CN108504763A - Identify primer sets and its application of cotton variety YM111 authenticities and seed purity - Google Patents
Identify primer sets and its application of cotton variety YM111 authenticities and seed purity Download PDFInfo
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Abstract
The invention discloses primer sets and its applications of identification cotton variety YM111 authenticities and seed purity.The primer sets of identification the cotton variety YM111 authenticities and seed purity of the present invention, the composition being made of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 this 8 kinds of primer pairs.The composition of this 8 kinds of primer pairs composition can be used for identifying the seed production purity of cotton crossbreed YM111, identify the authenticity of cotton crossbreed YM111 kinds, build the finger-print of cotton crossbreed YM111 kinds.The composition formed with this 8 kinds of primer pairs, can control the seed production quality of hybrid cotton YM111, be conducive to the popularization and protection of YM111.
Description
Technical field
The present invention relates to drawing for cotton variety YM111 authenticities and seed purity is identified in crop molecular genetic breeding field
Object group and its application.
Background technology
Cotton variety YM111 is the high-yield disease resisting transgenic hybrid cotton variety that the Handan Agriculture academy of sciences cultivates, and authorization is compiled
Number examine cotton 20170002 for state.YM111 is ripe Hybrid in transform insect-resistant gene made of the 6205 × Hanmian 802 selection and breeding of Handan.It is yellow
It sows in spring 120 days breeding times in river valley cotton region.Emergence is neat, and breeding time growing way is strong.Loosely, cane is more sturdy for plant type.Bell oval
Shape, median size, carpopodium is longer, and knot bell is strong, and blow-of-cottons are smooth and concentrate.Anti-blight (disease index 9.6), resistance to verticillium wilt (disease
Feelings index 28.0).Bollworm resisting.104.6 centimetres of plant height, the first fruit branch section 7.3 section.Boll per plant 22.2, Single boll weight 6.1
Gram.Son refers to 10.7 grams, and ginning outturn 41.4% spends rate 92.7% before white.28.1 millimeters of HVICC fibers upper half mean length, fracture
31.0 lis of ox/Tekes of specific strength, mic value 5.3, elongation at break 6.4%, reflectivity 74.8%, yellow depth 8.7 are whole
Neat degree index 84.8%, evenness index 138 of spinning.
Occurs shoddy cotton variety YM111 in the market in order to prevent, it is desirable to provide cotton variety YM111 authenticities
Supervision detection and breeding units seed quality of quick, precise Identification the method to serve seed law enforcement agency inside control
System.
Invention content
The technical problem to be solved by the present invention is to how identify the authenticity of cotton variety YM111 and/or purity.
In order to solve the above technical problems, the present invention provides identification or the primer pairs of auxiliary identification cotton variety YM111
Composition, be by NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 this 8
The composition of kind primer pair composition；
The NAU1071 two single stranded DNAs shown in SEQ ID No.1 in sequence table and SEQ ID No.2 form；
The NAU1085 two single stranded DNAs shown in SEQ ID No.3 in sequence table and SEQ ID No.4 form；
The NAU1102 two single stranded DNAs shown in SEQ ID No.5 in sequence table and SEQ ID No.6 form；
The NAU1186 two single stranded DNAs shown in SEQ ID No.7 in sequence table and SEQ ID No.8 form；
The NAU1042 two single stranded DNAs shown in SEQ ID No.9 in sequence table and SEQ ID No.10 form；
The NAU2026 two single stranded DNAs shown in SEQ ID No.11 in sequence table and SEQ ID No.12 form；
The NAU2274 two single stranded DNAs shown in SEQ ID No.13 in sequence table and SEQ ID No.14 form；
The NAU879 two single stranded DNAs shown in SEQ ID No.15 in sequence table and SEQ ID No.16 form.
In the primer pair composition of above-mentioned identification or auxiliary identification cotton variety YM111, the molar ratio of 8 kinds of primer pairs
Can be 1:1:1:1:1:1:1:1, the molar ratio of two single stranded DNAs in each primer pair can be 1：1.
In order to solve the above technical problems, the present invention provides the complete of the DNA fingerprinting for obtaining cotton variety YM111
Primer.
The primer set of the DNA fingerprinting provided by the present invention for obtaining cotton variety YM111 is above-mentioned identification or auxiliary
Help the primer pair composition of identification cotton variety YM111.
The present invention also provides identification or the reagent or kit of auxiliary identification cotton variety YM111 authenticities, the examinations
Agent or kit contain the primer pair composition of above-mentioned identification or auxiliary identification cotton variety YM111.
It is described the present invention also provides identification or the reagent or kit of auxiliary identification cotton variety YM111 seed purities
Reagent or kit contain the primer pair composition of above-mentioned identification or auxiliary identification cotton variety YM111.
Following any applications also belong to protection scope of the present invention：
X1) above-mentioned identification or auxiliary identify the primer pair composition of cotton variety YM111 or obtain cotton variety YM111's
Application of the primer set of DNA fingerprinting in the product for preparing identification or auxiliary identification cotton variety YM111 authenticities；
X2) above-mentioned identification or auxiliary identify the primer pair composition of cotton variety YM111 or obtain cotton variety YM111's
Application of the primer set of DNA fingerprinting in preparing identification or auxiliary identification cotton variety YM111 seed purity products；
X3) above-mentioned identification or auxiliary identify the primer pair composition of cotton variety YM111 or obtain cotton variety YM111's
Application of the primer set of DNA fingerprinting in identifying or assisting identification cotton variety YM111 authenticities；
X4) above-mentioned identification or auxiliary identify the primer pair composition of cotton variety YM111 or obtain cotton variety YM111's
Application of the primer set of DNA fingerprinting in identifying or assisting identification cotton variety YM111 seed purities；
X5) above-mentioned identification or auxiliary identify the primer pair composition of cotton variety YM111 or obtain cotton variety YM111's
Application of the primer set of DNA fingerprinting in cotton breeding；
X6) above-mentioned identification or auxiliary identify the primer pair composition of cotton variety YM111 or obtain cotton variety YM111's
Application of the primer set of DNA fingerprinting in the DNA fingerprinting of structure cotton variety YM111.
The present invention also provides the identification of cotton variety YM111 authenticities or auxiliary identification methods.
The identification of cotton variety YM111 authenticities provided by the present invention or auxiliary identification method, including：
1) respectively using the genomic DNA of cotton variety to be measured and cotton variety YM111 as template, distinguished with 8 kinds of primer pairs
PCR amplification is carried out, the cotton variety of the cotton variety PCR product to be measured and 8 kinds of primer pairs of 8 kinds of primer pairs is obtained
YM111PCR products；
2) by the cotton variety of the cotton variety PCR product to be measured of 8 kinds of primer pairs and 8 kinds of primer pairs
YM111PCR products carry out electrophoresis, determine whether are the cotton variety to be measured and the cotton variety YM111 according to electrophoresis banding pattern
For same kind；
8 kinds of primer pairs be the NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026,
This 8 kinds of primer pairs of NAU2274 and NAU879.
It is described that the cotton variety to be measured and the cotton are determined according to electrophoresis banding pattern in above-mentioned identification or auxiliary identification method
Whether flower variety YM111 is the method for same kind：If in the cotton variety PCR product to be measured of 8 kinds of primer pairs
The banding pattern of the cotton variety PCR product to be measured of each primer pair is produced with the cotton variety YM111PCR of 8 kinds of primer pairs
The cotton variety YM111PCR product banding patterns of corresponding primer pair are consistent in object, determine that cotton variety to be measured is cotton variety YM111
Or candidate is cotton variety YM11；If at least a kind of primer pair in the cotton variety PCR product to be measured of 8 kinds of primer pairs
The banding pattern of cotton variety PCR product to be measured and the cotton variety YM111PCR products of 8 kinds of primer pairs in corresponding primer pair
Cotton variety YM111PCR product banding patterns it is inconsistent, cotton variety to be measured is not cotton variety YM111 or candidate is not cotton
Kind YM11.
The present invention also provides the identification of cotton variety YM111 seed purities or auxiliary identification methods.
The identification of cotton variety YM111 seed purities provided by the present invention or auxiliary identification method, it may include：
1) respectively using the seed cdna group DNA of cotton seeds to be measured and cotton variety YM111 as template, with 8 kinds of primer pairs
PCR amplification is carried out respectively, obtains the cotton product of the cotton seeds PCR product to be measured and 8 kinds of primer pairs of 8 kinds of primer pairs
Kind YM111 seed PCR products；
2) by the cotton variety YM111 of the cotton seeds PCR product to be measured of 8 kinds of primer pairs and 8 kinds of primer pairs
Seed PCR product carries out electrophoresis, determines that the seed of cotton variety YM111 in the cotton seeds to be measured is pure according to electrophoresis banding pattern
Degree；
8 kinds of primer pairs be the NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026,
This 8 kinds of primer pairs of NAU2274 and NAU879.
It is described that cotton product in the cotton seeds to be measured are determined according to electrophoresis banding pattern in above-mentioned identification or auxiliary identification method
The method of the seed purity of kind YM111 may include：If each in the cotton seeds PCR product to be measured of 8 kinds of primer pairs
The equal cotton variety YM111 seed PCR products with 8 kinds of primer pairs of the banding pattern of the cotton seeds PCR product to be measured of primer pair
In corresponding primer pair cotton variety YM111 seed PCR product banding patterns it is consistent, determine that cotton seeds to be measured are cotton variety
The seed of YM111 or the candidate seed for being cotton variety YM111；If the cotton seeds PCR product to be measured of 8 kinds of primer pairs
In at least a kind of primer pair cotton seeds PCR product to be measured banding pattern and 8 kinds of primer pairs cotton variety YM111 kinds
The cotton variety YM111 seed PCR product banding patterns of corresponding primer pair are inconsistent in sub- PCR product, and cotton seeds to be measured are not cotton
The seed of flower variety YM111 or candidate are not the seed of cotton variety YM111.
Above-mentioned NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 this
Wantonly a kind in 8 kinds of primer pairs, 2 kinds wantonly, 3 kinds wantonly, 4 kinds wantonly, 5 kinds wantonly, wantonly 6 kinds or wantonly 7 kinds of primer pairs identifying or assisting identification
Application in cotton variety YM111 seed purities also belongs to protection scope of the present invention.
The present invention screen to have obtained by NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026,
The composition of NAU2274 and NAU879 this 8 kinds of primer pairs compositions.The composition of this 8 kinds of primer pairs composition can be used for identifying cotton
The seed production purity of flower cenospecies YM111, identifies the authenticity of cotton crossbreed YM111 kinds, builds cotton crossbreed YM111 product
The finger-print of kind.The composition formed with this 8 kinds of primer pairs, can control the seed production quality of hybrid cotton YM111, be conducive to
The popularization and protection of YM111.
Description of the drawings
Fig. 1 is the mother of cotton crossbreed YM111, the male parent Hanmian 802 of cotton crossbreed YM111, cotton crossbreed YM111
The electrophoresis pattern (finger-print) of the pcr amplification product of 8 kinds of primer pairs of this Handan 6205.In figure, M Marker, from top to bottom
7 stripe sizes be respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 6 respectively
A swimming lane is followed successively by the male parent of YM111, each two single plants of female parent and YM111 from left to right.
Fig. 2 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of LH4.In figure, M Marker, 7 bands from top to bottom
Size is respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 swimming lanes respectively,
Respectively two single plants.
Fig. 3 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of the emerging cotton No. 5 of silver.In figure, M Marker, from top to bottom
7 stripe sizes be respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 respectively
A swimming lane, respectively two single plants.
Fig. 4 is the electrophoresis pattern of the PCR product of middle 8 kinds of primer pairs for planting cotton 838.In figure, M Marker, from top to bottom
7 stripe sizes be respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 respectively
A swimming lane, respectively two single plants.
Fig. 5 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of large miscellaneous cotton No. 2.In figure, M Marker, from top to bottom
7 stripe sizes be respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 respectively
A swimming lane, respectively two single plants.
Fig. 6 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of bright and beautiful miscellaneous No. 10 of section.In figure, M Marker, from top to bottom
7 stripe sizes be respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 respectively
A swimming lane, respectively two single plants.
Fig. 7 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of cotton No. 8 difficult to understand.In figure, M Marker, from top to bottom 7
Stripe size is respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 swimming respectively
Road, respectively two single plants.
Fig. 8 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of SGKZ73.In figure, M Marker, from top to bottom 7
Stripe size is respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 swimming respectively
Road, respectively two single plants.
The electrophoresis pattern of the PCR product for 8 kinds of primer pairs that Fig. 9 is auspicious miscellaneous 816.In figure, M Marker, from top to bottom 7
Stripe size is respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 swimming respectively
Road, respectively two single plants.
Figure 10 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs in miscellaneous No. 3 of cotton township.In figure, M Marker, from top to bottom
7 stripe sizes be respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 respectively
A swimming lane, respectively two single plants.
Figure 11 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of Shandong miscellaneous 2138.In figure, M Marker, from top to bottom
7 stripe sizes be respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 respectively
A swimming lane, respectively two single plants.
The electrophoresis pattern of the PCR product for 8 kinds of primer pairs that Figure 12 is auspicious miscellaneous 818.In figure, M Marker, from top to bottom
7 stripe sizes are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 respectively
Swimming lane, respectively two single plants.
Figure 13 is the electrophoresis pattern of the PCR product of 8 kinds of primer pairs of YM111.In figure, M Marker, from top to bottom 7
Stripe size is respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp；Each primer pair has 2 swimming respectively
Road, respectively two single plants.
Figure 14 is the electrophoresis pattern for the seed purity that YM111 is detected with primer NAU1085.In Figure 14,1-24 YM111
24 single plants.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Examples provided below can be used as the art ordinary skill
The guide that personnel are further improved, is not construed as limiting the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.
Embodiment 1, identification cotton variety YM111
It is a set of for cotton crossbreed YM111 (hereinafter referred to as cotton variety YM111 or YM111) authenticity in order to screen
The core primers identified with purity, the hybrid cotton varieties that the present embodiment is participated in the experiment using 47 national areas' examinations as material, using point
Sub- labelling technique, to 102 pairs of cotton SSR primers by PCR amplification, by primary dcreening operation and final election, it is final obtain amplified band it is clear,
8 pairs of strong primers of reproducible and resolution capability.8 pairs of primers can be used for detecting the seed production purity of cotton crossbreed YM111,
Authenticity for detecting cotton crossbreed YM111 kinds, the finger-print for building cotton crossbreed YM111 kinds.Tool
The test method and result of body are as follows：
1, the primer pair composition of identification cotton variety YM111 is prepared
This step prepare identification cotton variety YM111 primer pair composition be by NAU1071, NAU1085,
The composition of NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 this 8 kinds of primer pairs composition；
NAU1071 two single stranded DNAs shown in SEQ ID No.1 in sequence table and SEQ ID No.2 form；NAU1085 is by sequence
Two single stranded DNA compositions shown in SEQ ID No.3 and SEQ ID No.4 in table；NAU1102 is by SEQ ID in sequence table
Two single stranded DNA compositions shown in No.5 and SEQ ID No.6；NAU1186 is by SEQ ID No.7 in sequence table and SEQ ID
Two single stranded DNA compositions shown in No.8；NAU1042 is two shown in SEQ ID No.9 in sequence table and SEQ ID No.10
Single stranded DNA composition；NAU2026 two single stranded DNA groups shown in SEQ ID No.11 in sequence table and SEQ ID No.12
At；NAU2274 two single stranded DNAs shown in SEQ ID No.13 in sequence table and SEQ ID No.14 form；NAU879 by
Two single stranded DNA compositions (table 1) shown in SEQ ID No.15 and SEQ ID No.16 in sequence table.Above-mentioned identification cotton variety
In the primer pair composition of YM111, the molar ratio of the equal independent packaging of each primer pair, 8 kinds of primer pairs is 1:1:1:1:1:1:
1:1, the molar ratio of two single stranded DNAs in each primer pair is 1：1.
Table 1. identifies the sequence of the primer pair composition of cotton variety YM111
2, the structure of the finger-print of the authenticity and purity and cotton crossbreed YM111 kinds of identification cotton variety YM111
It builds
Cotton crossbreed YM111 and its male parent Hanmian 802 and maternal Handan 6205, are all from the Handan Agriculture academy of sciences.Its
46 kinds of remaininging are that national area tries the hybrid cotton varieties participated in the experiment, and sample comes from each breeding units (table 3).Field sampling extraction
DNA。
Extraction cotton crossbreed YM111, the male parent Hanmian 802 of cotton crossbreed YM111, cotton crossbreed YM111 respectively
Maternal Handan 6205 and table 3 in the genomic DNA of each single plant of hybrid cotton varieties participated in the experiment of 46 national areas' examinations, with step 1
NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 this 8 kinds of primer pairs
In any primer pair expanded respectively, obtain the cotton crossbreed YM111's of each primer pair in above-mentioned 8 kinds of primer pairs
The maternal Handan 6205 of PCR product, the PCR product of male parent Hanmian 802 of cotton crossbreed YM111, cotton crossbreed YM111
The PCR product for the hybrid cotton varieties that 46 national area's examinations in PCR product and table 3 are participated in the experiment.Then it is solidifying to carry out 8% polyacrylamide
Gel electrophoresis and silver staining colour developing.
Wherein, NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879
The PCR system of this 8 kinds of primer pairs is 10.0 μ L, Taq enzyme containing 0.5U, and the concentration of forward primer and reverse primer is 0.5 μ
M。
This 8 kinds of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879
The amplification program of primer pair is：Then 94 DEG C of pre-degeneration 3min carry out following 30 cycles：94 DEG C of denaturation 45s, 55 DEG C of annealing
45s, 72 DEG C of extension 1min；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.
8 pairs of primers are verified in cotton variety all has polymorphism, is 3 kinds of banding patterns, generally will be same for visual representation
Classify according to the molecular size range of minimum amplified production in one primer pair amplifies collection of illustrative plates, when the molecular weight of minimum product is identical with
Secondary two molecular weight, and so on.The type definition of low molecular weight bands of a spectrum is 1, and the type definition of high molecular weight bands of a spectrum is
2, heterozygosis total banding type definition is 3.The corresponding equally unique coding of Tupu type conversion imaging identification card number.With
Coding distinguishes the difference of kind, from it is traditional there is band to read 1, that 0 is read without band is different, pronunciation is easy, it is quick [Fu little Qiong, 2011 years
The SSR finger-prints of Yellow River basin Cotton Region examination control kind, Cotton 2011.12:29-32].Cotton crossbreed YM111, cotton
The male parent Hanmian 802 of flower cenospecies YM111, the maternal Handan 6205 of cotton crossbreed YM111 8 pairs of primers pcr amplification product
Electrophoresis pattern as shown in Figure 1, its coding schedule is as shown in table 2.
The parent of 2 YM111 of table and cenospecies correspond to the coding schedule of collection of illustrative plates
2011-2016 participates in Yellow River basin cotton region country hybrid cotton novel wheat cultivars 47, is applying this 8 pairs of core primers
In, 46 corresponding Variety fingerprintings are different from YM111.This 8 pairs of core primers are to showing as YM111 codominance item
Band, other hybrid cotton varieties have 1-7 not show band (table 4) altogether to primer respectively.
3.47 parts of National Cotton regional testing hybrid cotton varieties sources of table
The coding of 4.47 parts of National Cotton regional testing Variety fingerprintings of table
In table 4, " 3 " in 8 kinds of primer pair banding patterns are that the PCR product of the corresponding primer pair to YM111's is identical aobvious altogether
Banding pattern, " 1 " and " 2 " is the banding pattern that the PCR product of corresponding primer pair is different from YM111's, and " 1 " and " 2 " is different
Banding pattern." showing primer number altogether " is the PCR product banding pattern of PCR product banding pattern primer pair corresponding to YM111's in 8 kinds of primer pairs
Identical primer pair number.
It is above-mentioned the experiment results show that by NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026,
The primer pair composition of NAU2274 and NAU879 this 8 kinds of primer pairs compositions can be used for building the fingerprint image of cotton variety YM111
Spectrum, identification cotton variety YM111 authenticities, identification cotton variety YM111 seed purities.
The identification method of cotton variety YM111 authenticities, specifically may include：
1) respectively with cotton variety to be measured (any cotton variety in serial number 1-46 in such as table 3) and cotton variety
The genomic DNA of YM111 (abbreviation YM111) be template, with NAU1071, NAU1085, NAU1102, NAU1186, NAU1042,
This 8 kinds of primer pairs of NAU2026, NAU2274 and NAU879 carry out PCR amplification respectively, obtain the cotton to be measured of 8 kinds of primer pairs
The YM111PCR products of kind PCR product and 8 kinds of primer pairs：The cotton variety PCR product to be measured of NAU1071 and
The YM111PCR products of NAU1071, the cotton variety PCR product to be measured of NAU1085 and NAU1085 YM111PCR products,
The cotton variety PCR to be measured of the cotton variety PCR product to be measured of NAU1102 and the YM111PCR products of NAU1102, NAU1186
The YM111PCR of the YM111PCR products of product and NAU1186, the cotton variety PCR product to be measured of NAU1042 and NAU1042 is produced
The cotton variety to be measured of object, the YM111PCR products of the cotton variety PCR product to be measured of NAU2026 and NAU2026, NAU2274
The YM111PCR of the cotton variety PCR product and NAU879 to be measured of the YM111PCR products of PCR product and NAU2274, NAU879
Product；
2) by the cotton variety of the cotton variety PCR product to be measured of 8 kinds of primer pairs and 8 kinds of primer pairs
YM111PCR products carry out electrophoresis, determine whether are the cotton variety to be measured and the cotton variety YM111 according to electrophoresis banding pattern
For same kind：If the cotton variety to be measured of each primer pair in the cotton variety PCR product to be measured of 8 kinds of primer pairs
The cotton variety of corresponding primer pair in the equal cotton variety YM111PCR products with 8 kinds of primer pairs of the banding pattern of PCR product
YM111PCR product banding patterns are consistent, determine that cotton variety to be measured is cotton variety YM111；If 8 kinds of primer pairs is to be measured
The banding pattern of the cotton variety PCR product to be measured of at least a kind of primer pair and 8 kinds of primer pairs in cotton variety PCR product
The cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent in cotton variety YM111PCR products, cotton variety to be measured
It is not cotton variety YM111.
It is above-mentioned the experimental results showed that, 8 kinds of primer pairs of all cotton varieties to be measured of serial number 1-46 in table 3
The cotton variety of the banding pattern of the cotton variety PCR product to be measured of at least a kind of primer pair and 8 kinds of primer pairs in PCR product
The cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent in YM111PCR products.Below with part cotton product to be measured
For kind, it is elaborated：In the PCR product of 8 kinds of primer pairs of LH4, a kind of this PCR production of primer pair of only NAU2026
The banding pattern of object is consistent with the cotton variety YM111PCR product banding patterns of NAU2026, the banding pattern of the PCR product of other 7 kinds of primer pairs
And the cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent (Fig. 2, Figure 13 and table 4)；
In the PCR product of 8 kinds of primer pairs of the emerging cotton No. 5 of silver, only NAU1071 and NAU2274 this 2 kinds of primer pairs
The banding pattern of PCR product is consistent with the cotton variety YM111PCR product banding patterns of 2 kinds of primer pairs, the PCR productions of other 6 kinds of primer pairs
The banding pattern of object is and the cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent (Fig. 3, Figure 13 and table 4)；
In the PCR product of the middle 8 kinds of primer pairs for planting cotton 838, only NAU1102 and NAU2026 this 2 kinds of primer pairs
The banding pattern of PCR product is consistent with the cotton variety YM111PCR product banding patterns of 2 kinds of primer pairs, the PCR productions of other 6 kinds of primer pairs
The banding pattern of object is and the cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent (Fig. 4, Figure 13 and table 4)；
In the PCR product of 8 kinds of primer pairs of large miscellaneous cotton No. 2, only NAU1102 and NAU1042 this 2 kinds of primer pairs
The banding pattern of PCR product is consistent with the cotton variety YM111PCR product banding patterns of 2 kinds of primer pairs, the PCR productions of other 6 kinds of primer pairs
The banding pattern of object is and the cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent (Fig. 5, Figure 13 and table 4)；
In the PCR product of 8 kinds of primer pairs of miscellaneous No. 10 of bright and beautiful section, only NAU1071 and NAU1085 this 2 kinds of primer pairs
PCR product banding pattern it is consistent with the cotton variety YM111PCR product banding patterns of 2 kinds of primer pairs, the PCR of other 6 kinds of primer pairs
The banding pattern of product is and the cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent (Fig. 6, Figure 13 and table 4)；
In the PCR product of 8 kinds of primer pairs of cotton No. 8 difficult to understand, only this 3 kinds of NAU1085, NAU1042 and NAU879 draws
The banding pattern of the PCR product of object pair is consistent with the cotton variety YM111PCR product banding patterns of 3 kinds of primer pairs, other 5 kinds of primer pairs
PCR product banding pattern and corresponding primer pair cotton variety YM111PCR product banding patterns it is inconsistent (Fig. 7, Figure 13 and table 4)；
In the PCR product of 8 kinds of primer pairs of SGKZ73, only this 3 kinds of NAU1085, NAU1102 and NAU1042 draws
The banding pattern of the PCR product of object pair is consistent with the cotton variety YM111PCR product banding patterns of 3 kinds of primer pairs, other 5 kinds of primer pairs
PCR product banding pattern and corresponding primer pair cotton variety YM111PCR product banding patterns it is inconsistent (Fig. 8, Figure 13 and table 4)；
In the PCR product of auspicious miscellaneous 816 8 kinds of primer pairs, only this 3 kinds of NAU1186, NAU2026 and NAU2274 draws
The banding pattern of the PCR product of object pair is consistent with the cotton variety YM111PCR product banding patterns of 3 kinds of primer pairs, other 5 kinds of primer pairs
PCR product banding pattern and corresponding primer pair cotton variety YM111PCR product banding patterns it is inconsistent (Fig. 9, Figure 13 and table 4)；
In the PCR product of 8 kinds of primer pairs in miscellaneous No. 3 of cotton township, only NAU1071, NAU1085, NAU1102 and
The banding pattern of the PCR product of this 4 kinds of primer pairs of NAU2026 is consistent with the cotton variety YM111PCR product banding patterns of 4 kinds of primer pairs,
The banding pattern of the PCR product of other 4 kinds of primer pairs is and the cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent (schemes
10, Figure 13 and table 4)；
In the PCR product of 8 kinds of primer pairs of Shandong miscellaneous 2138, only NAU1085, NAU1102, NAU1042,
The cotton variety YM111PCR products of the banding pattern of the PCR product of this 5 kinds of primer pairs of NAU2026 and NAU2274 and 5 kinds of primer pairs
Banding pattern is consistent, the equal cotton variety YM111PCR product banding patterns with corresponding primer pair of banding pattern of the PCR product of other 3 kinds of primer pairs
Inconsistent (Figure 11, Figure 13 and table 4)；
In the PCR product of auspicious miscellaneous 818 8 kinds of primer pairs, only NAU1071, NAU1102, NAU1186,
The cotton variety of the banding pattern and 6 kinds of primer pairs of the PCR product of this 6 kinds of primer pairs of NAU1042, NAU2026 and NAU879
YM111PCR product banding patterns are consistent, the equal cotton variety with corresponding primer pair of banding pattern of the PCR product of other 2 kinds of primer pairs
YM111PCR product banding patterns are inconsistent (Figure 12, Figure 13 and table 4).
By NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 this 8
The primer pair composition of kind primer pair composition can distinguish the cotton variety of serial number 1-46 in cotton variety YM111 and table 3
It opens, can be used for the identification of cotton variety YM111 authenticities, it can also be used to the identification of cotton variety YM111 seed purities.
The seed purity of embodiment 2, identification cotton variety YM111
Cotton seeds to being known as YM111 are detected, and detect certain a batch of cross breeding seed purity.In hybrid cotton
During the production of hybrid seeds, first the stamen of maternal (Handan 6205) removed, then award the pollen of male parent (Hanmian 802), a series of this work
Make to be at present manual operation, since personnel are limited during emasculation, the reasons such as hot weather lead to purity of hybrid not
It is good.The purity of detection cenospecies is conducive to control seed quality.Under normal circumstances, it is that maternal perforated is male the main reason for purity difference
Cause female parent self-cross ratio higher.According to the male parent Handan cotton of cotton crossbreed YM111, cotton crossbreed YM111 in embodiment 1
802, the NAU1071, NAU1085, NAU1102, NAU1186, NAU1042 of the maternal Handan 6205 of cotton crossbreed YM111,
The electrophoresis pattern (Fig. 1) of the pcr amplification product of this 8 kinds of primer pairs of NAU2026, NAU2274 and NAU879 determines this 8 kinds of primers
To all having polymorphism between YM111 and its Parent, banding pattern coding schedule is as shown in table 2.Illustrate in this 8 kinds of primer pairs
Wantonly a kind, 2 kinds wantonly, 3 kinds wantonly, 4 kinds wantonly, 5 kinds wantonly, 6 kinds wantonly, wantonly 7 kinds of primer pairs be used equally for the kind of identification cotton variety YM111
Sub- purity.Below by taking NAU1085 as an example, the specific method of the seed purity of identification cotton variety YM111 is illustrated.
Using the NAU1085 in embodiment 1, a kind of this primer pair detection purity, every batch of seed are sampled by national standard,
24 seeds are randomly selected in sample, extract DNA, carry out PCR amplification according to the method for embodiment 1, electrophoresis reads data, band
The definition of type is the same as embodiment 1.As a result as shown in figure 14, have in 24 seeds 4 seeds be maternal banding pattern (to be numbered in Figure 14 be 3,
7,12 and 22 seed), banding pattern 2, remaining is the hybridizing band pattern of YM111, and the variety SSR label of banding pattern 3, YM111 is pure
Degree is (24-4)/24*100%=83.3%.
The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and
Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in wider range
Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further.
In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen
Please in the open scope, and the change carried out with routine techniques known in the art.By the range of following attached claims,
It can carry out the application of some essential characteristics.
Sequence table
<110>The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute
<120>Identify primer sets and its application of cotton variety YM111 authenticities and seed purity
<130> GNCFH181161
<160> 16
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
accaacaatg gtgacctctt 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ccctccataa ccaaaagttg 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
agtcgcccct tctctaattt 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tgtaaaccga actcgttgtg 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
atctctctgt ctcccccttc 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gcatatctgg cgggtataat 20
<210> 7
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
aatggtcctg ctccagatt 19
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
aatcgtcgtc gtcgaattat 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
catgcaaatc catgctagag 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
ggtttctttg gtggtgaaac 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gaatctcgaa aaccccatct 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
atttggaagc gaagtaccag 20
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tcctcggatt atcaaaacct 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
tgaagaggac attgatgacg 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
aggaaccgat tcaaagctaa 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
tttccccatt cttggttaag 20
Claims (10)
1. the primer pair composition of identification or auxiliary identification cotton variety YM111, be by NAU1071, NAU1085, NAU1102,
The composition of NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 this 8 kinds of primer pairs composition；
The NAU1071 two single stranded DNAs shown in SEQ ID No.1 in sequence table and SEQ ID No.2 form；
The NAU1085 two single stranded DNAs shown in SEQ ID No.3 in sequence table and SEQ ID No.4 form；
The NAU1102 two single stranded DNAs shown in SEQ ID No.5 in sequence table and SEQ ID No.6 form；
The NAU1186 two single stranded DNAs shown in SEQ ID No.7 in sequence table and SEQ ID No.8 form；
The NAU1042 two single stranded DNAs shown in SEQ ID No.9 in sequence table and SEQ ID No.10 form；
The NAU2026 two single stranded DNAs shown in SEQ ID No.11 in sequence table and SEQ ID No.12 form；
The NAU2274 two single stranded DNAs shown in SEQ ID No.13 in sequence table and SEQ ID No.14 form；
The NAU879 two single stranded DNAs shown in SEQ ID No.15 in sequence table and SEQ ID No.16 form.
2. obtaining the primer set of the DNA fingerprinting of cotton variety YM111, it is characterised in that：The primer set is right
It is required that the primer pair composition described in 1.
3. the reagent or kit of identification or auxiliary identification cotton variety YM111 authenticities, it is characterised in that：The reagent or examination
Agent box contains primer pair composition described in claim 1.
4. the reagent or kit of identification or auxiliary identification cotton variety YM111 seed purities, it is characterised in that：The reagent or
Kit contains primer pair composition described in claim 1.
5. following any applications：
X1) primer set described in primer pair composition or claim 2 described in claim 1 is preparing identification or auxiliary mirror
Determine the application in the product of cotton variety YM111 authenticities；
X2) primer set described in primer pair composition or claim 2 described in claim 1 is preparing identification or auxiliary mirror
Determine the application in cotton variety YM111 seed purity products；
X3) identification cotton is being identified or assisted to the primer set described in primer pair composition or claim 2 described in claim 1
Application in flower variety YM111 authenticities；
X4) identification cotton is being identified or assisted to the primer set described in primer pair composition or claim 2 described in claim 1
Application in flower variety YM111 seed purities；
X5) primer set the answering in cotton breeding described in primer pair composition or claim 2 described in claim 1
With；
X6) application of the primer set described in claim 2 in the DNA fingerprinting of structure cotton variety YM111.
6. identification or the auxiliary identification method of cotton variety YM111 authenticities, including：
1) it respectively using the genomic DNA of cotton variety to be measured and cotton variety YM111 as template, is carried out respectively with 8 kinds of primer pairs
PCR amplification obtains the cotton variety of the cotton variety PCR product to be measured and 8 kinds of primer pairs of 8 kinds of primer pairs
YM111PCR products；
2) by the cotton variety YM111PCR productions of the cotton variety PCR product to be measured of 8 kinds of primer pairs and 8 kinds of primer pairs
Object carries out electrophoresis, determines whether the cotton variety to be measured and the cotton variety YM111 are same kind according to electrophoresis banding pattern；
8 kinds of primer pairs be claim 1 described in NAU1071, NAU1085, NAU1102, NAU1186, NAU1042,
This 8 kinds of primer pairs of NAU2026, NAU2274 and NAU879.
7. identification according to claim 6 or auxiliary identification method, it is characterised in that：It is described that institute is determined according to electrophoresis banding pattern
State whether cotton variety to be measured and the cotton variety YM111 are that the method for same kind is：If 8 kinds of primer pairs wait for
Survey the banding pattern of the cotton variety PCR product to be measured of each primer pair in cotton variety PCR product and 8 kinds of primer pairs
The cotton variety YM111PCR product banding patterns of corresponding primer pair are consistent in cotton variety YM111PCR products, determine cotton product to be measured
Kind is cotton variety YM111 or candidate is cotton variety YM11；If the cotton variety PCR product to be measured of 8 kinds of primer pairs
In at least a kind of primer pair cotton variety PCR product to be measured banding pattern and 8 kinds of primer pairs cotton variety
The cotton variety YM111PCR product banding patterns of corresponding primer pair are inconsistent in YM111PCR products, and cotton variety to be measured is not cotton
Kind YM111 or candidate is not cotton variety YM11.
8. identification or the auxiliary identification method of cotton variety YM111 seed purities, including：
1) respectively using the seed cdna group DNA of cotton seeds to be measured and cotton variety YM111 as template, distinguished with 8 kinds of primer pairs
PCR amplification is carried out, the cotton variety of the cotton seeds PCR product to be measured and 8 kinds of primer pairs of 8 kinds of primer pairs is obtained
YM111 seed PCR products；
2) by the cotton variety YM111 seeds of the cotton seeds PCR product to be measured of 8 kinds of primer pairs and 8 kinds of primer pairs
PCR product carries out electrophoresis, and the seed purity of cotton variety YM111 in the cotton seeds to be measured is determined according to electrophoresis banding pattern；
8 kinds of primer pairs be claim 1 described in NAU1071, NAU1085, NAU1102, NAU1186, NAU1042,
This 8 kinds of primer pairs of NAU2026, NAU2274 and NAU879.
9. identification according to claim 8 or auxiliary identification method, it is characterised in that：It is described that institute is determined according to electrophoresis banding pattern
The method for stating the seed purity of cotton variety YM111 in cotton seeds to be measured includes：If the cotton to be measured of 8 kinds of primer pairs
The equal cotton product with 8 kinds of primer pairs of the banding pattern of the cotton seeds PCR product to be measured of each primer pair in seed PCR product
The cotton variety YM111 seed PCR product banding patterns of corresponding primer pair are consistent in kind YM111 seed PCR products, determine cotton to be measured
The seed that the seed or candidate that seed is cotton variety YM111 are cotton variety YM111；If 8 kinds of primer pairs is to be measured
The banding pattern of the cotton seeds PCR product to be measured of at least a kind of primer pair and 8 kinds of primer pairs in cotton seeds PCR product
The cotton variety YM111 seed PCR product banding patterns of corresponding primer pair are inconsistent in cotton variety YM111 seed PCR products, to be measured
Cotton seeds be not cotton variety YM111 seed or candidate be not cotton variety YM111 seed.
10. NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 described in claim 1
It is being identified with wantonly a kind in NAU879 this 8 kinds of primer pairs, 2 kinds wantonly, 3 kinds wantonly, 4 kinds wantonly, 5 kinds wantonly, wantonly 6 kinds or wantonly 7 kinds of primer pairs
Or the application in auxiliary identification cotton variety YM111 seed purities.
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