CN108504763B - Primer group for identifying authenticity and seed purity of cotton variety YM111 and application thereof - Google Patents
Primer group for identifying authenticity and seed purity of cotton variety YM111 and application thereof Download PDFInfo
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Abstract
The invention discloses a primer group for identifying authenticity and seed purity of cotton variety YM111 and application thereof. The primer group for identifying the authenticity and the seed purity of the cotton variety YM111 is a composition consisting of 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU 879. The composition consisting of the 8 primer pairs can be used for identifying the seed production purity of the cotton hybrid YM111, identifying the authenticity of the cotton hybrid YM111 variety and constructing the fingerprint of the cotton hybrid YM111 variety. The seed production quality of hybrid cotton YM111 can be controlled by using the composition consisting of the 8 primer pairs, and the popularization and protection of YM111 are facilitated.
Description
Technical Field
The invention relates to a primer group for identifying the authenticity and the seed purity of a cotton variety YM111 in the field of crop molecular genetic breeding and application thereof.
Background
Cotton variety YM111 is a high-yield disease-resistant transgenic hybrid cotton variety cultivated by Handan agricultural academy of sciences, and has a certified number of national cotton 20170002. YM111 is a transgenic intermediate-maturing hybrid variety of Handan 6205X Handan cotton 802. The cotton area in the yellow river basin has 120 days of spring sowing growth period. The seedlings are neat, and the growth period is strong. The plant type is loose, and the stem is thick. Oval, medium size, long stalk, strong boll-forming property, smooth and concentrated boll opening. Resisting fusarium wilt (disease index of 9.6) and resisting verticillium wilt (disease index of 28.0). Is resistant to cotton bollworm. The plant height is 104.6 cm, and the first fruit branch is 7.3 nodes. The single plant bears 22.2 bolls, and the weight of the single boll is 6.1 g. The seed is 10.7 g, the score is 41.4%, and the blooming rate is 92.7%. The average length of the upper half part of HVICC fiber is 28.1 mm, the breaking specific strength is 31.0 cN/tex, the Marlon value is 5.3, the breaking elongation is 6.4%, the reflectivity is 74.8%, the yellow depth is 8.7, the uniformity index is 84.8%, and the spinning uniformity index is 138.
In order to prevent the appearance of the inferior cotton variety YM111 in the market, a method for rapidly and accurately identifying the authenticity of the cotton variety YM111 is required to serve the supervision and detection of a seed law enforcement unit and the internal control of the seed quality of a breeding unit.
Disclosure of Invention
The technical problem to be solved by the invention is how to identify the authenticity and/or purity of cotton variety YM 111.
In order to solve the technical problems, the invention provides a primer pair composition for identifying or assisting in identifying cotton variety YM111, which is a composition consisting of 8 primer pairs including NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU 879;
the NAU1071 consists of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table;
the NAU1085 is composed of two single-stranded DNAs shown by SEQ ID No.3 and SEQ ID No.4 in the sequence table;
the NAU1102 consists of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No.6 in a sequence table;
the NAU1186 consists of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No.8 in the sequence table;
the NAU1042 consists of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No.10 in a sequence table;
the NAU2026 is composed of two single-stranded DNAs shown by SEQ ID No.11 and SEQ ID No.12 in the sequence table;
the NAU2274 consists of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No.14 in the sequence table;
the NAU879 is composed of two single-stranded DNAs shown by SEQ ID No.15 and SEQ ID No.16 in the sequence table.
In the primer pair composition for identifying or assisting in identifying cotton variety YM111, the molar ratio of the 8 primer pairs can be 1:1:1:1:1:1:1:1, and the molar ratio of the two single-stranded DNAs in each primer pair can be 1: 1.
in order to solve the technical problems, the invention provides a set of primers for obtaining the DNA fingerprint of the cotton variety YM 111.
The primer set for obtaining the DNA fingerprint of the cotton variety YM111 provided by the invention is a primer pair composition for identifying or assisting in identifying the cotton variety YM 111.
The invention also provides a reagent or a kit for identifying or assisting in identifying the authenticity of cotton variety YM111, wherein the reagent or the kit contains the primer pair composition for identifying or assisting in identifying cotton variety YM 111.
The invention also provides a reagent or a kit for identifying or assisting in identifying the purity of the cotton variety YM111 seeds, and the reagent or the kit contains the primer pair composition for identifying or assisting in identifying the cotton variety YM 111.
Any of the following applications also fall within the scope of the present invention:
x1) application of the primer pair composition for identifying or assisting in identifying the cotton variety YM111 or the primer set for obtaining the DNA fingerprint of the cotton variety YM111 in preparation of products for identifying or assisting in identifying the authenticity of the cotton variety YM 111;
x2) application of the primer pair composition for identifying or assisting in identifying the cotton variety YM111 or the set of primers for obtaining the DNA fingerprint of the cotton variety YM111 in preparation of products for identifying or assisting in identifying the purity of the cotton variety YM111 seeds;
x3) application of the primer pair composition for identifying or assisting in identifying the cotton variety YM111 or the set of primers for obtaining the DNA fingerprint of the cotton variety YM111 in identifying or assisting in identifying the authenticity of the cotton variety YM 111;
x4) application of the primer pair composition for identifying or assisting in identifying the cotton variety YM111 or the set of primers for obtaining the DNA fingerprint of the cotton variety YM111 in identifying or assisting in identifying the purity of the cotton variety YM111 seeds;
x5) the application of the primer pair composition for identifying or assisting in identifying the cotton variety YM111 or the set of primers for obtaining the DNA fingerprint of the cotton variety YM111 in cotton breeding;
x6) application of the primer pair composition for identifying or assisting in identifying the cotton variety YM111 or the set of primers for obtaining the DNA fingerprint of the cotton variety YM111 in constructing the DNA fingerprint of the cotton variety YM 111.
The invention also provides a method for identifying or assisting in identifying the authenticity of cotton variety YM 111.
The method for identifying or assisting in identifying the authenticity of cotton variety YM111 provided by the invention comprises the following steps:
1) respectively carrying out PCR amplification by using genome DNA of a cotton variety to be detected and genome DNA of a cotton variety YM111 as templates and using 8 primer pairs to obtain a cotton variety PCR product to be detected of the 8 primer pairs and a cotton variety YM111PCR product of the 8 primer pairs;
2) carrying out electrophoresis on the PCR products of the cotton variety to be detected of the 8 primer pairs and the PCR products of the cotton variety YM111 of the 8 primer pairs, and determining whether the cotton variety to be detected and the cotton variety YM111 are the same variety or not according to the electrophoresis band type;
the 8 primer pairs are 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU 879.
In the above identification or auxiliary identification method, the method for determining whether the cotton variety to be detected and the cotton variety YM111 are the same variety according to the electrophoretic band pattern may be: if the banding patterns of the to-be-detected cotton variety PCR product of each primer pair in the to-be-detected cotton variety PCR products of the 8 primer pairs are consistent with the banding patterns of the cotton variety YM111PCR product of the corresponding primer pair in the cotton variety YM111PCR products of the 8 primer pairs, determining that the to-be-detected cotton variety is cotton variety YM111 or is a candidate of cotton variety YM 11; if the banding pattern of the to-be-detected cotton variety PCR product of at least one primer pair in the to-be-detected cotton variety PCR products of the 8 primer pairs is inconsistent with the banding pattern of the cotton variety YM111PCR product of the corresponding primer pair in the cotton variety YM111PCR products of the 8 primer pairs, the to-be-detected cotton variety is not cotton variety YM111 or the candidate is not cotton variety YM 11.
The invention also provides an identification or auxiliary identification method of the purity of the cotton variety YM111 seeds.
The identification or auxiliary identification method for the purity of the cotton variety YM111 seeds provided by the invention can comprise the following steps:
1) respectively taking the seed genome DNA of the cotton seed to be detected and the cotton variety YM111 as templates, and respectively carrying out PCR amplification by using 8 primer pairs to obtain the PCR product of the cotton seed to be detected of the 8 primer pairs and the PCR product of the cotton variety YM111 seed of the 8 primer pairs;
2) carrying out electrophoresis on the PCR products of the cotton seeds to be detected of the 8 primer pairs and the PCR products of the cotton variety YM111 seeds of the 8 primer pairs, and determining the seed purity of the cotton variety YM111 in the cotton seeds to be detected according to the electrophoresis band type;
the 8 primer pairs are 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU 879.
In the above identification or auxiliary identification method, the method for determining the seed purity of cotton variety YM111 in the cotton seed to be detected according to the electrophoretic band type may comprise: if the banding patterns of the to-be-detected cotton seed PCR product of each primer pair in the to-be-detected cotton seed PCR products of the 8 primer pairs are consistent with the banding patterns of the cotton variety YM111 seed PCR product of the corresponding primer pair in the cotton variety YM111 seed PCR products of the 8 primer pairs, determining that the to-be-detected cotton seed is the cotton variety YM111 seed or the candidate is the cotton variety YM111 seed; if the banding pattern of the PCR product of the cotton seed to be detected of at least one primer pair in the PCR products of the cotton seeds to be detected of the 8 primer pairs is inconsistent with the banding pattern of the PCR product of the cotton variety YM111 seed of the corresponding primer pair in the PCR products of the cotton variety YM111 seed of the 8 primer pairs, the cotton seed to be detected is not the seed of the cotton variety YM111 or is a candidate seed not the cotton variety YM 111.
The application of any 1, any 2, any 3, any 4, any 5, any 6 or any 7 primer pairs in the 8 primer pairs including the NAU1071, the NAU1085, the NAU1102, the NAU1186, the NAU1042, the NAU2026, the NAU2274 and the NAU879 in the identification or auxiliary identification of the purity of the cotton variety YM111 seeds also belongs to the protection scope of the invention.
The invention obtains the composition consisting of 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 through screening. The composition consisting of the 8 primer pairs can be used for identifying the seed production purity of the cotton hybrid YM111, identifying the authenticity of the cotton hybrid YM111 variety and constructing the fingerprint of the cotton hybrid YM111 variety. The seed production quality of hybrid cotton YM111 can be controlled by using the composition consisting of the 8 primer pairs, and the popularization and protection of YM111 are facilitated.
Drawings
FIG. 1 shows the electrophoresis patterns (finger prints) of PCR amplification products of 8 primer pairs of cotton hybrid YM111, male parent Handan cotton 802 of cotton hybrid YM111, and female parent Handan 6205 of cotton hybrid YM 111. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; each primer pair has 6 lanes, which are male parent and female parent of YM111 and two single strains of YM111 from left to right.
FIG. 2 is an electropherogram of PCR products of 8 primer pairs of LH 4. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 3 is an electropherogram of PCR products of 8 primer pairs of Yinxingcotton No. 5. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 4 is an electropherogram of PCR products of 8 primer pairs of Chinese plant cotton 838. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 5 is an electropherogram of PCR products of 8 primer pairs of Shuaoza 2. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 6 is an electropherogram of PCR products of 8 primer pairs of Kazakhstan No. 10. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 7 is an electropherogram of PCR products of 8 primer pairs of Ostwald No. 8. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 8 is an electropherogram of PCR products of 8 primer pairs of SGKZ 73. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
Fig. 9 is an electropherogram of PCR products of 8 primer pairs of reza 816. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 10 is an electropherogram of PCR products of 8 primer pairs of Gongxiangza No. 3. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 11 is an electropherogram of PCR products of 8 primer pairs of Luza 2138. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 12 is an electropherogram of PCR products of 8 primer pairs of Ruiz 818. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 13 is an electropherogram of PCR products of 8 primer pairs of YM 111. In the figure, M is Marker, and the sizes of 7 bands from top to bottom are respectively 500bp, 400bp, 300bp, 250bp, 200bp, 150bp and 100 bp; there are 2 lanes for each primer pair, two individuals.
FIG. 14 is an electropherogram of YM111 seed purity using primer NAU 1085. In FIG. 14, 1 to 24 are 24 individuals of YM 111.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified.
Example 1 identification of Cotton variety YM111
In order to screen a set of core primers for identifying the authenticity and purity of cotton hybrid YM111 (hereinafter referred to as cotton variety YM111 or YM111), in the embodiment, 47 hybrid cotton varieties which are tried in national regions are used as materials, a molecular marker technology is adopted, 102 pairs of cotton SSR primers are subjected to PCR amplification, and 8 pairs of primers with clear amplification bands, good repeatability and strong resolution capability are finally obtained through primary screening and check. The 8 pairs of primers can be used for detecting the seed production purity of the cotton hybrid YM111, detecting the authenticity of the cotton hybrid YM111 variety and constructing the fingerprint of the cotton hybrid YM111 variety. The specific test methods and results are as follows:
1. preparation of primer pair composition for identifying cotton variety YM111
The primer pair composition for identifying the cotton variety YM111 prepared in the step is a composition consisting of 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU 879; NAU1071 is composed of two single-stranded DNAs shown by SEQ ID No.1 and SEQ ID No.2 in the sequence table; NAU1085 is composed of two single-stranded DNAs shown by SEQ ID No.3 and SEQ ID No.4 in the sequence list; the NAU1102 consists of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No.6 in a sequence table; NAU1186 is composed of two single-stranded DNAs shown by SEQ ID No.7 and SEQ ID No.8 in the sequence table; NAU1042 consists of two single-stranded DNAs shown by SEQ ID No.9 and SEQ ID No.10 in the sequence table; NAU2026 is composed of two single-stranded DNAs shown by SEQ ID No.11 and SEQ ID No.12 in the sequence list; NAU2274 is composed of two single-stranded DNAs shown by SEQ ID No.13 and SEQ ID No.14 in the sequence list; NAU879 is composed of two single-stranded DNAs shown by SEQ ID No.15 and SEQ ID No.16 in the sequence Listing (Table 1). In the primer pair composition for identifying cotton variety YM111, each primer pair is independently packaged, the molar ratio of the 8 primer pairs is 1:1:1:1:1:1:1:1, and the molar ratio of the two single-stranded DNAs in each primer pair is 1: 1.
TABLE 1 sequence of primer set composition for identifying Cotton variety YM111
2. Identification of authenticity and purity of cotton variety YM111 and construction of fingerprint of cotton hybrid variety YM111
Cotton hybrid YM111 and its male parent Handan cotton 802 and female parent Handan 6205 are both from Handan's college of agricultural sciences. The remaining 46 varieties were cotton hybrids tested in the national district, and the samples were obtained from each breeding unit (Table 3). And (5) sampling in the field to extract DNA.
Genomic DNA of each individual plant of cotton hybrid YM111, male parent Handan cotton 802 of cotton hybrid YM111, female parent Handan 6205 of cotton hybrid YM111 and 46 national regional trial hybrid cotton varieties in Table 3 are respectively extracted, and any one of 8 primer pairs including NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 in step 1 is used for respective amplification, so as to obtain a PCR product of cotton hybrid YM111, a PCR product of male parent Handan cotton 802 of cotton hybrid YM111, a PCR product of female parent Handan 6205 of cotton hybrid YM111 and a PCR product of 46 national regional trial hybrid cotton varieties in Table 3 of each of the 8 primer pairs. Then, 8% polyacrylamide gel electrophoresis and silver staining were performed for color development.
Wherein, PCR systems of 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 are all 10.0 μ L, contain 0.5U Taq enzyme, and the concentrations of the forward primer and the reverse primer are both 0.5 μ M.
The amplification procedures for 8 primer pairs, NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879, were all as follows: pre-denaturation at 94 ℃ for 3min, followed by 30 cycles as follows: denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 1 min; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃.
The 8 pairs of primers are verified to have polymorphism in cotton varieties and are all 3 types of banding patterns, in order to be visually represented, the amplification maps of the same primer pair are generally classified according to the molecular weight of the minimum amplification product, and when the molecular weight of the minimum product is the same, the minimum product is classified according to the next second molecular weight, and so on. The type of the low molecular weight band is defined as 1, the type of the high molecular weight band is defined as 2, and the type of the hybrid co-occurrence band is defined as 3. The corresponding map type is converted into a unique code as the identity card number. The difference of varieties is distinguished by codes, and the reading method is simple, convenient and quick (Fuxiaoqiong, SSR finger print of cotton district test control species in yellow river basin in 2011, Chinese cotton 2011.12: 29-32) compared with the traditional reading method with reading 1 and reading method without reading 0. The electrophoresis patterns of the PCR amplification products of 8 pairs of primers for cotton hybrid YM111, male parent Handan cotton 802 of cotton hybrid YM111 and female parent Handan 6205 of cotton hybrid YM111 are shown in FIG. 1, and the code tables are shown in Table 2.
TABLE 2 encoding table of corresponding maps of parent and hybrid of YM111
2011-2016, 47 varieties are tested in the national cotton hybridization region in the yellow river basin, and in the 8 pairs of core primers, the fingerprint spectrums of 46 corresponding varieties are different from YM 111. These 8 pairs of core primer pairs YM111 showed a co-dominant band, and other hybrid cotton varieties had 1-7 primer pairs without co-dominant bands, respectively (Table 4).
TABLE 3.47 hybrid cotton variety sources for national cotton regional test
TABLE 4.47 coding of national cotton regional test variety fingerprints
In table 4, "3" of the 8 kinds of primer pair band patterns are each the same co-banding pattern as the PCR product of the corresponding primer pair of YM111, "1" and "2" are each a different band pattern from the PCR product of the corresponding primer pair of YM111, and "1" and "2" are different band patterns. The "number of co-appearing primers" is the number of primer pairs in which the band pattern of the PCR product is the same as that of the PCR product of the corresponding primer pair of YM111, among 8 primer pairs.
The above experimental results show that the primer pair composition consisting of 8 primer pairs, namely NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879, can be used for constructing a fingerprint of cotton variety YM111, identifying the authenticity of cotton variety YM111 and identifying the purity of cotton variety YM111 seeds.
The identification method of the authenticity of cotton variety YM111 specifically comprises the following steps:
1) respectively carrying out PCR amplification by using genome DNA of a cotton variety to be detected (such as any one of cotton varieties with serial numbers of 1-46 in Table 3) and genome DNA of a cotton variety YM111 (YM 111 for short) as templates and using 8 primer pairs including NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 to obtain PCR products of the cotton variety to be detected of the 8 primer pairs and YM111PCR products of the 8 primer pairs: the PCR product of the cotton variety to be detected of NAU1071, the YM111PCR product of NAU1071, the PCR product of the cotton variety to be detected of NAU1085, the YM111PCR product of NAU1085, the PCR product of the cotton variety to be detected of NAU1102, the YM111PCR product of NAU1102, the PCR product of the cotton variety to be detected of NAU1186, the YM111PCR product of NAU1186, the PCR product of the cotton variety to be detected of NAU1042, the YM111PCR product of NAU1042, the PCR product of the cotton variety to be detected of NAU2026, the YM111PCR product of NAU2026, the PCR product of the cotton variety to be detected of NAU2274, the YM111PCR product of NAU2274, the PCR product of the cotton variety to be detected of NAU879 and the YM111PCR product of NAU 879;
2) carrying out electrophoresis on the PCR products of the cotton variety to be detected of the 8 primer pairs and the PCR products of the cotton variety YM111 of the 8 primer pairs, and determining whether the cotton variety to be detected and the cotton variety YM111 are the same variety according to the electrophoresis band types: if the banding patterns of the to-be-detected cotton variety PCR product of each primer pair in the to-be-detected cotton variety PCR products of the 8 primer pairs are consistent with the banding patterns of the cotton variety YM111PCR product of the corresponding primer pair in the cotton variety YM111PCR products of the 8 primer pairs, determining that the to-be-detected cotton variety is cotton variety YM 111; if the banding pattern of the cotton variety PCR product to be detected of at least one primer pair in the cotton variety PCR products to be detected of the 8 primer pairs is different from the banding pattern of the cotton variety YM111PCR product of the corresponding primer pair in the cotton variety YM111PCR products of the 8 primer pairs, the cotton variety to be detected is not the cotton variety YM 111.
The above experiment results show that the banding patterns of the PCR products of the cotton variety to be tested of at least one primer pair in the PCR products of the 8 primer pairs of all the cotton varieties to be tested with the serial numbers of 1-46 in Table 3 are different from the banding patterns of the PCR products of the cotton variety YM111 of the corresponding primer pairs in the PCR products of the cotton variety YM111 of the 8 primer pairs. The following examples of some cotton varieties to be tested are explained in detail: of the PCR products of the 8 primer pairs of LH4, only the band pattern of the PCR product of the primer pair NAU2026 is consistent with that of the cotton variety YM111PCR product of NAU2026, and the band patterns of the PCR products of the other 7 primer pairs are all inconsistent with those of the cotton variety YM111PCR product of the corresponding primer pair (FIG. 2, FIG. 13 and Table 4);
in the PCR products of 8 primer pairs of Yinxing cotton No.5, the banding patterns of only the PCR products of 2 primer pairs of NAU1071 and NAU2274 are consistent with the banding patterns of the cotton variety YM111PCR products of the 2 primer pairs, and the banding patterns of the PCR products of other 6 primer pairs are not consistent with the banding patterns of the cotton variety YM111PCR products of the corresponding primer pairs (FIG. 3, FIG. 13 and Table 4);
in the 8 primer pairs of the medium plant cotton 838, only the band patterns of the 2 primer pairs, NAU1102 and NAU2026, were the same as those of the cotton variety YM111PCR product of the 2 primer pairs, and the band patterns of the PCR products of the other 6 primer pairs were not the same as those of the cotton variety YM111PCR product of the corresponding primer pair (FIG. 4, FIG. 13 and Table 4);
in the 8 primer pairs of the hybrid cotton No.2, only the band patterns of the 2 primer pairs including NAU1102 and NAU1042 are consistent with the band patterns of the cotton variety YM111PCR products of the 2 primer pairs, and the band patterns of the PCR products of the other 6 primer pairs are not consistent with the band patterns of the cotton variety YM111PCR products of the corresponding primer pairs (FIG. 5, FIG. 13 and Table 4);
in the PCR products of the 8 primer pairs of the Hetao 10 brocade, only the banding patterns of the PCR products of the 2 primer pairs of the NAU1071 and the NAU1085 are consistent with the banding patterns of the cotton variety YM111PCR products of the 2 primer pairs, and the banding patterns of the PCR products of the other 6 primer pairs are all inconsistent with the banding patterns of the cotton variety YM111PCR products of the corresponding primer pairs (FIG. 6, FIG. 13 and Table 4);
in the PCR products of the 8 primer pairs of Ostwald No.8, the band patterns of only the PCR products of 3 primer pairs, namely NAU1085, NAU1042 and NAU879, are consistent with those of the cotton variety YM111PCR products of the 3 primer pairs, and the band patterns of the PCR products of the other 5 primer pairs are not consistent with those of the cotton variety YM111PCR products of the corresponding primer pairs (FIG. 7, FIG. 13 and Table 4);
in the PCR products of the 8 primer pairs of SGKZ73, the band patterns of only the PCR products of 3 primer pairs of NAU1085, NAU1102 and NAU1042 are consistent with the band patterns of the cotton variety YM111PCR products of the 3 primer pairs, and the band patterns of the PCR products of the other 5 primer pairs are all inconsistent with the band patterns of the cotton variety YM111PCR products of the corresponding primer pairs (FIG. 8, FIG. 13 and Table 4);
of the 8 primer pairs of Ruizha 816, only the band patterns of the PCR products of 3 primer pairs, NAU1186, NAU2026 and NAU2274, were consistent with those of the cotton variety YM111PCR products of the 3 primer pairs, and the band patterns of the PCR products of the other 5 primer pairs were all inconsistent with those of the cotton variety YM111PCR products of the corresponding primer pairs (FIG. 9, FIG. 13 and Table 4);
the band patterns of only the PCR products of 4 primer pairs of NAU1071, NAU1085, NAU1102 and NAU2026 in the PCR products of 8 primer pairs of Miyazai No.3 are consistent with the band patterns of the cotton variety YM111PCR products of the 4 primer pairs, and the band patterns of the PCR products of the other 4 primer pairs are all inconsistent with the band patterns of the cotton variety YM111PCR products of the corresponding primer pairs (FIG. 10, FIG. 13 and Table 4);
of the PCR products of the 8 primer pairs of Luza 2138, only the band patterns of the PCR products of the 5 primer pairs of NAU1085, NAU1102, NAU1042, NAU2026 and NAU2274 were consistent with the band patterns of the PCR products of cotton variety YM111 of the 5 primer pairs, and the band patterns of the PCR products of the other 3 primer pairs were all inconsistent with the band patterns of the PCR products of cotton variety YM111 of the corresponding primer pairs (FIG. 11, FIG. 13 and Table 4);
of the 8 primer pairs of Ruiz 818, only the band patterns of the PCR products of the 6 primer pairs of NAU1071, NAU1102, NAU1186, NAU1042, NAU2026 and NAU879 were identical to the band pattern of the PCR product of cotton variety YM111 of the 6 primer pairs, and the band patterns of the PCR products of the other 2 primer pairs were not identical to the band pattern of the PCR product of cotton variety YM111 of the corresponding primer pair (FIG. 12, FIG. 13 and Table 4).
The primer pair composition consisting of 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 can distinguish cotton variety YM111 from cotton variety with serial numbers 1-46 in Table 3, can be used for authenticity identification of cotton variety YM111, and can also be used for purity identification of cotton variety YM111 seeds.
Example 2 identification of seed purity of Cotton variety YM111
Cotton seeds known as YM111 were tested for purity of a batch of hybrid seed. In the hybrid cotton seed production process, the stamen of the female parent (Handan 6205) is completely removed, then the pollen of the male parent (Handan cotton 802) is fertilized, the series of work is all manual operation at present, and the purity of the hybrid seed is poor due to limited personnel, hot weather and the like in the castration process. The purity of the hybrid is detected, which is beneficial to controlling the seed quality. Generally, the main reason for poor purity is that the female parent is missed to castrate, which results in higher proportion of the female parent inbred. Based on the electropherograms (FIG. 1) of the PCR amplification products of 8 primer pairs of Cotton hybrid YM111, Male Handan Cotton 802 of Cotton hybrid YM111, and female Handan 6205 of Cotton hybrid YM111, NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274, and NAU879 in example 1, it was determined that these 8 primer pairs all have polymorphisms between YM111 and their parents, and the band type code tables are shown in Table 2. Any 1, any 2, any 3, any 4, any 5, any 6 and any 7 primer pairs in the 8 primer pairs can be used for identifying the seed purity of the cotton variety YM 111. Specific methods for identifying the seed purity of cotton variety YM111 are illustrated below by taking NAU1085 as an example.
Purity was checked using the primer set NAU1085 of example 1, each batch of seeds was sampled according to the national standard, 24 seeds were randomly extracted from the sample, DNA was extracted, PCR amplification, electrophoresis, data reading, and band patterns were defined as in example 1 according to the method of example 1. As a result, as shown in fig. 14, 4 of the 24 seeds were the female parent banding pattern (seeds numbered 3, 7, 12 and 22 in fig. 14), the banding pattern was 2, and the remainder was the hybridization banding pattern of YM111, the banding pattern was 3, and the SSR marker purity of the variety of YM111 was (24-4)/24 × 100% — 83.3%.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
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Claims (10)
1. The primer pair composition for identifying or assisting in identifying cotton variety YM111 is a composition consisting of 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU 879;
the NAU1071 consists of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table;
the NAU1085 is composed of two single-stranded DNAs shown by SEQ ID No.3 and SEQ ID No.4 in the sequence table;
the NAU1102 consists of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No.6 in a sequence table;
the NAU1186 consists of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No.8 in the sequence table;
the NAU1042 consists of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No.10 in a sequence table;
the NAU2026 is composed of two single-stranded DNAs shown by SEQ ID No.11 and SEQ ID No.12 in the sequence table;
the NAU2274 consists of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No.14 in the sequence table;
the NAU879 is composed of two single-stranded DNAs shown by SEQ ID No.15 and SEQ ID No.16 in the sequence table.
2. The complete set of primers for obtaining the DNA fingerprint spectrum of the cotton variety YM111 is characterized in that: the primer set is the primer set composition of claim 1.
3. Reagent or kit for identifying or assisting in identifying authenticity of cotton variety YM111, which is characterized in that: the reagent or kit contains the primer pair composition of claim 1.
4. The reagent or the kit for identifying or assisting in identifying the purity of the cotton variety YM111 seeds is characterized in that: the reagent or kit contains the primer pair composition of claim 1.
5. Any of the following applications:
x1) use of the primer pair composition of claim 1 or the set of primers of claim 2 in the preparation of a product for identifying or assisting in identifying the authenticity of cotton variety YM 111;
x2) application of the primer pair composition of claim 1 or the primer set of claim 2 in preparation of products for identifying or assisting in identifying purity of cotton variety YM111 seeds;
x3) use of the primer pair composition of claim 1 or the set of primers of claim 2 for identifying or aiding in identifying the authenticity of cotton variety YM 111;
x4) application of the primer pair composition as claimed in claim 1 or the primer set as claimed in claim 2 in identification or auxiliary identification of purity of cotton variety YM111 seeds;
x5) use of the primer pair composition of claim 1 or the set of primers of claim 2 in cotton breeding;
x6) the use of the primer set of claim 2 in constructing DNA fingerprints of cotton variety YM 111.
6. A method of identifying or aiding in the identification of the authenticity of cotton variety YM111 comprising:
1) respectively carrying out PCR amplification by using genome DNA of a cotton variety to be detected and genome DNA of a cotton variety YM111 as templates and using 8 primer pairs to obtain a cotton variety PCR product to be detected of the 8 primer pairs and a cotton variety YM111PCR product of the 8 primer pairs;
2) carrying out electrophoresis on the PCR products of the cotton variety to be detected of the 8 primer pairs and the PCR products of the cotton variety YM111 of the 8 primer pairs, and determining whether the cotton variety to be detected and the cotton variety YM111 are the same variety or not according to the electrophoresis band type;
the 8 primer pairs are 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 described in claim 1.
7. The method of identification or assisted identification according to claim 6, characterized in that: the method for determining whether the cotton variety to be detected and the cotton variety YM111 are the same variety or not according to the electrophoresis band type comprises the following steps: if the banding patterns of the to-be-detected cotton variety PCR product of each primer pair in the to-be-detected cotton variety PCR products of the 8 primer pairs are consistent with the banding patterns of the cotton variety YM111PCR product of the corresponding primer pair in the cotton variety YM111PCR products of the 8 primer pairs, determining that the to-be-detected cotton variety is cotton variety YM111 or is a candidate of cotton variety YM 11; if the banding pattern of the to-be-detected cotton variety PCR product of at least one primer pair in the to-be-detected cotton variety PCR products of the 8 primer pairs is inconsistent with the banding pattern of the cotton variety YM111PCR product of the corresponding primer pair in the cotton variety YM111PCR products of the 8 primer pairs, the to-be-detected cotton variety is not cotton variety YM111 or the candidate is not cotton variety YM 11.
8. The identification or auxiliary identification method for the purity of the cotton variety YM111 seeds comprises the following steps:
1) respectively taking the seed genome DNA of the cotton seed to be detected and the cotton variety YM111 as templates, and respectively carrying out PCR amplification by using 8 primer pairs to obtain the PCR product of the cotton seed to be detected of the 8 primer pairs and the PCR product of the cotton variety YM111 seed of the 8 primer pairs;
2) carrying out electrophoresis on the PCR products of the cotton seeds to be detected of the 8 primer pairs and the PCR products of the cotton variety YM111 seeds of the 8 primer pairs, and determining the seed purity of the cotton variety YM111 in the cotton seeds to be detected according to the electrophoresis band type;
the 8 primer pairs are 8 primer pairs of NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879 described in claim 1.
9. The identification or assisted identification method of claim 8, characterized in that: the method for determining the seed purity of cotton variety YM111 in the cotton seeds to be detected according to the electrophoresis band type comprises the following steps: if the banding patterns of the to-be-detected cotton seed PCR product of each primer pair in the to-be-detected cotton seed PCR products of the 8 primer pairs are consistent with the banding patterns of the cotton variety YM111 seed PCR product of the corresponding primer pair in the cotton variety YM111 seed PCR products of the 8 primer pairs, determining that the to-be-detected cotton seed is the cotton variety YM111 seed or the candidate is the cotton variety YM111 seed; if the banding pattern of the PCR product of the cotton seed to be detected of at least one primer pair in the PCR products of the cotton seeds to be detected of the 8 primer pairs is inconsistent with the banding pattern of the PCR product of the cotton variety YM111 seed of the corresponding primer pair in the PCR products of the cotton variety YM111 seed of the 8 primer pairs, the cotton seed to be detected is not the seed of the cotton variety YM111 or is a candidate seed not the cotton variety YM 111.
10. The use of 8 primer pairs, NAU1071, NAU1085, NAU1102, NAU1186, NAU1042, NAU2026, NAU2274 and NAU879, as claimed in claim 1 for identifying or assisting in identifying the purity of cotton variety YM111 seed.
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| CN103555836B (en) * | 2013-10-25 | 2014-12-10 | 中国农业科学院棉花研究所 | Method for authenticating whether to-be-detected variety is Ruiza816 and special primer group thereof |
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| CN107099588B (en) * | 2017-04-28 | 2020-04-03 | 中国农业科学院棉花研究所 | Development and application of SSR (simple sequence repeat) marker for identifying earliness of upland cotton |
| CN107389871A (en) * | 2017-08-17 | 2017-11-24 | 邯郸市农业科学院 | A kind of structural overall merit selection of cotton strain |
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