461 10:25449801 T / C 462 10:26076414 A / C 463 10:26319509 N / . 464 10:27049951 G / C 465 10:27601202 C / T 466 10:28427917 G / A 467 10:34067973 Cl 468 10:38841082 A / T 469 10:41096175 G / C 470 10:43333554 A / T 471 10:45930241 C / T 472 10:52748219 A / G 473 10:53486886 G / A 474 10:54809742 T / A 475 10:56037674 G / A 476 10:57946372 T / C 477 10:62111107 T / C 478 10:63772670 A / G 479 10:65009596 G / A 480 10:66302419 AG 481 10:66561268 C / T 482 10:69729141 T / C 483 10:70129762 A / G 484 10:71435926 T / A 485 10:75186923 G / C 486 10:79121808 A / G 487 10:80956704 A / G 488 10:83347368 T / C 489 10:85054189 C / T490 10:87943825 G / T 491 11:1608860 Cl 492 11:3135701 C / T 493 11:5516300 A / C 494 11:7901494 G / C 495 11:8829287 T / C 496 11:9838649 G / C 497 11:10205394 G / A 498 11:14623780 G / A 499 11:15130619 A / G 500 11:16501843 T / C 501 11:17413536 C / T 502 11:20331918 C / T 503 11:21339192 T / C 504 11:26421211 A / G 505 11:28526023 T / Z^ 1 / Cj 506 11:29751453 T / C 507 11:31659044 T / A 508 11:37301201 T / C 509 11:39630736 G / A 510 11:41609139 T / C 511 11:43631504 T / C 512 11:47938099 T / C 513 11:48124614 G / C 514 11:50515439 G / C 515 11:51053452 C / T 516 11:51174695 C / T 517 11:51220969 A / G 518 11:51946761 G / C 519 11:54620229 G / A 520 11:61053496 T / C 521 11:62077306 A / C 522 11:71945501 T / G 523 11:76467241 G / A 524 11:85394050 T / C 525 12:3058123 C / T 526 12:3748492 G / A 527 12:9768907 A / G 528 12:9965523 C / T 529 12:13762495 C / A 530 12:15217226 T / C 531 12:23388972 T / C 532 12:24521328 G / A 533 12:28113129 A / G 534 12:28591983 A / G 535 12:33031304 G / A 536 12:35273984 A / G 537 12:36010557 A / T 538 12:39101062 C / T 539 12:39567199 A / C 540 12:40060233 C / T 541 12:42897837 G / A 542 12:43677883 G / A 543 12:44463207 G / T 544 12:47162341 A / G 545 12:47625982 T / C 546 12:48374842 G / A 547 12:49006527 G / A 548 12:52714208 A / G 549 12:53133484 C / A 550 12:55075301 T / C 551 12:55775640 A / G 552 12:55865055 T / C 553 12:56534771 G / A 554 12:56554141 T / C 555 12:60689518 A / C 556 12:61651013 A / G 557 12:69882933 C / T 558 12:70478193 C / T 559 12:73374955 A / C 560 12:81323134 G / A 561 12:83153060 A / G 562 12:89365894 A / G 563 12:91845453 A / C 564 12:93274173 C / A 565 12:93738705 G / A 566 12:93944591 C / A 567 12:94799128 T / C 568 12:95229835 G / A 569 12:97415996 G / A 570 12:99165142 C / G 571 12:99543819 C / A 572 12:101433824 G / A 573 12:103507645 A / G 574 12:103810973 T / C 575 12:105504707 C / A 576 13:4563768 G / T 577 13:4884657 G / A 578 13:8632006 C / G 579 13:22096703 A / T 580 13:36469964 T / C 581 13:38218039 G / A 582 13:43136454 T / A 583 13:43613004 C / A 584 13:43624023 A / G 585 13:44557631 C / G 586 13:44804002 G / T 587 13:55248457 C / G 588 13:56221963 T / C 589 13:56594417 G / A 590 13:57098334 G / C 591 13:57255793 C / T 592 13:62258737 G / A 593 13:62738028 T / C 594 14:496416 G / C 595 14:1776501 T / C 596 14:2482151 IC 597 14:3109667 C / T 598 14:4882139 A / G 599 14:11370708 T / C 600 14:13916867 C / T 601 14:16034688 C / A 602 14:18167998 G / A 603 14:20165579 A / G 604 14:20726665 C / T 605 14:22074017 A / G 606 14:32972186 C / G 607 14:34520480 T / C 608 14:35110036 G / A 609 14:37557425 T / C 610 14:39933904 G / A 611 14:43915281 T / C 612 15:2246407 A / C 613 15:5906146 T / C 614 15:7416771 A / C 615 15:9096693 G / A 616 15:9392633 C / T 617 15:11387306 A / G 618 15:11870421 G / A619 15:15837009 T / G 620 15:19495494 G / A 621 15:25408816 C / T 622 15:30643226 A / G 623 15:31992444 A / T 624 15:33443407 T / C 625 15:34559221 G / A 626 15:35541780 G / A 627 15:36065115 T / C 628 15:37870431 A / G 629 15:42354042 A / G 630 15:43536919 C / A 631 15:44315621 A / G 632 15:44413416 T / C 633 15:45889655 G / T 634 15:47673743 A / C 635 15:48286212 A / C 636 16:499263 A / G 637 16:3723679 T / C 638 16:6887230 C / T 639 16:7694581 T / C 640 16:7900661 G / C 641 16:8778140 T / G 642 16:10266338 C / T 643 16:10318086 C / T 644 16:12951849 G / A 645 16:13249028 A / C 646 16:13616110 A / G 647 16:15595003 C / T 648 16:16174612 A / G 649 16:17093159 G / A 650 16:19099047 G / A 651 16:24913554 T / C 652 16:32772441 G / C 653 16:35502412 T / C 654 16:37314520 G / T 655 16:39720328 A / G 656 16:40447923 C / T 657 16:41866488 A / G 658 16:44752407 C / G 659 16:45225818 T / C 660 16:47674505 G / T 661 16:48072521 C / G 662 16:48263025 T / C 663 16:49561912 C / G 664 16:49974351 G / C 665 17:4147675 A / G 666 17:4468825 T / C 667 17:5811859 C / T 668 17:10285809 A / G 669 17:11773265 C / A 670 17:12017498 C / G 671 17:12255767 C / T 672 17:18921554 G / A 673 17:27880117 G / A 674 17:28436811 G / C 675 17:29286412 A / C 676 17:30504863 C / A 677 17:32163790 G / T 678 17:33236962 A / C 679 17:33451338 C / T 680 17:33959025 C / T 681 17:34209687 G / C 682 17:34996026 A / G 683 17:35707041 C / T 684 17:36041512 C / T 685 17:40517576 A / G 686 17:42852297 C / T 687 17:43003291 G / A 688 17:44732926 G / A 689 17:45445540 C / T 690 17:45589844 G / A 691 17:46333362 C / A 692 18:2388633 T / G 693 18:3403977 C / T 694 18:3929143 G / A 695 18:8448312 C / T 696 18:8637951 C / G 697 18:8969962 N / . 698 18:13256995 G / A 699 18:13677773 A / G 700 18:18723573 A / T 701 18:19695906 G / A 702 18:23296279 C / A 703 18:24293007 T / C 704 18:24655619 A / G 705 18:26308843 G / A 706 18:27719882 T / C 707 18:31027266 A / G 708 19:270335 T / A 709 19:2114429 C / A 710 19:2688730 G / A 711 19:3822831 C / T 712 19:4823160 G / C 713 19:4939074 C / T 714 19:6586515 G / A 715 19:7465436 CT 716 19:7885892 G / T 717 19:8912993 A / G 718 19:13280204 G / C 719 19:15710560 G / T 720 19:15880188 A / C 721 19:20311234 C / T 722 19:20578931 C / T 723 19:21038538 C / T 724 19:26327677 T / C 725 19:26388739 AC 726 19:26979613 A / C 727 20:6339166 C / T 728 20:18789874 A / G 729 20:19028435 G / A 730 20:20544783 A / G 731 20:20587311 A / G 732 20:22094136 C / A 733 20:23981183 C / G 734 20:24190090 T / C 735 20:24255893 G / C 736 20:27972431 T / C 737 20:29400468 C / T 738 20:30812292 C / G 739 20:31308804 G / T 740 20:33307955 C / T 741 20:34956051 GA 742 20:35580553 A / C 743 20:35759881 T / C 744 20:37923934 T / C 745 20:39150517 C / G 746 20:39908284 G / A 747 20:46609310 G / A748 20:49146900 G / A 749 20:56420906 Cl 750 20:58613269 A / G 751 20:61815866 G / C 752 20:65670420 C / T 753 20:67529154 T / C 754 20:68081138 T / C 755 20:77758727 T / C 756 20:78620487 C / T 757 20:79165013 C / T 758 20:79438296 A / C 759 20:89153730 C / G 760 20:89488213 T / G 761 20:89682416 C / T 762 20:90101085 GC 763 20:91827736 A / G 764 20:91919844 C / G 765 20:93316651 G / A 766 20:94721217 T / G 767 20:98095802 C / T 768 20:99824776 C / A 769 21:3061812 A / G 770 21:5596362 C / T 771 21:10006507 T / C 772 21:11649291 T / C 773 21:11697961 G / A 774 21:12577280 T / C 775 21:14025462 G / A 776 21:14910709 A / G 777 21:15855493 A / G 778 21:18279939 C / T 779 21:18589104 A / G 780 21:19165597 A / T 781 21:19192497 C / T 782 21:19415640 G / A 783 21:21694365 G / A 784 21:22132508 A / G 785 21:23219115 T / C 786 21:26831360 G / A 787 21:30859844 G / A 788 21:31776920 C / T 789 21:33203955 T / C 790 21:36750047 G / C 791 21:38672493 A / G 792 21:39472741 T / C 793 21:41787491 T / C 794 21:43657013 A / G 795 21:45919522 C / G 796 21:52259783 A / C 797 21:68062875 G / A 798 21:68435856 C / A 799 21:70356379 A / G 800 21:76993977 T / C 801 21:80178714 C / T 802 21:81152642 T / C 803 21:81863949 G / A 804 21:82213523 G / T 805 21:82714747 T / C 806 21:84194566 G / C 807 21:84319127 G / A 808 21:85458891 C / T 809 21:86013145 T / G 810 21:86720773 C / T 811 21:92073932 G / A 812 21:94331915 A / G 813 21:98155322 C / T 814 22:2293346 G / T 815 22:2795330 G / A 816 22:5566092 G / C 817 22:6691812 T / C 818 22:10278062 A / C 819 22:12828416 T / C 820 22:22284782 A / G 821 22:22663170 A / G 822 22:23235076 C / G 823 22:23854673 G / T 824 22:25615574 T / A 825 22:28656358 C / T 826 22:31095926 G / A 827 22:31475818 G / A 828 22:32919826 C / G 829 22:35009865 G / A 830 22:37345539 A / G 831 23:2690505 A / G 832 23:7424233 T / C 833 23:7480437 T / A 834 23:13115937 G / T 835 23:17079265 T / C 836 23:17594967 T / C 837 23:22106645 T / C 838 23:22735071 T / G 839 23:23473586 T / G 840 23:25265734 T / C 841 23:25614597 C / T 842 23:26904772 G / A 843 23:30330134 A / G 844 23:30606759 G / A 845 23:33858483 C / A 846 23:36925287 A / C 847 23:37266882 A / G 848 23:41423630 T / G 849 23:41507322 G / A 850 23:43103465 C / G 851 23:43453190 T / G 852 23:44971381 A / G 853 24:6548872 C / A 854 24:7236806 Cl 855 24:11375375 C / T 856 24:16738616 A / C 857 24:19944593 G / A 858 24:21779446 A / G 859 24:22920106 A / T 860 24:23108642 C / T 861 24:26121521 T / C 862 24:26398180 A / G 863 24:28900111 T / C 864 24:36171465 A / C 865 24:42097099 T / A 866 24:43435423 C / T 867 25:655338 C / A 868 25:3861620 T / Z^ 1 / Cj 869 25:6487076 T / C 870 25:12398415 GT 871 25:13386116 G / A 872 25:15011422 A / G 873 25:16031288 G / A 874 25:17910131 C / T 875 25:24136111 T / C 876 25:25957634 A / G877 25:27659606 A / G 878 25:32508383 G / A 879 25:33043288 A / G 880 25:34716556 A / G 881 25:35536203 A / G 882 25:35815157 C / T 883 25:39089459 A / C 884 25:41396569 C / G 885 25:42022761 T / C 886 25:42435008 C / T 887 25:43275682 C / A 888 25:43568723 C / G 889 26:543339 G / A 890 26:987762 T / C 891 26:1532299 C T 892 26:2596041 T / C 893 26:2762116 C / G 894 26:3273611 T / G 895 26:4048102 G / A 896 26:4192497 A / G 897 26:6962380 C / T 898 26:9127939 C / T 899 26:9778216 T / C 900 26:10080357 G / A 901 26:11031917 G / C 902 26:13594405 C / T 903 26:16603632 T / C 904 26:17484721 T / A 905 26:18631616 C / A 906 26:19505902 C / T 907 26:23000168 G / C 908 26:23018587 T / C 909 26:26144816 G / A 910 27:2493363 T / C 911 27:5747383 A / G 912 27:6316822 G / A 913 27:7334613 C / T 914 27:8024389 A / C 915 27:10219720 G / C 916 27:11924865 T / C 917 27:14111391 G / C 918 27:14742472 A / G 919 27:21940923 A / G 920 27:27168242 G / A 921 27:28964361 C / G 922 28:4819906 G / A 923 28:6789996 C / A 924 28:9198597 A / T 925 28:14634869 T / A 926 28:21872936 A / G 927 28:22336033 C / T 928 28:22791960 T / C 929 28:22963896 C / T 930 28:23799071 C / T 931 28:25101440 G / C 932 28:26274706 T / C 933 28:29194191 C T 934 28:29327411 G / A 935 28:30262767 T / C 936 28:32112512 C / T 937 28:35525801 A / G 938 28:36907777 T / C 939 28:41029449 C / T 940 28:49935930 T / G 941 28:52033004 G / C 942 28:57907245 G / A 943 28:58812955 T / C 944 29:645525 A / G 945 29:3436373 A / T 946 29:6355613 T / C 947 29:6877652 T / C 948 29:8297899 C / G 949 29:8766933 A / G 950 29:9886954 A / G 951 29:14735581 T / C 952 29:17740514 G / C 953 29:22535355 T / A 954 29:23309465 T / C 955 29:28401101 A / G 956 29:29684014 G / A 957 29:30972968 C / T 958 29:31409121 G / T 959 29:31600907 G / C 960 29:35489791 C / T 961 30:1307858 G / A 962 30:4967300 A / G 963 30:5252281 G / A 964 30:20270917 A / G 965 30:21059240 G / A 966 30:24064333 A / G 967 30:25327725 T / C 968 30:25461446 C / G 969 30:26258301 C / T 970 30:27286231 A / C 971 30:28692644 C / G 972 X:2345264 G / T 973 X:6372012 T / G 974 X: 17070409 G / A 975 X: 31726773 T / G 976 X: 32142109 C / T 977 X:32707803 G / A 978 X:34385517 C / T 979 X:38962535 G / T 980 X:46079163 C / G 981 X:50308814 T / C 982 X: 57737117 A / G 983 X:66139797 G / A 984 X: 67449901 A / C 985 X:68229501 C / T 986 X:68620566 T / C 987 X:68765677 T / C 988 X:69951482 A / G 989 X:73801205 G / A 990 X: 74229727 G / A 991 X: 78248795 A / G 992 X:85950525 G / A 993 X:92003559 G / T 994 X:92491285 G / A 995 X:96618977 G / A 996 X: 97282210 G / A 997 X: 98984945 T / C 998 X:99438944 T / C 999 X:106641828 T / C 1000 Y:4995971 A / C2. The donkey liquid chip based on targeted sequencing according to claim 1, wherein thegenotyping object of the chip also comprises other SNP loci located on the donkey reference genome and matched by capture probes designed and synthesized according to the 1000 SNP loci.

3. The donkey liquid chip based on targeted sequencing according to claim 1, wherein the chip comprises a capture probe designed and synthesized according to each SNP locus screened, and the capture probe is two nucleic acid molecules overlapping each other by 60%-70% and both covering the SNP locus, wherein the length of each nucleic acid molecule is 100-120 nt.

4. The donkey liquid chip based on targeted sequencing according to claim 3, wherein the GC content of the capture probe is 30%-80%, and the selected areas do not contain a GAP area.

5. The donkey liquid chip based on targeted sequencing according to claim 3, wherein the SNP loci screened are evenly distributed on each chromosome of the donkey reference genome and have the heterozygosis rate less than 15%, and the MAF is greater than 0.35 in donkey breeds and wild donkey clusters.

6. The donkey liquid chip based on targeted sequencing according to claim 1, 2 or 5, wherein the donkey reference genome is the EquAsil.O version.

7. A donkey genotyping method based on targeted sequencing, comprising the following steps:SI preparing a donkey liquid chip based on targeted sequencing of any one of claims 1-5;S2 constructing a donkey DNA high-throughput sequencing library by using the genomic DNA of a donkey to be tested;S3 capturing a DNA fragment containing the corresponding SNP loci from the donkey DNA high-throughput sequencing library by using the prepared donkey liquid chip based on targeted sequencing;S4 amplifying and purifying the captured DNA fragment, carrying out high-throughput sequencing of the product, and comparing the sequencing result and the donkey reference genome, so as to acquire the genotyping data of the donkey to be tested.

8. The donkey genotyping method based on targeted sequencing according to claim 7, wherein the construction method of the donkey DNA high-throughput sequencing library specifically comprises the following step: fragmenting the genomic DNA of the donkey to be tested by ultrasonic interruption or enzyme cutting.claims 1-5 in molecular breeding of donkeys.

10. An application of a genotyping method for a SNP locus chrl 5:25408816, chrl5:23191566, chrl5:26258385 or chrl5:24168300 in a donkey genome based on targeted sequencing in molecular breeding of donkeys.36

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