CN116732026A

CN116732026A - Development method and application of oat SNP breeding chip

Info

Publication number: CN116732026A
Application number: CN202310319628.7A
Authority: CN
Inventors: 韩冰; 王婷; 张春林
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2023-03-29
Filing date: 2023-03-29
Publication date: 2023-09-12
Anticipated expiration: 2043-03-29
Also published as: CN116732026B

Abstract

The invention relates to the fields of genetics, molecular biology, bioinformatics and oat molecular breeding and the technical field of whole genome gene chips, in particular to a development method and application of an oat SNP breeding chip. The oat 50K SNP targeting capture probe comprises an oat 50K SNP locus probe, wherein the oat 50K SNP locus probe is single-stranded DNA synthesized according to oat 50K (58900) SNP molecular markers in total. The oat 50K SNP targeting capture probe can rapidly and effectively track the genetic material of oat, is beneficial to accelerating the breeding process of oat and improving the breeding efficiency of new varieties. Compared with the traditional 6K oat chip, the invention has the advantages of more sites, wide genetic diversity, more uniform chromosome coverage, more flexible chip, higher detection rate and better repeatability.

Description

Development method and application of oat SNP breeding chip

Technical Field

The invention relates to the fields of genetics, molecular biology, bioinformatics and oat molecular breeding and the technical field of whole genome gene chips, in particular to a development method of oat breeding chips developed based on a targeted sequencing technology and application thereof.

Background

Oat (Avena sativa l., 2n=6×= 42, aaccdd genome) belongs to the genus Avena of the family gramineae, is a food and feed dual-purpose crop planted globally, has important economic value, and is a key plant for molecular breeding research. The seeds are rich in dietary fibers, antioxidants, minerals and vitamins, have important functions on reducing blood sugar and preventing cardiovascular diseases, and have long been at a premium for consumers. However, oats are heterohexaploid, have a large genome (-11 GB), and have greatly limited the fundamental research of oats. Along with the rapid development of high-throughput sequencing technology and genome assembly technology, high-quality diploid, tetraploid and hexaploid oat genome sequence information is released, so that the understanding of oat basic biology is improved, and the method is of great importance to genetics, genomics, molecular breeding research and the like.

The single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is used as a genetic marker with wider distribution in genome, has the characteristics of high density, high genetic stability, easy automatic analysis and the like, and has been developed into the most common molecular marker in plant genetic variation research. Currently, genome-wide variant genotype acquisition is largely by resequencing and gene chip. The cost for obtaining mutation information by a whole genome or simplified genome sequencing (GBS) method is too high, site deletion is serious, the sequencing data volume is relatively large, and the software and hardware requirements for data storage and analysis calculation are high. The quality of detection of the mutation site is strongly dependent on the quality of the reference genome. SNP chips have good detection rate and stability in the aspect of sample site detection, and have been developed and applied in crops such as wheat, corn, rice, soybean and the like, and important research progress is made. Therefore, efficient, low-cost SNP genotyping techniques are the best option for developing sharing techniques and platforms.

In SNP chip technology, traditional solid phase chip is based on the complementary hybridization of probe and DNA sequence, and the fluorescent color signal of the marker is used for parting, and only SNP sites contained on the chip can be parting, so that the SNP chip cannot be added or deleted, has poor flexibility and high parting cost. The targeted capture probe is based on a high-throughput sequencing technology, specifically captures each target site, has the advantages of simple development technology, high detection accuracy, flexibility and large throughput, can stably obtain the same SNP marker in different laboratories, and provides a simple and reliable technical platform for accumulation, sharing, comparison and integration of detection data. The density of the target capture probe can be flexibly adjusted, can be reduced to low density 1K, can be expanded to high density 800K or even higher, has no limit on the test sample size, is suitable for all biological species, and has mature application in the aspects of whole genome association analysis, DNA fingerprint identification, molecular genetic map construction, QTL positioning, molecular marker assisted selection, whole genome selection and the like.

At present, based on complementary DNA (cDNA) of more than 20 oat varieties and SNP loci obtained in other modes, a first oat 6K Infnium iSelect solid-phase chip (Nicholas A.Tinker, shiaomanChao, gerard R.Lazo, rebekah E.Oliver, yung-Fen Huang, jesse A.Poland, eric N.Jellen, peter J.Maughan, andrzejKilian, eric W.Jackson.ASNP Genotyping Array for Hexaploid Oat [ J ]. The Plant Genome,2014,7 (3)) has been developed, but because of The very limited molecular markers of The chip, the proportion of coverage to Genome is too small, the application of The chip in basic research and genetic improvement of oat is limited, researchers usually use a simplified Genome sequencing (GBS) or wheat targeted capture chip to supplement insufficient resolution of oat 6K chip on mass materials, so that new chip markers are required to be increased to meet The requirements of developing novel oat quality identification resources. Therefore, an economic, high-density and high-flux oat genotyping chip is developed, which can provide important support for oat molecular breeding, accelerate the oat breeding process and better serve for oat industrialization application.

Disclosure of Invention

In order to meet the requirements of high-throughput and low-cost genotyping of oat and large-scale commercial breeding, the invention provides the oat 50K SNP targeting capture probe developed based on the targeting capture sequencing technology, the SNP loci used by the SNP targeting capture probe uniformly cover a chromosome set, and the SNP targeting capture probe has strong representativeness, high polymorphism, high specificity and strong universality, fills the bottleneck of the lack of the existing oat molecular marker, and provides an indispensable important tool for molecular breeding researches such as oat high-throughput genotyping, gene positioning, fingerprint, whole genome selection and the like.

The technical scheme of the invention is as follows:

the oat 50K SNP targeting capture probe comprises an oat 50K SNP locus probe, wherein the oat 50K SNP locus probe is single-stranded DNA synthesized according to oat 50K SNP molecular markers, and the 50K SNP locus comprises a core locus in the oat 50K SNP locus.

In a further embodiment, the 5' end of the probe carries a biotin group, and the probe is coupled to fluorescent microspheres through C12 molecular arm and amino modification, each fluorescent microsphere being coupled to one probe.

The preparation process of the oat 50K SNP targeting capture probe is as follows:

step 1: screening oat 50K SNP loci;

specifically, oat 50K SNP locus was obtained by screening by the following method:

step 1.1: detecting SNP loci on all sample genomes according to the resequencing data of a plurality of oat germplasm by comparing oat reference genomes for further selection; the above sites were selected according to the following criteria: MAF is more than or equal to 0.05, NA is less than 5%, heterozygosity is less than 5%, chromosome uniform distribution principle is combined, and simulation evaluation is carried out on each site.

Step 1.2: comparing the oat reference genome to detect SNP sites in all data for further selection according to open-source simplified genome sequencing (GBS) data; the above sites were selected according to the following criteria: MAF is more than or equal to 0.05, NA is less than 5%, heterozygosity is less than 5%, chromosome uniform distribution principle is combined, and simulation evaluation is carried out on each site.

Step 1.3: the oat 6K chip site (Nicholas A.Tinker, shiaomanChao, gerard R.Lazo, rebekah E.Oliver, yung-Fen Huang, jesse A.Poland, eric N.Jellen, peter J.Maughan, andrzejKilian, eric W.Jackson.ASNP Genotyping Array for Hexaploid Oat [ J ]. The Plant Genome,2014,7 (3)) was selected according to The following criteria: MAF is more than or equal to 0.05, NA is less than 5%, heterozygosity is less than 5%, chromosome uniform distribution principle is combined, and simulation evaluation is carried out on each site.

Step 1.4: SNP loci were screened for oat functional marker loci according to literature publications.

Step 2: designing and preparing an oat 50K SNP locus probe;

the design process of the oat 50K SNP locus probe comprises the following steps:

step 2.1: determining a probe design principle:

the length of the probe is 120bp, the GC content of the probe is 25% -69%, the average GC content is about 45%, the number of homologous regions is less than or equal to 3, and the selected region does not contain SSR and GAP regions to the maximum extent; combining with the principle of chromosome uniform distribution.

Step 2.2: then, taking the SNP locus obtained in the step 1 as a center, and screening a 120bp nucleotide sequence with the GC content closest to 45% in about 120bp as a probe;

step 2.3: and (2) synthesizing single-stranded DNA with biotin group modification at the 5' end according to the 120bp nucleotide sequence obtained by screening in the step (2.2), namely an oat 50K SNP locus probe.

Therefore, the oat 50K SNP molecular marker combination and the probe for detecting the SNP molecular marker combination (oat 50K SNP locus probe) can be used in oat 50K SNP targeting capture probe preparation. The oat 50K SNP molecular marker combination, the probe for detecting the SNP molecular marker combination (oat 50K SNP locus probe) and the oat 50K targeting capture probe can be also applied to oat genetic diversity analysis, molecular genetic map construction, whole genome association analysis, variety authenticity identification, molecular marker assisted selection breeding and whole genome selection breeding.

The beneficial effects of the invention are as follows:

(1) The invention adopts a liquid phase probe capturing technology to design and synthesize a set of target capturing probes which contain 50K SNP locus information and can be used for detecting oat background loci, and the target capturing probes can rapidly and effectively track genetic materials of oat, thereby being beneficial to accelerating the breeding process of oat and improving the breeding efficiency of new varieties.

(2) SNP loci related to the targeting capture probe are selected from 188 oat germplasm resequencing data, open-source simplified genome sequencing (GBS) data and partial loci of a 6K chip, and the material background of the data covers wider oat varieties, and is high in representativeness and rich in diversity information.

(3) SNP loci related to the target capture probe are uniformly distributed on a chromosome.

(4) Compared with the traditional 6K oat chip, the invention has the advantages of more sites, wide genetic diversity, more uniform chromosome coverage, more flexible chip, higher detection rate and better repeatability.

(5) Compared with a solid-phase chip, the chip provided by the invention has the advantages that the detection accuracy of the target capture probe is high, the effective information is more, the flexibility and the diversity are high, the detection cost is effectively reduced, and the scientific research and breeding efficiency is improved;

(6) In oat breeding research, compared with the existing resequencing technology, the targeting capture probe overcomes the defects of large resequencing data quantity, more useless information, large analysis difficulty and the like, and realizes the advantages of being beneficial to industrial development, such as rapid detection, cost reduction, simple data analysis and the like on the premise of reaching the same result; compared with simplified genome sequencing (GBS), the targeted capture probe avoids the defects of poor data repeatability in GBS analysis and the GBS marker data obtained from different materials, laboratories and platforms are difficult to compare and accumulate, so that the GBS data is difficult to utilize in large-scale populations; the target capture probe has the advantages of high data output consistency, long-term use of the same material data, relatively simple data analysis and the like;

(7) The oat 50KSNP targeted capture probe can be applied to oat genetic diversity analysis, variety identification, genetic relationship identification, DNA fingerprint construction and whole genome association analysis, so that the oat research and breeding process is accelerated.

Drawings

FIG. 1 is a schematic diagram showing the distribution of SNP sites in oat 50K SNP targeting capture probes on different chromosomes; namely, counting the number of target sites on different chromosomes; wherein a. The abscissa is chromosome ID; b. the left example is the number of sites/number of segments; c. the right example is chromosome length (in bp). The figure is plotted against the v2 version of the oat reference genome.

FIG. 2 is a SNP site uniformity distribution map (probe coverage density was calculated on a chromosome with a length of 1Mb as an observation window). The figure is plotted against the v2 version of the oat reference genome.

Fig. 3 is an AF distribution statistical chart. The figure is plotted against the v2 version of the oat reference genome.

Detailed Description

The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.

The oat reference genome data of the examples were from https:// heat. Pw. Usda. Gov/GG 3/content/pepco-ot 3098-hexaploid-oat-version-2-genome-assembly-release-collaboration-graingenes.

Example 1

The oat 50K SNP targeting capture probe of the embodiment comprises oat 50K SNP locus probe mixed solution and hybridization capture reagent.

step 1: screening oat 50K SNP loci;

step 1.1: according to the resequencing data of 188 oat germplasm (data source https:// www.ncbi.nlm.nih.gov/bioproject/.

Step 1.2: based on 659 copies of open source simplified genome sequencing (GBS) data (Peng Y, yan H, guo L, et al reference genome assemblies reveal the origin and evolution of allohexaploid oat. Nat Genet.2022;54 (8): 1248-1258.Doi:10.1038/s 41588-022-01127-7), a total of 30K SNP loci were obtained for oat reference genomes for selection of target loci; 1479 sites are obtained by screening according to MAF not less than 0.05, NA less than 5%, heterozygosity less than 5% and chromosome uniform distribution principle.

Step 1.3: 3204 sites were selected according to The oat 6K chip site (Nicholas A.Tinker, shiaomanChao, gerard R.Lazo, rebekah E.Oliver, yung-Fen Huang, jesse A.Poland, eric N.Jellen, peter J.Maughan, andrzejKilian, eric W.Jackson.ASNP Genotyping Array for Hexaploid Oat [ J ]. The Plant Genome,2014,7 (3)) according to MAF.gtoreq.0.05, NA <5%, heterozygosity <5% and chromosome uniformity profile.

Step 1.4: according to the literature publications (Peng Y, yanH, guo L, et al reference genome assemblies reveal the origin and evolution of allohexaploid oat. Nat Genet.2022;54 (8): 1248-1258.Doi:10.1038/s 41588-022-01127-7), 34 functionally labeled SNP loci were obtained;

the SNP loci constitute the oat 50K SNP locus, and comprise 58900 SNP loci in total.

Step 2: designing and preparing an oat 50K SNP locus probe;

step 2.1: determining a probe design principle:

the length of the probe is 120bp, the GC content of the probe is 25% -69%, the average GC content is about 45%, the number of homologous regions is less than or equal to 3, the selected region does not contain SSR and GAP regions to the maximum extent, and the chromosome uniform distribution principle is combined.

Step 2.2: taking the SNP locus obtained in the step 1 as a center, and screening a 120bp nucleotide sequence with the GC content closest to 45% in about 120bp as a probe;

step 2.3: synthesizing a single-stranded DNA nucleotide sequence with biotin group modification at the 5' end according to the 120bp nucleotide sequence obtained by screening in the step 2.2, and obtaining an oat 50K SNP locus probe;

specifically, the probes obtained in the step 2.3 are coupled to fluorescent microspheres through C12 molecular arms and amino modification, each fluorescent microsphere is coupled with one probe, and finally the probes are added into a targeting capture reagent to obtain the oat whole genome targeting capture probe, namely the oat 50K SNP targeting capture probe.

The target capture reagent comprises a general blocking solution I, a general blocking solution II, a TCGBS 2 Xhybrid Buffer, a TCGBS repeated sequence blocking solution Block, a TCGBS 2X Beads Wash Buffer, a TCGBS Wash Buffer I/Wash Buffer II/Wash Buffer III and TCGBS Stringent Wash Buffer which are independently packaged.

The core site information in the oat 50K SNP site obtained in this example is as follows: 1 to 104: chr1A: the method comprises the steps of (a) carrying out a sequence of steps of (b) carrying out a sequence of steps of (c). The method comprises the steps of (a) forming a first pattern on a first surface of a first substrate, and (b) forming a second pattern on a second surface of the second substrate, wherein the first pattern is a pattern on the first surface of the first substrate. 105 to 159: chr2A:2100930, 4239492, 10704631, 10938210, 13436858, 14441332, 18422955, 32282364, 39418692, 59982919, 60887086, 70607983, 75197819, 89599242, 104954798, 112783291, 130463628, 132068088, 135426392, 217334034, 242505005, 266261884, 274342752, 296150888, 316167952, 317795068, 330610398, 332882630, 335767331, 340794839, 344918901, 345951965, 348518457, 351045498, 354468810, 358393070, 359546799, 365230751, 367437482, 376887627, 381029377, 382692023, 386423751, 388053059, 392483868, 392798434, 396593342, 414088827, 417851447, 420594190, 420595093, 424692775, 427321507, 442730057, 446172245;

160～205：chr3A：1610216、6683114、8014312、10110259、10485169、14172173、14776263、17340560、20488584、21496967、26049655、32772758、33991651、35299853、38586125、44880561、54997130、58739452、62189906、85688337、94820162、114658260、116633323、128773290、151178376、200425079、280387479、351442042、363495278、375553019、383400710、390469516、396533215、397753012、401574339、401783978、406500645、408866831、409429625、412020155、413066356、416646002、417059585、420444403、421675699、425498293；

206～315：chr4A：5868933、8392393、19170174、19413350、25203745、25791291、28906638、30909572、31909139、36204577、37380881、37931499、51111451、56156245、59101899、61464368、73797069、74531320、89017088、90449403、92562971、104373841、111027202、111827939、111827957、115429197、117630111、121222702、135528254、136756326、137303694、139458951、159197158、161232316、163502837、168263215、170902690、171546185、177665690、187622384、195349317、197153086、207347401、208573136、222610526、222636708、231242768、231548094、235503941、241192849、241509123、247748826、248060825、248060830、248060889、252537773、254613235、257416765、258885798、259540777、262281263、272630002、277190662、281486838、281486849、286266765、288431566、292131290、292513321、295102035、297733585、297733643、300315368、300315381、300911283、301288818、303316574、306582903、314745375、314898344、315417088、315667578、321573717、337131944、350536261、350844103、359359746、359722506、359913387、362273696、362389355、373922766、374896108、378137144、378327427、396386903、401387124、406637920、413326636、425504487、426661755、430055295、438016051、445440228、446148292、448993225、457195088、460557686、462487598、463183142；

316～400：chr5A：1050490、6249598、7506416、8169576、11188916、14219787、16763198、19760395、23929408、36864940、37550369、45985582、69234885、78292272、82260577、83281867、95237393、97956121、104955266、110334622、120926145、121559563、132558985、133652751、147940962、148642661、151761466、154638018、159611159、162603151、182070963、188791902、192242039、203887430、217563535、222610538、231784772、240240115、252125842、257069571、261461273、262622135、271782223、284460811、287368033、292599093、298428508、305834482、316407299、317157082、322409847、322628089、324921979、325590714、330742245、340587172、344360993、351428308、361091415、377099210、380140169、384387808、384603300、396589627、401935107、403229343、403653144、410271145、415954782、416310066、420705569、430922569、441265600、449223765、450584555、450812613、462189418、462239914、464203725、468371869、468525614、475372538、476139251、478200125、479550901；

401～518：chr6A：62544、1012565、2455651、4470187、6997843、14405410、25578536、27379598、36452576、47261547、67456872、79634441、79883573、84507176、89303453、102016887、112552215、114422148、136429929、140069943、143659807、145601973、151055903、160914806、161540800、167462123、171606982、172156007、179427239、182498507、184441253、204321087、208540939、218759115、221171302、221171322、221371867、235141870、237187254、240828531、248879987、251864596、256825845、265718194、268002672、270603498、272991361、273394310、276951871、278083452、280051916、281852704、282437075、282736688、291049042、292332589、293675810、295680498、296239414、300620942、303761972、306859388、307122944、309068192、310869491、311384412、314918405、319443489、320079085、322530700、323175189、327428291、328029972、328227917、329136897、329906417、331238195、332637344、333020015、333340362、335833836、336030537、338431953、338607425、339431942、345968451、348836682、350217335、353989225、364663392、366460907、367790255、371604445、376988529、377585368、380245259、381323139、382680956、383227089、389290139、392079421、395939625、396134837、407009969、411379388、416884003、419204760、419212286、426934823、427650829、429461357、432083274、435221528、443546030、444718017、444743716、445852336、445852368；

519～589：chr7A：30299、797283、797351、5133297、7465119、9946073、11793802、15395939、17639035、19586651、26415871、43862820、45486520、47295123、49441855、54545600、55393233、57362711、58949123、61794447、62338021、63223946、64882556、65060462、65251259、71731013、84829439、86301312、86620598、93221864、94326878、100414766、103910393、114754034、120852435、125629877、135463951、136261080、139430971、140133058、147126394、154304327、161938874、162275342、162502546、222831530、230712620、230740407、251804505、252376556、253119579、296660241、361193782、388782436、395213388、396086697、400673360、423577618、448686669、452001562、461430759、468784757、474082282、480336404、481051356、485992955、487562251、488323297、490272719、490579940、491985052；

590～642：chr1C：794398、4966766、7517239、9316840、9358859、9447742、12025749、12728231、12975605、13286580、16386903、18440468、18644571、18829239、19622398、23813998、24351415、32333223、33013849、36097965、41500803、49149059、98433026、100225401、117648056、139862140、152181613、242734065、248887909、289524498、315674850、315921561、334542237、344894647、345768079、346038665、359261969、359832872、376528494、378345831、388388717、391800413、401337969、403086414、403786769、414634423、422023895、428386505、429393098、434959354、442827296、452742922、459071471；

643～722：chr2C：508638、1877188、2528207、3608980、6505327、8828258、9887223、16378340、16733830、21493372、30722539、32260907、34107248、42159271、47793038、55255692、58911330、67802606、76314393、79276831、88682059、91534141、91590749、130168941、130251230、144497831、161151777、161170416、161304339、173271478、179895203、189771395、223652458、247885154、254177797、259217358、271206331、277209877、282431123、320998183、325533604、336227470、336324599、336381796、345555907、349979246、361507264、367642842、373755255、379192574、381147253、382271246、396735565、400251049、401021912、408106436、412994796、428678286、431244629、449477697、466757749、493956422、499095023、501293309、502692609、508579710、519003218、520403117、522658623、531147422、533441899、534490497、535320919、537908978、543511967、550187082、554997859、558619184、560382933、562917658；

723～805：chr3C：61297、503695、598700、1201004、3394935、3394976、4103786、6165989、9922423、11627911、13906284、21931547、29629809、29955580、30791312、33847452、42483315、43861523、46984198、47170559、61358625、67823801、69552871、73650387、85817742、89054677、133070875、160667478、165150659、177812668、191447376、193827038、207598037、228857205、240224728、273109399、275506686、276817356、300505682、308926287、335084217、340272306、357039534、360384970、375228068、397621834、409004632、417466913、426577766、427727313、434921481、447033212、452211758、456779390、462944359、464786184、466302827、489683019、499309825、501522818、523737122、550827482、551112909、564770145、571627226、593909720、595004344、597544696、604657715、607082129、607902694、612599972、613269859、614722449、614823779、620371982、623382558、626791784、627443314、628928880、630346501、632651708、638133888；

806～918：chr4C：3156275、3156309、6151866、11154035、26834888、30694671、36293580、39086177、40288786、42236043、43109627、43848590、47392406、47641679、49158016、52760842、53598521、53939843、58316218、59961826、60096926、62416393、64784388、69604919、70450484、73378379、83748627、103386515、107244878、107330999、108482037、118792713、119239746、121940137、122327356、123510068、124053384、126900382、128226873、130464152、131337543、135871755、136394936、137398743、139983786、142766414、144128859、149693472、151667321、158587013、159537214、168296059、171843825、172869591、177332803、188589498、188815342、194106449、194117303、208025980、232058561、232313275、233414178、243942384、246993956、277770184、335855729、341048610、355301777、358590152、359548305、380344060、390159929、393180518、398088959、402898771、403502507、410722151、416930608、442743332、443020917、453953122、488144957、493824659、509539196、512906902、518164512、525011469、525018986、525845028、546203479、558810054、564981985、565345449、568926820、575890407、597642698、606728798、625266556、633582863、670582073、671502734、676162180、684407156、692454211、696599915、698128769、698161356、700308139、704691850、706603391、709770857、710256791；919～1044：chr5C：554202、3749792、3849936、7637563、9034502、12288161、12428473、15702114、16919102、19035849、19684941、34039963、38195971、41475312、47425662、53774061、53798541、53937809、60895738、62177980、63445568、64638626、64699517、68936987、87785914、90707249、93155853、97074885、98370861、98818134、105238445、107890566、108958735、115464154、116215262、126465885、130714668、146124820、148156344、150181347、156346016、161589949、170232433、170265452、181486109、181698549、190217791、212065553、224669563、240501203、249105387、264893757、275312253、290424574、313738041、325535102、338862900、340776106、346453345、346670057、360877555、369746847、370916238、376791265、377444427、380137077、383843868、391113723、399409221、400559622、407090768、408955951、409877500、411853930、412309560、422660477、427953795、429536327、433178666、437980316、438986627、439244384、443400619、449027771、459155181、469127358、471266768、475881780、485797166、490197664、493250155、502265146、504791556、505908673、506958259、508142521、511148919、513278340、514868949、517911508、519788490、523051612、533055170、544767033、549076111、550648593、553113006、553385567、557268792、563355183、564881004、569327026、572145755、572145791、581326964、584056057、590337215、595946882、601502958、606333257、606918814、610040984、611215306、612022343、612942538、613031851；

1045～1237：chr6C：3360302、4350869、4932310、5277621、7754285、8593394、23983484、25516860、27157177、28040770、32132844、35586904、35586974、36103399、37264411、47620214、51093616、53478089、61270790、62774418、62774508、65346528、66265528、68807067、71948109、74143042、86360213、90807037、100565028、105893326、107564980、111533955、113441009、115816527、123515378、123829932、125254623、128366562、131095916、141348221、144266889、149851686、153941728、154627558、163061787、163718401、168791300、175779829、180047936、182114010、182148927、185422948、185510831、185510842、187860000、188015604、200549497、202069843、208001681、208264410、208789832、209243891、209794718、211567909、211567992、212122148、214117823、228168286、229007541、231354909、231440504、236321429、241558979、242439816、242439849、243761709、247960808、253703197、259950029、267023629、269236695、279642437、280269608、282091119、285290859、290195093、294280244、296108433、296151607、296749043、297048385、297390218、298825148、299106924、312595097、314363926、314484553、320920928、322432467、325988513、326067226、331000408、333453880、333817680、333843521、341392424、341602867、342467150、352543596、352688195、354241895、361471158、363958670、365037839、386547357、388310966、391715451、395417260、398916813、402914237、412660307、413568227、424163261、429747892、442377446、442833450、445939991、453619415、454613425、461323879、463705554、466436641、466640395、470683625、485083599、490527343、494793165、498466125、499420835、505976028、510124456、510814068、511862138、513159581、523605012、523752016、524930246、526593071、528061670、529050405、533292145、533292168、533383364、533906881、537589325、540321220、540321291、541273513、543178314、545406727、545406821、555175028、561042608、566217156、568123645、570160049、570636165、575904696、580979024、581757331、581998214、587382947、595591783、601918078、603809955、605525359、605819832、606586959、608845375、610237906、610448989、610796208、612119763、614815629、615197400、620149276、622349425、623215985、623215987、624048923、624734309、624734322、625301507；

1238～1328：chr7C：1837898、2199518、3266542、4023199、7667076、8378814、19054935、24336059、26900380、38237201、41431078、43663497、44271391、50074554、52936464、54056524、57445019、60390318、61328257、74651850、75633576、80195974、80963688、82037659、84942319、87031454、88355213、89836426、90534162、90534234、91868420、92284011、95186313、96840627、97910653、100850974、103124523、108091562、142549489、144028476、152180231、155823992、165853418、184399176、192742152、224735147、262536122、271932318、272185938、272762809、293413597、294514478、303692756、314775121、336307083、339370302、343787557、347324923、365012556、366352205、383214632、385907184、394905236、402735312、443651331、452069295、453470984、456083674、481302865、497984044、515874361、529222932、530934528、533888689、535328997、537725597、537961172、541891943、541891949、542181045、543270953、544000557、544560923、545226352、545495454、546165378、548240499、549283342、550510685、551709679、551717893；

1329～1468：chr1D：2472108、2763296、6008789、9289749、10777024、13071780、15573885、22743758、28309513、28695982、29511718、34581984、40284363、40923696、42956217、43708591、44780684、45137946、51730344、54094919、55921214、61866244、64294194、65831128、68821548、72441042、74263335、76064584、78204798、81107048、82907066、85473604、87752359、90710231、93886779、101146104、105935958、109701762、118151136、120227503、125961341、126769804、129180085、132945644、132945664、135707309、141273940、144431592、149206540、150743912、153352539、154498503、162009883、162122649、164954116、167974831、168181978、170515053、173334238、177087356、180812692、181477217、182678841、185365277、187332483、190844598、208165315、238821603、246640567、249534966、269793107、279157022、297671371、305616514、306303753、308911527、310605052、316140032、317366109、323437400、326063088、327171559、327337249、333946245、335252519、337064441、344763160、346103369、347106295、348239378、349973298、352580647、355033090、355671612、362260658、362260792、370361856、373917305、375149492、376585987、378325233、380205234、385946522、386686765、386695328、387663636、393395423、394002709、398509223、399261806、406058505、417146864、420186032、425743135、433463172、433821555、434538143、436587940、437015611、437072201、437806624、439566446、440494209、446890370、447747018、447968126、451479068、453212346、455964242、461430019、465668254、470897750、473422693、475010970、475992716、478296618、479510735、479510759、479837889、484202525；

1469～1558：chr2D：4299081、6576682、7939906、8879735、25226872、25606770、28124073、29403920、31747759、35544923、36930852、38598148、39124581、40320810、60479331、61200152、61764990、63504731、66037015、68539636、72374382、72801418、75466908、85173718、97289693、97381351、100239493、102266911、104173506、111830406、124395524、125143812、127916002、129759198、139688996、147678453、149731658、150787737、154522423、156989872、160718930、160718983、164510104、167362478、168381966、168772578、169092119、173305350、176071219、188891939、196307317、196573569、200604526、204803324、217180387、221379579、223132394、229493777、245092057、258860376、266050457、269849936、304765446、313434878、316274959、316275047、318310631、341208220、397683112、425534524、429435773、439808678、470936426、476460687、477845930、495124480、508957795、510305795、513084570、514503617、518005448、521057369、521517068、523929499、523943553、524067993、527883400、531744996、532085894、532086148；

1559～1627：chr3D：7537059、7907346、9947897、10447064、14634248、15206447、19692269、28080785、35200774、45343435、48884333、52486495、54516812、65417650、74990279、76161058、93385358、107523050、107523132、118573098、119936647、149799762、199922500、224927766、236839180、287199373、303913397、324806982、326788707、337367716、345565226、347275925、360122324、362214749、364820238、377020875、382788379、383074462、393009205、399445661、406655585、408964857、420800669、429099312、434455330、439798196、444819830、449582558、453131736、455945456、456707829、458731719、461606368、462709025、463341020、463624031、464968253、465156231、465456066、467856832、467856844、469116316、474406617、474406681、474406761、474502067、477648265、477966906、479929633；

1628～1718：chr4D：11485004、14717658、15065859、31961722、32485235、33560371、35156356、39740867、42795157、44433053、59143960、61439239、78867509、83710252、94577427、97342443、99630391、102329405、114879597、135083886、138944406、139191630、145275183、158199248、183847307、197618490、202008461、208924184、214257428、217116193、218226761、220722019、224527103、226305656、242931934、249581054、255462900、264284840、267548200、268330950、271337080、272128244、274673552、277597999、284757400、292115085、295208268、307122231、312785806、315358650、320106105、321257483、322335201、331354099、347449985、349565426、355005406、355529054、357699853、363770741、375262472、377476161、380726205、385800811、391193802、394067252、394991049、398011595、398021682、402631607、403614105、404368909、407333043、410646224、412188909、414401279、418371869、418997245、421372071、421555572、428897486、430839695、432215688、440437664、441165072、443760645、446977976、448126463、451246460、453204068、454964761；1719～1790：chr5D：422491、2403544、2847753、8070862、9173118、15344875、20080029、20241303、23719531、24894899、42951694、44107567、48815715、49508914、51945197、56013951、62539497、149532814、182274396、224230643、244783821、258584112、274219604、300870135、312225241、324387095、334868009、336719110、346038010、346469081、346469183、354514206、366731773、378137934、379881301、380071905、392150519、396806532、401607567、401827548、403566921、407953083、414598189、418669755、418689444、420799211、425328381、436638413、438696800、438952723、445810673、450854676、455970239、459069234、460850708、460986821、471757307、472785728、480109072、484436332、484877489、487723676、489342416、490270563、491681200、492912927、494028859、495132865、495470919、495962366、497177907、498614358；

1791～1842：chr6D：2682247、4232838、4870848、5106374、5438355、8149967、9044749、16567192、21833434、25107483、30733743、31648517、35568291、37263838、38266044、42699055、47536590、82721650、111693433、111859680、122698189、143238944、150943424、162140158、167080037、167154453、192530085、193391439、199476613、206222045、213515826、230263674、239136051、243811626、244217054、253294894、256771000、259859605、272370825、273611234、283670850、285381472、286820765、289202530、290801536、291677712、292070963、292447424、294444168、294755480、296249886、298013338；

1843～2000：chr7D：1072783、1119801、1236244、1534371、2991626、6484731、8526949、12785153、16242070、17366942、17850661、22956474、24096794、28468246、36005342、36246561、38201748、44727899、51458782、52893291、54121226、57516030、57516038、67534447、70410781、71268375、74183904、77122480、80597019、80953710、91190332、97784606、101125798、101646423、103077178、107623530、109736013、109968936、109968972、115007259、118525929、122982073、128880373、129122344、130575310、144416893、144696534、147934872、151212412、156658652、159001645、159202650、161327586、163948311、181525332、182392553、185716424、192236268、197083938、200305527、200305588、203992071、205384321、207873222、211308312、213859261、215658345、221747688、227132133、237365568、238457399、240373853、240497344、251811510、252160256、257692302、265633476、276461950、281656816、281673877、281673955、282186605、288940800、289015491、295863972、297712659、300964765、301738058、302186316、305064006、305958757、309078989、309792985、310443347、311730518、317299513、318083871、320706656、327935646、330094148、334523638、334550921、335532754、339511013、341280805、348832923、348856568、362000098、371871465、374671605、379217936、381999320、388319071、392108332、403165236、405370611、412711051、412974598、423689553、425896659、434160829、434379013、435977775、437395621、445058667、449281896、452187250、455142358、462212286、466485714、469010673、469111622、469111697、474002520、476702195、479594709、480535689、483206258、488775954、493156510、493471148、495673345、497521926、503762348、506063835、507610286、508855720、511766848、512617462、514968304、515513948、516813821、519702746、520048073、521311574、524439726、525367002、527908773。

wherein, 1-2000 represents the serial numbers, chr1A, chr2A, chr3A, chr4A, chr5A, chr6A, chr7A, chr1B, chr2B, chr3B, chr4B, chr5B, chr6B, chr7B, chr1C, chr2C, chr3C, chr4C, chr5C, chr6C, chr7C, chr1D, chr2D, chr3D, chr4D, chr5D, chr6D, chr7D represents the chromosome ID where the SNP site is located, and the numbers behind the chromosome ID represent the position where the SNP site is located on the chromosome.

The distribution of SNP loci of oat 50K SNP targeting capture probes obtained in the embodiment in different chromosomes is shown in figure 1, and as can be seen from figure 1, the invention designs different numbers of probes according to the length of the chromosomes, so that the capture loci cover the whole genome.

The SNP locus uniformity distribution diagram of the oat 50K targeting capture probe is shown in figure 2, and as can be seen from figure 2, the coverage rate of a genome 1Mb window is 98.1%, the whole genome is better, and the condition that a large fragment is not covered is avoided.

The AF distribution of the oat 50K targeted capture probe is shown in FIG. 3, and as can be seen from FIG. 3, the AF values are symmetrically distributed about 0.5 as a center.

Example 2

The application method of the oat 50K targeted capture probe comprises the following steps:

(1) DNA extraction: extracting DNA from oat samples to be detected by adopting a high-throughput DNA extraction kit or a CTAB method;

(2) DNA quality inspection: analyzing the purity, the integrity and the pollution of the DNA by using 1% agarose gel electrophoresis; the DNA concentration is accurately quantified by using the Qubit; integrity was accurately checked using Agilent 2100. The standard of qualification of quality inspection is DNA with the total amount not less than 4 mug, the sample concentration less than 40 ng/. Mu.l, good sample integrity and no impurity pollution, (3) library construction: randomly breaking qualified sample DNA by using an ultrasonic breaker, and electrophoretically recovering DNA fragments with required length, and adding a connector at the tail end of the DNA fragments to form a library;

(4) Sequencing library construction: amplifying a sample library by using LM-PCR and purifying to obtain a sequencing library, which can be used for probe hybridization experiments;

(5) Hybridization capture library construction: taking 300ng of the sequencing library which is completely constructed, adding an oat 50K SNP targeting capture probe and a hybridization reagent after freeze-drying, and placing the mixture at 65 ℃ for incubation for 16 hours after denaturation to complete the hybridization reaction; washing the hybridization product with washing liquid, and performing 5-6 rounds of PCR to complete the construction of hybridization capture library;

(6) Hybridization capture library quality inspection: preliminary quantification is carried out by utilizing Qubit4.0, and the effective concentration of the library is accurately quantified by using a qPCR method so as to ensure the quality of the library;

(7) Sequencing: sequencing using a BGI high throughput sequencer and related reagents;

(8) Analysis: after obtaining the off-machine data, comparing the sequencing data with a reference genome by using BWA, and performing standard call SNP of the data by using GTAK; thereby obtaining the genome genotyping data to be tested.

Example 3

The oat 50K SNP targeted capture probe obtained in example 1 was applied as follows:

(1) Taking 24 oat leaves, wherein 20 samples are counted in the 24 oat leaves, wherein 4 samples (2 replicates of each sample) are contained in 8 samples, and specifically, the replicates of the WT1, the WT2, the WT3 and the WT4 are the replicates of the As-577, the As-542, the As-535 and the As-533 respectively; placing in a refrigerator at-20deg.C for preservation, and extracting DNA;

(2) DNA extraction, library construction, on-machine sequencing and obtaining final SNP data were performed according to the oat 50K SNP targeting capture probe application method of example 2;

(3) And calculating the detection rate. The product detection rate is an important index for measuring the quality of chips, and is generally measured in plants according to the ratio of the number of product detection sites to the number of development sites; the average detection rate of 24 samples in the embodiment is more than 99%. The detection site deletion rate is 0.09% -1.02%, which shows that the site design quality of the target capture probe and the probe capture efficiency are good. The specific detection rate results are shown in table 1.

Table 1.24 statistics of the detection results of oat samples

Sample of	Population site number	Number of missing sites	Loss rate	Detection rate of
					As-156	49516	328	0.66％	99.34％
As-191	49516	295	0.60％	99.40％
					As-195	49516	47	0.09％	99.91％
As-204	49516	36	0.07％	99.93％
					As-215	49516	95	0.19％	99.81％
As-296	49516	247	0.50％	99.50％
					As-299	49516	152	0.31％	99.69％
As-2	49516	180	0.36％	99.64％
					As-354	49516	215	0.43％	99.57％
As-35	49516	453	0.91％	99.09％
					As-37	49516	234	0.47％	99.53％
As-429	49516	150	0.30％	99.70％
					As-431	49516	322	0.65％	99.35％
As-440	49516	266	0.54％	99.46％
					As-443	49516	424	0.86％	99.14％
As-533	49516	158	0.32％	99.68％
					As-535	49516	505	1.02％	98.98％
As-542	49516	66	0.13％	99.87％
					As-577	49516	265	0.53％	99.47％
As-57	49516	244	0.49％	99.51％
					WT1	49516	174	0.35％	99.65％
WT2	49516	129	0.26％	99.74％
					WT3	49516	427	0.86％	99.14％
WT4	49516	149	0.30％	99.70％

(4) The repetition rate is calculated. And (3) carrying out sample repeatability statistics, wherein the average value of the overall repetition rate of the four groups of repeated samples is 98%, and the uniformity rate of homozygous sites is greater than 99%, which indicates that the detection stability of the target capture probe is good. The statistical results are shown in Table 2.

TABLE 2 test sample technique repeatability test statistics

Sample group	Effective site	Consensus site	Consistency rate of	Heterozygous site	Homozygosity consistency ratio
						WT1 vs As-577	49204	48026	97.61％	1176	100％
WT2 vs As-542	49334	48781	98.88％	552	100％
						WT3 vs As-535	48892	47877	97.92％	971	99.91％
WT4 vs As-533	49267	47829	97.08％	1428	99.98％

In general, the invention develops the oat high-throughput 50K SNP targeting capture probe based on the targeting capture sequencing technology, and the targeting capture probe can be effectively applied to oat genetic diversity analysis, molecular genetic map construction, whole genome association analysis, variety authenticity identification, molecular marker assisted selective breeding and whole genome selective breeding.

The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.

Claims

1. An oat 50K SNP targeted capture probe, comprising an oat 50K SNP site probe, wherein the oat 50K SNP site probe is single-stranded DNA synthesized from oat 50K SNP molecular markers, and the 50K SNP site comprises core SNP molecular markers as follows:

1～104：chr1A：5283582、7103302、10734851、10945761、11664332、14467396、15047396、16398486、21270926、25425155、29414961、75223307、101003855、115488498、135276592、148522929、202683424、205620149、222308771、229586249、234567116、240417456、253749877、281357546、293747404、298042526、298551987、304720100、308580466、315853787、316067487、317504915、323176649、323573314、326044569、331209279、331937768、331978174、340045388、342258714、346927231、352153597、352895025、353681133、353814760、354406639、356296360、357079009、359814991、360764207、361568511、364411537、366545162、368905078、370338339、370873141、371665959、371666020、378118472、378609831、381550595、381678547、382379106、383609412、385331788、387527211、387561633、388365359、390782899、391759928、392823534、396147375、401560695、405883242、407306848、407534418、408130557、415475150、420947480、430805639、451372954、461369888、462031762、472352616、484252481、488853414、495087041、499171795、502654285、506758067、507037483、509192469、509662645、515161916、515262213、517095456、522371782、525396862、527223945、528829545、533025127、539185410、540315630、540617233；

105～159：chr2A：2100930、4239492、10704631、10938210、13436858、14441332、18422955、32282364、39418692、59982919、60887086、70607983、75197819、89599242、104954798、112783291、130463628、132068088、135426392、217334034、242505005、266261884、274342752、296150888、316167952、317795068、330610398、332882630、335767331、340794839、344918901、345951965、348518457、351045498、354468810、358393070、359546799、365230751、367437482、376887627、381029377、382692023、386423751、388053059、392483868、392798434、396593342、414088827、417851447、420594190、420595093、424692775、427321507、442730057、446172245；

806～918：chr4C：3156275、3156309、6151866、11154035、26834888、30694671、36293580、39086177、40288786、42236043、43109627、43848590、47392406、47641679、49158016、52760842、53598521、53939843、58316218、59961826、60096926、62416393、64784388、69604919、70450484、73378379、83748627、103386515、107244878、107330999、108482037、118792713、119239746、121940137、122327356、123510068、124053384、126900382、128226873、130464152、131337543、135871755、136394936、137398743、139983786、142766414、144128859、149693472、151667321、158587013、159537214、168296059、171843825、172869591、177332803、188589498、188815342、194106449、194117303、208025980、232058561、232313275、233414178、243942384、246993956、277770184、335855729、341048610、355301777、358590152、359548305、380344060、390159929、393180518、398088959、402898771、403502507、410722151、416930608、442743332、443020917、453953122、488144957、493824659、509539196、512906902、518164512、525011469、525018986、525845028、546203479、558810054、564981985、565345449、568926820、575890407、597642698、606728798、625266556、633582863、670582073、671502734、676162180、684407156、692454211、696599915、698128769、698161356、700308139、704691850、706603391、709770857、710256791；

919～1044：chr5C：554202、3749792、3849936、7637563、9034502、12288161、12428473、15702114、16919102、19035849、19684941、34039963、38195971、41475312、47425662、53774061、53798541、53937809、60895738、62177980、63445568、64638626、64699517、68936987、87785914、90707249、93155853、97074885、98370861、98818134、105238445、107890566、108958735、115464154、116215262、126465885、130714668、146124820、148156344、150181347、156346016、161589949、170232433、170265452、181486109、181698549、190217791、212065553、224669563、240501203、249105387、264893757、275312253、290424574、313738041、325535102、338862900、340776106、346453345、346670057、360877555、369746847、370916238、376791265、377444427、380137077、383843868、391113723、399409221、400559622、407090768、408955951、409877500、411853930、412309560、422660477、427953795、429536327、433178666、437980316、438986627、439244384、443400619、449027771、459155181、469127358、471266768、475881780、485797166、490197664、493250155、502265146、504791556、505908673、506958259、508142521、511148919、513278340、514868949、517911508、519788490、523051612、533055170、544767033、549076111、550648593、553113006、553385567、557268792、563355183、564881004、569327026、572145755、572145791、581326964、584056057、590337215、595946882、601502958、606333257、606918814、610040984、611215306、612022343、612942538、613031851；

1628～1718：chr4D：11485004、14717658、15065859、31961722、32485235、33560371、35156356、39740867、42795157、44433053、59143960、61439239、78867509、83710252、94577427、97342443、99630391、102329405、114879597、135083886、138944406、139191630、145275183、158199248、183847307、197618490、202008461、208924184、214257428、217116193、218226761、220722019、224527103、226305656、242931934、249581054、255462900、264284840、267548200、268330950、271337080、272128244、274673552、277597999、284757400、292115085、295208268、307122231、312785806、315358650、320106105、321257483、322335201、331354099、347449985、349565426、355005406、355529054、357699853、363770741、375262472、377476161、380726205、385800811、391193802、394067252、394991049、398011595、398021682、402631607、403614105、404368909、407333043、410646224、412188909、414401279、418371869、418997245、421372071、421555572、428897486、430839695、432215688、440437664、441165072、443760645、446977976、448126463、451246460、453204068、454964761；

1719～1790：chr5D：422491、2403544、2847753、8070862、9173118、15344875、20080029、20241303、23719531、24894899、42951694、44107567、48815715、49508914、51945197、56013951、62539497、149532814、182274396、224230643、244783821、258584112、274219604、300870135、312225241、324387095、334868009、336719110、346038010、346469081、346469183、354514206、366731773、378137934、379881301、380071905、392150519、396806532、401607567、401827548、403566921、407953083、414598189、418669755、418689444、420799211、425328381、436638413、438696800、438952723、445810673、450854676、455970239、459069234、460850708、460986821、471757307、472785728、480109072、484436332、484877489、487723676、489342416、490270563、491681200、492912927、494028859、495132865、495470919、495962366、497177907、498614358；

1843～2000：chr7D：1072783、1119801、1236244、1534371、2991626、6484731、8526949、12785153、16242070、17366942、17850661、22956474、24096794、28468246、36005342、36246561、38201748、44727899、51458782、52893291、54121226、57516030、57516038、67534447、70410781、71268375、74183904、77122480、80597019、80953710、91190332、97784606、101125798、101646423、103077178、107623530、109736013、109968936、109968972、115007259、118525929、122982073、128880373、129122344、130575310、144416893、144696534、147934872、151212412、156658652、159001645、159202650、161327586、163948311、181525332、182392553、185716424、192236268、197083938、200305527、200305588、203992071、205384321、207873222、211308312、213859261、215658345、221747688、227132133、237365568、238457399、240373853、240497344、251811510、252160256、257692302、265633476、276461950、281656816、281673877、281673955、282186605、288940800、289015491、295863972、297712659、300964765、301738058、302186316、305064006、305958757、309078989、309792985、310443347、311730518、317299513、318083871、320706656、327935646、330094148、334523638、334550921、335532754、339511013、341280805、348832923、348856568、362000098、371871465、374671605、379217936、381999320、388319071、392108332、403165236、405370611、412711051、412974598、423689553、425896659、434160829、434379013、435977775、437395621、445058667、449281896、452187250、455142358、462212286、466485714、469010673、469111622、469111697、474002520、476702195、479594709、480535689、483206258、488775954、493156510、493471148、495673345、497521926、503762348、506063835、507610286、508855720、511766848、512617462、514968304、515513948、516813821、519702746、520048073、521311574、524439726、525367002、527908773；

wherein chr1A, chr2A, chr3A, chr4A, chr5A, chr6A, chr7A, chr1B, chr2B, chr3B, chr4B, chr5B, chr6B, chr7B, chr1C, chr2C, chr3C, chr4C, chr5C, chr6C, chr7C, chr1D, chr2D, chr3D, chr4D, chr5D, chr6D, chr D represents the chromosome ID where the SNP site is located, and the numbers following the chromosome ID represent the location of the SNP site on the chromosome.

2. The oat 50K SNP targeted capture probe of claim 1 wherein the 5' end of the probe carries a biotin group and the probe is coupled to fluorescent microspheres by C12 molecular arm and amino modification, one probe coupled to each fluorescent microsphere.

3. The method for preparing the oat 50K SNP targeted capture probe of claim 1, comprising:

step 1: screening oat 50K SNP loci;

oat 50K SNP locus is obtained by screening by the following method:

step 1.1: detecting SNP loci on all sample genomes by comparing oat reference genomes according to the resequencing data of a plurality of oat germplasm, and selecting target SNP loci;

step 1.2: according to the open-source simplified genome sequencing data, comparing the oat reference genome to detect SNP loci in all data, and selecting target SNP loci;

step 1.3: selecting a target SNP locus according to the oat 6K chip locus;

step 1.4: screening SNP loci according to published oat functional marker loci;

step 2: oat 50K SNP site probes were designed and prepared.

4. A process according to claim 3, wherein,

the target SNP locus is selected according to the following criteria: MAF is more than or equal to 0.05, NA is less than 5%, heterozygosity is less than 5%, and chromosomes are evenly distributed.

5. The method according to claim 3, wherein the design process of the oat 50K SNP site probe is as follows:

step 2.1: determining a probe design principle:

the probe length is 120bp, the GC content of the probe is 25% -69%, the number of homologous regions is less than or equal to 3, and the selected region does not contain SSR and GAP regions to the maximum extent; combining with a chromosome uniform distribution principle;

step 2.3: synthesizing single-stranded DNA with biotin group modification at the 5' end according to the 120bp nucleotide sequence obtained by screening in the step 2.2, and obtaining the oat 50K SNP locus probe.

6. Oat 50K SNP molecular marker and application of oat 50K SNP locus probe in oat 50K SNP targeting capture probe preparation, characterized in that the oat 50K SNP locus probe is single-stranded DNA synthesized according to oat 50K SNP molecular marker, and the 50K SNP locus comprises the core SNP molecular marker as set forth in claim 1.

7. Use of oat 50K SNP molecular markers, oat 50K SNP site probes in any of the following:

(1) Analysis of oat genetic diversity;

(2) Constructing an oat molecular genetic map;

(3) Oat whole genome association analysis;

(4) Identifying the authenticity of oat varieties;

(5) Auxiliary selection breeding of oat molecular markers;

(6) Selecting and breeding oat whole genome;

the oat 50K SNP site probe is single-stranded DNA synthesized according to oat 50K SNP molecular markers, and the 50K SNP site includes the core SNP molecular markers as set forth in claim 1.

8. Use of the oat 50K SNP targeted capture probe of claim 1 in any of the following:

(1) Analysis of oat genetic diversity;

(2) Constructing an oat molecular genetic map;

(3) Oat whole genome association analysis;

(4) Identifying the authenticity of oat varieties;

(5) Auxiliary selection breeding of oat molecular markers;

(6) And (5) whole genome selective breeding of oat.