CN103509869A - 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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- CN103509869A CN103509869A CN201310455292.3A CN201310455292A CN103509869A CN 103509869 A CN103509869 A CN 103509869A CN 201310455292 A CN201310455292 A CN 201310455292A CN 103509869 A CN103509869 A CN 103509869A
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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, a kind of method that relates to evaluation or assistant identification Juglans plants, particularly the nucleic acid molecule formula of a kind of utilization based on nucleus DNA E3 ubiquitin protein ligase gene sequence construct identified the method for Juglans plants kind.
Background technology
Juglans Juglans L.(Juglandaceae Juglandaceae) approximately there are 23 kinds in the world, divide 3 groups of (walnut group sect.Juglans, black walnut group sect.Rhysocaryon and juglans mandshurica group sect.Cardiocaryon) ， Asia, Europe, America and Africa to have distribution.Walnut J.regia(walnut group sect.Juglans) be the Important Economic seeds that produce 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 outward appearance is graceful, shell is thick hard, can be used as " ball for health " and plays, 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 of Walnut can be traced back to Chinese Han dynasty (220 years B.C. 202-Christian eras) with appreciating.
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 because quantity is rare, and it is superior that the matter of nut, shape, look all belong to, and at the superfine product that is regarded as literary composition object for appreciation walnut among the people, price is the highest.Fiber crops walnut is normal scattered in walnut and the mixed raw standing forest of juglans mandshurica under state of nature, and plant quantity is rare, is mainly distributed in the Di Hegou valley floor, low mountains and hills gentle slope on the ground such as Beijing, Tianjin, Hebei, Shanxi, Shaanxi.The leaflet profile of fiber crops 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.Owing to viewing and admiring nut price, skyrocketed in recent years, wild numb walnut is subject to immoderate collection, transplanting and felling, endangered.Fiber crops petal resource is in the urgent need to protection.
Literary composition is played walnut (numb walnut) as the Parents material Worth Expecting of breeding.Yet the nut edibleness that walnut (numb walnut) played in literary composition is not high, seed natural propagation ability is low, and literary composition is played the conservation of resources of walnut (numb walnut) and utilized and 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.Yet the origin of playing walnut due to literary composition is unclear, literary composition is played Walnut Breeding and is restricted always.
Plant variety general using phenotypic character is identified and identifies.But the phenotypic characteristic of plant variety is subject to the impact of environment, can be used for identifying and identify varietal character having calendar variation aspect performance significance.For example, the walnut class plant variety conventionally main nut morphological specificity that relies on is classified, and like this, just exist the problem of cultivar identification difficulty the season (as seedling stage) beyond the fruit phase.
If can find that a kind of announcement aspect Juglandaceae genetic diversity has the very good molecule marker of very high resolution in the levels such as kind, kind and genus; and further utilize it to develop the method for efficient evaluation Juglans plants and kind thereof; to promoting Walnut Germplasm Resources, particularly protection, utilization and the breeding of literary composition object for appreciation petal resource are significant.
Summary of the invention
The object of this invention is to provide the nucleic acid molecule formula of a kind of utilization based on nucleus DNA E3 ubiquitin protein ligase gene (Nuclear DNA E3ubiquitin-protein ligase gene, UBE3) sequence construct and identify the method for Juglans plants and kind thereof.
First object of the present invention is to provide a kind of method of evaluation or assistant identification Juglans plants to be measured.
The method of evaluation provided by the present invention or assistant identification Juglans plants to be measured, specifically can comprise the steps:
(1) take the genomic dna of Juglans plants to be measured is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table (selection area that can specific amplification goes out UBE3 gene) to carry out pcr amplification, obtains PCR product;
(2), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula of described Juglans plants to be measured;
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
666be illustrated in successively in the nucleotide sequence of described PCR product, the 56th base by the nucleotide sequence of described PCR product from 5 ' end is designated as the 1st base, 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 bases counting in turn to 3 ' extreme direction along the nucleotide sequence of described PCR product;
In described general formula, each B is one or both the stack in A, T, C and tetra-kinds of bases of G;
(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, 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, 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, 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, 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, described Juglans plants to be measured is or candidate is eastern united states 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, 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, described Juglans plants to be measured is or candidate is U.S. north California black walnut (Juglans hindsii).
In aforesaid method, described Juglans plants to be measured specifically can be any in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
Further, the method for above evaluation provided by the present invention or assistant identification Juglans plants to be measured, its operation steps can be converted into as follows:
(1) take the genomic dna of Juglans plants to be measured is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(2) by described PCR product order-checking, sequencing result and nucleotide sequence group I are compared;
Described nucleotide sequence group I in sequence table sequence 3-11 totally 9 sequences form;
(3), according to the comparison result of step (2), as follows described Juglans plants to be measured is identified:
If contain sequence 3 or sequence 4 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis);
If contain sequence 5 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is Juglans mandshurica (Juglans mandshurica);
If contain sequence 6 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is walnut (Juglans regia);
If contain sequence 7 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is complex peach (Juglans sigillata);
If contain sequence 8 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is eastern united states black walnut (Juglans nigra);
If contain sequence 9 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is stalwart walnut (Juglans major);
If contain sequence 10 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is U.S. north California black walnut (Juglans hindsii).
Second object of the present invention is to provide a kind of identify or whether assistant identification Juglans plants to be measured is the method for numb walnut (Juglans hopeiensis).
Provided by the present invention identify or whether assistant identification Juglans plants to be measured is the method for numb walnut (Juglans hopeiensis), specifically can comprise the steps:
(a) take the genomic dna of Juglans plants to be measured is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(b), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula of described Juglans plants to be measured;
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
666be illustrated in successively in the nucleotide sequence of described PCR product, the nucleotide sequence of described PCR product is designated as to the 1st base from 5 ' the 56th base of holding, 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 bases counting in turn to 3 ' extreme direction along the nucleotide sequence of described PCR product;
In described general formula, each B is one or both the stack in A, T, C and tetra-kinds of bases of G;
(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:
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), determine as follows whether described Juglans plants to be measured is numb walnut (Juglans hopeiensis):
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), described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis); Otherwise, described Juglans plants to be measured be not or candidate for numb walnut (Juglans hopeiensis).
In aforesaid method, described Juglans plants to be measured specifically can be any in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
Further, above provided by the present invention identify or whether assistant identification Juglans plants to be measured is the method for numb walnut (Juglans hopeiensis), its operation steps can be converted into as follows:
(a) take the genomic dna of Juglans plants to be measured is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(b) by described PCR product order-checking, sequencing result and nucleotide sequence group II are compared;
Described nucleotide sequence group II in sequence table sequence 3 and sequence 4 totally 2 sequences form;
(c) according to the comparison result of step (b), determine as follows whether described Juglans plants to be measured is numb walnut (Juglans hopeiensis): if contain sequence 3 or sequence 4 in the nucleotide sequence of described PCR product, described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis); Otherwise, described Juglans plants to be measured be not or candidate for numb walnut (Juglans hopeiensis).
The 3rd object of the present invention is to provide a kind of method of evaluation or assistant identification numb walnut to be measured (Juglans hopeiensis) kind.
The method of evaluation provided by the present invention or assistant identification numb walnut to be measured (Juglans hopeiensis) kind, specifically can comprise the steps:
(A) take the genomic dna of numb walnut to be measured (Juglans hopeiensis) is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(B), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis);
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
666be illustrated in successively in the nucleotide sequence of described PCR product, the nucleotide sequence of described PCR product is designated as to the 1st base from 5 ' the 56th base of holding, 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 bases counting in turn to 3 ' extreme direction along the nucleotide sequence of described PCR product;
In described general formula, each B is one or both the stack in A, T, C and tetra-kinds of bases of G;
(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), determine as follows the kind of described numb walnut to be measured (Juglans hopeiensis):
If the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) is b1) shown in nucleic acid molecule formula, 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, 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 as lower any: ' large meatball in vain ', ' high-ranking official's cap ', ' heart ';
Described S
238type fiber crops walnut (Juglans hopeiensis) kind be as lower any: ' Stem and leaf of Hongkong Pavetta ', ' apple is justified ', ' all the people present building '.
In aforesaid method, in step (D), also can comprise the steps: afterwards the described C that more described numb walnut to be measured (Juglans hopeiensis) is affiliated with 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, and 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 top vexed sharp type (shield point type) that is of the nut of ' high-ranking official's cap ' kind, base is flat and roomy, the whole full symmetry of nut, nut diameter is usually greater than 4.8cm, and ornamental value is high; The top vexed sharp type (shield point type) that is of nut of ' white large meatball ' kind, there is a rule the top side of nut and obvious shallow grooves, and base is flat, but not wide, the whole full symmetry of nut, nut typically has a diameter from 3.8-4cm left and right; The nut of ' heart ' kind is top obvious, and base is not bery flat, also not wide, and nut typically has a diameter from 4.2cm left and right.
At S
238in type fiber crops walnut (Juglans hopeiensis) kind, the nut of ' Stem and leaf of Hongkong Pavetta ' kind is top obviously, also has significantly longitudinally rib ridge between 2 rib ridges of nut, and nut is larger, typically has a diameter from 4.2-4.6cm left and right; The top vexed sharp type (shield point type) that is of nut of ' apple circle ' kind, the obvious shield of convex-concave surface between 2 rib ridges and put down, nut typically has a diameter from about 4.2cm; The top vexed sharp type (shield point type) that is of the nut of ' all the people present building ' kind, the convex-concave surface between 2 rib ridges is shield not obviously, the whole full symmetry of nut, nut typically has a diameter from 4.2cm left and right.
In aforesaid method, described numb walnut to be measured (Juglans hopeiensis) specifically can be any 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 evaluation 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) take the genomic dna of numb walnut to be measured (Juglans hopeiensis) is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(B) by described PCR product order-checking, sequencing result and nucleotide sequence group II are compared;
Described nucleotide sequence group II in sequence table sequence 3 and sequence 4 totally 2 sequences form;
(C), according to the comparison result of step (B), determine as follows the kind of described numb walnut to be measured (Juglans hopeiensis):
If the nucleotide sequence of described PCR product contains sequence 3, 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 product contains sequence 4, described numb walnut to be measured (Juglans hopeiensis) is described S
238type fiber crops walnut (Juglans hopeiensis) kind.
The 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 two single strand dnas shown in sequence 1 and sequence 2 in sequence table form;
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 in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
The 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 walnuts (Juglans hopeiensis).
Provided by the present invention for the identification of or the test kit of assistant identification fiber crops walnuts (Juglans hopeiensis), specifically can comprise primer pair, record the contrast card of nucleic acid molecule formula group II;
Described primer pair two single strand dnas shown in sequence 1 and sequence 2 in sequence table form;
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, identify or assistant identification Juglans plants to be measured in application also belong to protection scope of the present invention.
In described application, described Juglans plants to be measured is any in following 7 kinds: numb walnut (Juglanshopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
Above provided by the present invention for the identification of or the application at following (I) or (II) of the test kit of assistant identification fiber crops walnuts (Juglans hopeiensis) also belong to protection scope of the present invention:
(I) identify or whether assistant identification Juglans plants to be measured is numb walnut (Juglans hopeiensis);
(II) kind of evaluation or assistant identification numb walnut to be measured (Juglans hopeiensis).
In (I) described application, described Juglans plants to be measured can be any in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
In the application (II) Suo Shu, described numb walnut to be measured (Juglans hopeiensis) can be any 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, totally 9 sequences of sequence 3-11 forms by 700 Nucleotide, is a part for Juglans plants UBE3 gene coding region, and corresponding UBE3 gene coding region position consistency (being designated as UBE3-700 sequence).In all nucleic acid molecule formulas, the bottom right footmark numeral number of base is all according to sequence 3-11(UBE3-700 sequence above) in base sequencing mark in turn, with 5 ' of UBE3-700 sequence, hold the 1st base to be numbered 1.
The present invention utilizes one section of long sequence of 700bp of the coding region of nucleus DNA E3 ubiquitin protein ligase gene (UBE3), with the form of nucleic acid molecule formula, Juglans plants is identified.Experimental results show that, this research is found, with protein metabolism regulate and control one section of long sequence of the 700bp of relevant nucleus E3 ubiquitin ligase gene (UBE3) coding region in kind, plant and belong in 3 levels, disclosing aspect Juglandaceae genetic diversity and there is very high resolving power, be good molecule marker.The method of this molecular markers for identification Juglans plants of utilizing provided by the present invention is comparatively accurate, simple to operation, can improve production efficiency and the quality monitoring level of related industries, effectively make up the deficiency of previous methods, can make analysis precision reach the level of 1 nucleic acid molecule (nano level); In addition, the method has realized the accurate evaluation of non-fruit phase (as seedling stage) simultaneously.The present invention is for promoting that protection, utilization and the breeding of Walnut Germplasm Resources are all significant.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, 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 and really plants 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 and really plants 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 from Yi County, Hebei province Liu Jingxiang and builds Village), Wild jujube in Taihang Mountain Area, Beijing Botanical Garden.
The plant leaf sample of Yunnan complex peach (Juglans sigillata), gathers from forest-science academy, Yunnan Province (Kunming) and Dali prefecture.
The plant leaf sample of eastern united states black walnut (Juglans nigra), U.S.'s north California black walnut (Juglans hindsii) and 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 foundation and the application of the nucleic acid molecule formula of Juglans plants
In order to build the nucleic acid molecule formula that can be used in effective evaluation Juglans plants, 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 pleomorphism site has wherein been carried out to statistical study.Totally 12 kinds of related (for examination material) Juglans plants, specific as follows:
6 kinds of walnut 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 '.
6 kinds of walnuts---common edible walnut (Juglans regia) played in non-literary composition; Juglans mandshurica (Juglans mandshurica); Yunnan complex peach (Juglans sigillata); Eastern united states black walnut (Juglans nigra); U.S.'s north California black walnut (Juglans hindsii); U.S. Ya Lisangna state black walnut (stalwart walnut) (Juglans major).
One, design of primers
Conserved regions according to nucleus DNA E3 ubiquitin protein ligase gene (Nuclear DNA E3ubiquitin-protein ligase gene, UBE3), designs 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
The blade of 12 kinds of test samples of Juglans plants of take is experiment material, adopts ordinary method (as CTAB method or commercially available plant genome DNA extract test kit) to extract the genomic dna of each material.Take gained genomic dna as template, adopt the primer pair of step 1 design to carry out pcr amplification.Result demonstration, every kind of Juglans plants all can obtain the product that a size is 816bp.
Utilize reverse primer (H_nrDNA_UBE3_838R) to carry out unidirectional order-checking to each PCR product after purifying, obtain the DNA sequence dna (being designated as UBE3-700) of one section of 700bp that contains some polymorphic bases site.UBE3-700 sequence be described PCR product nucleotide sequence from 5 ' end 56-755 position.3 of every kind of plant sample sampling order-checkings or 3 above 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 of 6 kinds (Juglans hopeiensis)
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 eastern united states black walnut (Juglans nigra) is as shown in sequence in sequence table 8;
The UBE3-700 sequence of 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.'s north California black walnut (Juglans hindsii) is as shown in sequence in sequence table 10.
Three, sequence alignment
The UBE3-700 sequence of 12 kinds of Juglans plants of step 2 gained is compared, obtain 16 polymorphic base sites as shown in table 1.
16 polymorphic base sites that the UBE3-700 sequence of 12 kinds of Juglans plants of table 1 obtains
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.
In the situation that do not consider the 238th base loci polymorphism of UBE3-700 sequence, for walnut (numb walnut) played in literary composition, in table 1, in listed 16 polymorphic sites, there are 11 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, when order-checking, can obtain significantly stack 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, do not consider the situation of the 238th base loci polymorphism of UBE3-700 sequence
In the situation that do not consider the 238th base loci polymorphism of UBE3-700 sequence, according to every kind of Juglans plants listed in table 1 polymorphic base site, list corresponding nucleic acid molecule formula, specific as follows:
From 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 of the UBE3-700 sequence of fiber crops walnut (Juglans hopeiensis) 6 kinds 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 in UBE3-700 sequence Zhong position (from 5 ' end meter), list nucleic acid molecule formula general formula and be: W
14r
57r
97a
294w
296y
335y
369m
429y
431c
494r
518c
567y
594c
635y
666.
The UBE3-700 sequence of Juglans mandshurica (Juglans mandshurica) from 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 be respectively T, A, A, A, A, C, C, A, T, C, A, C, C, C, C, according to these Nucleotide and in UBE3-700 sequence Zhong position (from 5 ' end meter), list nucleic acid molecule formula and be: T
14a
57a
97a
294a
296c
335c
369a
429t
431c
494a
518c
567c
594c
635c
666.
The UBE3-700 sequence of common edible walnut (Juglans regia) from 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 be respectively A, G, G, A, T, T, T, C, C, C, G, C, T, C, T, according to these Nucleotide and in UBE3-700 sequence Zhong position (from 5 ' end meter), list nucleic acid molecule formula and be: A
14g
57g
97a
294t
296t
335t
369c
429c
431c
494g
518c
567t
594c
635t
666.
The UBE3-700 sequence of Yunnan complex peach (Juglans sigillata) from 5 ' end the 14th, 57,97,294,296,335,369,429,431,494,518,57,594,635 and 666 be respectively A, G, A, A, T, T, T, C, C, C, G, C, T, C, T, according to these Nucleotide and in UBE3-700 sequence Zhong position (from 5 ' end meter), list nucleic acid molecule formula and be: A
14g
57a
97a
294t
296t
335t
369c
429c
431c
494g
518c
567t
594c
635t
666.
The UBE3-700 sequence of eastern united states black walnut (Juglans nigra) 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, T, T, according to these Nucleotide and in UBE3-700 sequence Zhong position (from 5 ' end meter), list nucleic acid molecule formula and be: T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518c
567t
594t
635t
666.
The UBE3-700 sequence of U.S. Ya Lisangna state black walnut (stalwart walnut) (Juglans major) 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, T, T, T, T, according to these Nucleotide and in UBE3-700 sequence Zhong position (from 5 ' end meter), list nucleic acid molecule formula and be: T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518t
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 of U.S. north California black walnut (Juglans hindsii) are respectively T, G, A, G, T, T, C, A, C, T, G, C, T, C, T, according to these Nucleotide and in UBE3-700 sequence Zhong position (from 5 ' end meter), list nucleic acid molecule formula and be: T
14g
57a
97g
294t
296t
335c
369a
429c
431t
494g
518c
567t
594c
635t
666.
2, consider the situation of the 238th base loci polymorphism of UBE3-700 sequence
Consider UBE3
-in the situation of the 238th base loci 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 to Walnut Cultivars and further divide into 2 types.Specific as follows:
Type I (C
238type): comprise 3 literary composition object for appreciation walnut (numb walnut) kinds, i.e. ' white large meatball ', ' high-ranking official's caps ' 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 composition object for appreciation walnut (numb walnut) kinds, 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, by nucleic acid molecule formula, identify Juglans plants
1, do not consider the situation of the 238th base loci polymorphism of UBE3-700 sequence
(1) take the genomic dna of Juglans plants to be measured is template, with primer pair (sequence 1/ sequence 2), carries out pcr amplification, obtains PCR product;
(2), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula of described Juglans plants to be measured;
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
666be illustrated in successively in the nucleotide sequence of described PCR product, the nucleotide sequence of described PCR product is designated as to the 1st base, the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 bases counting in turn to 3 ' extreme direction along the nucleotide sequence of described PCR product (the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 bases of corresponding UBE3-700 sequence) from the 56th base of 5 ' end;
In described general formula, each B is one or both the stack in A, T, C and tetra-kinds of bases of G;
(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, 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, 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, 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, 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, described Juglans plants to be measured is or candidate is eastern united states 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, 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, described Juglans plants to be measured is or candidate is U.S. north California black walnut (Juglans hindsii).
2, consider the 238th base loci polymorphism of UBE3-700 sequence
(1) take the genomic dna of numb walnut to be measured (Juglans hopeiensis) is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(2), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula (method is the same) of described numb walnut to be measured (Juglans hopeiensis);
(3) determine as follows the kind of described numb walnut to be measured (Juglans hopeiensis):
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, 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, 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 can be by more described numb walnut to be measured (Juglans hopeiensis) the described C affiliated with 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, thereby the concrete kind of definite described numb walnut to be measured (Juglans hopeiensis).In detail as follows:
At C
238in type fiber crops walnut (Juglans hopeiensis) kind, the top vexed sharp type (shield point type) that is of the nut of ' high-ranking official's cap ' kind, base is flat and roomy, the whole full symmetry of nut, nut diameter is usually greater than 4.8cm, and ornamental value is high; The top vexed sharp type (shield point type) that is of nut of ' white large meatball ' kind, there is a rule the top side of nut and obvious shallow grooves, and base is flat, but not wide, the whole full symmetry of nut, the common 3.8-4cm of nut diameter left and right; The nut of ' heart ' kind is top obvious, and base is not bery flat, also not wide, and nut diameter is conventionally in 4.2cm left and right.
At S
238in type fiber crops walnut (Juglans hopeiensis) kind, the nut of ' Stem and leaf of Hongkong Pavetta ' kind is top obviously, also has significantly longitudinally rib ridge between 2 rib ridges of nut, and nut is larger, typically has a diameter from 4.2-4.6cm left and right; Top vexed sharp type (shield point type), the obvious shield of convex-concave surface between 2 rib ridges, the common 4.2cm of the nut diameter left and right of being of nut of ' apple circle ' kind; The top vexed sharp type (shield point type) that is of the nut of ' all the people present building ' kind, the convex-concave surface between 2 rib ridges is shield not obviously, the whole full symmetry of nut, nut typically has a diameter from 4.2cm left and right.
Embodiment 2, use nucleic acid molecule formula are identified other platymisciums of Juglandaceae
Cyclocarya paliurus (Cyclocarya paliurus): Juglandaceae, cyclocarya.
Chinese ash (Pterocarya stenoptera): Juglandaceae, Chinese ash belong to.
In order to verify that the nucleic acid molecule formula of embodiment 1 foundation is for the specificity of Juglans plant, the present inventor has also gathered 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, use the primer pair being formed by two single strand dnas shown in sequence 1 and sequence 2 to carry out pcr amplification, obtain respectively the UBE3-700 sequence (sequence 11) of Cyclocarya paliurus (Cyclocarya paliurus) and the UBE3-700 sequence (sequence 12) of Chinese ash (Pterocarya stenoptera).
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, the nucleic acid molecule formula of Cyclocarya paliurus (Cyclocarya paliurus) and Chinese ash (Pterocarya stenoptera) is all not identical with 7 kinds of nucleic acid molecule formulas (not considering the 238th bit base) of 12 kinds of Juglans plants in embodiment 1.Thereby validity and the specificity of the inventive method have been confirmed.
The present inventor studies and finds that the chloroplast gene group sequence of Juglans plants is maternal instinct monolepsis, coordinated the nucleic acid molecule formula from nuclear genome provided by the present invention to use, can be better for the effect of the Diversity Detection of Juglans plants.
Claims (10)
1. a method for evaluation or assistant identification Juglans plants to be measured, comprises the steps:
(1) take the genomic dna of Juglans plants to be measured is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(2), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula of described Juglans plants to be measured;
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
666be illustrated in successively in the nucleotide sequence of described PCR product, the nucleotide sequence of described PCR product is designated as to the 1st base, the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 bases counting in turn to 3 ' extreme direction along the nucleotide sequence of described PCR product from the 56th base of 5 ' end;
In described general formula, each B is one or both the stack in A, T, C and tetra-kinds of bases of G;
(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, 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, 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, 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, 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, described Juglans plants to be measured is or candidate is eastern united states 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, 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, described Juglans plants to be measured is or candidate is 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 in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
3. identify or whether assistant identification Juglans plants to be measured is a method of numb walnut (Juglans hopeiensis), comprise the steps:
(a) take the genomic dna of Juglans plants to be measured is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(b), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula of described Juglans plants to be measured;
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
666be illustrated in successively in the nucleotide sequence of described PCR product, the nucleotide sequence of described PCR product is designated as to the 1st base, the 14th, 57,97,294,296,335,369,429,431,494,518,567,594,635 and 666 bases counting in turn to 3 ' extreme direction along the nucleotide sequence of described PCR product from the 56th base of 5 ' end;
In described general formula, each B is one or both the stack in A, T, C and tetra-kinds of bases of G;
(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:
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), determine as follows whether described Juglans plants to be measured is numb walnut (Juglans hopeiensis):
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), described Juglans plants to be measured is or candidate is numb walnut (Juglans hopeiensis); Otherwise, described Juglans plants to be measured be not or candidate for numb walnut (Juglans hopeiensis).
4. a method for evaluation or assistant identification numb walnut to be measured (Juglans hopeiensis) kind, comprises the steps:
(A) take the genomic dna of numb walnut to be measured (Juglans hopeiensis) is template, uses the primer pair that two single strand dnas shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification, obtains PCR product;
(B), by described PCR product order-checking, according to sequencing result, list the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis);
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
666be illustrated in successively in the nucleotide sequence of described PCR product, the nucleotide sequence of described PCR product is designated as to the 1st base, the 14th, 57,97,238,294,296,335,369,429,431,494,518,567,594,635 and 666 bases counting in turn to 3 ' extreme direction along the nucleotide sequence of described PCR product from the 56th base of 5 ' end;
In described general formula, each B is one or both the stack in A, T, C and tetra-kinds of bases of G;
(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), determine as follows the kind of described numb walnut to be measured (Juglans hopeiensis):
If the nucleic acid molecule formula of described numb walnut to be measured (Juglans hopeiensis) is b1) shown in nucleic acid molecule formula, 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, 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 as lower any: ' large meatball in vain ', ' high-ranking official's cap ', ' heart ';
Described S
238type fiber crops walnut (Juglans hopeiensis) kind be as lower any: ' Stem and leaf of Hongkong Pavetta ', ' apple is justified ', ' all the people present building '.
5. method according to claim 4, is characterized in that: in step (D), also comprise the steps: afterwards the described C that more described numb walnut to be measured (Juglans hopeiensis) is affiliated with 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 phenotypic characteristic of a certain kind in type fiber crops walnut (Juglans hopeiensis) kind is identical, and described numb walnut to be measured (Juglans hopeiensis) is or candidate is corresponding kind.
6. according to the method described in claim 4 or 5, it is characterized in that: described numb walnut to be measured (Juglans hopeiensis) is any 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 '.
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 two single strand dnas shown in sequence 1 and sequence 2 in sequence table form;
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 in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
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 two single strand dnas shown in sequence 1 and sequence 2 in sequence table form;
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.
9. the application of test kit in evaluation or assistant identification Juglans plants to be measured described in claim 7;
Described Juglans plants to be measured is any in following 7 kinds: numb walnut (Juglans hopeiensis), Juglans mandshurica (Juglans mandshurica), walnut (Juglans regia), complex peach (Juglans sigillata), eastern united states black walnut (Juglans nigra), stalwart walnut (Juglans major), U.S.'s north California black walnut (Juglans hindsii).
10. the application of test kit at following (I) or (II) described in claim 8:
(I) identify or whether assistant identification Juglans plants to be measured is numb walnut (Juglans hopeiensis);
(II) kind of evaluation or assistant identification numb walnut to be measured (Juglans hopeiensis);
In (II), described numb walnut to be measured (Juglans hopeiensis) is any 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 '.
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Cited By (3)
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| CN104195243A (en) * | 2014-07-02 | 2014-12-10 | 中国科学院植物研究所 | Method for identifying plant variety by constructing nucleotide molecular formula by virtue of UBE3 gene |
| CN105296483A (en) * | 2015-12-04 | 2016-02-03 | 李凯兵 | Primer, kit and method for identifying walnut bark beetles |
| CN107699615A (en) * | 2017-10-27 | 2018-02-16 | 山东省林木种质资源中心 | A kind of method that nucleic acid molecule formula identification Malus spectabilis kind is built using DNA sequence dna |
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| CN102719543A (en) * | 2012-06-25 | 2012-10-10 | 中国科学院植物研究所 | Method for identifying plant varieties by utilizing chemical molecular formulas of nucleotides |
| CN103127212A (en) * | 2011-11-29 | 2013-06-05 | 吉林大学 | Application of walnut Cortex Juglandis Mandshuricae in medicine for gastric cancer |
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| CN103127212A (en) * | 2011-11-29 | 2013-06-05 | 吉林大学 | Application of walnut Cortex Juglandis Mandshuricae in medicine for gastric cancer |
| CN102719543A (en) * | 2012-06-25 | 2012-10-10 | 中国科学院植物研究所 | Method for identifying plant varieties by utilizing chemical molecular formulas of nucleotides |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195243A (en) * | 2014-07-02 | 2014-12-10 | 中国科学院植物研究所 | Method for identifying plant variety by constructing nucleotide molecular formula by virtue of UBE3 gene |
| CN104195243B (en) * | 2014-07-02 | 2016-03-30 | 中国科学院植物研究所 | Utilize the method for UBE3 gene constructed nucleic acid molecule formula plant identification kind |
| CN105296483A (en) * | 2015-12-04 | 2016-02-03 | 李凯兵 | Primer, kit and method for identifying walnut bark beetles |
| CN107699615A (en) * | 2017-10-27 | 2018-02-16 | 山东省林木种质资源中心 | A kind of method that nucleic acid molecule formula identification Malus spectabilis kind is built using DNA sequence dna |
| CN107699615B (en) * | 2017-10-27 | 2018-11-06 | 山东省林木种质资源中心 | A method of identifying Malus spectabilis kind using DNA sequence dna structure nucleic acid molecule formula |
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