CN105219858A - Grain Weight in Common Wheat gene TaGS5-3A single nucleotide polymorphism and application thereof - Google Patents
Grain Weight in Common Wheat gene TaGS5-3A single nucleotide polymorphism and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of Grain Weight in Common Wheat gene TaGS5-3A single nucleotide polymorphism and application thereof.The single nucleotide polymorphism that the present invention's application is specially following SNP site in Wheat volatiles qualification or assistant identification wheat grain to be measured correlated character (grain weight and/or grain length and/or grain is wide and/or grain thick) in application; Described SNP site is: with Wheat volatiles DNA for template, adopts primer pair to carry out the 718th Nucleotide from 5 ' end of the amplified production that pcr amplification obtains; The Nucleotide of described SNP site is T or G; Described primer pair is made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table.The present invention studies and finds TaGS5-3A-T allelotrope and Grain Weight in Common Wheat, grain length, grain is wide and grain is thick presents significantly or extremely significantly positive correlation, this SNP marker is codominant marker simultaneously, reproducible, cost is low, in wheat assisted Selection and Molecular design breeding, have good application prospect.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of Grain Weight in Common Wheat gene TaGS5-3A single nucleotide polymorphism and application thereof.
Background technology
Wheat, paddy rice, the food crop such as corn provide about 50% of necessary for human energy, but along with the growth of world population, the minimizing of cultivated area and the increase of grain-production cost, the output improving food crop is breeding men main purposes all the time.Wheat is the important food crop of China, and its cultivated area is only second to first food crop paddy rice, and the height of wheat yield directly affects standard of living and the national food security of our people.Therefore, utilize Protocols in Molecular Biology to clone the relevant functional gene of wheat yield, and develop corresponding molecule marker with this, for wheat molecular marker assistant breeding provides important genetic resources, to significantly improving China's wheat yield, accelerate China's wheat breeding process significant.
Grain is heavily one of three elements of output, and the key factor that decision grain is heavy comprises the grouting degree of a type and seed.In in the past 10 years, the research of Functional Genomics of Rice develops rapidly, accelerate to advance our understanding for rice grain shape, the gene of many grains type/grain heavy phase pass is obtained by the method for map based cloning, these genes take part in expansion and the division of cell mostly, play a role mainly through following approach: by participating in the ubiquitination pathway of proteolytic enzyme, by participating in the signal pathway of G-protein, by the Physiology and biochemistry approach (Zuo that the adjustment etc. participating in hormone is different, etal.Moleculargeneticdissectionofquantitativetraitlocire gulatingricegrainsize.AnnuRevGenet, 48:99-118).And in the heavy research field of the grain type/grain of wheat, genome structure with complexity huge due to wheat, although there is the QTL that a large amount of grain type/grain heavy phase is closed to be found, still little by the example of the heavy gene of the direct separation wheat of the method single grain type/grain of map based cloning.On the other hand, in recent years, having based on gramineous crop genomic level comparative genomics that this characteristic of good collinearity develops is that the clone of wheat important gene provides fundamental basis.The heavy gene of the wheat groat type be separated by the method/grain reported is comprised: the Starch synthesis gene (TaSus1) of wheat, the different allelic variations of this gene determine that the expression amount in differing materials is different, wherein excellent allelic variation is proved and closes (HouJ, etal.Globalselectiononsucrosesynthasehaplotypesduringace nturyofwheatbreeding.PlantPhysiol2014 with grain heavy phase; 164:1918-1929); The wide gene of wheat groat (TaGW2) is the homologous gene of the wide gene GW2 of rice grain, confirm that TaGW2 is wide negative regulatory factor (HongYT, the etal.TranscriptsuppressionofTaGW2increasedgrainwidthandw eightinbreadwheat.FunctIntegrGenomics2014 of wheat groat by the technology of RNAi; 14:341-349); Zhang etc. report a gene relevant to Grain Weight in Common Wheat and grain length, similar with the homologous gene GS3 in its paddy rice, the albumen of its coding all comprises the OSRdomain of a grain length negative correlation, the different allelic variations of this gene contain different OSRdomain, create different grain lengths/grain weight.Therefore, along with the announcement of wheat and sibling species draft genome thereof, the method for the homologous clone of genomics becomes the effective means of the heavy gene of clone's wheat groat type/grain based on the comparison.
Summary of the invention
The object of this invention is to provide a kind of Grain Weight in Common Wheat gene TaGS5-3A single nucleotide polymorphism and application thereof.
Application provided by the present invention, is specially the application of single nucleotide polymorphism in qualification or assistant identification wheat grain correlated character to be measured of following SNP site in Wheat volatiles; Described SNP site is: with Wheat volatiles DNA for template, adopts primer pair to carry out the 718th Nucleotide from 5 ' end of the amplified production that pcr amplification obtains; The Nucleotide of described SNP site is T or G; The primer pair of described primer pair for being made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table; Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
Certainly, the application of material in qualification or assistant identification wheat grain correlated character to be measured for detecting the single nucleotide polymorphism of following SNP site in Wheat volatiles also belongs to protection scope of the present invention; Described SNP site is: with Wheat volatiles DNA for template, adopts primer pair to carry out the 718th Nucleotide from 5 ' end of the amplified production that pcr amplification obtains; The Nucleotide of described SNP site is T or G; The primer pair of described primer pair for being made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table; Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
When the Nucleotide of described SNP site is T, the allelic variation type of TaGS5-3A gene is TaGS5-3A-T; When the Nucleotide of described SNP site is G, the allelic variation type of TaGS5-3A gene is TaGS5-3A-G.
Wherein, described " for detecting the material of the single nucleotide polymorphism of following SNP site in Wheat volatiles " specifically can be following test kit or primer pair:
Described test kit for containing, for example under the primer pair shown in (a) or (b), and the test kit of restriction enzyme Fnu4HI or its isoschizomers;
A primer pair that () is made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table;
(b) by by sequence in sequence table 1 and sequence 2 through the replacement of one or several Nucleotide and/or disappearance and/or after adding two single strand dnas shown in gained sequence form, and the primer pair identical with the function of primer pair described in (a).
Described primer pair is the primer pair shown in following (a) or (b):
A primer pair that () is made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table;
(b) by by sequence in sequence table 1 and sequence 2 through the replacement of one or several Nucleotide and/or disappearance and/or after adding two single strand dnas shown in gained sequence form, and the primer pair identical with the function of primer pair described in (a).
Described test kit and described primer pair also belong to protection scope of the present invention respectively.
Present invention also offers a kind of method of seed correlated character of more different wheat to be measured.
The method of the seed correlated character of more different wheat to be measured provided by the present invention, can be following A) or B):
A) utilize the method for the seed correlated character of the more different wheat to be measured of described test kit, specifically can comprise the step of following (a1)-(a3):
(a1) respectively with the genomic dna of each wheat to be measured for template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification;
(a2) restriction enzyme Fnu4HI or its isoschizomers is adopted to carry out complete degestion respectively to step (a1) gained PCR primer;
Described complete degestion refers to that described restriction enzyme Fnu4HI or its isoschizomers are enough, is enough to make all PCR primer can cut by it all cut;
(a3) the seed correlated character of more described each wheat to be measured as follows:
The DNA fragmentation cutting acquisition through step (a2) enzyme be the grain of the wheat described to be measured of following (1) heavy in or candidate overweight the wheat described to be measured that the DNA fragmentation cutting acquisition through step (a2) enzyme be (2) as follows;
The DNA fragmentation cutting acquisition through step (a2) enzyme is that the grain length of the wheat described to be measured of following (1) is longer than or candidate is longer than the wheat described to be measured that the DNA fragmentation cutting acquisition through step (a2) enzyme be (2) as follows;
The DNA fragmentation cutting acquisition through step (a2) enzyme be the grain of the wheat described to be measured of following (1) wide in or candidate be the wheat described to be measured of (2) as follows wider than the DNA fragmentation cutting acquisition through step (a2) enzyme;
The DNA fragmentation cutting acquisition through step (a2) enzyme be the grain of the wheat described to be measured of following (1) thick in or candidate be thicker than the wheat described to be measured that the DNA fragmentation cutting acquisition through step (a2) enzyme be (2) as follows;
(1) size is 863bp unique DNA fragment;
(2) size is respectively two DNA fragmentations of 719bp and 144bp;
B) utilize the method for the more different seed correlated character of wheat to be measured of described primer pair (sequence 1 and sequence 2), specifically comprise the step of following (b1)-(b2):
(b1) respectively with the genomic dna of each wheat to be measured for template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification;
(b2) the seed correlated character of more described each wheat to be measured is determined as follows:
The PCR primer obtained through step (b1) pcr amplification be the grain of the wheat described to be measured of following (3) heavy in or candidate overweight the wheat described to be measured that the PCR primer obtained through step (b1) pcr amplification is (4) as follows;
PCR primer through the acquisition of step (b1) pcr amplification is that the grain length of the wheat described to be measured of following (3) is longer than or candidate is longer than the wheat described to be measured that the PCR primer obtained through step (b1) pcr amplification is following (4);
The PCR primer obtained through step (b1) pcr amplification be the grain of the wheat described to be measured of following (3) wide in or candidate be the wheat described to be measured of (4) as follows wider than the PCR primer obtained through step (b1) pcr amplification;
The PCR primer obtained through step (b1) pcr amplification be the grain of the wheat described to be measured of following (3) thick in or candidate be thicker than the wheat described to be measured that the PCR primer obtained through step (b1) pcr amplification is (4) as follows;
(3) single PCR primer: the DNA fragmentation of nucleotide sequence as shown in sequence in sequence table 3;
(4) single PCR primer: the DNA fragmentation of nucleotide sequence as shown in sequence in sequence table 4;
Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
Present invention also offers a kind of grain of cultivating heavily to increase weight and/or grain length increases and/or the method for the wide broadening and/or thick wheat breed thickened of grain of grain.
Cultivation grain provided by the present invention heavily increases weight and/or grain length increases and/or the method for the wide broadening and/or thick wheat breed thickened of grain of grain, specifically can comprise the wheat chosen and meet following condition (I) or (II) carries out breeding step as parent:
(I) take genomic dna as template, described primer pair (sequence 1 and sequence 2) is adopted to carry out pcr amplification, gained PCR primer restriction enzyme Fnu4HI or its isoschizomers are carried out complete degestion, and obtaining size is 863bp unique DNA fragment;
(II) take genomic dna as template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification, obtain the unique DNA fragment as shown in sequence in sequence table 3.
In the above-mentioned methods, the annealing temperature adopted when carrying out described pcr amplification is 61 DEG C.
Further, the amplification program adopted when carrying out described pcr amplification is specially: 94 DEG C of 4min; 95 DEG C of 35s, 61 DEG C of 35s, 72 DEG C of 1min, 35 circulations; 72 DEG C of 10min; 10 DEG C of 10hr.
In actual applications, the mol ratio of two single stranded DNAs in PCR reaction system when carrying out described pcr amplification in described primer pair can be 1:1.
Further, described PCR reaction system is specially: template DNA 1.0 μ L; Mg
2+1 μ L; 10 × pcr amplification damping fluid 1.5 μ L; The each 0.5 μ L of upstream and downstream primer (10 μMs); DNTP (25 μMs) 0.12 μ L; Taq enzyme (5U/ μ L) 0.12 μ L; ddH
2o complements to 15 μ L.
The present invention also protects the DNA fragmentation of nucleotide sequence as shown in sequence in sequence table 3, and the allelic variation of the described SNP site that this DNA fragmentation is corresponding is TaGS5-3A-T.
The application of described DNA fragmentation in qualification or assistant identification wheat grain correlated character also belongs to protection scope of the present invention; Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
In the present invention, described grain heavily specifically embodies with " thousand seed weight ".
The present invention utilizes the method for molecule marker by the TaGS5-3A assignment of genes gene mapping on the 3A karyomit(e) of wheat, by being separated the sequence of TaGS5-3A gene and promoter region thereof, find that this gene the 6th exon exists a SNP (2334), make this gene there are two kinds of allelic variation TaGS5-3A-T and TaGS5-3A-G.SNP have developed a molecule marker accordingly, in China's wheat Mini core collection, the analysis of mark/trait associations is utilized to find, the extremely significantly positive correlation (P<0.01) of TaGS5-3A-T allelotrope and Grain Weight in Common Wheat, wide and grain is thick significantly or pole significant correlation (P<0.05 or P<0.01) with wheat grain length, grain, in addition, this excellent allelic variation receives strong selection in China's wheat breeding process.Meanwhile, this SNP marker is codominant marker, and reproducible, cost is low, in the molecular marker assisted selection improving Grain Weight in Common Wheat and Molecular design breeding, have good application prospect.
Accompanying drawing explanation
Fig. 1 is 36 parts of wheat lines TaGS5-3A sequence alignments (partial sequence).
Fig. 2 is the function SNP marker sepharose detected result of TaGS5-3A.
Fig. 3 be various years Bred Wheat Varieties thousand seed weight and the change of TaGS5-3A two kinds of gene frequencies.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Wheat breed in embodiment is all available from Crops In China kind matter Information Network, and the Unified number of table 2 is numbering in net.Network address: http://www.cgris.net/zhongzhidinggou/index.php.
The excavation of SNP site of embodiment 1, TaGS5-3A gene and the exploitation of certification mark thereof
One, the excavation of TaGS5-3A gene SNP site between wheat breed
1, for examination wheat lines
Selection is distributed in the different Mai Qu's of China, and 36 parts of wheat lines (numbering POL1-36) that grain characters differs greatly are as the excavation material (table 1) of pleomorphism site;
2, the sequence alignment of TaGS5-3A gene
Utilize conventional CTAB method to extract above each genomic dna for examination wheat lines, increase to the TaGS5-3A gene regions of 36 parts of wheat lines respectively, the amplimer of employing is TaGS5-3A-F and TaGS5-3A-R.
TaGS5-3A-F：5’-CCCTCCAACTTCACATG-3’；
TaGS5-3A-R：5’-TCATCGGTGTGTAGGAAG-3’。
Increase laggard row agarose gel electrophoresis, reclaims amplified fragments and check order; The Seqman in DNAStar is utilized to carry out sequence assembly, MegAlign carries out sequence alignment, result is as shown in Fig. 1 (partial sequence), only there is a SNP (being designated as SNP2334) at the 6th exon place in TaGS5-3A gene, the nucleotides sequence at this SNP site place is classified as T or G, thus two kinds of allelotrope are formed, respectively called after TaGS5-3A-T and TaGS5-3A-G.
Further, statistical study has been carried out to for the grain principal characteristic shape (thousand seed weight) of 36 parts of wheat lines tried and the genotype of TaGS5-3A, found that: wherein genotype is the thousand seed weight average of the wheat lines that TaGS5-3A-T isozygotys is 43.45g, it is the wheat lines (P<0.01) that TaGS5-3A-G isozygotys that pole is significantly higher than genotype, the thousand seed weight average 28.29g of the latter.
The thousand seed weight of 36 parts of wheat lines that table 1 is excavated for SNP and TaGS5-3A genotype statistics thereof
| Numbering | Title | Thousand seed weight | TaGS5-3A genotype | Numbering | Title | Thousand seed weight | TaGS5-3A genotype |
| POL1 | Zhongyou9507 | 51.7g | TaGS5-3A-T | POL19 | Shanxi wheat 11 | 46.35g | TaGS5-3A-T |
| POL2 | Shanxi wheat No. 8 | 41.3g | TaGS5-3A-T | POL20 | Shandong wheat No. 9 | 26.45g | TaGS5-3A-G |
| POL3 | No. 8, Beijing | 42.35g | TaGS5-3A-T | POL21 | Ji wheat 19 | 22.4g | TaGS5-3A-G/T |
| POL4 | Shijiazhuang 54 | 40.2g | TaGS5-3A-T | POL22 | 10000 years No. 4 | 21.05g | TaGS5-3A-T |
| POL5 | Tobacco grower 15 | 34.05g | TaGS5-3A-T | POL23 | Jiangxi early | 28.95g | TaGS5-3A-G |
| POL6 | Laishou 953 | 42.05g | TaGS5-3A-T | POL24 | Rob a wheat | 30.4g | TaGS5-3A-G |
| POL7 | Lu's drought 328 | 44.15g | TaGS5-3A-T | POL25 | Bai Maizi | 24.45g | TaGS5-3A-G |
| POL8 | Zheng wheat 9023 | 44.1g | TaGS5-3A-T | POL26 | China spring | 27.35g | TaGS5-3A-G |
| POL9 | Climb 86001-3 | 52.8g | TaGS5-3A-G | POL27 | Fried dough twist plate | 20.9g | TaGS5-3A-G |
| POL10 | Xuzhou 22 | 51.3g | TaGS5-3A-T | POL28 | Red golden wheat | 23.4g | TaGS5-3A-G |
| POL11 | Temperature wheat No. 8 | 51.7g | TaGS5-3A-T | POL29 | Orchid wheat | 28.6g | TaGS5-3A-G |
| POL12 | Lankao 906 | 51.7g | TaGS5-3A-T | POL30 | Bai Mangmai | 29.85g | TaGS5-3A-G |
| POL13 | Agricultural university 139 | 32.05g | TaGS5-3A-T | POL31 | March is yellow | 28.85g | TaGS5-3A-G |
| POL14 | Engrave virtuous 169 | 33.2g | TaGS5-3A-G | POL32 | Spend wheat in vain | 24.45g | TaGS5-3A-G |
| POL15 | Jingyang 60 | 27.3g | TaGS5-3A-G | POL33 | Bai Dongmai | 15.75g | TaGS5-3A-G |
| POL16 | Short rich No. 3 | 44.95g | TaGS5-3A-T | POL34 | Red winter wheat | 14.85g | TaGS5-3A-G |
| POL17 | Shandong wheat No. 1 | 53.4g | TaGS5-3A-T | POL35 | No. 8, Shijiazhuang | 46.26g | TaGS5-3A-T |
| POL18 | Beijing 15 | 28.55g | TaGS5-3A-G | POL36 | Stone 4185 | 43.04g | TaGS5-3A-G |
Note: in table, TaGS5-3A-G represents that TaGS5-3A-G isozygotys; TaGS5-3A-T represents that TaGS5-3A-T isozygotys; TaGS5-3A-G/T represents the heterozygosis (being also called for short TaGS5-3A-G/T heterozygosis) of TaGS5-3A-G and TaGS5-3A-T.
Two, the exploitation of the SNP marker of TaGS5-3A gene
According to the SNP2334 site that step one finds, find, compared with the large grain kind (TGAGC) that thousand seed weight average lower granule kind (GCAGC) is higher with thousand seed weight average, to there is a Fnu4HI restriction enzyme site further.Carry out the exploitation of SNP marker according to this difference, concrete steps are as follows:
1) amplification of specific fragment:
For trying the genomic dna of wheat breed as template, primer pair F/R is adopted to carry out pcr amplification.
F:5 '-AGACATGGTGGAGCAAGAGATG-3 ' (sequence 1);
R:5 '-GAACAACCTAATCCTCCTCCTGA-3 ' (sequence 2).
PCR reaction system
PCR response procedures
2) restriction enzyme Fnu4HI complete degestion (enzyme is enough) is carried out to specific fragment
It is as follows that enzyme cuts system, and 37 DEG C, enzyme cuts 30min
Note: restriction enzyme enzyme Fnu4HI is NEB product.
3) detection of endonuclease bamhi: use 1.5% sepharose to carry out the detection of endonuclease bamhi.
4) order-checking of object band and analysis.
5) genotype of the SNP2334 for examination wheat breed is determined according to electrophoresis and sequencing result:
If electrophoresis only obtains two object bands that size is respectively 719bp (the 1-719 position of sequence 4) and 144bp (the 720-863 position of sequence 4), then the corresponding genotype for examination wheat TaGS5-3A is that TaGS5-3A-G isozygotys; If electrophoresis only obtains the object band that size is 863bp (sequence 3), then the corresponding genotype TaGS5-3A-T for examination wheat TaGS5-3A isozygotys; If electrophoresis only obtains three object bands that size is 719bp (the 1-719 position of sequence 4), 144bp (the 720-863 position of sequence 4) and 863bp (sequence 3), the then corresponding genotype for examination wheat TaGS5-3A is TaGS5-3A-G/T heterozygosis, as shown in Figure 2.
Certainly, in actually operating, also restriction enzyme Fnu4HI can not be adopted to carry out enzyme to PCR primer cut, but directly PCR primer is checked order, genotype according to determining as follows for examination wheat breed TaGS5-3A: if PCR primer is single product---the DNA fragmentation (863bp) in sequence table shown in sequence 3, then the corresponding genotype for examination wheat TaGS5-3A is that TaGS5-3A-T isozygotys; If PCR primer is single product---the DNA fragmentation (863bp) in sequence table shown in sequence 4, then the corresponding genotype for examination wheat TaGS5-3A is that TaGS5-3A-G isozygotys; If PCR primer is two kinds of products---the DNA fragmentation (863bp) shown in sequence 4 in the DNA fragmentation (863bp) in sequence table shown in sequence 3 and sequence table, then the corresponding genotype for examination wheat TaGS5-3A is TaGS5-3A-G/T heterozygosis.
The functional verification of embodiment 2, TaGS5-3A gene SNP site
Application China wheat Mini core collection (HouJ, etal.Globalselectiononsucrosesynthasehaplotypesduringace nturyofwheatbreeding.PlantPhysiol2014; 164:1918-1929) carrying out the functional verification of TaGS5-3A gene, the phenotypic data in conjunction with multiple years such as thousand seed weight (TKW), grain length (KL), grain wide (KW), grains thick (KT) carries out marking/association analysis of proterties.Wherein, identify that the method for TaGS5-3A gene type is see embodiment 1.The essential information of the present embodiment whole wheat Mini core collection material and the seed phenotypic evaluation data in Luoyang, henan in 2002 refer to table 2 (in addition, in Luoyang, henan, having carried out corresponding measuring equally in 2010 in Shunyi, Beijing in 2005).When measuring seed phenotypic data, each sample at least arranges 3 replications, and result gets average.
Result shows: three annual thousand seed weight of the wheat lines (SNP2334 site is TT) of allelic variation TaGS5-3A-T are 38.17g, and the wheat lines of allelic variation TaGS5-3A-G (SNP2334 site is GG) is 31.26g, difference reaches pole conspicuous level (P<0.01); Equally, in grain length, grain is wide and grain is thick proterties, the wheat lines of allelic variation TaGS5-3A-T is larger compared with the wheat lines seed of allelic variation TaGS5-3A-G, difference reaches remarkable or pole conspicuous level (P<0.05 or P<0.01), and concrete outcome is in table 3.It can thus be appreciated that compared to allelic variation TaGS5-3A-G, allelic variation TaGS5-3A-T is the excellent allelic variation of TaGS5-3A gene.
The TaGS5-3A genotype of table 2 wheat Mini core collection material and seed phenotypic evaluation data
Note: in table 2, phenotypic character blank is not for measuring.
The association analysis of table 3TaGS5-3A and seed correlated character
Note: 2002LY represents Luoyang (2002); 2005LY represents Luoyang (2005); 2010SY represents Shunyi, Beijing (2010).*P<0.05，**P<0.01。
In addition, analysis discovery (Fig. 3) is carried out to the variation tendency of the thousand seed weight of various years improved variety and the frequency of TaGS5-3A two allelic variations, along with the passing in age, the thousand seed weight of China's Bred Wheat Varieties presents the trend of increase, consistent with this variation tendency is, the frequency of occurrences of excellent allelic variation TaGS5-3A-T in various years kind is also in the trend (Fig. 3) raised, this shows that modern breeding has carried out very strong selective action to this allelic variation, and this variation and the heavy significant correlation of grain.Therefore, this mark can be used as the functional label improving Grain Weight in Common Wheat, carry out improving yield of wheat molecular mark.
Claims (10)
1. in Wheat volatiles following SNP site single nucleotide polymorphism qualification or assistant identification wheat grain correlated character to be measured in application;
Described SNP site is: with Wheat volatiles DNA for template, adopts primer pair to carry out the 718th Nucleotide from 5 ' end of the amplified production that pcr amplification obtains; The Nucleotide of described SNP site is T or G; The primer pair of described primer pair for being made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table;
Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
2. for detecting the application of material in qualification or assistant identification wheat grain correlated character to be measured of the single nucleotide polymorphism of following SNP site in Wheat volatiles;
Described SNP site is: with Wheat volatiles DNA for template, adopts primer pair to carry out the 718th Nucleotide from 5 ' end of the amplified production that pcr amplification obtains; The Nucleotide of described SNP site is T or G; The primer pair of described primer pair for being made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table;
Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
3. application according to claim 2, is characterized in that: described " for detecting the material of the single nucleotide polymorphism of following SNP site in Wheat volatiles " is test kit according to claim 4 or primer pair according to claim 5.
4. for the identification of or the test kit of assistant identification wheat grain correlated character to be measured, containing, for example under the primer pair shown in (a) or (b), and restriction enzyme Fnu4HI or its isoschizomers;
A primer pair that () is made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table;
(b) by by sequence in sequence table 1 and sequence 2 through the replacement of one or several Nucleotide and/or disappearance and/or after adding two single strand dnas shown in gained sequence form, and the primer pair identical with the function of primer pair described in (a);
Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
5. for the identification of or the primer pair of assistant identification wheat grain correlated character to be measured, the primer pair for shown in following (a) or (b):
A primer pair that () is made up of the single stranded DNA shown in sequence 2 in the single stranded DNA shown in sequence in sequence table 1 and sequence table;
(b) by by sequence in sequence table 1 and sequence 2 through the replacement of one or several Nucleotide and/or disappearance and/or after adding two single strand dnas shown in gained sequence form, and the primer pair identical with the function of primer pair described in (a);
Described seed correlated character is grain weight, and/or grain length, and/or grain is wide, and/or grain is thick.
6. a method for the seed correlated character of more different wheat to be measured is following A) or B):
A) utilize the method for the seed correlated character of the more different wheat to be measured of test kit described in claim 4, comprise the step of following (a1)-(a3):
(a1) respectively with the genomic dna of each wheat to be measured for template, adopt the primer pair described in claim 4 carry out pcr amplification;
(a2) restriction enzyme Fnu4HI or its isoschizomers is adopted to carry out complete degestion respectively to step (a1) gained PCR primer;
(a3) the seed correlated character of more described each wheat to be measured as follows:
The DNA fragmentation cutting acquisition through step (a2) enzyme be the grain of the wheat described to be measured of following (1) heavy in or candidate overweight the wheat described to be measured that the DNA fragmentation cutting acquisition through step (a2) enzyme be (2) as follows; And/or
The DNA fragmentation cutting acquisition through step (a2) enzyme is that the grain length of the wheat described to be measured of following (1) is longer than or candidate is longer than the wheat described to be measured that the DNA fragmentation cutting acquisition through step (a2) enzyme be (2) as follows; And/or
The DNA fragmentation cutting acquisition through step (a2) enzyme be the grain of the wheat described to be measured of following (1) wide in or candidate be the wheat described to be measured of (2) as follows wider than the DNA fragmentation cutting acquisition through step (a2) enzyme; And/or
The DNA fragmentation cutting acquisition through step (a2) enzyme be the grain of the wheat described to be measured of following (1) thick in or candidate be thicker than the wheat described to be measured that the DNA fragmentation cutting acquisition through step (a2) enzyme be (2) as follows;
(1) size is 863bp unique DNA fragment;
(2) size is respectively two DNA fragmentations of 719bp and 144bp;
B) utilize the method for the seed correlated character of the more different wheat to be measured of primer pair described in claim 5, comprise the step of following (b1)-(b2):
(b1) respectively with the genomic dna of each wheat to be measured for template, adopt primer pair described in claim 5 to carry out pcr amplification;
(b2) the seed correlated character of more described each wheat to be measured is determined as follows:
The PCR primer obtained through step (b1) pcr amplification be the grain of the wheat described to be measured of following (3) heavy in or candidate overweight the wheat described to be measured that the PCR primer obtained through step (b1) pcr amplification is (4) as follows; And/or
PCR primer through the acquisition of step (b1) pcr amplification is that the grain length of the wheat described to be measured of following (3) is longer than or candidate is longer than the wheat described to be measured that the PCR primer obtained through step (b1) pcr amplification is following (4); And/or
The PCR primer obtained through step (b1) pcr amplification be the grain of the wheat described to be measured of following (3) wide in or candidate be the wheat described to be measured of (4) as follows wider than the PCR primer obtained through step (b1) pcr amplification; And/or
The PCR primer obtained through step (b1) pcr amplification be the grain of the wheat described to be measured of following (3) thick in or candidate be thicker than the wheat described to be measured that the PCR primer obtained through step (b1) pcr amplification is (4) as follows;
(3) single PCR primer: the DNA fragmentation of nucleotide sequence as shown in sequence in sequence table 3;
(4) single PCR primer: the DNA fragmentation of nucleotide sequence as shown in sequence in sequence table 4;
Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
7. cultivate grain heavily to increase weight and/or grain length increases and/or a method for the wide broadening and/or thick wheat breed thickened of grain of grain, comprise the wheat chosen and meet following condition (I) or (II) carries out breeding step as parent:
(I) take genomic dna as template, adopt the primer pair described in claim 4 to carry out pcr amplification, gained PCR primer restriction enzyme Fnu4HI or its isoschizomers are carried out complete degestion, and obtaining size is 863bp unique DNA fragment;
(II) take genomic dna as template, adopt primer pair described in claim 5 to carry out pcr amplification, obtain the unique DNA fragment as shown in sequence in sequence table 3.
8. the method according to claim 6 or 7, is characterized in that: the annealing temperature adopted when carrying out described pcr amplification is 61 DEG C.
9.DNA fragment, its nucleotide sequence is as shown in sequence in sequence table 3.
10. the application of DNA fragmentation described in claim 9 in qualification or assistant identification wheat grain correlated character;
Described seed correlated character is the heavy and/or grain length of grain and/or grain is wide and/or grain is thick.
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