CN109694919A - Four seed type cabbage hybrid Purity core primers and purity of hybrid InDel molecular markers for identification kit - Google Patents

Abstract

This disclosure relates to four seed type cabbage hybrid Purity core primers and purity of hybrid InDel molecular markers for identification kit, by screening related InDel molecular labeling extensively, a batch is excavated in the higher primer combination of heterozygosity in different type cabbage hybrid respectively, these primer sets are shared to be identified in respective type Chinese cabbage parents, the characteristic DNA fingerprint of parents can be constructed, it is combined with the primer or primer for having polymorphism between wherein parents, and can identify purity of hybrid.It combines these primers and constitutes a kit, greatly reduce the cost of enterprise or breeding units self-developing label, be with a wide range of applications.

Description

Four seed type cabbage hybrid Purity core primers and purity of hybrid InDel molecular markers for identification kit

Technical field

The disclosure belongs to agriculture vegetable variety identification field, and in particular to four seed type cabbage hybrid Purity cores Heart primer and purity of hybrid InDel molecular markers for identification kit.

Background technique

Here statement only provides background information related with the disclosure, without necessarily constituting the prior art.

Dabaicai zaipei area occupies first of all kinds of vegetables in China, and sowing quantity is huge.Mainly make in Chinese cabbage production at present With cenospecies, the seed purity of cenospecies is one of important indicator of seed quality, and the commodity and yield to Chinese cabbage have weight The influence wanted.Shandong is cabbage hybrid mainly for kind of an area, every year to the cabbage hybrid of all parts of the country supply up to 10⁵ Kilogram, more than half of national total sowing quantity is accounted for, the output value surpasses 100,000,000 yuan, therefore the production of cabbage hybrid and operation are to our province The development of seed industry has great influence.Field shape identification is usually used in traditional cabbage hybrid Purity Method, since the Chinese Cabbage in Shandong Province production of hybrid seeds mostly carries out in spring, the summer seed of harvest generally requires to sow identification in the fall, identifies Time needs a complete nutrient growth season, and duration is longer, and a sale has been had already passed by when identifying result Busy season, the cenospecies for causing current year to harvest can only be sold next year, and the storage of seeds cost of enterprise is increased.In addition field shape Identification is also easy to be influenced by various environmental factors, it is therefore desirable to develop a kind of time ahead of time, it is easy to operate, quick and precisely and The cabbage hybrid Purity method consistent with general survey result.

Using molecular labeling method identification of seed purity not by environmental restrictions, quick and precisely, have been developed as a kind of comparison Mature technology, as (Yan Huiling, Yan Shucheng, Yang Huimin, draw Jiang Ke to SRAP label, and Jiang Yanxia utilizes SRAP molecular marking technique Identify Chinese cabbage seeds purity [J] tropical agricultural science, 2011,31 (06): 50-52+60), SSR marker (Guan Zhikun, Luo Shuan Rosy clouds, Li Yanxia, old snow is flat, Wang Yanhua, Gu Aixia, and Shen Shuxing identifies glossy dark green No. 3 Chinese cabbage cultivar purity using SSR marker [J] seed, 2011,30 (09): 34-35+39. Yang Xiaoyun, Tian Shumei, Zhang Qingxia, Zhang Shuxia take charge of towards light, Wang Yuan, Wang Desen Blueness grinds serial cabbage hybrid and parent's fingerprint map construction and hybrids seed purity test [J] Molecular Plant Breeding, and 2013,11 (01):107-112；The SSR of Ma Xiaopeng, Liu Menglong, Zhang Lugang New Chinese Cabbage Variety " Qiu Bai 80 " purity identify [J] northwest agriculture Industry journal, 2013,22 (10): 131-134.), EST-SSR label (Zhao Xin, Wang Yong, Lan Qing are wealthy, He Changzheng, Chen Rui, Li Oujing, Liu Na, Zhu Zhu, Guo Yongze obtain [J] China based on the compound EST-SSR Chinese cabbage seeds Purity marked and SNP site Vegetables, 2013 (14): 31-38.) etc. be respectively used to the Purity of different cultivars cabbage hybrid.In above-mentioned pass In the research of molecular markers for identification cabbage hybrid purity, researchers only screen 1-4 to only suitable specific cross kind The primer or primer of Purity combine, these primers are not suitable for other Hybrid seed purity tests, can not be used to identify seed Authenticity.In comparison the form for the SSR primer combination that Yang Xiao cloud etc. (2011) uses can not only be identified more miscellaneous Kind or parent material are handed over, can be used for the identification of cenospecies authenticity.But to there are no blanket primer sets so far Conjunction can be used for cabbage hybrid Purity.

Summary of the invention

The disclosure is directed to the shortcoming in terms of different type cabbage hybrid purity molecular Marker Identification, by screening extensively Related InDel molecular labeling excavates a batch in the higher primer sets of heterozygosity in different type cabbage hybrid respectively It closes, these primer sets are shared to be identified in respective type Chinese cabbage parents, can construct the characteristic DNA fingerprint of parents, use is wherein double There are primer or the primer combination of polymorphism between parent, and can identify purity of hybrid.It combines these primers and constitutes a reagent Box greatly reduces the cost of enterprise or breeding units self-developing label, is with a wide range of applications.About utilize SSR The patent of molecular markers for identification cabbage hybrid purity is existing, but SSR marker type is excessively complicated, and pair of primers often produces Raw 3-4 allele site, even if may also occur the case where amplifying two or more band in self-mating system Occur, needs when Hybridization identification to carry out PCR amplification and electrophoresis detection simultaneously with parent, comparative electrophoresis map judges that cenospecies is pure Degree, this test operation is more complicated, experimentation cost is higher.In comparison, what InDel label then will be succinct is more, in gene Polymorphism in group is relatively low, and only there are two allele sites for tool for most of InDel labels, accidentally there is 3-4 polymorphic position Point, only occur from different material in resource identification and show different equipotential states, for single pure lines material according to So only have single band to be amplified, and heterozygosis state is exactly two clean bands of comparison, is as a result easier interpretation.Therefore, it develops Cabbage hybrid Purity kit advantage based on InDel label is significant.

The disclosure specifically uses following technical scheme:

In first typical embodiment of the disclosure, a kind of cabbage hybrid Purity core primers are provided To group, the core primers are to group by any one group or any several groups of combination in following primer pair: such as SEQ ID NO.1-2 Primer pair, such as SEQ ID NO.3-4 primer pair, such as SEQ ID NO.5-6 primer pair, such as SEQ ID NO.7-8 primer pair, such as SEQ ID NO.9-10 primer pair, such as SEQ ID NO.11-12 primer pair, such as SEQ ID NO.13-14 primer pair, such as SEQ IDNO.15-16 primer pair, such as SEQ ID NO.17-18 primer pair, such as SEQ ID NO.19-20 primer pair, such as SEQ ID NO.21-22 primer pair, such as SEQ ID NO.23-24 primer pair, such as SEQ ID NO.25-26 primer pair, such as SEQ ID NO.27-28 primer pair, such as SEQ IDNO.29-30 primer pair, such as SEQ ID NO.31-32 primer pair, such as SEQ ID NO.33- 34 primer pairs, such as SEQ ID NO.35-36 primer pair, such as SEQ ID NO.37-38 primer pair, such as SEQ ID NO.39-40 draw Object to, such as SEQ ID NO.41-42 primer pair, such as SEQ IDNO.43-44 primer pair, such as SEQ ID NO.45-46 primer pair, Such as SEQ ID NO.47-48 primer pair, such as SEQ ID NO.49-50 primer pair, SEQ ID NO.51-52 primer pair, such as SEQ ID NO.53-54 primer pair, such as SEQ ID NO.55-56 primer pair, such as SEQ ID NO.57-58 primer pair, such as SEQ ID NO.59-60 primer pair, such as SEQ ID NO.61-62 primer pair, such as SEQ ID NO.63-64 primer pair, such as SEQ IDNO.65- 66 primer pairs, such as SEQ ID NO.67-68 primer pair, such as SEQ ID NO.69-70 primer pair, such as SEQ ID NO.71-72 draw Object to, such as SEQ ID NO.73-74 primer pair, such as SEQ ID NO.75-76 primer pair, such as SEQ ID NO.77-78 primer pair, Such as SEQ IDNO.79-80 primer pair, such as SEQ ID NO.81-382 primer pair, such as SEQ ID NO.83-84 primer pair, such as SEQ ID NO.85-86 primer pair, such as SEQ ID NO.87-88 primer pair, such as SEQ ID NO.89-90 primer pair and such as SEQ ID NO.91-92 primer pair.

In the disclosure, described any several groups refer to any two, three, four ... four ten six groups of combination.

In the disclosure, each primer pair is known as one group.

In one of the disclosure or some embodiments, the type of the Chinese cabbage includes folded armful of class, straight tube class, import Four class of encircle-class and domestic encircle-class.

Wherein, the Chinese cabbage for embracing that class Chinese cabbage includes following any or any above trade name: Shandong six, Shandong seven is folded Number, ox board 19, ox early autumn No. 1, tackling key problem two, the white No.1 in Shandong, Lu Bai six, fine work be imperial No. five expensive, rich anti-60, rich anti-70, the Qin White No. two, moral it is high 16, it is No. 5 precocious, newly select Yuyuan Garden two, highly resistance two etc..

Wherein, straight tube class Chinese cabbage includes the Chinese cabbage of following any or any above trade name: Tianjin Indian mallow leaf, saliva are educated 75F1, saliva educate 60F1, in white 76, in white No. 62, improvement two blueness of Taiyuan, two blueness of Taiyuan, Shanxi dish three, preferably small miscellaneous 56, it is white 75, special Japan continues dish etc..

Wherein, import encircle-class Chinese cabbage is Japan and Korea S's import kind, including following any or any above trade name Chinese cabbage: yellow core morning 45, Han Chun, early Chinese cabbage advantage, chrysanthemum brocade, Jin Jin, autumn Huang 70, spring month yellow baby cabbage etc..

Wherein, domestic encircle-class Chinese cabbage includes the Chinese cabbage of following any or any above trade name: Beijing is No. three new, Rich anti-78, spring and autumn king, the high CR117 of moral, improvement be miscellaneous No. three green, small justice and autumn, the positive Exocarpium Citri Rubrum 65 in day etc..

It further, include in following 20 groups to group to identify the folded core primers for embracing class cabbage hybrid purity Any one group or any several groups of combination: such as SEQ ID No.7-8 primer pair, such as SEQ ID No.25-26 primer pair, such as SEQ ID No.27-28 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.79-80, SEQ ID No.5-6 primer pair, such as SEQ ID No.11-12 primer pair, As SEQ ID No.15-16 primer pair, such as SEQ ID No.17-18 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.31-32 primer pair, such as SEQ ID No.53-54 primer pair, such as SEQ ID No.61-62 primer pair, such as SEQ ID No.69-70 primer pair, such as SEQ ID No.73-74 primer pair.

Preferably, to identify that the folded core primers for embracing class cabbage hybrid purity include following 10 groups to group: such as SEQ ID No.7-8 primer pair, such as SEQ ID No.25-26 primer pair, such as SEQ ID No.27-28 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair and such as SEQ ID No.79-80.I.e. above-mentioned preceding 10 groups of primer pair.

It is furthermore preferred that identify that the folded core primers for embracing class cabbage hybrid purity include following combinations to group: on State 20 groups of primer pair.

Further, to identify that the core primers of straight tube class cabbage hybrid purity include following 20 groups any to group One group or any several groups of combination: such as SEQ ID No.27-28 primer pair, such as SEQ ID No.33-34 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.37-38 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.69-70 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.75-76 primer pair, such as SEQ ID No.3-4 primer pair, such as SEQ ID No.11- 12 primer pairs, such as SEQ ID No.19-20 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.43-44 draw Object to, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.57-58 primer pair, such as SEQ ID No.83-84 primer pair, Such as SEQ ID No.89-90 primer pair, such as SEQ ID No.91-92 primer pair.

Preferably, to identify that the core primers of straight tube class cabbage hybrid purity include following combinations to group: above-mentioned Preceding 10 groups of primer pair.

It is furthermore preferred that identify that the core primers of straight tube class cabbage hybrid purity include following combinations to group: on State 20 groups of primer pair.

Further, to identify that the core primers of import encircle-class Chinese cabbage cabbage hybrid purity include following to group 20 groups any one group or any several groups of combination: such as SEQ ID No.1-2 primer pair, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.7-8 primer pair, such as SEQ ID No.13-14 primer pair, such as SEQ ID No.23-24 primer pair, such as SEQ ID No.39-40 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.53-54 primer pair, such as SEQ ID No.59-60 primer pair, such as SEQ ID No.65-66 primer pair, such as SEQ ID No.9-10 primer pair, such as SEQ ID No.17- 18 primer pairs, such as SEQ ID No.21-22 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.41-42 draw Object to, such as SEQ ID No.51-52 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.67-68 primer pair, Such as SEQ ID No.81-82 primer pair, such as SEQ ID No.87-88 primer pair.

Preferably, to identify that the core primers of import encircle-class cabbage hybrid purity include following combinations to group: Above-mentioned preceding 10 groups of primer pair.

It is furthermore preferred that identify that the core primers of import encircle-class cabbage hybrid purity include following groups to group It closes: above-mentioned 20 groups of primer pair.

Further, to identify that the core primers of domestic encircle-class Chinese cabbage cabbage hybrid purity include following to group 20 groups any one group or any several groups of combination:

Such as SEQ ID No.3-4 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.45-46 primer To, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.49-50 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.85-86 primer pair, such as SEQ ID No.91-92 primer pair, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.9-10 primer pair, such as SEQ ID No.17-18 primer pair, such as SEQ ID No.19-20 primer pair, such as SEQ ID No.31-32 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.57-58 primer pair, such as SEQ ID No.65-66 primer pair, such as SEQ ID No.73-74 primer pair, such as SEQ ID No.77-78 primer pair.

Preferably, to identify that the core primers of domestic encircle-class cabbage hybrid purity include following combinations to group: Above-mentioned preceding 10 groups of primer pair.

It is furthermore preferred that identify that the core primers of domestic encircle-class cabbage hybrid purity include following groups to group It closes: 20 groups above-mentioned of primer pair.

In one of the disclosure or some embodiments, a kind of cabbage hybrid Purity core primers pair are provided Group, its main feature is that:

It is divided into four sets, one is to identify the folded core primers for embracing class cabbage hybrid purity to group, including 10 groups Core primers pair, it may be assumed that such as SEQ ID No.7-8 primer pair, such as SEQ ID No.25-26 primer pair, such as SEQ ID No.27-28 Primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.45-46 primer To, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair and Such as SEQ ID No.79-80；

Secondly for identify the core primers of straight tube class cabbage hybrid purity to group, including 10 groups of core primers It is right, it may be assumed that such as SEQ ID No.27-28 primer pair, such as SEQ ID No.33-34 primer pair, such as SEQ ID No.35-36 primer To, such as SEQ ID No.37-38 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.69-70 primer pair, such as SEQ ID No.71-72 primer pair and such as SEQ ID No.75-76 primer pair；

Thirdly for identify the core primers of import encircle-class Chinese cabbage cabbage hybrid purity to group, including 10 groups of cores Heart primer pair, it may be assumed that such as SEQ ID No.1-2 primer pair, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.7-8 primer To, such as SEQ ID No.13-14 primer pair, such as SEQ ID No.23-24 primer pair, such as SEQ ID No.39-40 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.53-54 primer pair, such as SEQ ID No.59-60 primer pair and such as SEQ ID No.65-66 primer pair

It is four for identify the core primers of domestic encircle-class Chinese cabbage cabbage hybrid purity to group, including 10 groups of cores Heart primer pair, it may be assumed that as SEQ ID No.3-4 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.45-46 are drawn Object to, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.49-50 primer pair, such as SEQ ID No.63-64 primer pair, Such as SEQ ID No.67-68 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.85-86 primer pair and such as SEQ ID No.91-92 primer pair.

In one of the disclosure or some specific embodiments, a kind of cabbage hybrid Purity core is provided and is drawn Object to group, its main feature is that: be divided into four sets, one be to identify the folded core primers for embracing class cabbage hybrid purity to group, Including 10 groups of core primers to and 10 groups of spare core primer pairs, 10 groups of core primers draw to SEQ ID No.7-8 such as is respectively as follows: Object to, such as SEQ ID No.25-26 primer pair, such as SEQ ID No.27-28 primer pair, such as SEQ ID No.35-36 primer pair, Such as SEQ ID No.41-42 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair and such as SEQ ID No.79-80；10 groups of spare core primers To being respectively as follows: SEQ ID No.5-6 primer pair, such as SEQ ID No.11-12 primer pair, such as SEQ ID No.15-16 primer To, as SEQ ID No.17-18 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.31-32 primer pair, Such as SEQ ID No.53-54 primer pair, such as SEQ ID No.61-62 primer pair, such as SEQ ID No.69-70 primer pair and such as SEQ ID No.73-74 primer pair；

Secondly for identify the core primers of straight tube class cabbage hybrid purity to group, including 10 groups of core primers pair With 10 groups of spare core primer pairs, 10 groups of core primers are to being respectively as follows: such as SEQ ID No.27-28 primer pair, such as SEQ ID No.33-34 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.37-38 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.69-70 primer pair, such as SEQ ID No.71-72 primer pair and such as SEQ ID No.75-76 primer pair, 10 groups of spare cores Primer pair is respectively as follows: such as SEQ ID No.3-4 primer pair, such as SEQ ID No.11-12 primer pair, such as SEQ ID No.19-20 Primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.43-44 primer pair, such as SEQ ID No.45-46 primer To, such as SEQ ID No.57-58 primer pair, such as SEQ ID No.83-84 primer pair, such as SEQ ID No.89-90 primer pair, such as SEQ ID No.91-92 primer pair；

Thirdly for identify the core primers of import encircle-class Chinese cabbage cabbage hybrid purity to group, including 10 groups of cores Heart primer pair and 10 groups of spare core primer pairs, 10 groups of core primers are to being respectively as follows: such as SEQ ID No.1-2 primer pair, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.7-8 primer pair, such as SEQ ID No.13-14 primer pair, such as SEQ ID No.23- 24 primer pairs, such as SEQ ID No.39-40 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.53-54 draw Object is to, such as SEQ ID No.59-60 primer pair and such as SEQ ID No.65-66 primer pair, 10 groups of spare core primer pairs difference Are as follows: such as SEQ ID No.9-10 primer pair, such as SEQ ID No.17-18 primer pair, such as SEQ ID No.21-22 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.51-52 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.81-82 primer pair, such as SEQ ID No.87-88 primer pair.

It is four for identify the core primers of domestic encircle-class Chinese cabbage cabbage hybrid purity to group, including 10 groups of cores Heart primer pair and 10 groups of spare core primer pairs, 10 groups of core primers are to being respectively as follows: such as SEQ ID No.3-4 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.49-50 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.85-86 primer pair and such as SEQ ID No.91-92 primer pair, 10 groups of spare cores Primer pair is respectively as follows: such as SEQ ID No.5-6 primer pair, such as SEQ ID No.9-10 primer pair, such as SEQ ID No.17-18 and draws Object to, such as SEQ ID No.19-20 primer pair, such as SEQ ID No.31-32 primer pair, such as SEQ ID No.41-42 primer pair, Such as SEQ ID No.57-58 primer pair, such as SEQ ID No.65-66 primer pair, such as SEQ ID No.73-74 primer pair and such as SEQ ID No.77-78 primer pair.

In identification, first choice uses core primers, is secondly spare core primer, may insure to provide from kit in this way Primer in screen identification purity of hybrid needed for primer.

In second typical embodiment of the disclosure, provide a kind of for identifying the examination of cabbage hybrid purity Agent box, the kit include the primer pair group.

Further, the kit includes a folded armful of class, straight tube class, import encircle-class and four sets of domestic encircle-class.

Further, the kit further includes polyacrylamide gel detection architecture or fluorescent capillary electrophoresis tube detection body It is that the reagent for including is that those skilled in the art routinely know that details are not described herein in the system.

In the typical embodiment of third of the disclosure, it is big in identification to provide the primer pair group or the kit Application in cabbage hybrid purity.

In the 4th typical embodiment of the disclosure, a kind of method of cabbage hybrid Purity is provided, This method includes the steps that being identified using the primer pair, the primer pair group or the kit.

In one of the disclosure or some specific embodiments, the method for the cabbage hybrid Purity includes Following steps:

(1) if randomly selecting dry granular seed from Chinese cabbage seeds to be detected, seed broadcasting is carried out in container for plant growth Growth, after cotyledon expansion, single seedling cotyledon of clip is spare；

(2) in extraction step (1) material DNA；

(3) using the genomic DNA extracted in step (2) as template, PCR amplification is carried out using the primer pair group, is obtained Pcr amplification product；

(4) pcr amplification product for obtaining step (3) carries out electrophoresis detection, the number occurred by counting single band Calculate seed purity.

Further, in step (1), for statistical significance and higher accuracy, at least select 100, 200 or 300 seeds are detected.

Further, in step (3), PCR amplification system are as follows: include 2 × Taq Plus PCR in 25 μ L reaction systems Each 1 μ L of the 12.5 forward and reverse primer of μ L, 10pmol of Master Mix, template (50ng μ L-1) 1 μ L, 9.5 μ L of deionized water.

PCR amplification program is 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 10s, 58.5 DEG C of annealing 20s, 72 DEG C of extension 10s, is followed Ring 35 times, last 72 DEG C of extensions 5min.

Compared with the relevant technologies that the present inventor knows, the one of technical solution of the disclosure has following beneficial to effect Fruit:

The disclosure with it is commercially available sell it is representational it is folded embrace, straight tube, import fill the span of a man's arms it is miscellaneous with domestic four seed type Chinese cabbages of filling the span of a man's arms Handing over kind is material, by screening primer combination extensively, obtains the core primers 10 of suitable four seed types Hybrid seed purity test Right to, spare primer 10, the ratio that heterozygous state occurs in respective type cenospecies in the primer screened is largely higher than 50%, using these primers as core component, exploitation is used for different type cabbage hybrid Purity, benefit at kit Locate fairly obvious.

Present disclose provides four sets of primers to combine, and is respectively suitable for four kinds of different type cabbage hybrid Purities, then Four seed types i.e. folded armful, straight tube, import is filled the span of a man's arms, domestic fill the span of a man's arms substantially covers most of Chinese cabbage type, even if going out now Existing fast vegetables type, sowing in the fall can also be with balling, so can also judge that certain fast vegetable kind is to belong to that above-mentioned type Chinese cabbage, be similarly a fast dish Hybrid seed purity test.

Detailed description of the invention

The Figure of description for constituting disclosure a part is used to provide further understanding of the disclosure, the signal of the disclosure Property embodiment and its explanation for explaining the disclosure, do not constitute the improper restriction to the disclosure.

Fig. 1 is that (P1 and P2 are two parents of ox board 19 to amplification of first group of primer between parents, and 1-10 is primer Number, M is DNA molecular amount standard 50bp ladder).

Fig. 2 be 1,2,7 primer of number in partial hybridization kind qualification result (P1 and P2 are two parents of ox board 19, 1-60 respectively represents 60 seeds, and M is DNA molecular amount standard 50bp ladder).

Specific embodiment

It is noted that described further below be all exemplary, it is intended to provide further instruction to the disclosure.Unless another It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field The identical meanings of understanding.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation and/or their combination.

In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool The technical solution of the disclosure is described in detail in the embodiment of body.

This kit is obtained by following method:

1.1 test material

Test material includes 40 commercially available cabbage hybrids, these kinds are divided into four classes according to obvolvent type, respectively It is folded armful class (15), straight tube class (11), typical encircle-class Japan and Korea S's import kind (7), the new No. three type encircle-class in Beijing Domestic kind (7), shown in details following table.These seeds are bought from market, and 5-10 nursery of cenospecies are taken, emergence It is spare after taking the leaf tissue of at least 5 plants seedling to mix afterwards.

1.2 test method

1.2.1DNA extract: DNA extracts the method referring to Zhang Zhigang etc..Specifically, it in 2ml screw socket grinding pipe, is added TPS reagent (100mmolL-1 Tris-HCl, 1molL of 65 DEG C of 800ul preheatings^-1KCl、10mmol·L^-1EDTA, 200mg·L^-1RNA enzyme, pH 8.0) and 3-4 diameter 5mm ceramic grinding pearl, in automatic grinding instrument (France, Precellys24 5000rmin on)^-1Concussion grinding 30sec；It is homogenized in 65 DEG C of incubators or water-bath inside holding 30min；To even It starches cool to 10000rmin after room temperature^-1Room temperature is centrifuged 10min；Supernatant is transferred in the centrifuge tube of a new 1.5mL, is added Enter isometric chloroform and carefully mixes, 12000rmin^-14 DEG C of centrifugation 10min；It is new that supernatant is again transferred to one It in the centrifuge tube of 1.5mL and isometric isopropanol is added carefully mixes, room temperature makes DNA precipitate 5min；In 4 DEG C of 12000rmin^-1 It is centrifuged 10min, abandons supernatant；75% ethanol washing of 1ml precipitating, 7500r/min is added^-1Be centrifuged 1min, abandon supernatant after it is short in room temperature Temporary dry DNA；100 μ L 10mmolL are added^-14 DEG C of Tris-HCl (pH 8.0) overnight to dissolve genomic DNA；Next day takes out 3000r·min^-1Supernatant is transferred in a new centrifuge tube after centrifugation 1min.With UltroSpec 3300pro (GE Healthcare) spectrophotometric measures DNA concentration, by genomic DNA concentration dilution to 50ngul^-1It is saved backup in -20 DEG C.

1.2.2 primer screening

It has carried out screening in the sequencing result of the material 06-247 and He102 of genome sequencing from early period, screen altogether 100 polymorphic sites, every chromosome have 10, obtain polymorphic site two sides sequence, according to these primers, The primer numbers of design are shown in Table 1, and primer entrusts Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthesis.

1.2.3PCR amplification

PCR amplification with reference to Liu Shuantao etc. document, specifically, using Tiangeng biotechnology (Beijing) Co., Ltd 2 × Taq Plus PCR MasterMiH (KT205-01) includes 2 × Taq Plus PCR Master in 25 μ L reaction systems Each 1 μ L of the 12.5 forward and reverse primer of μ L, 10pmol of Mix, template (50ng μ L-1) 1 μ L, 9.5 μ L of deionized water.PCR amplification It is carried out in Bio-Rad T100TM grads PCR instrument (production of Bio-Rad company, the U.S.), amplification program is 94 DEG C of initial denaturations 3min, 94 DEG C of denaturation 10s, 58.5 DEG C of annealing 20s, 72 DEG C of extension 10s are recycled 35 times, last 72 DEG C of extensions 5min.PCR product With 6% non-deformed polypropylene glutamine gel electrophoresis separation, using 50bp ladder as DNA molecular amount standard, silver staining develops the color, Taking photograph of intelligent mobile phone.

1.2.4 result counts

According to amplification, amplification heterozygosis or homozygotic state of each primer in each kind are recorded, counts each pair of and draws Object amplifies the percentage of heterozygous state, referred to as site heterozygosis rate in all types of, is expressed as a percentage, and 0 certain site of expression exists It is all homozygotic state in the cenospecies of respective type.In Excel table by all types of primer amplification heterozygosis rate results respectively by First group primer of the preceding 10 pairs of primers as respective type Hybridization identification is screened in high to Low arrangement, and 11-20 is to primer conduct Second group, referred to as spare primer.It is constructed for kit.

2 interpretations of result:

Site heterozygosis rate of the primer selected by 2.1 in different type cabbage hybrid

Site heterozygosis rate of the selected primer in all types of cenospecies is shown in Table 1, these primers it is folded embrace a class, straight tube class, into Heterozygosis rate in mouthful encircle-class (Japan and Korea S's class), domestic encircle-class (be not true to type encircle-class) is respectively interposed in 0~73.33%, 0~ 72.73%, 0~85.71% and 0~100.00%.Site heterozygosis rate difference pole of most primers in four type cenospecies Significantly, such as A01001 in domestic type heterozygosis rate 42.86%, in import encircle-class be 0 type the phenomenon that it is very universal.Table Bright different primers are suitable for different type Hybrid seed purity test, it is necessary to which exploitation is reflected for various types cabbage hybrid purity Fixed core primers.

Site heterozygosis rate in primer different type cenospecies selected by table 1

2 primer sequence of table

The screening of 2.2 different type Hybrid seed purity test core primers

According to method 1.2.4, the site heterozygosis rate of primer in all types of cenospecies is arranged from high to low, filters out and comes Preceding 10 primer constitutes the core primers of respective type Hybrid seed purity test, and first group of primer, number are known as in kit From 1-10.The primer for coming 11-20 is screened again, alternately primer, number 11-20.Detailed results are by table 3- table 6.From table Middle data can be seen that the site heterozygosis rate of all types of first group of core primers 50% or more, i.e., hybridizes in respective type The probability for occurring heterozygous state in kind is greater than 50%.Even if the second group of spare primer provided, most heterozygosis rates are all higher than 50%, it is only a small number of (it is folded embrace class 5 to, only 1 pair of straight tube class, remaining does not have) primer detection result heterozygosis rate less than 50%, then may be used To ensure the primer needed for screening identification purity of hybrid in the primer that kit provides.

In four class cores (first group) primer, do not find to appear in the primer in four seed types simultaneously, i.e., it is general to draw The screening of object be it is not applicable, only find that 2 appear in primer in three types to (A07008 and A06002) simultaneously.By two groups If primer merges, also only a pair of primer having in four seed types of discovery, is A05002, it is folded embrace a class, straight tube class, into Site heterozygosis rate is 60.00%, 63.64%, 57.14% and 57.14% respectively in mouthful encircle-class and domestic encircle-class, can be with Referred to as universal primer.But pair of primers solve all mixing breed kind Purities be obviously it is inadequate, so universal primer ratio It is more difficult to look for, and it is directed to different types of cabbage hybrid Purity, developing different primers, to be combined into power ratio higher.Cause This this kit will include four sections.

The composition of 2.3 kits

This disclosure relates to cabbage hybrid Purity kit include four sections i.e. cabbage hybrid Purity Kit (folded to embrace class, straight tube class, import encircle-class, domestic encircle-class), each kit includes two groups of primers, and number 1-10 is First group, number 11-20 be second group.Positive DNA control is provided simultaneously, it is ensured that each pair of primer can expand under given conditions Increase two difference purpose bands out.Each kit includes polyacrylamide gel detection type and the detection of fluorescent capillary electrophoresis tube Type, client can select corresponding type according to self-condition.

Kit provides operational manual, provides operating procedure and points for attention in detail.Below with reference to 19 great Bai of ox board Dish Hybrid seed purity test, provides using example.

Table 3 is folded to embrace class Hybridization identification core primers

4 straight tube class Hybridization identification core primers of table

5 Japan and Korea S's class Hybridization identification core primers of table

Table 6 is not true to type encircle-class Hybridization identification core primers

2.4 cabbage hybrid purity InDel Marker Identification kits (folded to embrace type) are in " ox board 19 " Chinese cabbage purity Application (polyacrylamide gel electrophoresis) in identification

2.4.1 DNA is extracted

First choice acquires parents' leaf tissue from field, and it is spare that each parent at least acquires five plants of leaf tissue equivalent mixings. Simultaneously before seed storage, at least 300 seeds are randomly selected from the seed of each " ox board 19 " Chinese cabbage seed breeding base, it will Seed broadcasting is in seedlings nursing plate, Routine Management, and after cotyledon expansion, single seedling cotyledon of clip is spare.One seedling of every clip Afterwards, scissors or blade all disinfect in alcohol, avoid cross contamination.DNA extracts reference method 1.2.1.

2.4.2 it is used for the primer screening of Hybrid seed purity test

Respectively using parents as template, PCR amplification and product detection, preferred detection primer 1- are carried out according to the method for 1.2.3 10, if not finding there is polymorphism primer in parents, it is further continued for carrying out the screening of 11-20 primer, until finding polymorphic Property primer.Fig. 1 is amplification of first group of primer in parents, primers of 5 pairs of the discovery with polymorphism, primer numbers are 1, 2,3,7 and 9.For the sake of with caution, select 2-4 therein to for Hybrid seed purity test.

2.4.2 19 Hybrid seed purity test of ox board is identified

Purity is carried out to 19 cenospecies of (number 1,2,7) primer pair ox board using 3, three pairs of primer results are kissed completely It closes, Fig. 2 is that qualification result of 3 pairs of primers in partial hybridization kind compares, it can be seen from the figure that the seed of number 5 is parent The selfed seed of P1, the seed of number 58 are the selfed seeds of parent P2.The shared DNA from 283 seeds is obtained clearly Amplified band, wherein parents' selfed seed only has 7 (P1 selfed seed 3, P2 selfed seeds 4), and purity of hybrid is (283-7)/283*100%=97.5%.Meet 96% purity requirement of national Specification, it can direct packing and selling.

When carrying out Hybrid seed purity test in reality, maker and parents' control can also be not added in electrophoresis, only do and hybridize Kind sample, the number energy calculated purity occurred by counting single band, can simplify operation in this way.Because being just suitable for For the primer for identifying purity, primer screening step have been known for band molecular weight that parent in parents expands it is big or It is small, it is only necessary to which that the pillar location occurred according to selfed seed is it may determine that selfed seed comes from that parent, to judge that A parent is easier self-pollination, and such result can also instruct the harvest of coming year cenospecies, it may be assumed that if P1 selfed seed goes out Existing ratio is high, seriously affects purity of hybrid, then can carry out Purity in advance, can directly distinguish from seed breeding base Male parent or maternal upper immature seed are acquired, seedling is obtained by way of embryo culture and extracts DNA progress Purity, if certain The seed purity harvested on one parent cannot be met the requirements, then can harvest the seed on another parent, only to drop to greatest extent The loss of low production of hybrid seeds process.In addition the primer combination that kit provides can also carry out Chinese-cabbage Germplasm identification, seed money The building of source DNA fingerprint, cenospecies authenticity identification etc., application range is very extensive.

3. result

The disclosure with it is commercially available sell it is representational it is folded embrace, straight tube, import are filled the span of a man's arms, the domestic four seed type Chinese cabbages such as fill the span of a man's arms are miscellaneous Handing over kind is material, by screening primer combination extensively, obtains the core primers 10 of suitable four seed types Hybrid seed purity test Right to, spare primer 10, the ratio that heterozygous state occurs in respective type cenospecies in the primer screened is largely higher than 50%, using these primers as core component, exploitation is used for different type cabbage hybrid Purity, benefit at kit Locate fairly obvious.Since Chinese cabbage causes frequently with the not affine production of hybrid seeds, self-incompatibility easy fluctuation affected by environment is selfed Purity of hybrid difficulty or ease guarantee.And purity of hybrid is the important indicator of seed quality, the side of conventional identification Chinese cabbage purity Method is still field planting character observation method, this needs a lasting season of growth, current year harvest seed belt planted in fall into Row field test can only wait second years ability packing and selling, this virtually increases the inventory cost of enterprise.If can work as Just carry out Purity before being put in storage in year, when even harvesting, on the one hand underproof seed may not necessarily harvest, and reduce labour Cost, another aspect, qualified seed current year can reduce the pressure of business inventory fund with packing and selling, will significantly improve The effectiveness of operation of enterprise.

Although before this about the SSR molecular marker kit of cabbage hybrid Purity (in fastening storehouse, Su Tongbing, Sui Guanglei Chinese cabbage SSR core primers and kind detection kit), which is not used widely, the reason is that SSR The polymorphism of label is excessively abundant, and the polymorphic site that it is generated in a certain number of species populations is much larger than 2, such as Li Guoqiang (building of Li Guoqiang .2008. Chinese cabbage Core Germplasms and evaluation Beijing: Chinese Academy of Agricultural Sciences's master thesis, 18- 38.) and Yang Jinxue (Yang Jinxue .2013. for Chinese cabbage cultivar nucleic acid fingerprint base building SSR core primers screen the Yangzhou: Yangzhou University's master thesis, 18-42.) with SSR marker detect Chinese-cabbage Germplasm, most pair of primers has 10~ 12 polymorphic sites, it is average then can produce 4.00~5.16, or even in pure lines all may there are two even two with On seat, this is unfavorable for Hybrid seed purity test.In comparison, InDel label is based on full-length genome weight sequencing technologies The New molecular marker developed, its development process resurvey the sequence difference of sequence discovery based entirely on full-length genome, and other Molecular labeling is compared, and the probability that different length size InDel mutation occurs for the same site of genome is smaller, existing research channel syndrome Bright, most InDel labels only generate 2 polymorphic sites, and the InDel label more than 2 polymorphic sites accounts in genome It is fewer that (Zhu Dongxu, Wang Yanhua, Zhao Jianjun, Liu Bo Lee, the pavilion Xiao Feng is refined glad, and Shen Shuxing .2014. cabbage is relative to great Bai The foundation and apply gardening journal, 41 (8): 1699-1706 that the special InDel of dish linkage group is marked；Wei Libin, Miao Hong plum, Lee Spring, Duan Yinghui, Xu Fangfang, Zhang Haiyang .2017. sesame SNP and InDel mark genetic diversity, group structure and chain injustice Weighing apparatus analysis Molecular Plant Breeding, 15 (8): 3070-3079；Liu Shuantao, Zhang Zhigang, Si Liying, Wang Ronghua, Li Qiaoyun, Wang Li China, Zhao Zhizhong, Liang Shuimei, Zhang Quanfang walk Relationship iden- tification [J] of fast based on the InDel Chinese cabbage breeding material marked Plant genetic resources journal, 2018,19 (04): 657-667).Such label has natural for Hybrid seed purity test Advantage.Present disclose provides four sets of primers to combine, respectively suitable four kinds of different type cabbage hybrid Purities, then and four kinds Type i.e. folded armful, straight tube, import is filled the span of a man's arms, domestic fill the span of a man's arms substantially covers most of Chinese cabbage type, even if occur now Fast vegetables type, sowing in the fall can also be with balling, so can also judge that certain fast vegetable kind is belong to that above-mentioned type big Chinese cabbage is similarly a fast dish Hybrid seed purity test.

Each kit can be polyacrylamide gel detection type or capillary electrophoresis detection type (such as fluorescent capillary pipe electricity Swimming detection type), fluorescent capillary electrophoresis tube detection type only needs to carry out fluorescent marker to primer, and corresponding cost can be higher.Client can To select corresponding type according to oneself laboratory condition.

Above-described embodiment is the preferable embodiment of the disclosure, but embodiment of the present disclosure is not by above-described embodiment It limits, made changes, modifications, substitutions, combinations, simplifications under other any spiritual essence and principles without departing from the disclosure, It should be equivalent substitute mode, be included within the protection scope of the disclosure.

SEQUENCE LISTING

<110>Shandong Academy of Agricultural Sciences's vegetable or flower research institute

<120>four seed type cabbage hybrid Purity core primers and purity of hybrid InDel molecular markers for identification

Kit

<130> 2018

<160> 92

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> DNA

<213>A01004 forward primer

<400> 1

agtggtgatg ctaacggtat gaa 23

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<212> DNA

<213>A01004 reverse primer

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cgaggaggag tttgaatgag gt 22

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<212> DNA

<213>A02001 forward primer

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agttgtgcga cttgagatgg c 21

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<212> DNA

<213>A02001 reverse primer

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ccgtagacga cagtataaga accc 24

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<212> DNA

<213>A02002 forward primer

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cttgtgcgct ctcactcact tc 22

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<213>A02002 reverse primer

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acctcctcta ctccgtatgt tcg 23

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<213>A02003 forward primer

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catgtcatca tacttcaagt tccct 25

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<213>A02003 reverse primer

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tcggttccgt gtgagttgc 19

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<212> DNA

<213>A02005 forward primer

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cttttaaggg acgctttcgg 20

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<212> DNA

<213>A02005 reverse primer

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ctcgccgttt aacctcacc 19

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<212> DNA

<213>A02007 forward primer

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gagaagagag agcgcaagcc 20

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<213>A02007 reverse primer

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tccgacgcaa acccagtg 18

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<212> DNA

<213>A02008 forward primer

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tttctttcgc ctctcagttg g 21

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<212> DNA

<213>A02008 reverse primer

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gatttcaatc ggatgtagta aaacg 25

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<212> DNA

<213>A02010 forward primer

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caacttctta gtgtacgttc gttcct 26

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<212> DNA

<213>A02010 reverse primer

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caacttctta gtgtacgttc gttcct 26

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<212> DNA

<213>A03002 forward primer

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atccagggag agccaaacg 19

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<213>A03002 reverse primer

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ttgggtggtc ttcttgatgg t 21

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<213>A03003 forward primer

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accttgaact tgaagctggt cc 22

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<213>A03003 reverse primer

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cagcagccac aaccttacgc 20

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<213>A03004 forward primer

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ctggcagcgt tgctttatca c 21

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<212> DNA

<213>A03004 reverse primer

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gaagaagaga aagaagagga gagaaaa 27

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<213>A03005 forward primer

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aagcggagag gaagctaagg a 21

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<213>A03005 reverse primer

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aatgcgtcag ctaactgcaa ca 22

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<213>A03007 forward primer

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gccctcatat acattacatc accac 25

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<213>A03007 reverse primer

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agaaagttct gaaggcgctt g 21

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<213>A03008 forward primer

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cgaaaaggaa gtgggattgg 20

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<212> DNA

<213>A03008 reverse primer

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ggcacagaac tcacagtaac agc 23

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<213>A03009 forward primer

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gccggagaat ccatcgtca 19

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<213>A03009 reverse primer

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agagggaagc tgtcttgcgt g 21

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<213>A04001 forward primer

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tagaatttaa ggatccgtgt gacat 25

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<213>A04001 reverse primer

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taagcttcgt gagaatggtc tattt 25

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<212> DNA

<213>A04004 forward primer

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gccagacggt cggattttc 19

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<213>A04004 reverse primer

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ttcacggact ctttggaggt tt 22

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<213>A04005 forward primer

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tgaacttggt tatggtcatt tgg 23

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<213>A04005 reverse primer

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catcgcgtgt agatatgctc ct 22

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<213>A04006 forward primer

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tcaaacctaa accctaacaa gcc 23

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<212> DNA

<213>A04006 reverse primer

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ggtaacggta ttttggagtc gg 22

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<212> DNA

<213>A05001 forward primer

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cggacaactt tcttatctcc acg 23

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<212> DNA

<213>A05001 reverse primer

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cggtttcaat ttttcaggct ct 22

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<213>A05002 forward primer

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agaaaaaggg cagacccagt t 21

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<212> DNA

<213>A05002 reverse primer

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ccgatgacga cggaatcag 19

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<213>A05006 forward primer

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gcttccctag caaacccatt t 21

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<212> DNA

<213>A05006 reverse primer

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ttggagcaat acaaccaaga gc 22

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<212> DNA

<213>A05007 forward primer

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taacggtgac atctgaggcg 20

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<212> DNA

<213>A05007 reverse primer

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acaggtgcag gaccaaggat 20

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<212> DNA

<213>A06002 forward primer

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ataaccagaa agttctttgg atctacc 27

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<212> DNA

<213>A06002 reverse primer

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aaactcacgg caccctatca t 21

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<213>A06004 forward primer

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aagtcaaagg cgatcattgt atc 23

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<212> DNA

<213>A06004 reverse primer

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cacatctgaa tttcttgtta gtgga 25

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<212> DNA

<213>A07001 forward primer

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aagtgcggac aaccaaagga 20

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<213>A07001 reverse primer

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ttagagaaag ggagtaaaga accaag 26

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<212> DNA

<213>A07002 forward primer

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gctgagaagg agacggtcga 20

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<212> DNA

<213>A07002 reverse primer

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ggtttggggt tttgagatgg a 21

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<212> DNA

<213>A07003 forward primer

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ttgaactgct catccttccg tc 22

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<212> DNA

<213>A07003 reverse primer

<400> 56

ttatgaactc accagaattt tgtagg 26

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<212> DNA

<213>A07004 forward primer

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gattgaagga ggaagatcca acg 23

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<212> DNA

<213>A07004 reverse primer

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caacatttca ccctcaagca cc 22

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<213>A07006 forward primer

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tttgagagaa tgctggagga gg 22

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aatcagtgga agcccggtta 20

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taagttttca gttgaaactg gcaaa 25

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<213>A07007 reverse primer

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ttttctgtat cttcttggat gtcga 25

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<213>A07008 forward primer

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cacgacgagt aaaaccagtg aga 23

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<212> DNA

<213>A07008 reverse primer

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tctgcctgcc ttggtcctat 20

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<212> DNA

<213>A08001 forward primer

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tgcgtgagat ggtcttttgg ta 22

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<212> DNA

<213>A08001 reverse primer

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cggctctctc ttttcaggga ta 22

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<212> DNA

<213>A08003 forward primer

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cattctgaaa aatgggaatt gc 22

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<212> DNA

<213>A08003 reverse primer

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agtcatctcc atcgtcttgc tct 23

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<213>A08004 forward primer

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aagttgttgc tcaaacaaac taggac 26

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<212> DNA

<213>A08004 reverse primer

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ggtggagaag atagtgaatg tgtaaag 27

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<212> DNA

<213>A08005 forward primer

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tggcgtcgaa cagcagaga 19

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<212> DNA

<213>A08005 reverse primer

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ctagtgcgga gaggagattg aga 23

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<213>A09002 forward primer

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gcttctgtgt ctggtttgag ga 22

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<212> DNA

<213>A09002 reverse primer

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tcttggctta ggacgatgag gt 22

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<213>A09003 forward primer

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gaagtacaag accatcattc accc 24

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ggactatcag tatgagtagc aacaatct 28

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<213>A09004 forward primer

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tctttgactg gaagttgcct gat 23

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<212> DNA

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<400> 78

ccattttttg ccagagagtg tatt 24

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<212> DNA

<213>A09006 forward primer

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aggtgacaaa ttaccagtgg gtg 23

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<212> DNA

<213>A09006 reverse primer

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tgttgcttcc agtaatctgc cat 23

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<212> DNA

<213>A09008 forward primer

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atcagcgaac ggcttcaagt t 21

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<212> DNA

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cctggctatg aggtgtgtgg tat 23

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<213>A10001 forward primer

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tgagatgttg ctcttggtgt tgt 23

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<212> DNA

<213>A10001 reverse primer

<400> 84

ccagtgggga tattatacag gga 23

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<212> DNA

<213>A10002 forward primer

<400> 85

aaataacaac attctctcgc tctca 25

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<212> DNA

<213>A10002 reverse primer

<400> 86

gcatcaacta cattgcctcc tc 22

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<212> DNA

<213>A10003 forward primer

<400> 87

ctgagaatgt gcctctaaca acaga 25

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<212> DNA

<213>A10003 reverse primer

<400> 88

aggcaaaaga ttgggacggt 20

<210> 89

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<212> DNA

<213>A10008 forward primer

<400> 89

cctactctgg ttttcatggt catc 24

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<211> 24

<212> DNA

<213>A10008 reverse primer

<400> 90

ccttgtggtt ttgatacttt cctc 24

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<212> DNA

<213>A10009 forward primer

<400> 91

catgttgctg cctcttcttg c 21

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<212> DNA

<213>A10009 reverse primer

<400> 92

aaactaaaac gcttgcttgg tga 23

Claims (10)

1. a kind of cabbage hybrid Purity core primers are to group, characterized in that the core primers are drawn group by following Any one group of object centering or any several groups of combination: as SEQ ID NO.1-2 primer pair, such as SEQ ID NO.3-4 primer pair, Such as SEQ ID NO.5-6 primer pair, such as SEQ ID NO.7-8 primer pair, such as SEQ ID NO.9-10 primer pair, such as SEQ ID NO.11-12 primer pair, such as SEQ ID NO.13-14 primer pair, such as SEQ IDNO.15-16 primer pair, such as SEQ ID NO.17- 18 primer pairs, such as SEQ ID NO.19-20 primer pair, such as SEQ ID NO.21-22 primer pair, such as SEQ ID NO.23-24 draw Object to, such as SEQ ID NO.25-26 primer pair, such as SEQ ID NO.27-28 primer pair, such as SEQ IDNO.29-30 primer pair, Such as SEQ ID NO.31-32 primer pair, such as SEQ ID NO.33-34 primer pair, such as SEQ ID NO.35-36 primer pair, such as SEQ ID NO.37-38 primer pair, such as SEQ ID NO.39-40 primer pair, such as SEQ ID NO.41-42 primer pair, such as SEQ IDNO.43-44 primer pair, such as SEQ ID NO.45-46 primer pair, such as SEQ ID NO.47-48 primer pair, such as SEQ ID NO.49-50 primer pair, SEQ ID NO.51-52 primer pair, such as SEQ ID NO.53-54 primer pair, such as SEQ ID NO.55- 56 primer pairs, such as SEQ ID NO.57-58 primer pair, such as SEQ ID NO.59-60 primer pair, such as SEQ ID NO.61-62 draw Object to, such as SEQ ID NO.63-64 primer pair, such as SEQ IDNO.65-66 primer pair, such as SEQ ID NO.67-68 primer pair, Such as SEQ ID NO.69-70 primer pair, such as SEQ ID NO.71-72 primer pair, such as SEQ ID NO.73-74 primer pair, such as SEQ ID NO.75-76 primer pair, such as SEQ ID NO.77-78 primer pair, such as SEQ IDNO.79-80 primer pair, such as SEQ ID NO.81-382 primer pair, such as SEQ ID NO.83-84 primer pair, such as SEQ ID NO.85-86 primer pair, such as SEQ ID NO.87-88 primer pair, such as SEQ ID NO.89-90 primer pair and such as SEQ ID NO.91-92 primer pair.

2. core primers as described in claim 1 are to group, characterized in that identify folded armful of class cabbage hybrid purity Core primers are to any one group or any several groups of the combination that group includes in following 20 groups: such as SEQ ID No.7-8 primer pair, such as SEQ ID No.25-26 primer pair, such as SEQ ID No.27-28 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.79-80, SEQ ID No.5-6 primer pair, As SEQ ID No.11-12 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.17-18 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.31-32 primer pair, such as SEQ ID No.53-54 primer pair, such as SEQ ID No.61-62 primer pair, such as SEQ ID No.69-70 primer pair, such as SEQ ID No.73-74 primer pair；

Preferably, draw to identify that the folded core primers for embracing class cabbage hybrid purity include following 10 groups: first 10 groups to group Object pair；

It is furthermore preferred that identify that the folded core primers for embracing class cabbage hybrid purity include following 20 groups to group: whole 20 Group primer pair.

3. core primers as described in claim 1 are to group, characterized in that identify straight tube class cabbage hybrid purity Core primers include following 20 groups any one group or any several groups of combination to group: such as SEQ ID No.27-28 primer pair, such as SEQ ID No.33-34 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.37-38 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.69-70 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.75-76 primer pair, such as SEQ ID No.3- 4 primer pairs, such as SEQ ID No.11-12 primer pair, such as SEQ ID No.19-20 primer pair, such as SEQ ID No.29-30 primer To, such as SEQ ID No.43-44 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.57-58 primer pair, such as SEQ ID No.83-84 primer pair, such as SEQ ID No.89-90 primer pair, such as SEQ ID No.91-92 primer pair；

Preferably, to identify that the core primers of straight tube class cabbage hybrid purity include following combinations to group: first 10 groups Primer pair；

It is furthermore preferred that identify that the core primers of straight tube class cabbage hybrid purity include following combinations to group: whole 20 Group primer pair.

4. core primers as described in claim 1 are to group, characterized in that identify import encircle-class Chinese cabbage Chinese cabbage crossing The core primers of kind purity include following 20 groups any one group or any several groups of combination to group: such as SEQ ID No.1-2 primer To, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.7-8 primer pair, such as SEQ ID No.13-14 primer pair, such as SEQ ID No.23-24 primer pair, such as SEQ ID No.39-40 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.53-54 primer pair, such as SEQ ID No.59-60 primer pair, such as SEQ ID No.65-66 primer pair, such as SEQ ID No.9- 10 primer pairs, such as SEQ ID No.17-18 primer pair, such as SEQ ID No.21-22 primer pair, such as SEQ ID No.29-30 draw Object to, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.51-52 primer pair, such as SEQ ID No.55-56 primer pair, Such as SEQ ID No.67-68 primer pair, such as SEQ ID No.81-82 primer pair, such as SEQ ID No.87-88 primer pair；

Preferably, to identify that the core primers of import encircle-class cabbage hybrid purity include following combinations to group: preceding 10 The primer pair of group；

It is furthermore preferred that identify that the core primers of import encircle-class cabbage hybrid purity include following combinations to group: complete 20 groups of portion primer pair.

5. core primers as described in claim 1 are to group, characterized in that identify domestic encircle-class Chinese cabbage Chinese cabbage crossing The core primers of kind purity include following 20 groups any one group or any several groups of combination to group:

Such as SEQ ID No.3-4 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.49-50 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.85-86 primer pair, such as SEQ ID No.91-92 primer pair, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.9-10 primer pair, such as SEQ ID No.17-18 Primer pair, such as SEQ ID No.19-20 primer pair, such as SEQ ID No.31-32 primer pair, such as SEQ ID No.41-42 primer To, such as SEQ ID No.57-58 primer pair, such as SEQ ID No.65-66 primer pair, such as SEQ ID No.73-74 primer pair, such as SEQ ID No.77-78 primer pair；

Preferably, to identify that the core primers of domestic encircle-class cabbage hybrid purity include following combinations to group: preceding 10 The primer pair of group；

It is furthermore preferred that identify that the core primers of domestic encircle-class cabbage hybrid purity include following combinations to group: complete 20 groups of portion primer pair.

6. a kind of cabbage hybrid Purity core primers are to group, characterized in that be divided into four sets, one is folded to identify The core primers of class cabbage hybrid purity are embraced to group, including 10 groups of core primers pair, it may be assumed that such as SEQ ID No.7-8 primer To, such as SEQ ID No.25-26 primer pair, such as SEQ ID No.27-28 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair and such as SEQ ID No.79-80；

Secondly for identify the core primers of straight tube class cabbage hybrid purity to group, including 10 groups of core primers pair, it may be assumed that Such as SEQ ID No.27-28 primer pair, such as SEQ ID No.33-34 primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.37-38 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.69-70 primer pair, such as SEQ ID No.71-72 primer pair and such as SEQ ID No.75-76 primer pair；

Thirdly for identify that the core primers of import encircle-class Chinese cabbage cabbage hybrid purity draw group, including 10 groups of cores Object pair, it may be assumed that such as SEQ ID No.1-2 primer pair, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.7-8 primer pair, such as SEQ ID No.13-14 primer pair, such as SEQ ID No.23-24 primer pair, such as SEQ ID No.39-40 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.53-54 primer pair, such as SEQ ID No.59-60 primer pair and such as SEQ ID No.65-66 primer pair；

It is four for identify that the core primers of domestic encircle-class Chinese cabbage cabbage hybrid purity draw group, including 10 groups of cores Object pair, it may be assumed that such as SEQ ID No.3-4 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.45-46 primer To, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.49-50 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.85-86 primer pair and such as SEQ ID No.91-92 primer pair.

7. core primers as claimed in claim 6 are to group, characterized in that be divided into four sets, one is to identify that folded armful of class is big The core primers of cabbage hybrid purity to group, including 10 groups of core primers to and 10 groups of spare core primer pairs, 10 groups of cores Primer pair is respectively as follows: such as SEQ ID No.7-8 primer pair, such as SEQ ID No.25-26 primer pair, such as SEQ ID No.27-28 Primer pair, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.45-46 primer To, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair and Such as SEQ ID No.79-80；10 groups of spare core primer pairs are respectively as follows: SEQ ID No.5-6 primer pair, such as SEQ ID No.11-12 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.17-18 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.31-32 primer pair, such as SEQ ID No.53-54 primer pair, such as SEQ ID No.61-62 primer pair, such as SEQ ID No.69-70 primer pair and such as SEQ ID No.73-74 primer pair；

Secondly for identify the core primers of straight tube class cabbage hybrid purity to group, including 10 groups of core primers to and 10 Group spare core primer pair, 10 groups of core primers are to being respectively as follows: such as SEQ ID No.27-28 primer pair, such as SEQ ID No.33- 34 primer pairs, such as SEQ ID No.35-36 primer pair, such as SEQ ID No.37-38 primer pair, such as SEQ ID No.41-42 draw Object to, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.69-70 primer pair, Such as SEQ ID No.71-72 primer pair and such as SEQ ID No.75-76 primer pair, 10 groups of spare core primer pairs be respectively as follows: as SEQ ID No.3-4 primer pair, such as SEQ ID No.11-12 primer pair, such as SEQ ID No.19-20 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.43-44 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.57-58 primer pair, such as SEQ ID No.83-84 primer pair, such as SEQ ID No.89-90 primer pair, such as SEQ ID No.91-92 primer pair；

Thirdly for identify that the core primers of import encircle-class Chinese cabbage cabbage hybrid purity draw group, including 10 groups of cores Object to and 10 groups of spare core primer pairs, 10 groups of core primers are to being respectively as follows: such as SEQ ID No.1-2 primer pair, such as SEQ ID No.5-6 primer pair, such as SEQ ID No.7-8 primer pair, such as SEQ ID No.13-14 primer pair, such as SEQ ID No.23-24 Primer pair, such as SEQ ID No.39-40 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.53-54 primer To, such as SEQ ID No.59-60 primer pair and such as SEQ ID No.65-66 primer pair, 10 groups of spare core primer pairs are respectively as follows: Such as SEQ ID No.9-10 primer pair, such as SEQ ID No.17-18 primer pair, such as SEQ ID No.21-22 primer pair, such as SEQ ID No.29-30 primer pair, such as SEQ ID No.41-42 primer pair, such as SEQ ID No.51-52 primer pair, such as SEQ ID No.55-56 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.81-82 primer pair, such as SEQ ID No.87-88 primer pair；

It is four for identify that the core primers of domestic encircle-class Chinese cabbage cabbage hybrid purity draw group, including 10 groups of cores Object to and 10 groups of spare core primer pairs, 10 groups of core primers are to being respectively as follows: such as SEQ ID No.3-4 primer pair, such as SEQ ID No.15-16 primer pair, such as SEQ ID No.45-46 primer pair, such as SEQ ID No.47-48 primer pair, such as SEQ ID No.49-50 primer pair, such as SEQ ID No.63-64 primer pair, such as SEQ ID No.67-68 primer pair, such as SEQ ID No.71-72 primer pair, such as SEQ ID No.85-86 primer pair and such as SEQ ID No.91-92 primer pair, 10 groups of spare cores Primer pair is respectively as follows: such as SEQ ID No.5-6 primer pair, such as SEQ ID No.9-10 primer pair, such as SEQ ID No.17-18 and draws Object to, such as SEQ ID No.19-20 primer pair, such as SEQ ID No.31-32 primer pair, such as SEQ ID No.41-42 primer pair, Such as SEQ ID No.57-58 primer pair, such as SEQ ID No.65-66 primer pair, such as SEQ ID No.73-74 primer pair and such as SEQ ID No.77-78 primer pair.

8. a kind of for identifying the kit of cabbage hybrid purity, characterized in that the kit includes claim 1~7 Any one of described in primer pair group；

Preferably, the kit further includes polyacrylamide gel detection architecture or fluorescent capillary electrophoresis tube detection architecture.

9. primer pair group according to any one of claims 1 to 7 or kit according to any one of claims 8 are miscellaneous in identification Chinese cabbage The application in kind of purity is handed over, it is characterized in that: the type of the Chinese cabbage includes folded embracing a class, straight tube class, import encircle-class and domestic Four class of encircle-class；

Preferably, the Chinese cabbage for embracing that class Chinese cabbage includes following any or any above trade name: Shandong six, Shandong seven is folded Number, ox board 19, ox early autumn No. 1, tackling key problem two, the white No.1 in Shandong, Lu Bai six, fine work be imperial No. five expensive, rich anti-60, rich anti-70, the Qin White No. two, moral it is high 16, it is No. 5 precocious, newly select Yuyuan Garden two, highly resistance two；

Straight tube class Chinese cabbage includes the Chinese cabbage of following any or any above trade name: Tianjin Indian mallow leaf, saliva educate 75F1, saliva is educated 60F1, in white 76, in white No. 62, improvement two blueness of Taiyuan, two blueness of Taiyuan, Shanxi dish three, preferably small miscellaneous 56, day be white 75, special day Originally continue dish；

Import encircle-class Chinese cabbage is Japan and Korea S's import kind, the Chinese cabbage including following any or any above trade name: Huang Xin Early 45, Han Chun, early Chinese cabbage advantage, chrysanthemum brocade, Jin Jin, autumn Huang 70, spring month yellow baby cabbage；

Domestic encircle-class Chinese cabbage includes the Chinese cabbage of following any or any above trade name: Beijing is No. three new, rich anti-78, spring Autumn king, the high CR117 of moral, green miscellaneous No. three, small adopted and autumn, the positive Exocarpium Citri Rubrum 65 in day are improved.

10. a kind of method of cabbage hybrid Purity, characterized in that method includes the following steps:

(1) if randomly selecting dry granular seed from Chinese cabbage seeds to be detected, seed broadcasting is grown in container for plant growth, After cotyledon expansion, single seedling cotyledon of clip is spare；

(2) in extraction step (1) material DNA；

(3) using the genomic DNA extracted in step (2) as template, using the described in any item primer pair groups of claim 1~7 PCR amplification is carried out, pcr amplification product is obtained；

(4) pcr amplification product for obtaining step (3) carries out electrophoresis detection, is calculated by counting the number that single band occurs Seed purity.