CN103509869B - Method for identifying juglans plant by using UBE3 gene-based nucleotide formula - Google Patents
Method for identifying juglans plant by using UBE3 gene-based nucleotide formula Download PDFInfo
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- CN103509869B CN103509869B CN201310455292.3A CN201310455292A CN103509869B CN 103509869 B CN103509869 B CN 103509869B CN 201310455292 A CN201310455292 A CN 201310455292A CN 103509869 B CN103509869 B CN 103509869B
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Abstract
The invention discloses a method for identifying or assisting in identifying of a to-be-detected juglans plant. The method comprises the following steps: 1), using a genomic DNA of the to-be-detected juglans plant as a template and conducting PCR (polymerase chain reaction) amplification with a primer pair consisting of two single-chain DNA molecules as shown in sequence 1 and sequence 2 in a sequence table to obtain a PCR product; 2), sequencing the PCR product and listing the nucleotide formula of the to-be-detected juglans plant according to the sequencing result; 3) comparing the nucleotide formula of the to-be-detected juglans plant with a plurality of corresponding species of nucleotide formulas so as to determine the specie of the to-be-detected juglans plant. Experiments prove that the method is accurate and also simple in operation, the production efficiency and the quality monitoring level of related industry are improved, and the defects of a previous method are effectively overcome. In addition, the method achieves the identification of the juglans plant in a non-fruit stage (like a seedling stage) at the same time. The method is of great significance for promoting protection, utilization and breeding of genetic resources of juglans.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of qualification or assistant identification Juglans plants, particularly a kind of method utilizing the nucleic acid molecule formula based on nucleus DNA E3 ubiquitin protein ligase gene sequence construct to identify Juglans plants kind.
Background technology
Juglans Juglans L.(Juglandaceae Juglandaceae) about there are 23 kinds in the world, point 3 groups (walnut group sect.Juglans, black walnut group sect.Rhysocaryon and juglans mandshurica group sect.Cardiocaryon), in Asia, there is distribution in Europe, America and Africa.Walnut J.regia(walnut group sect.Juglans) be the Important Economic seeds producing edible nut.Juglans mandshurica J.mandshurica(juglans mandshurica group sect.Cardiocaryon) be the precious arbor species of production high-quality timber.The excavation of economic plants and utilization are the bases of Walnut Breeding and Sustainable Development of Walnut Industry.
Walnut played in literary composition is special product of China, and for admiring, its nut attractive appearance, shell are thick hard, can be used as " ball for health " and play, and the engraving of nut has very high viewing and admiring and reserve value.Art walnut is described as cheap, the most popular body-building apparatus.Playing and appreciating of Walnut can trace back to Chinese Han dynasty (220 years B.C. 202-Christian eras).
The nut of fiber crops walnut J.hopeiensis, walnut J.regia, juglans mandshurica J.mandshurica, complex peach J.sigillatta all can play.Fiber crops walnut is due to rare numbers, and the matter of nut, shape, look all belong to superior, and be regarded as among the people the superfine product that walnut played in literary composition, price is the highest.Walnut is often scattered in its natural state mixes raw standing forest in walnut and juglans mandshurica, plant rare numbers for fiber crops, with being mainly distributed in the low mountains and hills gentle slope on the ground such as Beijing, Tianjin, Hebei, Shanxi, Shaanxi and gully karness.The fiber crops leaflet profile of walnut, leaflet logarithm, nut external appearance characteristic are all between walnut and juglans mandshurica, and the thick benevolence of nutshell is little, and fruiting quantities is few.For a long time, Chinese mainly pick up from the fruit of the natural mixed raw standing forest of juglans mandshurica and walnut that form slection state is graceful, the thick hard rare Walnut of shell.In recent years owing to viewing and admiring nut rapid rise of price, wild flax walnut was subject to immoderate collection, transplanting and felling, endangered.Fiber crops petal resource is in the urgent need to protection.
The Parents material Worth Expecting of walnut (numb walnut) as breeding played in literary composition.But the nut edibleness that walnut (numb walnut) played in literary composition is not high, and seed natural propagation ability is low, the resources protection and utilization that walnut (numb walnut) played in literary composition is never subject to due attention.Literary composition object for appreciation walnut (numb walnut) new variety of cultivating fast growing are one of important topics.But the origin of playing walnut due to literary composition is unclear, literary composition is played Walnut Breeding and is restricted always.
Plant variety general phenotypic character carries out identifying and identifying.But the phenotypic characteristic of plant variety, by the impact of environment, can be used for qualification and identifies that varietal character exists calendar variation in performance significance.Such as, walnut class plant variety usually mainly relies on nut morphological specificity and classifies, and like this, the season (as seedling stage) beyond the fruit phase just also exists the problem of cultivar identification difficulty.
If can find a kind ofly to disclose the excellent molecule marker in Juglandaceae genetic diversity with very very high resolution in the levels such as kind, kind and genus; and utilize it to develop the method for efficient qualification Juglans plants and kind thereof further; the protection of petal resource, utilization and breeding are played to promotion Walnut Germplasm Resources, particularly literary composition significant.
Summary of the invention
The object of this invention is to provide a kind of method utilizing the nucleic acid molecule formula based on nucleus DNA E3 ubiquitin protein ligase gene (Nuclear DNA E3ubiquitin-protein ligase gene, UBE3) sequence construct to identify Juglans plants and kind thereof.
First object of the present invention is to provide a kind of method of qualification or assistant identification Juglans plants to be measured.
The method of qualification provided by the present invention or assistant identification Juglans plants to be measured, specifically can comprise the steps:
(1) with the genomic dna of Juglans plants to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2 (can specific amplification go out the selection area of UBE3 gene), obtain PCR primer;
(2) described PCR primer is checked order, list the nucleic acid molecule formula of described Juglans plants to be measured according to sequencing result;
The general formula of the nucleic acid molecule formula of described Juglans plants to be measured is:
B
14B
57B
97B
294B
296B
335B
369B
429B
431B
494B
518B
567B
594B
635B
666；
In described general formula, described B
14, described B
57, described B
97, described B
294, described B
296, described B
335, described B
369, described B
429, described B
431, described B
494, described B
518, described B
567, described B
594, described B
635with described B
666represent successively in the nucleotide sequence of described PCR primer, (front 55 base sequences are due to conservative in Juglans the 56th base of the nucleotide sequence of described PCR primer from 5 ' end to be designated as the 1st base, consistent between kind, and delete, only retain variable region sequences), the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 base counted in turn along the nucleotide sequence of described PCR primer to 3 ' extreme direction;
In described general formula, each B is one or both the superposition in A, T, C and G tetra-kinds of bases;
(3) the nucleic acid molecule formula of Juglans plants to be measured described in step (2) gained and nucleic acid molecule formula group I are compared;
Described nucleic acid molecule formula group I is by following a1)-a7) totally 7 kinds of nucleic acid molecule formulas form:
a1）W
14R
57R
97A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
a2）T
14A
57A
97A
294A
296C
335C
369A
429T
431C
494A
518C
567C
594C
635C
666；
a3）A
14G
57G
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a4）A
14G
57A
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a5）T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594T
635T
666；
a6）T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518T
567T
594T
635T
666；
a7）T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594C
635T
666；
At a1) in, W represents base A and T; R represents base A and G; Y represents base T and C; M represents base A and C;
(4) according to the comparison result of step (3), as follows described Juglans plants to be measured is identified:
If the nucleic acid molecule formula of described Juglans plants to be measured is a1) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis);
If the nucleic acid molecule formula of described Juglans plants to be measured is a2) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is Juglans mandshurica (Juglans mandshurica);
If the nucleic acid molecule formula of described Juglans plants to be measured is a3) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is walnut (Juglans regia);
If the nucleic acid molecule formula of described Juglans plants to be measured is a4) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is complex peach (Juglans sigillata);
If the nucleic acid molecule formula of described Juglans plants to be measured is a5) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is American black walnut (Juglans nigra);
If the nucleic acid molecule formula of described Juglans plants to be measured is a6) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is stalwart walnut (Juglans major);
If the nucleic acid molecule formula of described Juglans plants to be measured is a7) shown in nucleic acid molecule formula, then described Juglans plants to be measured be or candidate for the U.S. north California black walnut (Juglans hindsii).
In the above-mentioned methods, described Juglans plants to be measured specifically can be any one in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
Further, the method for above qualification provided by the present invention or assistant identification Juglans plants to be measured, its operation steps can be converted into as follows:
(1) with the genomic dna of Juglans plants to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(2) described PCR primer is checked order, sequencing result and nucleotide sequence group I are compared;
Described nucleotide sequence group I by sequence 3-11 in sequence table totally 9 sequences form;
(3) according to the comparison result of step (2), as follows described Juglans plants to be measured is identified:
If containing sequence 3 or sequence 4 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis);
If containing sequence 5 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured is or candidate is Juglans mandshurica (Juglans mandshurica);
If containing sequence 6 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured is or candidate is walnut (Juglans regia);
If containing sequence 7 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured is or candidate is complex peach (Juglans sigillata);
If containing sequence 8 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured is or candidate is American black walnut (Juglans nigra);
If containing sequence 9 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured is or candidate is stalwart walnut (Juglans major);
If containing sequence 10 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured be or candidate is northern California black walnut (Juglans hindsii) of the U.S..
Second object of the present invention is to provide one and identifies or whether assistant identification Juglans plants to be measured is the method for numb walnut (Juglans hopeiensis).
Provided by the present inventionly to identify or whether assistant identification Juglans plants to be measured is the method for numb walnut (Juglans hopeiensis), specifically can comprise the steps:
A () for template with the genomic dna of Juglans plants to be measured, is carried out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, is obtained PCR primer;
B described PCR primer checks order by (), list the nucleic acid molecule formula of described Juglans plants to be measured according to sequencing result;
The general formula of the nucleic acid molecule formula of described Juglans plants to be measured is:
B
14B
57B
97B
294B
296B
335B
369B
429B
431B
494B
518B
567B
594B
635B
666；
In described general formula, described B
14, described B
57, described B
97, described B
294, described B
296, described B
335, described B
369, described B
429, described B
431, described B
494, described B
518, described B
567, described B
594, described B
635with described B
666represent successively in the nucleotide sequence of described PCR primer, the nucleotide sequence of described PCR primer is designated as the 1st base from the 56th base that 5 ' holds, and (front 55 base sequences are due to conservative in Juglans, consistent between kind, and delete, only retain variable region sequences), the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 base counted in turn along the nucleotide sequence of described PCR primer to 3 ' extreme direction;
In described general formula, each B is one or both the superposition in A, T, C and G tetra-kinds of bases;
C the nucleic acid molecule formula of the nucleic acid molecule formula of Juglans plants to be measured described in step (b) gained and following numb walnut (Juglans hopeiensis) is compared by ():
W
14R
57R
97A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
In formula, W represents base A and T; R represents base A and G; Y represents base T and C; M represents base A and C;
D (), according to the comparison result of step (c), determines whether described Juglans plants to be measured is numb walnut (Juglans hopeiensis) as follows:
If the nucleic acid molecule formula of described Juglans plants to be measured is identical with the nucleic acid molecule formula of described numb walnut (Juglans hopeiensis), then described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis); Otherwise, then described Juglans plants to be measured be not or candidate for numb walnut (Juglans hopeiensis).
In the above-mentioned methods, described Juglans plants to be measured specifically can be any one in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
Further, provided by the present inventionly to identify or whether assistant identification Juglans plants to be measured is the method for numb walnut (Juglans hopeiensis) above, its operation steps can be converted into as follows:
A () for template with the genomic dna of Juglans plants to be measured, is carried out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, is obtained PCR primer;
B described PCR primer checks order by (), sequencing result and nucleotide sequence group II are compared;
Described nucleotide sequence group II by sequence in sequence table 3 and sequence 4 totally 2 sequences form;
C () is according to the comparison result of step (b), determine whether described Juglans plants to be measured is numb walnut (Juglans hopeiensis) as follows: if containing sequence 3 or sequence 4 in the nucleotide sequence of described PCR primer, then described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis); Otherwise, then described Juglans plants to be measured be not or candidate for numb walnut (Juglans hopeiensis).
3rd object of the present invention is to provide a kind of method of qualification or assistant identification numb walnut to be measured (Juglans hopeiensis) kind.
The method of qualification provided by the present invention or assistant identification numb walnut to be measured (Juglans hopeiensis) kind, specifically can comprise the steps:
(A) with the genomic dna of numb walnut to be measured (Juglans hopeiensis) for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(B) described PCR primer is checked order, list the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) according to sequencing result;
The general formula of the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) is:
B
14B
57B
97B
238B
294B
296B
335B
369B
429B
431B
494B
518B
567B
594B
635B
666；
In described general formula, described B
14, described B
57, described B
97, described B
294, described B
238, described B
296, described B
335, described B
369, described B
429, described B
431, described B
494, described B
518, described B
567, described B
594, described B
635with described B
666represent successively in the nucleotide sequence of described PCR primer, the nucleotide sequence of described PCR primer is designated as the 1st base from the 56th base that 5 ' holds, and (front 55 base sequences are due to conservative in Juglans, consistent between kind, and delete, only retain variable region sequences), the 14th, 57,97,238,294,296,335,369,429,431,494,518,567,594,635 and 666 base counted in turn along the nucleotide sequence of described PCR primer to 3 ' extreme direction;
In described general formula, each B is one or both the superposition in A, T, C and G tetra-kinds of bases;
(C) the nucleic acid molecule formula of numb walnut to be measured (Juglans hopeiensis) described in step (B) gained and nucleic acid molecule formula group II are compared:
Described nucleic acid molecule formula group II is by following b1) and b2) totally 2 kinds of nucleic acid molecule formulas form:
b1）W
14R
57R
97C
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
b2）W
14R
57R
97S
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
In formula, W represents base A and T; R represents base A and G; S represents C and G; Y represents base T and C; M represents base A and C;
(D) according to the comparison result of step (C), the kind of described numb walnut to be measured (Juglans hopeiensis) is determined as follows:
If the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) is b1) shown in nucleic acid molecule formula, then described numb walnut to be measured (Juglans hopeiensis) is C
238type fiber crops walnut (Juglans hopeiensis) kind; If the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) is b2) shown in nucleic acid molecule formula, then described numb walnut to be measured (Juglans hopeiensis) is S
238type fiber crops walnut (Juglans hopeiensis) kind;
Described C
238type fiber crops walnut (Juglans hopeiensis) kind be following in any one: ' white large meatball ', ' high-ranking official's cap ', ' heart ';
Described S
238type fiber crops walnut (Juglans hopeiensis) kind be following in any one: ' Stem and leaf of Hongkong Pavetta ', ' apple circle ', ' all the people present building '.
In the above-mentioned methods, after step (D), also can comprise the steps: more described numb walnut to be measured (Juglans hopeiensis) and the described C belonging to it
238type fiber crops walnut (Juglans hopeiensis) kind or described S
238the phenotypic characteristic of each kind in type fiber crops walnut (Juglans hopeiensis) kind; If described numb walnut to be measured (Juglans hopeiensis) and the described C described in it
238type fiber crops walnut (Juglans hopeiensis) kind or described S
238the nut phenotypic characteristic of a certain kind in type fiber crops walnut (Juglans hopeiensis) kind is identical, then described numb walnut to be measured (Juglans hopeiensis) is or candidate is corresponding kind.
At C
238in type fiber crops walnut (Juglans hopeiensis) kind, the nut of ' high-ranking official's cap ' kind is top is vexed tip (shield tip), and base is flat and roomy, the overall full symmetry of nut, and nut diameter is usually greater than 4.8cm, and ornamental value is high; The nut of ' white large meatball ' kind is top in vexed tip (shield tip), and there are a rule and obvious shallow grooves in the top side of nut, and base is put down, but not wide, the overall full symmetry of nut, and nut typically has a diameter from about 3.8-4cm; The nut of ' heart ' kind is obviously top, and base is not bery flat, and also not wide, nut typically has a diameter from about 4.2cm.
At S
238in type fiber crops walnut (Juglans hopeiensis) kind, the nut of ' Stem and leaf of Hongkong Pavetta ' kind is obviously top, also has obvious longitudinal rib ridge between 2 rib ridges of nut, and nut is comparatively large, typically has a diameter from about 4.2-4.6cm; The nut of ' apple circle ' kind is top in vexed tip (shield tip), the obvious shield of convex-concave surface between 2 rib ridges and putting down, and nut typically has a diameter from about 4.2cm; The nut of ' all the people present building ' kind is top in vexed tip (shield tip), the obvious not shield of the convex-concave surface between 2 rib ridges, and the overall full symmetry of nut, nut typically has a diameter from about 4.2cm.
In the above-mentioned methods, described numb walnut to be measured (Juglans hopeiensis) specifically can be any one in following six kinds: ' white large meatball ', ' high-ranking official's cap ', ' heart ', ' Stem and leaf of Hongkong Pavetta ', ' apple circle ' and ' all the people present building '.
Further, the method for above qualification provided by the present invention or assistant identification numb walnut to be measured (Juglans hopeiensis) kind, its operation steps can be converted into as follows:
(A) with the genomic dna of numb walnut to be measured (Juglans hopeiensis) for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(B) described PCR primer is checked order, sequencing result and nucleotide sequence group II are compared;
Described nucleotide sequence group II by sequence in sequence table 3 and sequence 4 totally 2 sequences form;
(C) according to the comparison result of step (B), the kind of described numb walnut to be measured (Juglans hopeiensis) is determined as follows:
If the nucleotide sequence of described PCR primer contains sequence 3, then described numb walnut to be measured (Juglans hopeiensis) is described C
238type fiber crops walnut (Juglans hopeiensis) kind; If the nucleotide sequence of described PCR primer contains sequence 4, then described numb walnut to be measured (Juglans hopeiensis) is described S
238type fiber crops walnut (Juglans hopeiensis) kind.
4th object of the present invention be to provide a kind of for the identification of or the test kit of assistant identification Juglans plants to be measured.
Provided by the present invention for the identification of or the test kit of assistant identification Juglans plants to be measured, specifically can comprise primer pair, record the contrast card of nucleic acid molecule formula group I;
Described primer pair is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
Described nucleic acid molecule formula group I is by following a1)-a7) totally 7 kinds of nucleic acid molecule formulas form:
a1）W
14R
57R
97A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
a2）T
14A
57A
97A
294A
296C
335C
369A
429T
431C
494A
518C
567C
594C
635C
666；
a3）A
14G
57G
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a4）A
14G
57A
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a5）T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594T
635T
666；
a6）T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518T
567T
594T
635T
666；
a7）T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594C
635T
666；
At a1) in, W represents base A and T; R represents base A and G; Y represents base T and C; M represents base A and C;
Described Juglans plants to be measured is any one in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
5th object of the present invention be to provide a kind of for the identification of or the test kit of assistant identification fiber crops walnut (Juglans hopeiensis).
Provided by the present invention for the identification of or assistant identification fiber crops walnut (Juglans hopeiensis) test kit, specifically can comprise primer pair, record the contrast card of nucleic acid molecule formula group II;
Described primer pair is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
Described nucleic acid molecule formula group II is by following b1) and b2) totally 2 kinds of nucleic acid molecule formulas form:
b1）W
14R
57R
97C
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
b2）W
14R
57R
97S
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
In formula, W represents base A and T; R represents base A and G; S represents C and G; Y represents base T and C; M represents base A and C.
Above provided by the present invention for the identification of or the test kit of assistant identification Juglans plants to be measured, the application in qualification or assistant identification Juglans plants to be measured also belongs to protection scope of the present invention.
In the application, described Juglans plants to be measured is any one in following 7 kinds: numb walnut (Juglanshopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
Above provided by the present invention for the identification of or the application of test kit in following (I) or (II) of assistant identification fiber crops walnut (Juglans hopeiensis) also belong to protection scope of the present invention:
(I) to identify or whether assistant identification Juglans plants to be measured is numb walnut (Juglans hopeiensis);
(II) kind of qualification or assistant identification numb walnut to be measured (Juglans hopeiensis).
In (I) described application, described Juglans plants to be measured can be any one in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
In the application described in (II), described numb walnut to be measured (Juglans hopeiensis) can be any one in following six kinds: ' white large meatball ', ' high-ranking official's cap ', ' heart ', ' Stem and leaf of Hongkong Pavetta ', ' apple circle ' and ' all the people present building '.
In the present invention, sequence 3-11 totally 9 sequences forms by 700 Nucleotide, is a part for Juglans plants UBE3 gene coding region, and the UBE3 gene coding region position consistency (being designated as UBE3-700 sequence) of correspondence.In nucleic acid molecule formulas all above, the bottom right footmark numeral number of base is all according to sequence 3-11(UBE3-700 sequence) in base sequencing carry out in turn marking, with 5 ' of UBE3-700 sequence end the 1st base number for 1.
One section of sequence that the present invention utilizes the 700bp of the coding region of nucleus DNA E3 ubiquitin protein ligase gene (UBE3) long, identifies Juglans plants with the form of nucleic acid molecule formula.Experiment proves, this research finds, regulating and controlling one section of long sequence of the 700bp of relevant nucleus E3 ubiquitin ligase gene (UBE3) coding region in kind, kind and belong to 3 levels disclose in Juglandaceae genetic diversity and have very high resolving power with protein metabolism, is excellent molecule marker.The method of this molecular markers for identification Juglans plants that utilizes provided by the present invention is comparatively accurate, simple to operation, production efficiency and the quality monitoring level of related industries can be improved, effectively make up the deficiency of previous methods, analysis precision can be made to reach the level of 1 nucleic acid molecule (nano level); In addition, the method achieves the accurate qualification of non-fruit phase (as seedling stage) simultaneously.The present invention is for promoting that the protection of Walnut Germplasm Resources, utilization and breeding are all significant.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The plant leaf sample of fiber crops walnut (Juglans hopeiensis) kind ' white large meatball ', ' high-ranking official's cap ', ' heart ', ' Stem and leaf of Hongkong Pavetta ', ' apple circle ' and ' all the people present building ', all gathers from profit source, Yi County, Hebei province woods fruit plantation company limited fiber crops walnut base (being positioned at Liu Jingxiang Xi Baoquan village, Yi County).
The plant leaf sample of common edible walnut (Juglans regia), all gathers from profit source, Yi County, Hebei province woods fruit plantation company limited fiber crops walnut base (being positioned at Liu Jingxiang Xi Baoquan village, Yi County), Wild jujube in Taihang Mountain Area, Luoning County, Henan Province forestry bureau.
The plant leaf sample of Juglans mandshurica (Juglans mandshurica), all gathers and builds Village from Yi County, Hebei province Liu Jingxiang), Wild jujube in Taihang Mountain Area, Beijing Botanical Garden.
The plant leaf sample of Yunnan complex peach (Juglans sigillata), gathers from forest-science academy of Yunnan Province (Kunming) and Dali prefecture.
The plant leaf sample of American black walnut (Juglans nigra), U.S. California black walnut, north (Juglans hindsii) and the U.S. Ya Lisangna state black walnut (stalwart walnut) (Juglans major), all gathers from Luoning County, Henan Province forestry bureau.
The plant leaf sample of Cyclocarya paliurus (Cyclocarya paliurus) and Chinese ash (Pterocarya stenoptera), all gathers from Beijing Botanical Garden.
Embodiment 1, for the identification of the foundation of the nucleic acid molecule formula of Juglans plants and application
The nucleic acid molecule formula of Juglans plants is can be used in effectively identifying in order to build, the present inventor obtains nucleus DNA E3 ubiquitin protein ligase gene (Nuclear DNA E3ubiquitin-protein ligase gene from following serial Juglans plants, UBE3) coding region partial sequence, and statistical study has been carried out to pleomorphism site wherein.Involved (for examination material) Juglans plants totally 12 kinds, specific as follows:
Walnut 6 kinds played in literary composition---numb walnut (Juglans hopeiensis) kind ' white large meatball ', ' high-ranking official's cap ', ' heart ', ' Stem and leaf of Hongkong Pavetta ', ' apple circle ' and ' all the people present building '.
Walnut 6 kinds---common edible walnut (Juglans regia) played in non-literary composition; Juglans mandshurica (Juglans mandshurica); Yunnan complex peach (Juglans sigillata); American black walnut (Juglans nigra); U.S. California black walnut, north (Juglans hindsii); U.S. Ya Lisangna state black walnut (stalwart walnut) (Juglans major).
One, design of primers
According to the conserved regions of nucleus DNA E3 ubiquitin protein ligase gene (Nuclear DNA E3ubiquitin-protein ligase gene, UBE3), design following primer:
Forward primer (title: H_nrDNA_UBE3_23F): 5 ’ – TCGCCTCCAAGTTCAGTG – 3 ' (sequence 1);
Reverse primer (title: H_nrDNA_UBE3_838R): 5 ’ – CTCCCATAGGTGTAGTTCCA – 3 ' (sequence 2).
Two, the sequencing of pcr amplification and product
With the blade of 12 of Juglans plants kinds of test samples for experiment material, ordinary method (as CTAB method or commercially available plant genome DNA extract test kit) is adopted to extract the genomic dna of each material.With gained genomic dna for template, the primer pair adopting step one to design carries out pcr amplification.Result shows, and it is the product of 816bp that often kind of Juglans plants all can obtain a size.
Utilize reverse primer (H_nrDNA_UBE3_838R) to carry out unidirectional order-checking to each PCR primer after purifying, obtain the DNA sequence dna (being designated as UBE3-700) of one section of 700bp containing some polymorphic base position.UBE3-700 sequence is the 56-755 position of nucleotide sequence from 5 ' end of described PCR primer.Every kind of plant sample check order more than 3 or 3 individual, found that, between individuality, the sequencing result of UBE3-700 sequence is consistent in the kind of 12 kind of plant and in kind.
Wherein, the numb walnut (Juglans hopeiensis) of 6 kinds
The UBE3-700 sequence of fiber crops walnut (Juglans hopeiensis) kind ' white large meatball ', ' high-ranking official's cap ' and ' heart ' these 3 kinds of Juglans plants is as shown in sequence in sequence table 3;
The UBE3-700 sequence of fiber crops walnut (Juglans hopeiensis) kind ' Stem and leaf of Hongkong Pavetta ', ' apple circle ' and ' all the people present building ' these 3 kinds of Juglans plants is as shown in sequence in sequence table 4;
The UBE3-700 sequence of Juglans mandshurica (Juglans mandshurica) is as shown in sequence in sequence table 5;
The UBE3-700 sequence of common edible walnut (Juglans regia) is as shown in sequence in sequence table 6;
The UBE3-700 sequence of Yunnan complex peach (Juglans sigillata) is as shown in sequence in sequence table 7;
The UBE3-700 sequence of American black walnut (Juglans nigra) is as shown in sequence in sequence table 8;
The UBE3-700 sequence in U.S. Ya Lisangna state black walnut (stalwart walnut) (Juglans major) is as shown in sequence in sequence table 9;
The UBE3-700 sequence of U.S. California black walnut, north (Juglans hindsii) is as shown in sequence in sequence table 10.
Three, sequence alignment
The UBE3-700 sequence of 12 of step 2 gained kinds of Juglans plants is compared, obtains 16 polymorphic base position as shown in table 1.
16 polymorphic base position of the UBE3-700 sequence acquisition of table 1 12 kinds of Juglans plants
Note: W represents A and T; R represents A and G; Y represents T and C; M represents A and C; S represents C and G.
When not considering the 238th base position polymorphism of UBE3-700 sequence, play for walnut (numb walnut) for literary composition, have 11 in 16 polymorphic sites listed in table 1 for heterozygous polymorphic site (the 14th, 57,97,296,335,369,429,431,518,594 and 666 Nucleotide).And for other 7 kinds of Juglans plants, 16 polymorphic sites listed in table 1 are mononucleotide type polymorphic site.For heterozygous polymorphic site, significantly superposition can be obtained when checking order bimodal, and mononucleotide type polymorphic site shows as simple spike.
Four, for the identification of the structure of the nucleic acid molecule formula of Juglans plants
1, the situation of the 238th base position polymorphism of UBE3-700 sequence is not considered
When not considering the 238th base position polymorphism of UBE3-700 sequence, according to often kind of polymorphic base position of Juglans plants listed in table 1, list corresponding nucleic acid molecule formula, specific as follows:
Fiber crops walnut (Juglans hopeiensis) 6 kinds UBE3-700 sequence from 5 ' end the 14th, 57, 97, 294, 296, 335, 369, 429, 431, 494, 518, 567, 594, 635 and 666 are respectively W (=A+T), R (=A+G), R (=A+G), A, W (=A+T), Y (=T+C), Y (=T+C), M (=A+C), Y (=T+C), C, R (=A+G), C, Y (=T+C), C, Y (=T+C), according to these Nucleotide and the position in UBE3-700 sequence (from 5 ' end meter) thereof, listing nucleic acid molecule formula general formula is: W
14r
57r
97a
294w
296y
335y
369m
429y
431c
494r
518c
567y
594c
635y
666.
From 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 of the UBE3-700 sequence of Juglans mandshurica (Juglans mandshurica) is respectively T, A, A, A, A, C, C, A, T, C, A, C, C, C, C, according to these Nucleotide and the position in UBE3-700 sequence (from 5 ' end meter) thereof, listing nucleic acid molecule formula is: T
14a
57a
97a
294a
296c
335c
369a
429t
431c
494a
518c
567c
594c
635c
666.
From 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 of the UBE3-700 sequence of common edible walnut (Juglans regia) is respectively A, G, G, A, T, T, T, C, C, C, G, C, T, C, T, according to these Nucleotide and the position in UBE3-700 sequence (from 5 ' end meter) thereof, listing nucleic acid molecule formula is: A
14g
57g
97a
294t
296t
335t
369c
429c
431c
494g
518c
567t
594c
635t
666.
From 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 of the UBE3-700 sequence of Yunnan complex peach (Juglans sigillata) is respectively A, G, A, A, T, T, T, C, C, C, G, C, T, C, T, according to these Nucleotide and the position in UBE3-700 sequence (from 5 ' end meter) thereof, listing nucleic acid molecule formula is: A
14g
57a
97a
294t
296t
335t
369c
429c
431c
494g
518c
567t
594c
635t
666.
From 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 of the UBE3-700 sequence of American black walnut (Juglans nigra) is respectively T, G, A, G, T, T, C, A, C, T, G, C, T, T, T, according to these Nucleotide and the position in UBE3-700 sequence (from 5 ' end meter) thereof, listing nucleic acid molecule formula is: T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518c
567t
594t
635t
666.
From 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 of the UBE3-700 sequence in U.S. Ya Lisangna state black walnut (stalwart walnut) (Juglans major) is respectively T, G, A, G, T, T, C, A, C, T, G, T, T, T, T, according to these Nucleotide and the position in UBE3-700 sequence (from 5 ' end meter) thereof, listing nucleic acid molecule formula is: T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518t
567t
594t
635t
666.
The U.S. north California black walnut (Juglans hindsii) UBE3-700 sequence from 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 be respectively T, G, A, G, T, T, C, A, C, T, G, C, T, C, T, according to these Nucleotide and the position in UBE3-700 sequence (from 5 ' end meter) thereof, listing nucleic acid molecule formula is: T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518c
567t
594c
635t
666.
2, the situation of the 238th base position polymorphism of UBE3-700 sequence is considered
Consider UBE3
-when the 238th base position polymorphism of 700 sequences, each literary composition is played Walnut Cultivars and can be represented by longer molecular formula, and literary composition can be played Walnut Cultivars and divide into 2 types further.Specific as follows:
Type i (C
238type): comprise 3 literary compositions and play walnut (numb walnut) kind, i.e. ' white large meatball ', ' high-ranking official's cap ' and ' heart '.UBE3-700 sequence from 5 ' end the 238th be C.Its nucleic acid molecule formula is:
W
14R
57R
97C
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666。
Type II (S
238type): comprise 3 literary compositions and play walnut (numb walnut) kind, be i.e. ' Stem and leaf of Hongkong Pavetta ', ' apple circle ' and ' all the people present building '.UBE3-700 sequence from 5 ' end the 238th be S (=C+G).Its nucleic acid molecule formula is:
W
14R
57R
97S
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666。
Five, with nucleic acid molecule formula qualification Juglans plants
1, the situation of the 238th base position polymorphism of UBE3-700 sequence is not considered
(1) with the genomic dna of Juglans plants to be measured for template, carry out pcr amplification with primer pair (sequence 1/ sequence 2), obtain PCR primer;
(2) described PCR primer is checked order, list the nucleic acid molecule formula of described Juglans plants to be measured according to sequencing result;
The general formula of the nucleic acid molecule formula of described Juglans plants to be measured is:
B
14B
57B
97B
294B
296B
335B
369B
429B
431B
494B
518B
567B
594B
635B
666；
In described general formula, described B
14, described B
57, described B
97, described B
294, described B
296, described B
335, described B
369, described B
429, described B
431, described B
494, described B
518, described B
567, described B
594, described B
635with described B
666represent successively in the nucleotide sequence of described PCR primer, the nucleotide sequence of described PCR primer is designated as the 1st base from the 56th base that 5 ' holds, the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 base (the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 base of corresponding UBE3-700 sequence) counted in turn along the nucleotide sequence of described PCR primer to 3 ' extreme direction;
In described general formula, each B is one or both the superposition in A, T, C and G tetra-kinds of bases;
(3) as follows described Juglans plants to be measured is identified:
If the nucleic acid molecule formula of described Juglans plants to be measured is W
14r
57r
97a
294w
296y
335y
369m
429y
431c
494r
518c
567y
594c
635y
666, then described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis);
If the nucleic acid molecule formula of described Juglans plants to be measured is T
14a
57a
97a
294a
296c
335c
369a
429t
431c
494a
518c
567c
594c
635c
666, then described Juglans plants to be measured is or candidate is Juglans mandshurica (Juglans mandshurica);
If the nucleic acid molecule formula of described Juglans plants to be measured is A
14g
57g
97a
294t
296t
335t
369c
429c
431c
494g
518c
567t
594c
635t
666, then described Juglans plants to be measured is or candidate is walnut (Juglans regia);
If the nucleic acid molecule formula of described Juglans plants to be measured is A
14g
57a
97a
294t
296t
335t
369c
429c
431c
494g
518c
567t
594c
635t
666, then described Juglans plants to be measured is or candidate is complex peach (Juglans sigillata);
If the nucleic acid molecule formula of described Juglans plants to be measured is T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518c
567t
594t
635t
666, then described Juglans plants to be measured is or candidate is American black walnut (Juglans nigra);
If the nucleic acid molecule formula of described Juglans plants to be measured is T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518t
567t
594t
635t
666, then described Juglans plants to be measured is or candidate is stalwart walnut (Juglans major);
If the nucleic acid molecule formula of described Juglans plants to be measured is T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518c
567t
594c
635t
666, then described Juglans plants to be measured be or candidate for the U.S. north California black walnut (Juglans hindsii).
2, the 238th base position polymorphism of UBE3-700 sequence is considered
(1) with the genomic dna of numb walnut to be measured (Juglans hopeiensis) for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(2) described PCR primer is checked order, list the nucleic acid molecule formula (method is the same) of described numb walnut to be measured (Juglans hopeiensis) according to sequencing result;
(3) kind of described numb walnut to be measured (Juglans hopeiensis) is determined as follows:
If the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) is W
14r
57r
97c
238a
294w
296y
335y
369m
429y
431c
494r
518c
567y
594c
635y
666, then described numb walnut to be measured (Juglans hopeiensis) is C
238type fiber crops walnut (Juglans hopeiensis) kind (as ' white large meatball ', ' high-ranking official's cap ', ' heart ');
If the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) is W
14r
57r
97s
238a
294w
296y
335y
369m
429y
431c
494r
518c
567y
594c
635y
666, then described numb walnut to be measured (Juglans hopeiensis) is S
238type fiber crops walnut (Juglans hopeiensis) kind (as ' Stem and leaf of Hongkong Pavetta ', ' apple circle ', ' all the people present building ').
Further, also by more described numb walnut to be measured (Juglans hopeiensis) and the described C belonging to it
238type fiber crops walnut (Juglans hopeiensis) kind or described S
238the nut phenotypic characteristic of each kind in type fiber crops walnut (Juglans hopeiensis) kind, thus determine the concrete kind of described numb walnut to be measured (Juglans hopeiensis).In detail as follows:
At C
238in type fiber crops walnut (Juglans hopeiensis) kind, the nut of ' high-ranking official's cap ' kind is top is vexed tip (shield tip), and base is flat and roomy, the overall full symmetry of nut, and nut diameter is usually greater than 4.8cm, and ornamental value is high; The nut of ' white large meatball ' kind is top in vexed tip (shield tip), and there are a rule and obvious shallow grooves in the top side of nut, and base is put down, but not wide, the overall full symmetry of nut, usual about the 3.8-4cm of nut diameter; The nut of ' heart ' kind is obviously top, and base is not bery flat, and also not wide, nut diameter is usually at about 4.2cm.
At S
238in type fiber crops walnut (Juglans hopeiensis) kind, the nut of ' Stem and leaf of Hongkong Pavetta ' kind is obviously top, also has obvious longitudinal rib ridge between 2 rib ridges of nut, and nut is comparatively large, typically has a diameter from about 4.2-4.6cm; The nut of ' apple circle ' kind is top in vexed tip (shield tip), the obvious shield of convex-concave surface between 2 rib ridges, usual about the 4.2cm of nut diameter; The nut of ' all the people present building ' kind is top in vexed tip (shield tip), the obvious not shield of the convex-concave surface between 2 rib ridges, and the overall full symmetry of nut, nut typically has a diameter from about 4.2cm.
Embodiment 2, other platymisciums of use nucleic acid molecule formula qualification Juglandaceae
Cyclocarya paliurus (Cyclocarya paliurus): Juglandaceae, cyclocarya.
Chinese ash (Pterocarya stenoptera): Juglandaceae, Chinese ash belong to.
In order to verify nucleic acid molecule formula that embodiment 1 the sets up specificity for Juglans plant, the present inventor also acquires the plant leaf of Cyclocarya paliurus (Cyclocarya paliurus) and Chinese ash (Pterocarya stenoptera), and according to the method for embodiment 1, extract its genomic dna, pcr amplification is carried out with the primer pair be made up of two single strand dnas shown in sequence 1 and sequence 2, obtain the UBE3-700 sequence (sequence 11) of Cyclocarya paliurus (Cyclocarya paliurus) and the UBE3-700 sequence (sequence 12) of Chinese ash (Pterocarya stenoptera) respectively.
Further, list the nucleic acid molecule formula (not considering the 238th bit base) of Cyclocarya paliurus (Cyclocarya paliurus) and Chinese ash (Pterocarya stenoptera), as follows:
Cyclocarya paliurus: T
14g
57a
97a
294t
296t
335c
369a
429c
431c
494g
518c
567t
594c
635t
666;
Chinese ash: T
14g
57a
97a
294t
296t
335c
369a
429c
431c
494g
518c
567t
594c
635t
666.
Visible, Cyclocarya paliurus (Cyclocarya paliurus) is all not identical with 7 kinds of nucleic acid molecule formulas (not considering the 238th bit base) of kind of the Juglans plants of 12 in embodiment 1 with the nucleic acid molecule formula of Chinese ash (Pterocarya stenoptera).Thus confirm validity and the specificity of the inventive method.
It is maternal instinct monolepsis that the present inventor studies the Chloroplast gene sequence finding Juglans plants, coordinated the nucleic acid molecule formula from nuclear genome provided by the present invention to use, the effect for the Diversity Detection of Juglans plants can be better.
Claims (7)
1. a method for qualification or assistant identification Juglans plants to be measured, comprises the steps:
(1) with the genomic dna of Juglans plants to be measured for template, carry out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, obtain PCR primer;
(2) described PCR primer is checked order, list the nucleic acid molecule formula of described Juglans plants to be measured according to sequencing result;
The general formula of the nucleic acid molecule formula of described Juglans plants to be measured is:
B
14B
57B
97B
294B
296B
335B
369B
429B
431B
494B
518B
567B
594B
635B
666；
In described general formula, described B
14, described B
57, described B
97, described B
294, described B
296, described B
335, described B
369, described B
429, described B
431, described B
494, described B
518, described B
567, described B
594, described B
635with described B
666represent successively in the nucleotide sequence of described PCR primer, the nucleotide sequence of described PCR primer is designated as the 1st base from the 56th base that 5 ' holds, the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 base counted in turn along the nucleotide sequence of described PCR primer to 3 ' extreme direction;
In described general formula, each B is one or both the superposition in A, T, C and G tetra-kinds of bases;
(3) the nucleic acid molecule formula of Juglans plants to be measured described in step (2) gained and nucleic acid molecule formula group I are compared;
Described nucleic acid molecule formula group I is by following a1)-a7) totally 7 kinds of nucleic acid molecule formulas form:
a1)W
14R
57R
97A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
a2)T
14A
57A
97A
294A
296C
335C
369A
429T
431C
494A
518C
567C
594C
635C
666；
a3)A
14G
57G
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a4)A
14G
57A
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a5)T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594T
635T
666；
a6)T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518T
567T
594T
635T
666；
a7)T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594C
635T
666；
At a1) in, W represents base A and T; R represents base A and G; Y represents base T and C; M represents base A and C;
(4) according to the comparison result of step (3), as follows described Juglans plants to be measured is identified:
If the nucleic acid molecule formula of described Juglans plants to be measured is a1) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis);
If the nucleic acid molecule formula of described Juglans plants to be measured is a2) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is Juglans mandshurica (Juglans mandshurica);
If the nucleic acid molecule formula of described Juglans plants to be measured is a3) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is walnut (Juglans regia);
If the nucleic acid molecule formula of described Juglans plants to be measured is a4) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is complex peach (Juglans sigillata);
If the nucleic acid molecule formula of described Juglans plants to be measured is a5) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is American black walnut (Juglans nigra);
If the nucleic acid molecule formula of described Juglans plants to be measured is a6) shown in nucleic acid molecule formula, then described Juglans plants to be measured is or candidate is stalwart walnut (Juglans major);
If the nucleic acid molecule formula of described Juglans plants to be measured is a7) shown in nucleic acid molecule formula, then described Juglans plants to be measured be or candidate for the U.S. north California black walnut (Juglans hindsii).
2. method according to claim 1, is characterized in that: described Juglans plants to be measured is any one in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
3. to identify or whether assistant identification Juglans plants to be measured is a method of numb walnut (Juglans hopeiensis), comprise the steps:
A () for template with the genomic dna of Juglans plants to be measured, is carried out pcr amplification with the primer pair be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2, is obtained PCR primer;
B described PCR primer checks order by (), list the nucleic acid molecule formula of described Juglans plants to be measured according to sequencing result;
The general formula of the nucleic acid molecule formula of described Juglans plants to be measured is:
B
14B
57B
97B
294B
296B
335B
369B
429B
431B
494B
518B
567B
594B
635B
666；
In described general formula, described B
14, described B
57, described B
97, described B
294, described B
296, described B
335, described B
369, described B
429, described B
431, described B
494, described B
518, described B
567, described B
594, described B
635with described B
666represent successively in the nucleotide sequence of described PCR primer, the nucleotide sequence of described PCR primer is designated as the 1st base from the 56th base that 5 ' holds, the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 base counted in turn along the nucleotide sequence of described PCR primer to 3 ' extreme direction;
In described general formula, each B is one or both the superposition in A, T, C and G tetra-kinds of bases;
C the nucleic acid molecule formula of the nucleic acid molecule formula of Juglans plants to be measured described in step (b) gained and following numb walnut (Juglans hopeiensis) is compared by ():
W
14R
57R
97A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
In formula, W represents base A and T; R represents base A and G; Y represents base T and C; M represents base A and C;
D (), according to the comparison result of step (c), determines whether described Juglans plants to be measured is numb walnut (Juglans hopeiensis) as follows:
If the nucleic acid molecule formula of described Juglans plants to be measured is identical with the nucleic acid molecule formula of described numb walnut (Juglans hopeiensis), then described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis); Otherwise, then described Juglans plants to be measured be not or candidate for numb walnut (Juglans hopeiensis).
4. for the identification of or the test kit of assistant identification Juglans plants to be measured, comprise primer pair, record the contrast card of nucleic acid molecule formula group I;
Described primer pair is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
Described nucleic acid molecule formula group I is by following a1)-a7) totally 7 kinds of nucleic acid molecule formulas form:
a1)W
14R
57R
97A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
a2)T
14A
57A
97A
294A
296C
335C
369A
429T
431C
494A
518C
567C
594C
635C
666；
a3)A
14G
57G
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a4)A
14G
57A
97A
294T
296T
335T
369C
429C
431C
494G
518C
567T
594C
635T
666；
a5)T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594T
635T
666；
a6)T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518T
567T
594T
635T
666；
a7)T
14G
57A
97G
294T
296T
335C
369A
429C
431T
494G
518C
567T
594C
635T
666；
At a1) in, W represents base A and T; R represents base A and G; Y represents base T and C; M represents base A and C;
Described Juglans plants to be measured is any one in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
5. for the identification of or the test kit of assistant identification numb walnut to be measured (Juglans hopeiensis), comprise primer pair, record the contrast card of nucleic acid molecule formula group II;
Described primer pair is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
Described nucleic acid molecule formula group II is by following b1) and b2) totally 2 kinds of nucleic acid molecule formulas form:
b1)W
14R
57R
97C
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
b2)W
14R
57R
97S
238A
294W
296Y
335Y
369M
429Y
431C
494R
518C
567Y
594C
635Y
666；
In formula, W represents base A and T; R represents base A and G; S represents C and G; Y represents base T and C; M represents base A and C.
6. the application of test kit described in claim 4 in qualification or assistant identification Juglans plants to be measured;
Described Juglans plants to be measured is any one in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), American black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S. California black walnut, north (Juglans hindsii).
7. test kit described in claim 5 is being identified or whether assistant identification Juglans plants to be measured is application in numb walnut (Juglans hopeiensis).
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