CN105950615B - A kind of method and kit detecting TaAGPL Allelic Variation - Google Patents

Abstract

The invention discloses a kind of detectionsTaAGPLThe primer pair and kit and detection method of Allelic Variation.Primer pair be it is following 1) or 2) shown in primer pair: 1) SEQ ID:1, SEQ ID:3；2) SEQ ID:2, SEQ ID:3.PCR reaction is carried out using the primer pair, it can allelic variation that is easy and effectively identifying common wheat wheat plasmotype ADP-glucose pyrophosphorylase gene.The method of the present invention can be completed by conventional PCR instrument and DNA analysis instrument, easily operated, easy and have very high flux.

Description

A kind of method and kit detecting TaAGPL Allelic Variation

Technical field

The invention belongs to genetic engineering fields, and in particular to a kind of method and reagent for detecting TaAGPL Allelic Variation Box.

Background technique

Wheat is allohexaploid, is hybridized in evolution by 3 genomes and is formed, and includes A, B and D totally 3 sets of chromosome Group (AABBDD, 2n=6x=42).But have the repetition of lots of genes between A, B and D genome, in addition the repetition in wheat Sequence accounts for genome about 80%, especially for the separation identification and functional study for controlling a certain important character gene be easy by The influence of the homologous gene of different chromosomes hinders the development of wheat genetic and breeding improvement research.Wheat is in the world One of most important Three major grain crops (wheat, rice and corn), annual total output reaches 600,000,000 tons or more in the world (http://faostat.fao.org/).Although wheat occupies first place in the world in China's cultivated area with total output, wheat year Equal consumption figure and import volume are also to account for significant proportion.Therefore, be still during China's wheat breeding one of yield is important Measurement index.

The yield forming of wheat in addition to the external conditions such as cultivation (including illumination, temperature, fertility) influence other than, wheat itself Genotype plays decisive role；And constituting the important ingredient of wheat flour is starch, starch when seed is initially formed It is gradually accumulated in endosperm, therefore the number of Starch synthesis has with kernel weight and directly contacts.Adenosine diphosphate glucose coke phosphorus Phosphorylase (ADP-glucose pyrophosphorylase, AGPase) is catalyzed the glucose-obtained by sucrose in plant 1- phosphoric acid (G-l-P) reacts to form adenosine diphosphate glucose (ADP-glucose) and discharge Pi with ATP, and ADP-glucose The synthesis that amylose and amylopectin are participated in as the substrate of amylosynthease is that rice, wheat, corn, potato etc. make The key enzyme of object Starch synthesis, active size are positively correlated with starch accumulation rate and filling rate.Wheaten starch synthesis is closed Key enzymatic activity and its also indicated that with the research of Starch Accumulation of Grain Rate Relationship: AGPase activity and amylose, amylopectin, The accumulation rate and filling rate of total starch are in extremely significant positive correlation.Yu Zhenwen etc. is studies have shown that Post flowering 7 days, 14 days, 2l It, 28 days and 35 days wheat seed AGPase activity and each phase total starch accumulation rate be in significant or extremely significant positive correlation.

There are two types of various forms of AGPase in the endosperm of the cereal crops such as wheat, they are cytoplasm and plastid respectively , wherein cytoplasmatic forms account for 65%~95% activity, therefore the AGPase of cytoplasm figure plays important work in Starch synthesis With.AGPase is made of two identical large subunits and two identical small subunits, wherein large subunit (AGPase Large Subunit, AGPL) it is that main catalytic action adjusts center, directly affect the rate of Starch synthesis substrate formation.Olive etc. The AGPL gene order in wheat is obtained using the method in probe screening by hybridization library, Ainsworth etc. utilizes Southern Hybridizing method is by the wheat AGPL assignment of genes gene mapping to 1A, 1B and 1D chromosome, and Smidansky etc. is by control synthesis corn AGPL's Shunken2 (Sh2r6hs) gene is modified, and is changed the allosteric modulating sites amino acid of AGPL albumen, is enhanced The combination stability of the big small subunit of AGPase, after being transferred to wheat, wheat yield and total biomass are increased.Early stage mainly for TaAGPL Genetic food, expression the correlation of height and content of starch and synthesis rate studied, make In Developing Grain of Wheat dynamic change and there must be understanding abundant to TaAGPL gene expression.And to TaAGPL in wheat Difference of the gene in tri- genomes of A, B and D and in population material the distribution of allelic variation and correlation function research it is very few, This greatly hinders the research of the different allelic variations and Correlated Yield Characters to wheaten starch synthesis key gene TaAGPL.

Summary of the invention

It is detected the purpose of the present invention is the allelic variation to solve AGPase gene in common wheat Starch synthesis approach Technical problem provides a kind of method for detecting wheat TaAGPL Allelic Variation and provides advantage allelic variation for molecule mark Remember assisted selection.

The method of auxiliary detection wheat TaAGPL Allelic Variation provided by the present invention, includes the following steps:

1) drawn according to the area exon I of TaAGPL gene nucleotide series and the conservative region design of exon II region sequence Object pair, the introne I sequence for including between the area exon I and the area exon II have polymorphism between different chromosomes group, A sequence in the primer pair is located in the area the exon I, and another primer sequence of the primer pair is located at described outer In the area Xian Zi II；

2) PCR amplification is carried out to detected materials with the primer pair that step 1) obtains, obtains pcr amplification product；

3) polymorphism for detecting the pcr amplification product, determines described to be measured according to the polymorphism of the pcr amplification product TaAGPL Allelic Variation type in material.

In the above method, the TaAGPL gene is wheat ADP-glucose pyrophosphorylase gene.

In the above method, the polymorphism is length polymorphism.

In the above method, the wheat ADP-glucose pyrophosphorylase gene is two phosphorus of wheat plasmotype adenosine Sour grapes sugar pyrophosphorylation enzyme gene.

In the above method, the sequence between the area the exon I and the area exon II has length caused by insertion and deletion Diversity sequence.

In the above method, the detected materials are wheat.

In the above method, the primer pair is following 1) or 2) shown:

1) SEQ ID:1 and SEQ ID:3；

2) SEQ ID:2 and SEQ ID:3.

In the above method, the polymorphism according to pcr amplification product determines TaAGPL base to be measured in the detected materials The method of the allelic variation of cause are as follows: using the length of the pcr amplification product as the allelic variation type of the TaAGPL gene (allelic variation that i.e. a kind of pcr amplification product of length corresponds to a kind of gene).

The method of the length polymorphism of the detection pcr amplification product is shown in following I or II:

I, the pcr amplification product length is detected with DNA analysis instrument, the position of the signal peak detected is i.e. For the length of the pcr amplification product；The primer pair is marked with fluorophor；

II, the pcr amplification product is sequenced.

It is a further object to provide a kind of kits for detecting TaAGPL Allelic Variation

The kit of detection TaAGPL Allelic Variation provided by the present invention, for it is following 1) or 2) shown in primer It is right:

1) SEQ ID:1, SEQ ID:3；

2) SEQ ID:2, SEQ ID:3；

In mentioned reagent box, the TaAGPL gene is wheat ADP-glucose pyrophosphorylase gene.

In mentioned reagent box, the wheat ADP-glucose pyrophosphorylase gene is wheat plasmotype adenosine two Glucose 1-phosphate1- pyrophosphorylation enzyme gene.

The invention has the following advantages: the present invention is to solve AGPase gene in common wheat Starch synthesis approach The technical issues of allelic variation detects, provides a kind of detection method, helps quickly to identify wheat TaAGPL genetic mutation class Type and be applied to molecular mark.This method is based on the characteristics of TaAGPL gene in Common Wheat Varieties: area between sequence (i.e. the sequence of exon has very high conservative, and the sequence of introne has very high again for domain conservative and length polymorphism Length polymorphism), in the PCR primer of exon region over-designed, expand to obtain TaGAPL gene by the method for PCR DNA fragmentation, shows the polymorphism between these segments by certain identification method (3730DNA analyzer), and then can be with Achieve the purpose that identify TaAGPL Allelic Variation in a wheat breed.It can establish gene molecule marker using the strategy Detection architecture carries out TaAGPL gene to efficiently separate identification, can also TaAGPL gene to other filial generation groups Allelic variation type identified；This molecular labeling system construction strategy can also be with further genralrlization to similar The separation of the allelic variation of the functional gene of sequence conservation and length polymorphism is identified.

In the method for the present invention, according to the TaAGPL gene order institute in the result of laboratory early-stage study and Genbank The primer of design demonstrates the conservative of the primer with the verification test that conservative primer carries out TaAGPL gene identification and has Effect property, also shows the accuracy and reliability of the molecular labeling system.

The method of the present invention, can be complete by conventional PCR instrument and 3730DNA analyzer without expensive instrument and reagent At easily operated.The method of the present invention sensitivity and resolution ratio high based on 3730DNA analyzer can efficiently separate and quasi- The really different allelic variation of identification TaAGPL gene.In the methods of the invention, a wheat can detecte by a PCR reaction The gene of A, B and D group in kind, and PCR instrument and 3730DNA analyzer are automated and high-throughput instrument, a people one It can analyze nearly 300 wheat breeds.The method of the present invention greatly improves the efficiency of TaAGPL identified for genes and accurate Property.

Molecular labeling construction strategy based on sequence conservation and length polymorphism of the invention, is applicable not only to TaAGPL The building of gene molecule marker detection architecture is equally applicable to the control important character with sequence conservation and length polymorphism The building of the Markers for Detection system of gene.The invention by directly facilitate TaAGPL gene allelic variation excavate and The researchs such as the synthesis of TaAGPL gene pairs wheaten starch, contribution to yield are carried out in a deep going way.

Detailed description of the invention

The nucleotide sequence comparison schematic diagram of the small 54TaAGPL that lays down of Fig. 1 common wheat.

Different background color indicates different nucleotide similarities.In figure background color more deeply feel show nucleotide similarity more It is high.Black: nucleotide similarity 100%；Dark grey: nucleotide similarity 50%；Grey: nucleotide similarity is 30%；White: nucleotide similarity 0%.

The position of primer on the small 54TaAGPL exon I and exon II that lays down of Fig. 2 common wheat.

The area Exon I, exon I；The area Intron I, introne I；The area Exon II, exon II；Introne I (Intron I there are length variations caused by insertion and deletion in).

Conservative primer pair are as follows: TaAGPL1 is made of F1g-FAM and R2a-common；TaAGPL2 is by F1h-FAM and R2a- Common composition.

Fig. 3 utilizes 3730DNA analyzer using conservative product fragment analysis of the primer in four body material of China spring nullisomic Detect PCR product, the longitudinal axis indicate DNA fragmentation size, horizontal axis indicate signal strength, i.e., PCR product amount number.Arrow in figure The specific DNA fragment showing in PCR product, corresponds to a special TaAGPL allelic variation, and remaining peak indicates molecule Amount standard (LIZ500Standard internal standard).

CS, wheat breed China spring；Tetra- body 1D of CSN1AT1D, wheat breed China spring nullisomic 1A；CSN1BT1D, wheat product Kind tetra- body 1D of China spring nullisomic 1B；CSN1DT1B, wheat breed China spring nullisomic 1D tetra- body 1B, 1A, 1B and 1D respectively indicate small Wheat chromosome 1A, 1B and 1D.

The allelic variation for the TaAGPL gene that Fig. 4 is detected in wheat Mini core collection library using conservative primer.It utilizes 3730DNA analyzer detects PCR product.Horizontal axis indicates DNA fragmentation size, and the longitudinal axis indicates signal strength, the i.e. amount of PCR product How much；Arrow in figure indicates the specific DNA fragment in PCR product, corresponds to a special TaAGPL Allelic Variation, The peak that length is 314 and 320 indicates molecular weight standard (LIZ500Standard internal standard).

The sequence verification and multisequencing of five kinds of allelic variations of TaAGPL-1B are detected in Fig. 5 wheat Mini core collection library It compares.

The sequence framed that breaks is a tandem repetitive sequence (A)_n, in addition a kind of a kind of TaAGPL-1A and TaAGPL-1D Allelic variation, the allelic variation of totally 7 kinds of TaAGPL genes.

Every kind of allelic variation of TaAGPL-1B gene is lived with wire frame respectively.

Title material: Xiaoyan54 is small to lay down 54；Dahuangpi, big Calusena lansium；The Ji Jimai10-2893 wheat No. 10； Heshangmai-Yu8679RIL93, Buddhist monk wheat/Henan 8679RIL-93；Heshangmai-Yu8679RIL110, Buddhist monk wheat/Henan 8679RIL-110；Shannong01-35, mountain agriculture 01-35；Yu8679, Henan 8679；Heshangmai-Yu8679RIL22, Buddhist monk Wheat/Henan 8679RIL-22；Heshangmai-Yu8679RIL41, Buddhist monk wheat/Henan 8679RIL-41；Linyou145, Linyou 145； HanF5-4, Korea Spro F5-4；Hongqiangchang is red to rob field；Taikongmai, space wheat；Luyuang212, Shandong original 212； B1924, B1924；E_en1, Hubei Province grace 1；Lanhuamai, orchid wheat；Beijingyemaizi2, Beijing wild oat fruit 2； Hengshuiyemaizi1, Hengshui wild oat fruit 1；Baodingyemaizi2, Baoding wild oat fruit 2；Jinchun3, Jin Chun No. 3 numbers； Baihuamai, white flower wheat；Yunong202, Henan agriculture 202.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

TaAGPL Allelic Variation and progress chromosome mapping in embodiment 1, detection wheat breed China spring

One, design of primers

First with MegAlign software by common wheat it is small lay down 54 TaAGPL nucleotide (including cDNA and genome DNA) sequence carries out Multiple Sequence Alignment, and in these gene orders, there are two features: (1) three homologous gene TaAGPL- for discovery 1A, TaAGPL-1B and TaAGPL-1D, exon region are very conservative；(2) including subregion includes length polymorphism abundant With SNP difference (Fig. 1).Found by Fig. 1, in First Intron (Intron I) in gene TaAGPL-1A, TaAGPL-1B and There are difference in length (tables 1) between TaAGPL-1D.Based on the two features, a kind of identification TaAGPL genetic test plan is proposed Slightly: designing the PCR primer guarded between gene according to the coded sequence in conservative region, expand to obtain these by the method for PCR The DNA fragmentation of TaAGPL gene, then detection (utilizing 3730DNA analyzer) shows the polymorphism between these DNA fragmentations, into And it realizes the separation to all TaAGPL genes in a wheat breed and identifies.

The small TaAGPL gene laid down in 54 Multiple Sequence Alignment is subjected to, according to its conservative region (exon I and outer aobvious Sub- II) coded sequence design PCR primer.In order to improve the repeatability and accuracy of experiment, 2 sets of PCR primers are devised, respectively It is TaAGPL1 (for upstream primer nucleotide sequence as shown in SEQ ID:1, downstream primer nucleotide sequence is as shown in SEQ ID:3) With TaAGPL2 (for upstream primer nucleotide sequence as shown in SEQ ID:2, downstream primer nucleotide sequence is as shown in SEQ ID:3). TaAGPL1 and TaAGPL2 has a universal primer and upstream primer respectively, all crosses over Intron I (Fig. 2), utilizes two pairs Primer 54 is expanded in small lay down, and for TaAGPL-1A gene, target fragment length is respectively 330bp and 253bp, 3730DNA The actually detected length of analyzer is 323bp and 248bp；For TaGAPL-1D gene, target fragment theoretical length be 335bp and The actually detected length of 258bp, 3730DNA analyzer is 327bp and 253bp；For TaAGPL-1B gene, target fragment is theoretical Long fragment length is 320bp and 243bp, and the actually detected length of 3730DNA analyzer is 312bp and 238bp (table 2).This two The obtained testing result for covering conservative primer can be mutually authenticated.Below with fragment length name allelic variation all with The length of TaAGPL1 combination amplification is foundation.

In order to verify the conservative of primer, by the AGPL in the small TaAGPL gene and barley, corn and rice laid down in 54 Multiple Sequence Alignment has been carried out, search is compared to primer sequence, finds primers F 1g-FAM, F1h-FAM and barley, Wheat Species Sequence it is consistent, there are SNP differences with the AGPL sequence of corn and rice；And R2a-common in all sequences used all Equally.Illustrate that this two sets of primers have very high representativeness on identification wheat TaAGPL gene.Finally obtained primer sequence It arranges (table 3).

Table 1 is small lay down TaAGPL gene in 54 Intron I length between three genomes polymorphism

Primer location and theoretical fragment length of the table 2 based on small 54TaAGPL gene order design of laying down.Primer initial position It is to be counted since ATG.

3 TaAGPL gene of table guards primer sequence

Note: what * was indicated is that the primer 5 ' end G base is marked by 6FAM.

Two, PCR amplification

Wheat breed China spring and its four body material of nullisomic are planted from Inst. of Genetics and Development Biology, CAS Object cell and chromosome engineering National Key Laboratory, wherein China spring is purchased from seeds company of Wuyang Region, Henan Province county.

CS, wheat breed China spring；Tetra- body of CSN1AT1D, wheat breed China spring 1A nullisomic 1D；CSN1BT1D, wheat product Kind tetra- body of China spring 1B nullisomic 1D；Tetra- body of CSN1DT1B, wheat breed China spring 1D nullisomic 1B.

PCR is carried out by template of the genomic DNA of four body material seedling of China spring and its nullisomic using the primer in table 3, Reaction system is as follows:

Since the resolution ratio of agarose electrophoresis is lower, it is unable to fully show the length polymorphism of PCR product.In order into one The composition of step detection PCR product, using 3730DNA analyzer, which has very high resolution ratio, extensive up to 1bp Applied in DNA fragmentation analysis and research, such as microsatellite marker, amplified fragment length polymorphism and DNA footprint analysis.

Three, A, B and D genome positioning of pcr amplification product

PCR product is diluted to the 1/40 of original content with distilled water, is taken in μ L to the 96 new orifice plates of cut back 5, The EDTA, the sodium acetate of 5 μ L 900mM and the ethyl alcohol of 37.5 μ L100% of 5 μ L 37.5mM is added, 15 points of standing is protected from light after mixing Clock, 2000g are centrifuged 35 minutes, are inverted PCR plate and are centrifuged to 180g stopping, the ethyl alcohol of 52.5 μ L70% is added, are inverted PCR plate centrifugation Stop to 180g, add the ethyl alcohol of 52.5 μ L70%, is inverted PCR plate and is centrifuged to 180g stopping, after air-drying, every hole adds 8 μ L formamide (containing 0.1 μ L LIZ-500size standard) (ABI Products), mixture is denaturalized under the conditions of 95 DEG C 5 minutes, then stand 10 minutes on ice.

It is detected using PCR product of the 3730DNA analyzer to denaturation, testing result such as Fig. 3.Utilize GeneMapper DNA fragmentation size (table 4) corresponding to the signal peak that v4.0 software analysis detection arrives.It can be found that conservative primer in China spring 3 signal peaks can be detected in the PCR product of TaAGPL1 and TaAGPL2 respectively, and two sets of primer PCR results correspond to each other, i.e., There are very high consistency between the PCR product test map of two sets of conservative primers.It can be from CS using method of the invention Detect 3 signal peaks, i.e. 3 DNA fragmentations；It can respectively detect 2 respectively from CSN1AT1D, CSN1BT1D and CSN1DT1B A signal peak, the chromosome that is lacked of peak respective material of each system's missing, so by 312 and 238,323 and 248,327 and 253 navigate on 1B, 1A and 1D (table) respectively, and the clip size expanded in testing result and small 54 materials of laying down is corresponding (table).

Table 4 utilizes the actually detected verifying to DNA fragmentation from four body material of China spring and its nullisomic of molecular labeling system

Note: N indicates DNA purpose band missing

Embodiment 2 detects other wheat breeds TaAGPL Allelic Variation

Effectively analyzed using the method for the present invention backbone parent (more than 200 parts) in wheat breeding, modern varieties and The TaAGPL allelic variation type of strain, for instructing molecular breeding to practice, the material of 23 parts of picking different allelic variations is carried out The sequence verification of displaying and based on PCR.

One, design of primers

Primer (table 3) is guarded using two pairs of TaAGPL genes designed in embodiment 1.

Two, PCR amplification

Genomic DNA is extracted using conventional CTAB method.

PCR is carried out by template of genomic DNA using the primer of table 1, reaction system is as follows:

Three, pcr amplification product species detection

PCR product is diluted to the 1/40 of original content with ddH2O, takes in μ L to the 96 new orifice plates of cut back 5, adds The EDTA, the sodium acetate of 5 μ L 900mM and the ethyl alcohol of 37.5 μ L100% for entering 5 μ L 37.5mM, are protected from light standing 15 minutes after mixing, 2000g be centrifuged 35 minutes, be inverted PCR plate be centrifuged to 180g stop, be added 52.5 μ L70% ethyl alcohol, be inverted PCR plate be centrifuged to 180g stops, and adds the ethyl alcohol of 52.5 μ L70%, is inverted PCR plate and is centrifuged to 180g stopping, after air-drying, every hole adds 8 μ L Formamide (containing 0.1 μ L LIZ-500size standard) (ABI Products), is denaturalized 5 under the conditions of 95 DEG C for mixture Minute, then stand 10 minutes on ice.

Detected using PCR product of the 3730DNA analyzer to denaturation, testing result with GeneMapper v4.0 into Row analysis.It detects 5 kinds of B group allelic variation altogether in 262 parts of backbone parents, TaAGPL-1B-307 is named as with length, TaAGPL-1B-308, TaAGPL-1B-311, TaAGPL-1B-312, TaAGPL-1B-313, A and D group respectively detect a kind respectively Allelic variation, TaAGPL-1A-323, TaAGPL-1B-327.45 parts of modern varieties or strain are screened, 23 kinds of picking as displaying Different allelic variations, as shown in table 5.Thus also illustrate, the polymorphism of TaAGPL-1A and TaAGPL-1D in wheat compares It is low；And then there is allelic variation abundant in TaAGPL-1B.

There are table 5 picking, 23 parts of materials 5 kinds of different allelic variation results to be shown

Four, it is analyzed using the PCR-Sanger method sequence verification of TaAGPL difference allelic variation

DNA fragmentation size corresponding to the signal peak detected arranges in table 5.The PCR product of these materials is carried out Multiple Sequence Alignment (Fig. 5) after sequencing.As a result, it has been found that the polymorphism of length is by adenyl-deoxyribonucleotide A in different materials Caused by tandem sequence repeats number is different, determine what the length polymorphism of gene and 3730DNA analyzer determined according to sequencing result The result of genetic fragment length polymorphism is consistent, i.e., the gene groups number result detected with sequencing approach and uses 3730DNA The gene groups number that analyzer method detects is consistent.Wheat lines can accurately be identified through the invention by demonstrating In TaAGPL gene type.

Five, the association analysis of different allelic variations and mass of 1000 kernel

TaAGPL is the gene for controlling the key enzyme of wheaten starch synthesis, and the accumulation part of starch determines small in seed The mass of 1000 kernel of wheat seed.In order to which whether the allelic variation for studying this gene is related to thousand grain weight properties, to 262 parts of wheat backbone 3 years mass of 1000 kernel data of material have carried out association analysis.Because gene TaAGPL-1A and TaAGPL-1D allelic variation is in group Respectively there was only a kind of allelic variation, so the allelic variation of following analysis is both from gene TaAGPL-1B.

Genotype identification is carried out to the method in the material use present invention first, to 5 kinds of allelic variations in population material The frequency that type occurs is analyzed.As a result, it has been found that the material frequency of occurrences highest with TaAGPL-1B-312 allelic variation (143 parts), and the material frequency of occurrences with TaAGPL-1B-307 and TaAGPL-1B-313 allelic variation is minimum (each 9 parts). It will test after material classified according to local varieties and modern varieties, be found to have TaAGPL-1B-308 allelic variation The ratio highest (86.67%) that modern varieties account in material, while being found that while with TaAGPL-1B-312 allelic variation The ratio that modern varieties account in material is not high (34.34%), but due to the material number with TaAGPL-1B-312 allelic variation Mesh is also very much, so the number of modern varieties is most；Modern product in material with TaAGPL-1B-311 allelic variation The ratio accounted in kind minimum (19.35%) (table 6).The above analysis result explanation: 1) TaAGPL-1B-308 is TaAGPL-1B base One excellent allele of cause, is selected in modern breeding process, and TaAGPL-1B-311 is a disadvantage equipotential Gene, the phenotype with TaAGPL-1B-311 allelic variation are fallen in breeding by artificial screening, this in modern varieties is caused The frequency of a allelic variation is very low；2) there is likely to be other in addition to length polymorphism in TaAGPL-1B-312 allelic variation Variation type.In short, having already been through artificial selection during TaAGPL gene breeding described above.

According to allelic variation classification of type, 3 years mass of 1000 kernel data of group are analyzed, different year is compared Under, the average difference of the mass of 1000 kernel between different allelic variation types, use method for the least significant difference (LSD) into Row Multiple range test.As a result, it has been found that three annual datas of the mass of 1000 kernel of the material with TaAGPL-1B-308 allelic variation are all shown as Higher than the material with other allelic variations.And it is significantly higher than the material with TaAGPL-1B-311 allelic variation.And have Having the mass of 1000 kernel of other allelic variation materials to be also all generally higher than has TaAGPL-1B-311 material (table 7).Above it may be said that Bright, 1) TaAGPL-1B-308 allelic variation is manually selected as the excellent allelic variation of control mass of 1000 kernel and hands down by heredity, and TaAGPL-1B-311 allelic variation is eliminated by gradually artificial selection；2) although TaAGPL-1B-308 allelic variation is not in the modern times Occur very high frequency in kind, but this point just illustrates that mass of 1000 kernel is a complicated character, may not be only by The control of TaAGPL gene, and with constitute the factor of mass of 1000 kernel under the comprehensive function of other gene locis, but TaAGPL gene Crucial effect is played in numerous genes, this point can be manually selected in breeding process by TaAGPL to be explained.

The conservative primer pair for devising two sets of TaAGPL genes first using the present invention in a word, passes through high-throughput 3730DNA Analyzer can navigate to respectively amplified fragments 323,312 and 327 on A, B and D genome, this two sets of primers mutually may be used With verifying；Utilize conservative Primer Analysis wheat backbone parent material and modern varieties or strain material, it was found that TaAGPL- 1A, TaAGPL-1B and TaAGPL-1D have 1,5 and 1 allelic variation respectively, and by different allelic variation materials Length polymorphism is verified in the sequencing of PCR-Sanger method.The duplicate mass of 1000 kernel data in 3 years of analysis detection material, card It is related to high mass of 1000 kernel that TaAGPL-1B-308 allelic variation is illustrated, receives positive selection in breeding process, and TaAGPL-1B- 311 receive negative selection, illustrate that developed label system can be used for instructing the relevant molecular breeding practice of mass of 1000 kernel, to educate Kind man provides a kind of efficient detection method.

The distribution frequency of the different allelic variations of table 6

Note :-indicate genotype missing.

The association analysis of table 7 TaAGPL-1B allelic variation and mass of 1000 kernel

Note: the subscript lowercase of each " mean+SD " indicates the Multiple range test under the level of signifiance of α=0.05 As a result.

<110>Inst. of Genetics and Development Biology, CAS

<120>a kind of detectionTaAGPLThe method and kit of Allelic Variation

<160> 3

<210> 1

<211> 18

<212> DNA

<400> 1

GACAGCAGCAGCATCAGG

<210> 2

<211> 21

<212> DNA

<400> 2

GCAGTGCGTGCTCACCTC

<210> 3

<211> 21

<212> DNA

<400> 3

GGCCCTTGTGCTTGTGAG

Claims (8)

1. one kind is for detectingTaAGPLThe primer pair of Allelic Variation, which is characterized in that the primer pair sequence is as follows:

1) SEQ ID:1 and SEQ ID:3 or 2) shown in SEQ ID:2 and SEQ ID:3.

2. one kind is for detectingTaAGPLThe kit of Allelic Variation, which is characterized in that it includes following 1) or 2) shown Primer pair:

1) SEQ ID:1 and SEQ ID:3 or 2) SEQ ID:2 and SEQ ID:3.

3. kit according to claim 2, it is characterised in that: describedTaAGPLGene is wheat adenosine diphosphate grape Sugared pyrophosphorylation enzyme gene.

4. kit according to claim 3, it is characterised in that: the wheat ADP-glucose pyrophosphorylase Gene is wheat plasmotype ADP-glucose pyrophosphorylase gene.

5. a kind of detectionTaAGPLThe method of Allelic Variation, which is characterized in that it includes the following steps:

1) basisTaAGPLThe area exon I of gene nucleotide series and the conservative region design primer of exon II region sequence It is right；

2) PCR amplification is carried out to detected materials with the primer pair that step 1) obtains, obtains pcr amplification product；

3) polymorphism for detecting the pcr amplification product, determines the detected materials according to the polymorphism of the pcr amplification product In it is to be measuredTaAGPLThe allelic variation type of gene；

Wherein, the primer pair is following 1. or 2. shown:

1. SEQ ID:1 and SEQ ID:3；

2. SEQ ID:2 and SEQ ID:3.

6. according to the method described in claim 5, it is characterized by: describedTaAGPLGene is wheat adenosine diphosphate glucose Pyrophosphorylation enzyme gene；The polymorphism is length polymorphism.

7. method according to claim 5 or 6, it is characterised in that: the wheat adenosine diphosphate glucose pyrophosphorylation Enzyme gene is wheat plasmotype ADP-glucose pyrophosphorylase gene.

8. according to the method any in claim 5-6, it is characterised in that: the detected materials are wheat.