CN110373492A - Without the hidden sub- grass LTR-RT molecular labeling primer of awns and the application in Germplasm Identification - Google Patents
Without the hidden sub- grass LTR-RT molecular labeling primer of awns and the application in Germplasm Identification Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to hidden sub- grass LTR-RT (the long end repetition retrotransposon) molecular labeling primer of no awns and the application in the hidden son grass category Germplasm Identification of no awns.It is specific to be distinguished using 80 pairs 23 kinds of primer pair without the hidden sub- grass seeds matter of awns, identification method of the invention can be combined using less primer to be completed to identify for trying the comparison that hidden son grass belongs to germplasm within 4h, with the advantages such as accurate, efficient, quick, at low cost and easy to operate, the application sensitivity with higher and accuracy.
Description
Technical field
The invention belongs to field of biotechnology, the hidden sub grass LTR-RT molecular labeling primer of no awns and belongs to germplasm in hidden son grass and reflect
Application in fixed.
Background technique
The no hidden son of awns careless (Cleistogenes songorica) is that the hidden son grass of grass family belongs to perennial grass, is born in dry more
Non-irrigated grassland, desert or half-desert are constructive species and the sociales of desert steppe sandyly.It, can be with stronger drought tolerance
The growth of the area annual rainfall 120mm, cold resistance, heat resistance are strong, and more resistance to trample, Green stage is long, cannot be only used for Animal husbandry production,
Also act as general lawn plant.In northwest China, arid, semiarid zone, arid-desert areas ecosystem stabilization are played
Important function has the important ecological value and genetic worth.Hidden son grass belongs to there are about more than 20, is distributed in Central Asia and north
There is 12 kind of 2 mutation in portion and south of europe, China.Simultaneously as China's landforms are wide, the various life formed in different niches
State type, different niches include different longitudes and latitudes, height above sea level etc. element, these all constitute the heredity that hidden son grass belongs to germ plasm resource
Diversity.Previously, domestic and foreign scholars were based primarily upon morphology label and identified hidden son grass category germ plasm resource, division kind and kind
Lower grade (including subspecies, mutation, modification) relies primarily on some quantitative characters, such as the length of awns, lemma and grain husk.But due to
These characters are closely similar in inter-species, bring to the division of kind difficult and uncertain, include subspecies, become as lower grade is planted
Kind, modification are just more difficult to distinguish by morphology.Therefore, it establishes a set of hidden son grass quickly, accurate, cheap and belongs to germ plasm resource essence
Quasi- identification technology is very important for screening excellent resources assistant breeding.
With the development of modern molecular biology, molecular labeling is more and more applied in cultivar identification, such as with PCR
SSR, ILP, LTR-RT related molecular marker (RBIP, IRAP, ISBP, REMAP) based on reaction etc..LTR-RT sequence is being planted
Widely distributed, transferable, stronger sequence conservation in object genome all makes it suitable as molecular labeling.With other molecules
Label is compared, and LTR-RT mark of correlation has the advantages that many uniquenesses, with stronger rich, specific, codominance, reliably
Property.It can be used for cultivar identification, Phylogenetic Analysis, analysis of genetic diversity and genetic linkage mapping etc..
As a kind of novel molecular labeling, so far, LTR-RT mark of correlation is mainly in wheat, barley, millet etc.
Analysis of genetic diversity and genetic linkage maps building are developed and are mainly used in a small number of plants.Such as Surinder
Singh etc. has studied 47 kinds of wild and Cultivate berley genetic diversities (referring to " The Crop using LTR-RT mark of correlation
Journal ", 2017, " Transposon-based genetic diversity assessment in wild and
cultivated barley").But in non-mode plant without awns in hidden son grass, the exploitation of LTR-RT mark of correlation and in kind
There is not been reported for application in identification.Therefore, it is developed without the hidden son grass LTR-RT related molecular marker of awns simultaneously in full-length genome level
It studies it and belongs to the application in Germplasm Identification in hidden son grass, belonging to Germplasm Identification and molecular mark to hidden son grass has weight
Want meaning.
Summary of the invention
It is an object of that present invention to provide 80 pairs without the hidden sub- grass LTR-RT molecular labeling primer of awns, for belonging to germplasm to hidden son grass
It is identified.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
Without the hidden sub- grass LTR-RT related molecular marker primer of awns, the molecular labeling primer includes as be shown in the examples
80 pairs of molecular labeling primers.
The hidden sub- grass LTR-RT related molecular marker primer of no awns belongs to the application in Germplasm Identification in hidden son grass, wherein described
It includes that hidden son grass listed in table 1 belongs to germplasm that hidden son grass, which belongs to germplasm,.
The hidden son grass of table 1 belongs to germplasm information
A kind of kit identified hidden son grass and belong to germplasm draws comprising the hidden sub- grass LTR-RT related molecular marker of the no awns
Object.
The beneficial effects of the present invention are:
The present invention can be to the various ecotype that hidden son grass is formed in different niches (including different longitudes and latitudes, height above sea level
Etc. element) be rapidly performed by identification, for by quantitative character, as the length character of awns, lemma and grain husk is difficult to differentiate between
Hidden sub- grass seeds and plant that lower grade includes subspecies, mutation, modification these hidden son grass belong to germ plasm resources further to be segmented, it is right
It is had a very important significance in screening excellent resources assistant breeding.
In the case where not calculating the time consumed by DNA extraction process, detection method of the invention can utilize less
Primer combination within 4h complete to for try it is hidden son grass belong to germplasm comparison identify, have it is accurate, efficient, quick, at low cost with
And the advantages such as easy to operate, the application sensitivity with higher and accuracy.
Detailed description of the invention
Fig. 1 is specific implementation step of the present invention flow chart；
Fig. 2 is amplification figure of the primer Cs_LTR_13 to 23 parts of germplasm；
Fig. 3 is amplification figure of the primer Cs_LTR_19 to 23 parts of germplasm；
Fig. 4 is amplification figure of the primer Cs_LTR_59 to 23 parts of germplasm；
Fig. 5 is amplification figure of the primer Cs_LTR_216 to 23 parts of germplasm；
Fig. 6 is amplification figure of the primer Cs_LTR_338 to 23 parts of germplasm.
Specific embodiment
It is described in more detail below specific embodiments of the present invention.It should be appreciated that may be realized in various forms the present invention
And it should not be limited by the embodiments set forth herein.It is to be able to thoroughly understand this hair on the contrary, providing these embodiments
It is bright, and the scope of the present invention can be fully disclosed to those skilled in the art.
"comprising" or " comprising " as mentioned throughout the specification and claims are an open language, therefore are answered
It is construed to " including but not limited to ".Specification subsequent descriptions are to implement better embodiment of the invention, and so description is
For the purpose of the rule of specification, the range that is not intended to limit the invention.Protection scope of the present invention is when the appended power of view
Benefit requires subject to institute's defender.
Embodiment:
Specific implementation step flow chart of the invention referring to Fig.1, specifically includes:
1. expert evidence DNA is extracted
Referring to SDS method, specific steps are as follows:
1) 50 full, healthy seeds are chosen, 20 DEG C of sprouting 72h of double-layer filter paper method are utilized；
2) the above seed is mixed in mortar and is fully ground, 600 μ L DNA buffer are added, continue to be ground.It will
Lapping liquid is transferred to 2.0mL centrifuge tube；
3) above-mentioned centrifuge tube is transferred to 65 DEG C of water-bath 10min, this shakes up 1-2 times in the process；
4) chloroform is added in ventilating kitchen: the 600 μ L of mixed liquor that isoamyl alcohol volume ratio is 24: 1, mixing of turning upside down,
12000 turns of centrifugation 10min under room temperature draw 360 μ L supernatants and are transferred in 1.5ml centrifuge tube；
5) it is uniformly mixed, is placed at room temperature for after the 240 μ L of 36 μ L of 3mol/LNaAc (pH=5.2) and isopropanol of pre-cooling is added
After 10min, 12000 turns of centrifugation 10min of room temperature abandon supernatant, are centrifuged 30s, residual night is sucked out with 10 μ L liquid-transfering guns；
6) 75% ethyl alcohol of 500 μ L is added, precipitating is bounced, 12000 turns of centrifugation 5min pour out ethyl alcohol.It is centrifuged 30s, is used
Residual night is sucked out 10 μ L liquid-transfering guns, this step is repeated 2 times, and residual ethanol is dried up in ventilating kitchen；
7) 20 μ L sterile deionized waters are added, after completely dissolution, detect DNA molecular amount size, benefit with 1% agarose electrophoresis
DNA concentration is measured with ultramicron ultraviolet specrophotometer, and is diluted to 25ng/ μ L, 4 DEG C save for use.
2. the molecular labeling PCR amplification of expert evidence DNA
This seminar carried out without awns it is hidden son grass genome sequencing, sequence be placed in hundred mikey cloud platforms (http: //
Www.biocloud.net), using RepeatModeler and LTR_FINDER tool, 299079 LTR-RT sequences are identified altogether
Column.By online tool RepeatMasker (Masker) with rice to compare DNA source file, the length of identification LTR-RT sequence is last
The region (LTR) is held, DNAMA N design primer is used.
As can be seen from the above description, identification is very more without the hidden sub- grass LTR-RT sequence of awns.In the hidden 20 pairs of dyes of son grass of no awns
Equally distributed condition selects LTR-RT sequence on colour solid, devises totally 350 pairs of tetra- seed type of RBIP, IRAP, ISBP, REMAP
Primer, and polymorphism height, reproducible primer by virtue of experience are finally obtained with the creative screening of a large amount of test, it is designed
80 pairs of primers out are as shown in table 2.
2 80 pairs of primer information of table
The 80 pairs of primer pairs material DNA to be identified provided using this method carries out PCR amplification, and reaction system and program are such as
Under:
1) reaction system: 10 μ L systems include expert evidence DNA profiling 25ng, forward and reverse primer each 0.4 μm of ol/L, 5 μ
L2 × power Taq MasterMix (hundred Tykes), residual volume is supplied with ultrapure water；
2) touchdown PCR, first 94 DEG C of initial denaturation 3min response procedures: are used；Then, 94 DEG C of denaturation 30s, 60-50 DEG C is moved back
Fiery 30s, 72 DEG C of extension 30s, totally 11 recycle；Then 94 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 25 are followed
Ring；72 DEG C of extension 7min, 4 DEG C of preservations.
3. modacrylic acyl ammonia gel electrophoresis and silver staining detect
Operating procedure is as follows:
1) it cleans glass plate: glass plate being cleaned up with tap water, is dried after being rinsed with deionized water.With 95% ethyl alcohol
It cleans twice, blotting paper is dried.Prevent two pieces of glass plates from polluting mutually in operating process.
2) it assembles glass plate: after glass plate is thoroughly dried, assembling them into electrophoresis offset plate using adhesive tape.
3) glue: 10% ammonium persulfate for being separately added into 150 μ L TEMED in 200mL 6.0%PAGE glue and newly preparing
2000 μ L of solution can be used for filling 4 plate glue.After above-mentioned rapid mixing fall glue, the glue made is fallen along the side of offset plate rapidly
Enter, to prevent gelling solid, comb is put well and fixed in advance, excludes the bubble generated.Offset plate stands 30min, solidifies it sufficiently,
The glue that offset plate surface is overflowed is cleared up, comb is gently extracted, is washed with water clean spare.
5) sample preparation: 2 μ 6 × sample loading buffers of L are added in 10 μ L PCR products, after mixing, in water-bath or PCR
95 DEG C of denaturation 5min, 4 DEG C of cooling 10min or more on instrument.
6) electrophoresis: drawing buffer with syringe and rinse gel top several times, removes bubble and gel pieces, while by glue
Item sets right.Each well clicks and enters the 1 μ L sample of μ L~2, selects suitable Marker according to primer size.Under 200V constant pressure
Electrophoresis, electrophoresis 90min (magnitude range that electrophoresis time depends on amplified fragments).After electrophoresis, two blocks of glass are carefully separated
Plate, the separating gel from glass plate.
7) silver staining: glue is put into the plastic casing equipped with distilled water by a, cleans 10s, pours out distilled water, prepares silver staining.B. to
0.08% silver nitrate solution is added in plastic casing equipped with film, so that solution was not had film, is shaken for 60 revs/min on shaking table
10min.C. silver nitrate is poured into waste liquid barrel, with distilled water rinsed clean film, film is transferred to the hydroxide containing 1.5%
Sodium, 0.4% formaldehyde developer solution in, on shaking table 60 revs/min shake several minutes and clear band line occur until.D. film is shifted
To lamp box, takes pictures and save photo.Wherein, such as primer Cs_LTR_13 is shown in Fig. 2, primer Cs_ to the amplification of 23 parts of germplasm
LTR_19 is shown in that Fig. 3, primer Cs_LTR_59 are shown in Fig. 4 to the amplification figure of 23 parts of germplasm to the amplification of 23 parts of germplasm；Primer
Cs_LTR_216 is shown in Fig. 5 to the amplification of 23 parts of germplasm；Primer Cs_LTR_338 is shown in Fig. 6 to the amplification of 23 parts of germplasm.
8) banding pattern of each identification same site amplified fragments of germplasm and migration position are compared, expand banding pattern with 0,1
It indicates, on identical migration position, have band to be denoted as " 1 ", no band is denoted as " 0 ", constructs " 1 ", " 0 " matrix.23 parts of kinds of each primer pair
The differentiation information of matter is shown in Table 3.
3 23 parts of table hidden son grass belong to " 0 ", " 1 " matrix that germplasm is constructed based on 80 LTR-RT related molecular markers
For the allele in table 3, by taking Cs_LTR_14 as an example, corresponding display is that have 10 allele, but in fact
The case where being all the corresponding gene of Cs_LTR_14 of amplification, why will appear multiple allele, be hidden because of no awns
Sub- grass is allotetraploid, and LTR-RT is transferable in genome, generates multiple copies, and it is possible to a plurality of item occur
The case where band (allele).
The case where can be seen that each primer amplification result in table 3,80 pairs without the hidden sub- grass LTR-RT relevant molecule mark of awns
Note can amplify polymorphic bands for planting experimentally at 23 in matter, and stable amplification result, have repeatability.Wherein, 80 pairs
The hidden sub- grass LTR-RT related molecular marker of no awns goes out 336 allele bands for planting experimentally coamplification in matter at 23, average every
A site is 4.2 allele bands.Expection heterozygosity (He) range of 80 pairs of LTR-RT related molecular markers is 0.08
(Cs_LTR_331) to 0.88 (Cs_LTR_117), average value 0.55.Polymorphic sex index (PIC) range is 0.08 (Cs_LTR_
331) to 0.87 (Cs_LTR_117), average value 0.49.
9) for statistical analysis to the qualification result of each pair of primer, difference number of sites >=1 item determines between difference identification germplasm
Two parts of germplasm can be distinguished, and germ plasm resource can be refined identification.Nearly all primer pair can complete the area of kind of grade
Point, more than inferior grade also can be used primer pair combination mode distinguish one by one.Specific Germplasm Identification the results are shown in Table 4.
4 80 LTR-RT related molecular markers of table distinguish germplasm information (germplasm numbers corresponding germplasm information and is shown in Table 1)
The monoid of 4 germplasm of table belongs to germplasm without the hidden son grass of awns to 23 parts and carries out PCR amplification by taking Cs_LTR_85 as an example, according to
23 parts of germplasm can be divided into 8 monoids (situation) by the band difference that each kind amplifies.Wherein, germplasm 1 and 3 belongs to
Monoid 1 is because their amplified band is the same；Germplasm 2,4,5,6,7,8 belongs to monoid 2 as 9 amplified band,
But their type of strip and the type of strip of monoid 1 have difference again.Thereafter similarly.Therefore, if only utilizing Cs_LTR_85
If primer, it is only capable of distinguishing 23 parts of germplasm parts.And if distinguishing 23 parts of germplasm completely, it is necessary to using not
With the combination of primer.For example, germplasm 1 and 3 we can not use Cs_LTR_85, but if result in conjunction with Cs_LTR_10
Words, so that it may which germplasm 1 and 3 is distinguished.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (4)
1. without the hidden sub- grass LTR-RT molecular labeling primer of awns, which is characterized in that the molecular labeling primer includes following 80 pairs,
The nucleotide sequence of forward primer and reverse primer is specific as follows:
(1) Cs_LTR_10: forward primer sequence is SEQ ID NO.1, and reverse primer sequences are SEQ ID NO.2；
(2) Cs_LTR_11: forward primer sequence is SEQ ID NO.3, and reverse primer sequences are SEQ ID NO.4；
(3) Cs_LTR_13: forward primer sequence is SEQ ID NO.5, and reverse primer sequences are SEQ ID NO.6；
(4) Cs_LTR_14: forward primer sequence is SEQ ID NO.7, and reverse primer sequences are SEQ ID NO.8；
(5) Cs_LTR_16: forward primer sequence is SEQ ID NO.9, and reverse primer sequences are SEQ ID NO.10；
(6) Cs_LTR_17: forward primer sequence is SEQ ID NO.11, and reverse primer sequences are SEQ ID NO.12；
(7) Cs_LTR_19: forward primer sequence is SEQ ID NO.13, and reverse primer sequences are SEQ D NO.14；
(8) Cs_LTR_27: forward primer sequence is SEQ ID NO.15, and reverse primer sequences are SEQ ID NO.16；
(9) Cs_LTR_35: forward primer sequence is SEQ ID NO.17, and reverse primer sequences are SEQ D NO.18；
(10) Cs_LTR_36: forward primer sequence is SEQ ID NO.19, and reverse primer sequences are SEQ ID NO.20；
(11) Cs_LTR_39: forward primer sequence is SEQ ID NO.21, and reverse primer sequences are SEQ ID NO.22；
(12) Cs_LTR_40: forward primer sequence is SEQ ID NO.23, and reverse primer sequences are SEQ ID NO.24；
(13) Cs_LTR_41: forward primer sequence is SEQ ID NO.25, and reverse primer sequences are SEQ ID NO.26；
(14) Cs_LTR_50: forward primer sequence is SEQ ID NO.27, and reverse primer sequences are SEQ ID NO.28；
(15) Cs_LTR_51: forward primer sequence is SEQ ID NO.29, and reverse primer sequences are SEQ ID NO.30；
(16) Cs_LTR_53: forward primer sequence is SEQ ID NO.31, and reverse primer sequences are SEQ ID NO.32；
(17) Cs_LTR_59: forward primer sequence is SEQ ID NO.33, and reverse primer sequences are SEQ ID NO.34；
(18) Cs_LTR_66: forward primer sequence is SEQ ID NO.35, and reverse primer sequences are SEQ ID NO.36；
(19) Cs_LTR_68: forward primer sequence is SEQ ID NO.37, and reverse primer sequences are SEQ ID NO.38；
(20) Cs_LTR_69: forward primer sequence is SEQ ID NO.39, and reverse primer sequences are SEQ ID NO.40；
(21) Cs_LTR_84: forward primer sequence is SEQ ID NO.41, and reverse primer sequences are SEQ ID NO.42；
(22) Cs_LTR_85: forward primer sequence is SEQ ID NO.43, and reverse primer sequences are SEQ ID NO.44；
(23) Cs_LTR_94: forward primer sequence is SEQ ID NO.45, and reverse primer sequences are SEQ ID NO.46；
(24) Cs_LTR_106: forward primer sequence is SEQ ID NO.47, and reverse primer sequences are SEQ ID NO.48；
(25) Cs_LTR_107: forward primer sequence is SEQ ID NO.49, and reverse primer sequences are SEQ ID NO.50；
(26) Cs_LTR_110: forward primer sequence is SEQ ID NO.51, and reverse primer sequences are SEQ ID NO.52；
(27) Cs_LTR_115: forward primer sequence is SEQ ID NO.53, and reverse primer sequences are SEQ ID NO.54；
(28) Cs_LTR_117: forward primer sequence is SEQ ID NO.55, and reverse primer sequences are SEQ ID NO.56；
(29) Cs_LTR_122: forward primer sequence is SEQ ID NO.57, and reverse primer sequences are SEQ ID NO.58；
(30) Cs_LTR_126: forward primer sequence is SEQ ID NO.59, and reverse primer sequences are SEQ ID NO.60；
(31) Cs_LTR_137: forward primer sequence is SEQ ID NO.61, and reverse primer sequences are SEQ ID NO.62；
(32) Cs_LTR_138: forward primer sequence is SEQ ID NO.63, and reverse primer sequences are SEQ ID NO.64；
(33) Cs_LTR_141: forward primer sequence is SEQ ID NO.65, and reverse primer sequences are SEQ ID NO.66；
(34) Cs_LTR_142: forward primer sequence is SEQ ID NO.67, and reverse primer sequences are SEQ ID NO.68；
(35) Cs_LTR_144: forward primer sequence is SEQ ID NO.69, and reverse primer sequences are SEQ ID NO.70；
(36) Cs_LTR_149: forward primer sequence is SEQ ID NO.71, and reverse primer sequences are SEQ ID NO.72.
(37) Cs_LTR_155: forward primer sequence is SEQ ID NO.73, and reverse primer sequences are SEQ ID NO.74；
(38) Cs_LTR_156: forward primer sequence is SEQ ID NO.75, and reverse primer sequences are SEQ ID NO.76；
(39) Cs_LTR_159: forward primer sequence is SEQ ID NO.77, and reverse primer sequences are SEQ ID NO.78；
(40) Cs_LTR_161: forward primer sequence is SEQ ID NO.79, and reverse primer sequences are SEQ ID NO.80；
(41) Cs_LTR_166: forward primer sequence is SEQ ID NO.81, and reverse primer sequences are SEQ ID NO.82；
(42) Cs_LTR_168: forward primer sequence is SEQ ID NO.83, and reverse primer sequences are SEQ ID NO.84；
(43) Cs_LTR_173: forward primer sequence is SEQ ID NO.85, and reverse primer sequences are SEQ ID NO.86；
(44) Cs_LTR_187: forward primer sequence is SEQ ID NO.87, and reverse primer sequences are SEQ ID NO.88；
(45) Cs_LTR_188: forward primer sequence is SEQ ID NO.89, and reverse primer sequences are SEQ ID NO.90；
(46) Cs_LTR_190: forward primer sequence is SEQ ID NO.91, and reverse primer sequences are SEQ ID NO.92；
(47) Cs_LTR_191: forward primer sequence is SEQ ID NO.93, and reverse primer sequences are SEQ ID NO.94；
(48) Cs_LTR_199: forward primer sequence is SEQ ID NO.95, and reverse primer sequences are SEQ ID NO.96；
(49) Cs_LTR_205: forward primer sequence is SEQ ID NO.97, and reverse primer sequences are SEQ ID NO.98；
(50) Cs_LTR_207: forward primer sequence is SEQ ID NO.99, and reverse primer sequences are SEQ ID NO.100；
(51) Cs_LTR_208: forward primer sequence is SEQ ID NO.101, and reverse primer sequences are SEQ ID NO.102；
(52) Cs_LTR_214: forward primer sequence is SEQ ID NO.103, and reverse primer sequences are SEQ ID NO.104；
(53) Cs_LTR_216: forward primer sequence is SEQ ID NO.105, and reverse primer sequences are SEQ ID NO.106；
(54) Cs_LTR_220: forward primer sequence is SEQ ID NO.107, and reverse primer sequences are SEQ ID NO.108；
(55) Cs_LTR_225: forward primer sequence is SEQ ID NO.109, and reverse primer sequences are SEQ ID NO.110；
(56) Cs_LTR_230: forward primer sequence is SEQ ID NO.111, and reverse primer sequences are SEQ ID NO.112；
(57) Cs_LTR_231: forward primer sequence is SEQ ID NO.113, and reverse primer sequences are SEQ ID NO.114；
(58) Cs_LTR_235: forward primer sequence is SEQ ID NO.115, and reverse primer sequences are SEQ ID NO.116；
(59) Cs_LTR_237: forward primer sequence is SEQ ID NO.117, and reverse primer sequences are SEQ ID NO.118；
(60) Cs_LTR_238: forward primer sequence is SEQ ID NO.119, and reverse primer sequences are SEQ ID NO.120；
(61) Cs_LTR_241: forward primer sequence is SEQ ID NO.121, and reverse primer sequences are SEQ ID NO.122；
(62) Cs_LTR_246: forward primer sequence is SEQ ID NO.123, and reverse primer sequences are SEQ ID NO.124；
(63) Cs_LTR_250: forward primer sequence is SEQ ID NO.125, and reverse primer sequences are SEQ ID NO.126；
(64) Cs_LTR_253: forward primer sequence is SEQ ID NO.127, and reverse primer sequences are SEQ ID NO.128；
(65) Cs_LTR_258: forward primer sequence is SEQ ID NO.129, and reverse primer sequences are SEQ ID NO.130；
(66) Cs_LTR_260: forward primer sequence is SEQ ID NO.131, and reverse primer sequences are SEQ ID NO.132；
(67) Cs_LTR_275: forward primer sequence is SEQ ID NO.133, and reverse primer sequences are SEQ ID NO.134；
(68) Cs_LTR_285: forward primer sequence is SEQ ID NO.135, and reverse primer sequences are SEQ ID NO.136；
(69) Cs_LTR_298: forward primer sequence is SEQ ID NO.137, and reverse primer sequences are SEQ ID NO.138；
(70) Cs_LTR_300: forward primer sequence is SEQ ID NO.139, and reverse primer sequences are SEQ ID NO.140；
(71) Cs_LTR_303: forward primer sequence is SEQ ID NO.141, and reverse primer sequences are SEQ ID NO.142；
(72) Cs_LTR_309: forward primer sequence is SEQ ID NO.143, and reverse primer sequences are SEQ ID NO.144；
(73) Cs_LTR_313: forward primer sequence is SEQ ID NO.145, and reverse primer sequences are SEQ ID NO.146；
(74) Cs_LTR_319: forward primer sequence is SEQ ID NO.147, and reverse primer sequences are SEQ ID NO.148；
(75) Cs_LTR_324: forward primer sequence is SEQ ID NO.149, and reverse primer sequences are SEQ ID NO.150；
(76) Cs_LTR_331: forward primer sequence is SEQ ID NO.151, and reverse primer sequences are SEQ ID NO.152；
(77) Cs_LTR_333: forward primer sequence is SEQ ID NO.153, and reverse primer sequences are SEQ ID NO.154；
(78) Cs_LTR_338: forward primer sequence is SEQ ID NO.155, and reverse primer sequences are SEQ ID NO.156；
(79) Cs_LTR_339: forward primer sequence is SEQ ID NO.157, and reverse primer sequences are SEQ ID NO.158；
(80) Cs_LTR_342: forward primer sequence is SEQ ID NO.159, and reverse primer sequences are SEQ ID NO.160.
2. belonging to answering in Germplasm Identification in hidden son grass without the hidden sub- grass LTR-RT molecular labeling primer of awns as described in claim 1
With.
3. application as claimed in claim 2, wherein the hidden son grass belongs to germplasm include the following:
4. a kind of kit identified hidden son grass and belong to germplasm, which is characterized in that include the no hidden son grass of awns described in claim 1
LTR-RT molecular labeling primer.
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