CN109136402B - CAPS mark for detecting Chinese wheat 895 flag leaf included angle QTL and application - Google Patents

Abstract

The invention discloses a CAPS mark for detecting a Chinese wheat 895 flag leaf included angle QTL and application thereof. The method carries out field identification on the flag leaf included angle of 174 family of poplar 16/medium wheat 895DH colony in 2016-2017 years, carries out genotype analysis on the colony by using a 660K SNP chip, constructs a high-density linkage map, finds a main effective site which is positioned on 1Ds and used for controlling the flag leaf included angle, has an LOD value of 3.4-10.8 and 6.1-9.1% of explained Phenotypic Variation (PVE), and marks the site as QFla.caas-1 Ds. The CAPS marker Fla-1Ds-CAPS was developed based on its linkage marker AX-94664699. The invention not only provides a good tool for auxiliary selective breeding, but also creates conditions for the flag leaf included angle control gene of the map-based cloning site.

Description

CAPS mark for detecting Chinese wheat 895 flag leaf included angle QTL and application

Technical Field

The invention relates to the technical field of biology, in particular to a CAPS mark for detecting a wheat 895 flag leaf included angle QTL and application thereof.

Background

Wheat is one of the most important food crops in the world, and China is the largest wheat producing and consuming country in the world. In the face of double pressure of population growth and land reduction, further improvement of wheat yield is an urgent and long-term task for ensuring food safety. Due to land and water resource limitations, the potential for increasing total wheat yield by expanding planting area is very limited, and thus only by increasing and maintaining higher yield per unit levels.

The plant type is a general term of the whole plant form related to the crop yield and the arrangement characteristics of the plant in space, and mainly comprises the characters of tillering number, tillering angle, spike shape, plant height, leaf size, leaf angle and the like, and also comprises physiological characters and ecological characters which influence the utilization efficiency of the group light energy. The plant type affects the photosynthetic efficiency, lodging resistance, resistance to some diseases and insect pests and the like of crops, and finally affects the crop yield. The wheat plant type is always a very interesting character for breeders and is closely related to the crop yield. The Ideal Plant Type (Ideal Plant Type) of wheat has the characteristics of short Plant, compact Plant Type, upright leaves, awn, high harvest index, independent and strong stems and the like. Improving plant type and constructing reasonable high-light-efficiency population is an effective way for realizing wheat yield breakthrough.

Leaves are the main organs for the production of photosynthetic products. In the later growth stage of wheat, the flag leaves have the greatest effect on the formation of grains and yield. The included angle between the flag leaves and the stalks has great influence on the light receiving condition of the wheat plants. The light receiving area of the flag leaf can be influenced by the small included angle of the flag leaf, so that the photosynthetic rate of the plant is reduced; the too large included angle also shields the light receiving area of the lower leaves, which is also not beneficial to the photosynthetic efficiency of the whole leaves. Therefore, the research on the flag leaf included angle regulation gene and the construction of a reasonable plant type have important significance for increasing and stabilizing the yield of wheat.

Disclosure of Invention

The invention aims to provide a CAPS mark for detecting the Chinese dwarf 895 flag leaf included angle QTL and application thereof.

The invention firstly protects a specific primer pair; the specific primer pair consists of a primer PF and a primer PR;

the primer PF is (a1) or (a 2):

(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

(a2) DNA molecule which is obtained by substituting one or more nucleotides in the sequence 1 and has the same function as the sequence 1;

the primer PR is (a3) or (a4) as follows:

(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

(a4) and (b) a DNA molecule which is obtained by substituting the sequence 2 by one or more nucleotides and has the same function as the sequence 2.

The molar ratio of the primer PF to the primer PR is 1: 1.

The specific primer pair is used for at least one of the following (b1) - (b 8):

(b1) identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(b2) breeding wheat with relatively small included angle of flag leaves;

(b3) breeding wheat with relatively large included angle of flag leaves;

(b4) detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla. caas-1 Ds;

(b5) preparing a kit for identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(b6) preparing a kit for breeding wheat with relatively small flag leaf included angle;

(b7) preparing a kit for breeding wheat with relatively large flag leaf included angle;

(b8) preparing a kit for detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla.

The invention also protects the application of the specific primer pair, which is at least one of the following (b1) - (b 8):

(b1) identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(b2) breeding wheat with relatively small included angle of flag leaves;

(b3) breeding wheat with relatively large included angle of flag leaves;

(b4) detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla. caas-1 Ds;

(b5) preparing a kit for identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(b6) preparing a kit for breeding wheat with relatively small flag leaf included angle;

(b7) preparing a kit for breeding wheat with relatively large flag leaf included angle;

(b8) preparing a kit for detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla.

The invention also protects a kit containing the specific primer pair; the application of the kit is at least one of the following (c1) - (c 4):

(c1) identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(c2) breeding wheat with relatively small included angle of flag leaves;

(c3) breeding wheat with relatively large included angle of flag leaves;

(c4) detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla.

The invention also discloses a method for identifying or assisting in identifying the wheat flag leaf angle character, which is the method A or the method B.

The method A comprises the following steps: taking the genome DNA of the wheat to be detected as a template, carrying out PCR amplification by adopting the specific primer pair, carrying out enzyme digestion on the amplification product by using SfaN I, and if the enzyme digestion product has a DNA fragment of 400bp and does not have a DNA fragment of 265bp, the included angle of the flag leaf of the wheat to be detected is relatively small; if the enzyme digestion product has a 265bp DNA fragment and does not have a 400bp DNA fragment, the included angle of the flag leaf of the wheat to be detected is relatively large.

The method B comprises the following steps: detecting whether the genome DNA of the wheat to be detected contains a specific DNA fragment A and a specific DNA fragment B, if the genome DNA contains the specific DNA fragment A and does not contain the specific DNA fragment B, the included angle of flag leaves of the wheat to be detected is relatively small; if the genome DNA contains the specific DNA fragment B and does not contain the specific DNA fragment A, the included angle of the flag leaves of the wheat to be detected is relatively large;

the specific DNA fragment A is a sequence 4 or a reverse complementary sequence thereof;

the specific DNA fragment B is sequence 5 or a reverse complementary sequence thereof.

The invention also discloses a method (method C) for identifying or assisting in identifying the wheat flag leaf included angle character, which comprises the following steps: detecting the genotype of the wheat flag leaf included angle QTL QFla.caas-1Ds to be detected, wherein the flag leaf included angle of the wheat carrying the dominant allele of the flag leaf included angle QTL QFla.caas-1Ds is relatively smaller; the flag leaf angle of wheat carrying the recessive allele of flag leaf angle QTL QFla. caas-1Ds is relatively large.

The invention also discloses a method (method D) for protecting, detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla.caas-1Ds, which comprises the following steps: taking the genome DNA of wheat to be detected as a template, carrying out PCR amplification by adopting the specific primer pair, carrying out enzyme digestion on an amplification product by using SfaN I, and if the enzyme digestion product has a 400bp DNA fragment and does not have a 265bp DNA fragment, determining that the wheat to be detected is or is a candidate of the wheat carrying dominant allele of the flag leaf included angle QTL QFla.caas-1 Ds; if the enzyme digestion product has a 265bp DNA fragment and does not have a 400bp DNA fragment, the wheat to be detected is or is candidate to be the wheat carrying the flag leaf included angle QTL QFla.

The invention also discloses a method (method E) for protecting, detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla.caas-1Ds, which comprises the following steps: detecting whether the genome DNA of the wheat to be detected contains a specific DNA fragment A and a specific DNA fragment B, if the genome DNA contains the specific DNA fragment A and does not contain the specific DNA fragment B, the wheat to be detected is or is a candidate of the wheat carrying dominant allele of the flag leaf included angle QTL QFla.caas-1 Ds; if the genome DNA contains a specific DNA fragment B and does not contain a specific DNA fragment A, the wheat to be detected is or is candidate to be the wheat carrying the recessive allele of the flag leaf included angle QTL QFla.caas-1 Ds;

the specific DNA fragment A is a sequence 4 or a reverse complementary sequence thereof;

the specific DNA fragment B is sequence 5 or a reverse complementary sequence thereof.

In any of the above methods, the reaction system for PCR amplification may specifically be: template DNA (30 ng/. mu.L) 2. mu.L, primer PF (10. mu.M) 0.5. mu.L, primer PR (10. mu.M) 0.5. mu.L, 2 XPCR Mix (0.1U. mu.L)^-1Taq enzyme, 500. mu. mol L^-1dNTPs、20mmol L^-1Tri-HCl、10mmol L^-1KCl、3mmol L^-1MgCl₂)10μL，ddH₂O 7μL。

In any of the above methods, the PCR procedure may specifically be: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 30s for 35 cycles; extending for 5min at 72 ℃; storing at 4 ℃.

The invention also protects the specific DNA fragment A and/or the specific DNA fragment B.

The invention also protects the application of any one of the methods in wheat breeding.

The invention also protects the application of any one of the specific DNA fragment A and the specific DNA fragment B in wheat breeding.

The invention also protects a wheat breeding method, which is any one of the methods A-D;

the method A comprises the following steps: selecting wheat with a relatively small included angle of flag leaves obtained by screening according to the method A, the method B or the method C as a breeding material;

the method B comprises the following steps: selecting the wheat carrying dominant allele of the flag leaf included angle QTL QFla.caas-1Ds obtained by screening according to the method E or the method D as a breeding material;

the method C comprises the following steps: selecting wheat with a relatively large included angle of flag leaves obtained by screening according to the method A, the method B or the method C as a breeding material;

the method comprises the following steps: wheat carrying the recessive allele of the flag leaf included angle QTL QFla. caas-1Ds obtained by screening according to the method E or the method D is used as a breeding material.

Any one of the above described flag leaf included angles is relatively small, which means any one of the following (a) - (c):

(a) when the effects of other sites influencing the size of the flag leaf included angle on the compared wheat genome are equal, the flag leaf included angle of the wheat carrying the dominant allele of the flag leaf included angle QTL QFla.caas-1Ds is smaller relative to the flag leaf included angle of the wheat carrying the recessive allele of the flag leaf included angle QTL QFla.caas-1 Ds;

(b) when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat which is identified by the method A and has the DNA fragment of 400bp and does not have the DNA fragment of 265bp in the enzyme digestion product is smaller than the flag leaf included angle of the wheat which has the DNA fragment of 265bp and does not have the DNA fragment of 400bp in the enzyme digestion product;

(c) when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat without the specific DNA fragment B and containing the specific DNA fragment A in the genome DNA is smaller than the flag leaf included angle of the wheat without the specific DNA fragment A and containing the specific DNA fragment B in the genome DNA.

Any one of the above flag leaves with a relatively large included angle refers to any one of the following (d) - (f):

(d) when the effects of other sites influencing the size of the flag leaf included angle on the compared wheat genome are equal, the flag leaf included angle of the wheat carrying the recessive allele of the flag leaf included angle QTL QFla.caas-1Ds is larger relative to the flag leaf included angle of the wheat carrying the dominant allele of the flag leaf included angle QTL QFla.caas-1 Ds;

(e) when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat which is identified by the method A and has the 265bp DNA fragment and does not have the 400bp DNA fragment in the enzyme digestion product is larger than the flag leaf included angle of the wheat which has the 400bp DNA fragment and does not have the 265bp DNA fragment in the enzyme digestion product;

(f) when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat which contains the specific DNA segment B in the genome DNA and does not contain the specific DNA segment A is larger than the flag leaf included angle of the wheat which contains the specific DNA segment A in the genome DNA and does not contain the specific DNA segment B.

Any one of the 265bp DNA fragments is a sequence 6 or a reverse complementary sequence thereof.

Any one of the above 400bp DNA fragments is sequence 7 or its reverse complementary sequence.

Any one of the above wheat can be any one or any several of the following varieties: yangmai 16, Migmai 895, Yangmai 16/Migmai 895DH populations, and 118 wheat varieties shown in Table 2 of the examples section.

Any one of the QTL QFla, caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

According to the invention, the included angle of the flag leaves of 174 families of populus 16/Zhongmai 895DH groups is identified in the fields of Henan New and rural areas, Shangqiu, 2017-2018 in Henan New and rural areas, Lowe and Wuhan Hubei in Henan, the group is subjected to genotype analysis by using a 660K SNP chip, a high-density linkage map is constructed, QTL positioning finds a main effective site which is positioned on 1Ds and used for controlling the included angle of the flag leaves, the LOD value is 3.4-10.8, and the explained Phenotypic Variation (PVE) is 6.1-9.1%, and the site is marked as QFLA. The CAPS marker Fla-1Ds-CAPS was developed based on its linkage marker AX-94664699 (FIG. 1). Experiments prove that: the molecular marker Fla-1Ds-CAPS can be effectively used for breeding and utilization of Chinese wheat 895QFla. The invention not only provides a good tool for auxiliary selective breeding, but also creates conditions for the flag leaf included angle control gene of the map-based cloning site.

Drawings

FIG. 1 is a graph of QFla. caas-1Ds mapped by composite interval mapping method in the Poplar 16X Zhongmai 895DH population.

FIG. 2 shows the results of the electrophoresis detection of the PCR products of the species and after the enzyme digestion. 1, Marker; 2. 3: wheat 895 and poplar wheat 16 in PCR products; 4-12: the enzyme-digested Chinese barley 895, poplar barley 16 and other varieties.

FIG. 3 shows flag leaf types of mature Hordeum vulgare 16 and Hordeum vulgare 895.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Flag leaf angle (Fls): the included angle between the flag leaf and the plant stem, the included angle of the flag leaf is divided into 1-6 grades, and the included angle is 1 grade: less than or equal to 30 degrees; and 2, stage: 30-60 degrees; and 3, level: 60-90 degrees; 4, level: 90-120 parts of; and 5, stage: 120-150 °; and 6, level: not less than 150 degrees.

Chinese wheat 895: the Chinese agricultural academy of sciences crop scientific research institute and the Chinese agricultural academy of sciences cotton research institute cooperate to breed a new wheat variety with high yield, disease resistance and eurytopic property. Reference documents: the key points of seed selection and cultivation of Hayagi, Yanjun, Zhang Yong, high-yield dwarf lodging-resistant Guangzhi wheat new variety Mimai 895 are [ J ] crop journal, 2013(5):154 and 155. The Chinese wheat 895 has a flag leaf included angle of 2 grades.

Wheat winnowing 16: a new species of spring medium gluten wheat bred by the research institute of agricultural science in the region of Ri and river of Jiangsu. Reference documents: land-grown Bin, Chengxiang, Zenbo bridge, etc. the characteristic features and high yield cultivation technique of Yangmai 16, a new high-quality medium-gluten wheat variety [ J ] Jiangsu agricultural science, 2006(3):112. the public can be obtained from the institute of crop science of Chinese academy of agricultural sciences. The included angle of the Yangmai 16 flag leaves is 6 grades.

The flag leaf type of mature Hordeum vulgare 16 and Hordeum vulgare 895 is shown in figure 3.

Example 1 obtaining of wheat flag leaf included angle QFla. caas-1Ds and linkage marker thereof

Test materials: zhongmai 895, Yangmai 16 and DH colony (containing 174 families) constructed by using Yangmai 16 as female parent and Zhongmai 895 as male parent and adopting wheat and corn hybridization technology.

Field identification of the included angle of flag leaves: new country in Henan in 2016-2017, Shangqiu, new country in Henan in 2017-2018, Luzhou and Wuhan in Hubei. The test adopts a completely random block design, 3 times of repetition, 6m of cell length and 0.25m of line spacing, and 6 lines in total. And (5) when the flag leaves of each family are drawn for 15-20d, investigating the included angle of the flag leaves of each family.

And (3) field data analysis: and calculating the Pearson correlation coefficient of the flag leaf by using a PROC CORR model of SAS9.2 software. Correlation analysis shows that the correlation coefficients between 2016-2017 new country and Shangqiu repeats are 0.83 and 0.78(P <0.01), the correlation coefficients between 2017-2018 new country, Luohao and Wuhan repeats are 0.79, 0.76 and 0.68(P <0.01), and the correlation coefficients of the leaf pinch angle under different environments are 0.70(P <0.01), so that the effectiveness of the group field phenotype data is shown.

And (3) genetic map construction: removing DH line with deletion rate of marker higher than 20%, removing segregation marker with no polymorphism, deletion rate higher than 10% and two genotype ratio higher than 7:3 or lower than 3: 7. The remaining polymorphic markers were optimized for genetic Mapping using the BIN-Mapping function of IcMapping V4.0. The construction of genetic map was carried out using the JoinMap V4.0 and MSTmap Online.

QTL positioning: QTL positioning is carried out on the leaf included angle of the Yangmai 16/Zhongmai 895DH population by QTLICiMapping V4.0 Composite Interval Mapping (CIM), and an LOD value of 2.5 is selected as a threshold value. QTL location finds the major effective site (figure 1) which is located on 1Ds and controls the included angle of flag leaves, the two sides are marked as AX-109378629 and AX-110977092, the LOD value is 3-11, the explanation phenotype variation is 6.1% -9.1%, and the site is marked as QFla. The markers on both sides are 39.66cM and 45.24cM, respectively, and the physical location on the Chinese spring reference genome (IWGSC Refseq v1.0) is about 10-103 Mb. And selecting a plurality of SNP markers near the site for developing CAPS markers closely linked with QFla.

Example 2 establishment of the Fla-1Ds-CAPS labelling method

First, genome specific primer design

The genetic position of AX-94664699 on the linkage diagram 1 is 19.3cM, the physical position on the Chinese spring reference genome (IWGSC Refseq v1.0) is 16.2Mb, and the genetic positions and the physical positions of markers AX-109378629 and AX-110977092 positioned at both sides of QFla.caas-1Ds are very close to each other, so that the method can be used for marker development. The flanking sequences of SNP AX-94664699 (SEQ ID NO: 3) were aligned with the Ensemblplants database (http:// plants. ensembl. org.) to obtain homologous sequences, and 1D chromosome-specific primers P (SEQ ID NO: 1 and SEQ ID NO: 2) were designed and synthesized by Beijing Hua Biotech, Inc.

PF: 5' -CTTGCATTGCATTGCTTCT (SEQ ID NO: 1);

PR: 5' -GCTTGCATAAATCACGGCTC (SEQ ID NO: 2).

Second, the establishment of detection method

Test materials: zhongmai 895, Yangmai 16 and DH colony (containing 174 families) constructed by wheat and corn hybridization technology with Yangmai 16 as female parent and Zhongmai 895 as male parent.

1. Extracting genome DNA of wheat to be detected, adding ddH₂O diluted to a concentration of 30 ng/. mu.L.

2. And (3) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and adopting a primer pair consisting of a primer PF and a primer PR to obtain a PCR product.

The PCR system is shown in Table 1.

PCR program, pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 30s for 35 cycles; extending for 5min at 72 ℃; storing at 4 ℃.

TABLE 1 PCR System

3. The PCR product obtained in step 2 was detected by electrophoresis on a 1.5% agarose gel and sequenced. The lengths of the amplified fragments of Zhongmai 895 and Yangma 16 are both 827bp (figure 2), the amplified fragment of the Zhongmai 895 sequence is shown as a sequence 4 after sequencing, the amplified fragment of Yangma 16 is shown as a sequence 5, and a G/T SNP site exists at the 429bp site.

4. The PCR products of Yangmai 16 and Mianmai 895 were digested with SfaN I endonuclease at 37 ℃ for 1 h. The enzyme digestion reaction system is 10 mu L, and contains 4 mu L of PCR product, 10 XBuffer 1 mu L, 0.3 mu L of SfaN I and ddH₂O 4.7μL。

5. The cleavage product of step 4 was detected by 1.5% agarose gel electrophoresis and sequenced. The results show that: the Yangmai 16PCR product was digested with SfaN I to obtain 265bp fragments (SEQ ID NO: 6), 160bp fragments, 130bp fragments and 100bp fragments, and did not contain 400bp fragments, while the Zhongmai 895PCR product was digested with SfaN I to obtain 400bp fragments (SEQ ID NO: 7), 160bp fragments and 100bp fragments, and did not contain 265bp fragments (FIG. 2). The enzyme digestion product has different electrophoretic bands to distinguish the genotypes of Yangmai 16 and Miyama 895 at the site, and the mark is Fla-1 Ds-CAPS.

The genotype of the 174 pedigrees Fla-1Ds-CAPS markers is different from that of the AX-94664699 chip in 6 pedigrees, and the genotype is similar to that of the chip marker AX-94664699 when the chip marker is placed in the original spectrogram, which indicates that the chip marker AX-94664699 is successfully converted into the CAPS marker.

Therefore, the allele of the wheat flag leaf included angle QTL QFla.caas-1Ds to be detected can be judged by the following method:

amplifying the genome DNA of wheat to be detected by using a primer consisting of a primer PF and a primer PR to obtain an amplification product, carrying out enzyme digestion on the amplification product by using SfaN I to obtain an enzyme digestion product, detecting the enzyme digestion product by agarose gel electrophoresis, and judging the allele of the wheat flag leaf included angle QTL QFla.caas-1Ds to be detected according to the electrophoresis result:

if the amplification product of the material to be detected is cut by SfaN I enzyme, the electrophoresis contains a 400bp strip and does not contain a 265bp strip, the site genotype of the material to be detected is the same as that of Chinese wheat 895, the gene is presumed to contain a QFla.caas-1Ds dominant allele, and the included angle of flag leaves is smaller;

if the amplification product of the material to be detected is cut by SfaN I enzyme, the electrophoresis contains 265bp bands and does not contain 400bp bands, the site genotype of the material to be detected is the same as Yangmai 16, the material to be detected is presumed to contain QFla.caas-1Ds recessive allele, and the included angle of flag leaves is larger.

Example 3 actual sample detection

Test materials: in China, 118 parts of wheat varieties (germplasm library of the institute of crop science of Chinese academy of agricultural sciences) in different provinces are shown in Table 2.

1. Leaf angle phenotype identification

And planting 118 wheat varieties in 2016-2017 years and 2017-2018 years in Xingxiang and Luo, and performing field identification on the included angle of the flag leaves. The test adopts a completely random block design, three times of repetition, a double-row area, a row length of 1.5m, a row spacing of 0.25m and 60 seeds sowed in each row. And (5) surveying the leaf included angle of each variety when the colony draws for 15-20 days later. The two-year flag leaf angle mean value was used for subsequent analysis.

2. Fla-1Ds-CAPS marker detection

The assay was performed as in example 2, with medium wheat 895 and Yangmai 16 as controls.

The detection results of the 118 wheat varieties and the average value of the flag leaf included angles are shown in table 2. 93 shares of gene type identical to Zhongmai 895, which is presumed to contain QFla. caas-1Ds dominant allele, and Fls has an average value of 2.1; 25 shares are the same as Yangmai 16 genotype, presumably containing the QFla. caas-1Ds recessive allele, with an average value of Fls of 4.3. The mean value of the variety Fls containing the dominant allele of QFla.caas-1Ds was 2.2 lower (approximately 66 ℃ lower) than the variety containing the recessive allele of QFla.caas-1 Ds. T-test was performed using the PROC TTEST model of SAS9.2 statistical software, and the difference was significant at 0.05 level (P ═ 0.011).

TABLE 2118 wheat variety Fla-1Ds-CAPS marker test results

Note: 1, dividing the included angle of the flag leaves into 1-6 grades, and 1: less than or equal to 30 degrees; 2: 30-60 degrees; 3: 60-90 degrees; 4: 90-120 parts of; 5: 120-150 °; 6: 150 degrees; 2: "+" indicates that the site genotype is the same as that of Zhongmai 895, and the allele containing the dominant flag leaf included angle of QFla. caas-1Ds is presumed to reduce the flag leaf included angle; "-" indicates that the site genotype is the same as Yangmai 16, and the allele of negative flag leaf included angle of QFla. caas-1Ds is presumed to contain, so that the flag leaf included angle cannot be reduced.

Sequence listing

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gtgtgcattt acatgagtga attcatgtgc atatgtctga gcgtctacgt atgtactgtg 720

ttaaaaaaac tattgtatct tcattaggat ttactagttt tttcctaaac ctatttttta 780

ttttctgtta gcttgttctt ttagcgtgag ccgtgattta tgcaagc 827

<210> 6

<211> 265

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 6

tggcgccgag ctcgccaagg ggcacgtcca tggcttccgg atgtcgctct ggcacgagca 60

cctcggcaag acgcacgatg acttcctgcg cccgggaagc ctagagtgcg tgcagagggt 120

caacaagatg gccgatgagt actggagcct ctacgtcggc gaccagctgc cggaagacct 180

cccaggccac ctgctcacct accccgtctc ggtagacaag gccggcaatg tctcagcggt 240

cacgggattt gaattcttcc ctgac 265

<210> 7

<211> 400

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 7

tggcgccgag ctcgccaagg ggcacgtcca tggcttccgg atgtcgctct ggcacgagca 60

cctcggcaag acgcacgatg acttcctgcg cccgggaagc ctagagtgcg tgcagagggt 120

caacaagatg gccgatgagt actggagcct ctacgtcggc gaccagctgc cggaagacct 180

cccaggccac ctgctcacct accccgtctc ggtagacaag gccggcaatg tctcagcggt 240

cacgggattt gaattcttcc ctgacacaga ggctcgcgtg cttggtgagc caacgggaat 300

aaaagattat tttctgagca catagctcca ggtttgccgc tcctagtttg tgttgattaa 360

tcctggcacc tccgggtttt ttttttttgc gcatcgtgtt 400

Claims (12)

1. A specific primer pair; the specific primer pair consists of a primer PF and a primer PR;

the primer PF is a single-stranded DNA molecule shown in a sequence 1 of a sequence table;

the primer PR is a single-stranded DNA molecule shown in a sequence 2 of a sequence table.

2. The use of the specific primer pair according to claim 1, which is at least one of the following (b1) - (b 8):

(b1) identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(b2) breeding wheat with relatively small included angle of flag leaves;

(b3) breeding wheat with relatively large included angle of flag leaves;

(b4) detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla. caas-1 Ds;

(b5) preparing a kit for identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(b6) preparing a kit for breeding wheat with relatively small flag leaf included angle;

(b7) preparing a kit for breeding wheat with relatively large flag leaf included angle;

(b8) preparing a kit for detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla. caas-1 Ds;

the fact that the included angle of the flag leaves is relatively small means that when the effects of other sites influencing the size of the included angle of the flag leaves on the compared wheat genomes are equal, the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves is smaller relative to the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the relatively large included angle of the flag leaves means that when the effects of other sites influencing the size of the included angle of the flag leaves on the genome of the compared wheat are equal, the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves is larger than the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

3. A kit comprising a specific primer pair according to claim 1; the application of the kit is at least one of the following (c1) - (c 4):

(c1) identifying or assisting in identifying the characteristic of the wheat flag leaf included angle;

(c2) breeding wheat with relatively small included angle of flag leaves;

(c3) breeding wheat with relatively large included angle of flag leaves;

(c4) detecting or assisting in detecting the genotype of the wheat flag leaf included angle QTL QFla. caas-1 Ds;

the fact that the included angle of the flag leaves is relatively small means that when the effects of other sites influencing the size of the included angle of the flag leaves on the compared wheat genomes are equal, the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves is smaller relative to the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the relatively large included angle of the flag leaves means that when the effects of other sites influencing the size of the included angle of the flag leaves on the genome of the compared wheat are equal, the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves is larger than the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

4. The method for identifying or assisting in identifying the characteristic of the wheat flag leaf included angle is a method A or a method B;

the method A comprises the following steps: taking genome DNA of wheat to be detected as a template, carrying out PCR amplification by adopting the specific primer pair of claim 1, carrying out enzyme digestion on an amplification product by using SfaN I, and if the enzyme digestion product has a DNA fragment of 400bp and does not have a DNA fragment of 265bp, the included angle of flag leaves of the wheat to be detected is relatively small; if the enzyme digestion product has a 265bp DNA fragment and does not have a 400bp DNA fragment, the included angle of the flag leaf of the wheat to be detected is relatively large;

the fact that the flag leaf included angle is relatively small means that when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat which is identified by the method A to have the DNA fragment of 400bp and does not have the DNA fragment of 265bp in the enzyme digestion product is smaller than the flag leaf included angle of the wheat which is identified by the method A to have the DNA fragment of 265bp and does not have the DNA fragment of 400bp in the enzyme digestion product;

the fact that the flag leaf included angle is relatively large means that when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat which is identified by the method A and has the 265bp DNA fragment and does not have the 400bp DNA fragment in the enzyme digestion product is larger than the flag leaf included angle of the wheat which has the 400bp DNA fragment and does not have the 265bp DNA fragment in the enzyme digestion product;

the method B comprises the following steps: detecting whether the genome DNA of the wheat to be detected contains a specific DNA fragment A and a specific DNA fragment B, if the genome DNA contains the specific DNA fragment A and does not contain the specific DNA fragment B, the included angle of flag leaves of the wheat to be detected is relatively small; if the genome DNA contains the specific DNA fragment B and does not contain the specific DNA fragment A, the included angle of the flag leaves of the wheat to be detected is relatively large;

the specific DNA fragment A is a sequence 4 or a reverse complementary sequence thereof;

the specific DNA fragment B is a sequence 5 or a reverse complementary sequence thereof;

the fact that the flag leaf included angle is relatively small means that when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat without the specific DNA fragment B through the specific DNA fragment A in the genome DNA is smaller than the flag leaf included angle of the wheat without the specific DNA fragment A through the flag leaf included angle of the wheat with the specific DNA fragment B in the genome DNA;

the fact that the flag leaf included angle is relatively large means that when the effects of other sites influencing the size of the flag leaf included angle on the genome of the compared wheat are equal, the flag leaf included angle of the wheat without the specific DNA fragment A through the specific DNA fragment B in the genome DNA is larger than the flag leaf included angle of the wheat without the specific DNA fragment B through the flag leaf included angle of the wheat with the specific DNA fragment A in the genome DNA.

5. The method for identifying or assisting in identifying the wheat flag leaf included angle character comprises the following steps: detecting the genotype of the wheat flag leaf included angle QTL QFla.caas-1Ds to be detected, wherein the flag leaf included angle of the wheat carrying the dominant allele of the flag leaf included angle QTL QFla.caas-1Ds is relatively smaller; the flag leaf angle of wheat carrying the recessive allele of flag leaf angle QTL QFla. caas-1Ds is relatively large;

the fact that the included angle of the flag leaves is relatively small means that when the effects of other sites influencing the size of the included angle of the flag leaves on the compared wheat genomes are equal, the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves is smaller relative to the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the relatively large included angle of the flag leaves means that when the effects of other sites influencing the size of the included angle of the flag leaves on the genome of the compared wheat are equal, the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves is larger than the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

6. The method for detecting or assisting in detecting the genotype of wheat flag leaf included angle QTL QFla. caas-1Ds comprises the following steps: taking genome DNA of wheat to be detected as a template, carrying out PCR amplification by adopting the specific primer pair as claimed in claim 1, carrying out enzyme digestion on an amplification product by SfaN I, and if the enzyme digestion product has a DNA fragment of 400bp and does not have a DNA fragment of 265bp, carrying out PCR amplification on the genome DNA of the wheat to be detected or selecting the wheat to be detected as the wheat carrying dominant allele of the flag leaf included angle QTL QFla.caas-1 Ds; if the enzyme digestion product has a 265bp DNA fragment and does not have a 400bp DNA fragment, the wheat to be detected is or is candidate to be the wheat carrying the flag leaf included angle QTL QFla.caas-1Ds recessive allele;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

7. The method for detecting or assisting in detecting the genotype of wheat flag leaf included angle QTL QFla. caas-1Ds comprises the following steps: detecting whether the genome DNA of the wheat to be detected contains a specific DNA fragment A and a specific DNA fragment B, if the genome DNA contains the specific DNA fragment A and does not contain the specific DNA fragment B, the wheat to be detected is or is a candidate of the wheat carrying dominant allele of the flag leaf included angle QTL QFla.caas-1 Ds; if the genome DNA contains a specific DNA fragment B and does not contain a specific DNA fragment A, the wheat to be detected is or is candidate to be the wheat carrying the recessive allele of the flag leaf included angle QTL QFla.caas-1 Ds;

the specific DNA fragment A is a sequence 4 or a reverse complementary sequence thereof;

the specific DNA fragment B is a sequence 5 or a reverse complementary sequence thereof;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

8. Use of the method of any one of claims 4 to 7 in wheat breeding.

9. A method of breeding wheat, the method comprising the steps of: wheat with a relatively small included angle of flag leaves obtained by screening according to the method of claim 4 or 5 is used as a breeding material;

the fact that the included angle of the flag leaves is relatively small means that when the effects of other sites influencing the size of the included angle of the flag leaves on the compared wheat genomes are equal, the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves is smaller relative to the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

10. A method of breeding wheat, the method comprising the steps of: wheat carrying dominant allele of the flag leaf included angle QTL QFla.caas-1Ds screened according to the method of claim 6 or 7 is used as breeding material;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

11. A method of breeding wheat, the method comprising the steps of: the wheat with a relatively large included angle of flag leaves obtained by screening according to the method of claim 4 or 5 is used as a breeding material;

the relatively large included angle of the flag leaves means that when the effects of other sites influencing the size of the included angle of the flag leaves on the genome of the compared wheat are equal, the included angle of the flag leaves of the wheat carrying the recessive allele of the included angle of the flag leaves is larger than the included angle of the flag leaves of the wheat carrying the dominant allele of the included angle of the flag leaves QTL QFla.caas-1 Ds;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

12. A method of breeding wheat, the method comprising the steps of: wheat carrying recessive alleles of the flag leaf included angle QTL QFla.caas-1Ds screened according to the method of claim 6 or 7 is used as a breeding material;

the QTL QFla.caas-1Ds is positioned on the wheat 1Ds, the LOD value is 3-11, and the explained phenotypic variation is 6.1% -9.1%.

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