Screening method for breeding parent variety of high-quality wheat and primer group combination used by same

文档序号：1320948 发布日期：2020-07-14 浏览：4次中文

阅读说明：本技术 一种用于培育优质小麦的亲本品种的筛选方法及其使用的引物组组合 (Screening method for breeding parent variety of high-quality wheat and primer group combination used by same ) 是由武玉莹夏先春何中虎于 2020-05-07 设计创作，主要内容包括：本发明公开了一种用于培育优质小麦的亲本品种的筛选方法及其使用的引物组组合。引物组组合由引物组1—引物组21组成,用于扩增21个STARP标记,每个引物组包括3条引物,63条引物的核苷酸序列依次如SEQ ID NO:1—SEQ ID NO:63所示。本发明开发的STARP标记,PCR扩增后不用酶切和电泳,可以高通量检测多个样品,大大提高了检测效率,实现了高通量、低成本快速检测的目的,便于筛选用于培育优质小麦的亲本品种,加快育种进程。本发明在辅助筛选小麦品种中具有重要的理论意义和经济价值。(The invention discloses a screening method for breeding parent varieties of high-quality wheat and a primer group combination used by the same. The primer group combination consists of a primer group 1-a primer group 21 and is used for amplifying 21 STARP markers, each primer group comprises 3 primers, and the nucleotide sequences of the 63 primers are sequentially shown as SEQ ID NO. 1-SEQ ID NO. 63. The STARP marker developed by the invention does not need enzyme digestion and electrophoresis after PCR amplification, can detect a plurality of samples at high flux, greatly improves the detection efficiency, realizes the purpose of high-flux, low-cost and quick detection, is convenient for screening parent varieties for cultivating high-quality wheat and accelerates the breeding process. The invention has important theoretical significance and economic value in auxiliary screening of wheat varieties.)

1. A primer set combination consisting of a primer set 1 for amplifying GluA1-Indel, a primer set 2 for amplifying GluA1-SNP, a primer set 3 for amplifying GluD1-SNP, a primer set 4 for amplifying Glu-A3 e-490-SNP, a primer set 5 for amplifying Glu-A3g-SNP, a primer set 6 for amplifying Glu-B3B-SNP, a primer set 7 for amplifying Glu-B3c-SNP, a primer set 8 for amplifying PpoA1-SNP, a primer set 9 for amplifying PpoD1-Indel, a primer set 10 for amplifying PsyA1-Indel, a primer set 11 for amplifying yB1-SNP, a primer set 12 for amplifying PsyD1-SNP, a primer set 68613 for amplifying ZDsA 8-Indel, a primer set 6-Indel, a primer set 3514-SNP, a primer set for amplifying PinB 354619-SNP, a primer set for amplifying PinB 3619-PdN19-SNP, a primer set for amplifying PpndNb4619-SNP, a primer set for amplifying PnPinB 3619-PdNb4619-SNP;

GluA1-Indel and GluA1-SNP are STARP markers developed based on the wheat Glu-A1 gene;

GluD1-SNP is a STARP marker developed based on wheat Glu-D1 gene;

Glu-A3e-490SNP and Glu-A3g-SNP are STARP markers developed based on the wheat Glu-A3 gene;

Glu-B3B-SNP and Glu-B3c-SNP are STARP markers developed based on the wheat Glu-B3 gene;

the PpoA1-SNP is a STARP marker developed based on the wheat PpoA-A1 gene;

PpoD1-Indel is a STARP marker developed based on the wheat Ppo-D1 gene;

PsyA1-Indel is a STARP marker developed based on the wheat Psy-A1 gene;

the PsyB1-SNP is a STARP marker developed based on the wheat Psy-B1 gene;

the PsyD1-SNP is a STARP marker developed based on the wheat Psy-D1 gene;

ZDSA1-Indel is a STARP marker developed based on the wheat TaZDs-A1 gene;

l oxB1-SNP is a STARP marker developed based on wheat Ta L ox-B1 gene;

l cyA1-SNP is a STARP marker developed based on wheat Ta L cy-A1 gene;

l cyB1-SNP is a STARP marker developed based on wheat Ta L cy-B1 gene;

the PdsB1-SNP is a STARP marker developed based on the wheat TaPds-B1 gene;

PodA1-SNP is a STARP marker developed based on the wheat Tapod-A1 gene;

pina D1-Indel is a STARP marker developed based on the wheat Pina-D1 gene;

the PinbD1-SNP is a STARP marker developed based on the wheat Pinb-D1 gene;

PinbB2-Indel is a STARP marker developed based on the wheat Pinb-B2 gene;

the primer group 1 comprises a primer 1, a primer 2 and a primer 3;

the primer group 2 comprises a primer 4, a primer 5 and a primer 6;

the primer group 3 comprises a primer 7, a primer 8 and a primer 9;

the primer group 4 comprises a primer 10, a primer 11 and a primer 12;

the primer group 5 comprises a primer 13, a primer 14 and a primer 15;

the primer group 6 comprises a primer 16, a primer 17 and a primer 18;

the primer group 7 comprises a primer 19, a primer 20 and a primer 21;

the primer group 8 comprises a primer 22, a primer 23 and a primer 24;

the primer group 9 includes a primer 25, a primer 26 and a primer 27;

the primer group 10 includes a primer 28, a primer 29 and a primer 30;

the primer group 11 includes a primer 31, a primer 32 and a primer 33;

the primer group 12 includes a primer 34, a primer 35 and a primer 36;

the primer group 13 includes a primer 37, a primer 38 and a primer 39;

the primer group 14 includes a primer 40, a primer 41, and a primer 42;

the primer group 15 includes a primer 43, a primer 44 and a primer 45;

the primer group 16 includes a primer 46, a primer 47, and a primer 48;

the primer group 17 includes a primer 49, a primer 50 and a primer 51;

the primer group 18 includes a primer 52, a primer 53 and a primer 54;

the primer group 19 includes a primer 55, a primer 56 and a primer 57;

the primer set 20 includes a primer 58, a primer 59, and a primer 60;

the primer group 21 includes a primer 61, a primer 62 and a primer 63;

the nucleotide sequence of primer 1 consists of tag sequence B and SEQ ID NO:1 from position 22 to 39 from the 5' end;

the nucleotide sequence of primer 2 consists of tag sequence a and SEQ ID NO:2 from the 5' end to positions 22 to 39;

the nucleotide sequence of the primer 3 is shown as SEQ ID NO: 3 is shown in the specification;

the nucleotide sequence of primer 4 consists of tag sequence B and SEQ ID NO: 4 from position 22 to 42 from the 5' end;

the nucleotide sequence of primer 5 consists of tag sequence a and SEQ ID NO: 5 from the 5' end to positions 22 to 42;

the nucleotide sequence of the primer 6 is shown as SEQ ID NO:6 is shown in the specification;

the nucleotide sequence of primer 7 consists of tag sequence B and SEQ ID NO: 7 from position 22 to 45 from the 5' end;

the nucleotide sequence of primer 8 consists of tag sequence a and SEQ ID NO: 8 from the 5' end to positions 22 to 45;

the nucleotide sequence of the primer 9 is shown as SEQ ID NO: 9 is shown in the figure;

the nucleotide sequence of primer 10 consists of tag sequence a and SEQ ID NO: 10 from the 5' end to positions 22 to 42;

the nucleotide sequence of primer 11 consists of tag sequence B and SEQ ID NO: 11 from position 22 to 42 from the 5' end;

the nucleotide sequence of the primer 12 is shown as SEQ ID NO: 12 is shown in the specification;

the nucleotide sequence of primer 13 consists of tag sequence B and SEQ ID NO: 13 from the 22 nd to the 44 th nucleotide sequence from the 5' end;

the nucleotide sequence of primer 14 consists of tag sequence a and SEQ ID NO: 14 from the 5' end to positions 22 to 44;

the nucleotide sequence of the primer 15 is shown as SEQ ID NO: 15 is shown in the figure;

the nucleotide sequence of primer 16 consists of tag sequence a and SEQ ID NO: 16 from position 22 to 46 from the 5' end;

the nucleotide sequence of primer 17 consists of tag sequence B and SEQ ID NO: 17 from the 5' end to positions 22 to 46;

the nucleotide sequence of the primer 18 is shown as SEQ ID NO: 18 is shown in the figure;

the nucleotide sequence of primer 19 consists of tag sequence B and SEQ ID NO: 19 from the 5' end to positions 22 to 40;

the nucleotide sequence of primer 20 consists of tag sequence a and SEQ ID NO: 20 from position 22 to 40 from the 5' terminus;

the nucleotide sequence of the primer 21 is shown as SEQ ID NO: 21 is shown in the figure;

the nucleotide sequence of primer 22 consists of tag sequence a and SEQ ID NO: 22 from position 22 to 40 from the 5' end;

the nucleotide sequence of primer 23 consists of tag sequence B and SEQ ID NO: 23 from the 5' end to positions 22 to 40;

the nucleotide sequence of the primer 24 is shown as SEQ ID NO: shown at 24;

the nucleotide sequence of primer 25 consists of tag sequence a and SEQ ID NO: 25 from the 5' end to positions 22 to 47;

the nucleotide sequence of primer 26 consists of tag sequence B and SEQ ID NO: 26 from position 22 to 48 from the 5' terminus;

the nucleotide sequence of the primer 27 is shown as SEQ ID NO: 27 is shown;

the nucleotide sequence of primer 28 consists of tag sequence B and SEQ ID NO: 28 from position 22 to 46 from the 5' end;

the nucleotide sequence of primer 29 consists of tag sequence a and SEQ ID NO: 29 from position 22 to 46 from the 5' terminus;

the nucleotide sequence of the primer 30 is shown as SEQ ID NO: 30 is shown in the figure;

the nucleotide sequence of primer 31 consists of tag sequence B and SEQ ID NO: 31 from the 5' end to positions 22 to 43;

the nucleotide sequence of primer 32 consists of tag sequence a and SEQ ID NO: 32 from the 5' end to positions 22 to 43;

the nucleotide sequence of the primer 33 is shown as SEQ ID NO: 33;

the nucleotide sequence of primer 34 consists of tag sequence B and SEQ ID NO: 34 from the 5' end to positions 22 to 41;

the nucleotide sequence of primer 35 consists of tag sequence a and SEQ ID NO: 35 from the 5' end to positions 22 to 41;

the nucleotide sequence of the primer 36 is shown as SEQ ID NO: 36 is shown;

the nucleotide sequence of primer 37 consists of tag sequence B and SEQ ID NO: 37 from the 5' end to positions 22 to 47;

the nucleotide sequence of primer 38 consists of tag sequence a and SEQ ID NO: 38 from the 5' end to positions 22 to 47;

the nucleotide sequence of the primer 39 is shown as SEQ ID NO: 39;

the nucleotide sequence of primer 40 consists of tag sequence a and SEQ ID NO: 40 from the 5' end to positions 22 to 39;

the nucleotide sequence of primer 41 consists of tag sequence B and SEQ ID NO: 41 from the 5' end to positions 22 to 39;

the nucleotide sequence of the primer 42 is shown as SEQ ID NO: 42 is shown;

the nucleotide sequence of primer 43 consists of tag sequence a and SEQ ID NO: 43 from the 5' end to positions 22 to 39;

the nucleotide sequence of primer 44 consists of tag sequence B and SEQ ID NO: 44 from the 5' end to positions 22 to 39;

the nucleotide sequence of primer 45 is shown in SEQ ID NO: 45 is shown;

the nucleotide sequence of primer 46 consists of tag sequence B and SEQ ID NO: 46 from the 5' end to positions 22 to 39;

the nucleotide sequence of primer 47 consists of tag sequence a and SEQ ID NO: 47 from position 22 to 39 from the 5' terminus;

the nucleotide sequence of the primer 48 is shown as SEQ ID NO: 48 is shown;

the nucleotide sequence of primer 49 consists of tag sequence B and SEQ ID NO: 49 from the 5' end to positions 22 to 46;

the nucleotide sequence of primer 50 consists of tag sequence a and SEQ ID NO: 50 from position 22 to 46 from the 5' terminus;

the nucleotide sequence of the primer 51 is shown as SEQ ID NO: 51 is shown;

the nucleotide sequence of primer 52 consists of tag sequence a and SEQ ID NO: 52 from the 5' end to positions 22 to 40;

the nucleotide sequence of primer 53 consists of tag sequence B and SEQ ID NO: 53 from position 22 to 40 from the 5' end;

the nucleotide sequence of the primer 54 is shown as SEQ ID NO: 54 is shown;

the nucleotide sequence of primer 55 consists of tag sequence B and SEQ ID NO: 55 from the 5' end to positions 22 to 41;

the nucleotide sequence of primer 56 consists of tag sequence a and SEQ ID NO: 56 from the 5' end to positions 22 to 41;

the nucleotide sequence of the primer 57 is shown as SEQ ID NO: shown as 57;

the nucleotide sequence of primer 58 consists of tag sequence B and SEQ ID NO: 58 from the 5' end to positions 22 to 42;

the nucleotide sequence of primer 59 consists of tag sequence a and SEQ ID NO: 59 from the 5' end to positions 22 to 41;

the nucleotide sequence of the primer 60 is shown as SEQ ID NO: 60 is shown;

the nucleotide sequence of primer 61 consists of tag sequence B and SEQ ID NO: 61 from position 22 to 46 from the 5' terminus;

the nucleotide sequence of primer 62 consists of tag sequence a and SEQ ID NO: 62 from position 22 to 48 from the 5' terminus;

the nucleotide sequence of the primer 63 is shown as SEQ ID NO:63, respectively.

2. The primer set combination of claim 1, wherein: the primer group 1-the primer group 21 also comprise a primer A and/or a primer B;

the primer A sequentially comprises a DNA fragment A, a PCR blocker and a DNA fragment B from 5 'to 3'; the nucleotide sequence of the DNA fragment A is shown as SEQ ID NO: 64; the nucleotide sequence of the DNA fragment B is shown as1 st to 21 st from the 5' end of SEQ ID NO 2;

the primer B sequentially comprises a DNA fragment C, a PCR blocker and a DNA fragment D from 5 'to 3'; the nucleotide sequence of the DNA fragment C is shown as SEQ ID NO. 65; the nucleotide sequence of the DNA fragment D is shown as1 st to 21 st from the 5' end of SEQ ID NO 1.

3. The primer set combination of claim 2, wherein:

the 5' end of the DNA fragment A is provided with a fluorescence label A; carrying out quenching group modification on the DNA fragment B;

the 5' end of the DNA fragment C is provided with a fluorescent label B; and carrying out quenching group modification on the DNA fragment.

4. The primer set combination of claim 3, wherein:

the fluorescence label A is a FAM fluorescence label; the nucleotide sequence of the tag sequence A is shown as1 st to 21 st positions from the 5' end of SEQ ID NO 2;

the fluorescent label B is a HEX fluorescent label; the nucleotide sequence of the tag sequence B is shown as the 1 st to 21 st positions from the 5' end of SEQ ID NO. 1.

5. The use of the primer set combination according to any one of claims 1 to 4, which is any one of x1) to x 6):

x1) detecting the genotype of the wheat to be tested based on the 21 STARP markers according to claim 1;

x2) preparing a kit for detecting the genotype of wheat to be tested based on the 21 STARP markers according to claim 1;

x3) detecting at least one of gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of the wheat to be detected;

x4) preparing a kit for detecting at least one of gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of wheat to be detected;

x5) wheat breeding;

x6) preparing a kit for wheat breeding.

6. A kit comprising the primer set combination according to any one of claims 1 to 4; the use of the kit is any one of x1), x3) and x 5):

x1) detecting the genotype of the wheat to be tested based on the 21 STARP markers according to claim 1;

x5) wheat breeding.

7. A method for detecting at least one of gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, suitability for making noodles, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of wheat to be detected comprises the following steps: the genotype of wheat to be tested based on the 21 STARP markers according to claim 1 is determined separately, and then the following judgment is made:

the gluten strength of the wheat to be tested with the genotype of the GluA1-Indel Del and/or the genotype of the GluA1-SNP TT and/or the genotype of the GluD1-SNP TT is stronger than that of the wheat to be tested with the genotype of the GluA1-Indel Ins and/or the genotype of the GluA1-SNP CC and/or the genotype of the GluD1-SNP CC;

the activity of polyphenol oxidase of the wheat to be detected with the genotype of PpoA1-SNP being TT and/or the genotype of PpoD1-Indel being Del is lower than that of the wheat to be detected with the genotype of PpoA1-SNP being CC and/or the genotype of PpoD1-Indel being Ins;

the browning degree of a product of the wheat to be detected with the genotype of PpoA1-SNP being TT and/or the genotype of PpoD1-Indel being Del is less than that of the wheat to be detected with the genotype of PpoA1-SNP being CC and/or the genotype of PpoD1-Indel being Ins;

the yellow pigment content of the wheat to be detected is higher than that of the wheat to be detected, wherein the genotype of the PsyA1-Indel is Ins, the genotype of the PsyB1-SNP is TT, and/or the genotype of the PsyD1-SNP is AA, and/or the genotype of the ZDSA1-Indel is Ins, and/or the genotype of the PsyB 1-Indel is 1, and/or the genotype of the PsyA1-Indel is CC, and/or the genotype of the PsyB1-SNP is TT, and/or the genotype of the PsyD1-SNP is AA, and/or the genotype of the ZDSA1-Indel is Ins, and/or the genotype of the ZDSA1-Indel is CC, and/or the genotype of the wheat to be detected;

"the wheat to be tested with the genotype of PsyA1-Indel being Del and/or the genotype of PsyB1-SNP being GG and/or the genotype of PsyD1-SNP being GG and/or the genotype of ZDSA1-Indel being Del and/or the genotype of L oxB1-SNP being CC and/or the genotype of L cyA1-SNP being AA and/or the genotype of L cyB1-SNP being GG and/or the genotype of PdsB1-SNP being GG" is compared with the wheat to be tested with the genotype of PsyA1-Indel being Ins and/or the genotype of PsyB1-SNP being TT and/or the genotype of PsyD1-SNP being AA and/or the genotype of ZDSA1-Indel being Ins and/or the genotype of L oxB 1-Indel being GG and/or the genotype of L cyA 1-PdSNP and/or the genotype of L cyB1-SNP and/or the genotype of ZDSA L cyB 1-Indel being CC and/or the genotype of PdsB1-SNP "is a noodle suitable for preparing wheat to be a noodle;

the peroxidase activity of the wheat to be detected with the genotype of PodA1-SNP AA is higher than that of the wheat to be detected with the genotype of PodA1-SNP GG;

the flour whiteness of the wheat to be detected with the genotype of PodA1-SNP AA is higher than that of the wheat to be detected with the genotype of PodA1-SNP GG;

the flour viscosity of the wheat to be detected with the genotype of PodA1-SNP AA is lower than that of the wheat to be detected with the genotype of PodA1-SNP GG;

the hardness of grains of the wheat to be detected with the genotype of PinaD1-Indel as Del and/or the genotype of PinbD1-SNP as AA and/or the genotype of PinbB2-Indel as Del is higher than that of the wheat to be detected with the genotype of PinaD1-Indel as Ins and/or the genotype of PinbD1-SNP as GG and/or the genotype of PinbB2-Indel as Ins.

Any one of (A) - (E):

(A) a screening method for breeding parent varieties of wheat with enhanced gluten strength comprises the following steps: detecting the genotype of the wheat based on GluA1-Indel, GluA1-SNP and/or GluD1-SNP, wherein the wheat with the genotype of the GluA1-Indel being Del and/or the genotype of the GluA1-SNP being TT and/or the genotype of the GluD1-SNP being TT is a parent variety for cultivating wheat with enhanced gluten strength;

(B) a screening method for breeding a parent variety of wheat with reduced polyphenol oxidase activity and/or reduced browning of wheat products, comprising the steps of: detecting the genotype of the wheat based on PpoA1-SNP and/or PpoD1-Indel, wherein the wheat with the genotype of 'PpoA 1-SNP being TT and/or the genotype of PpoD1-Indel being Del' is the parent variety for cultivating the wheat with reduced polyphenol oxidase activity and/or reduced browning degree of wheat products;

(C) detecting the genotype of the wheat based on PsyA1-Indel, PsyB1-SNP, PsyD1-SNP, ZDsA1-Indel, L oxB1-SNP, L cyA1-SNP, L cyB1-SNP and/or PdsB1-SNP, and determining that the genotype of the PsyA1-Indel is Del and/or the genotype of the PsyB1-SNP is GG and/or the genotype of the PsyD1-SNP is GG and/or the genotype of the ZDSA1-Indel is Del and/or the genotype of the L oxB1-SNP is CC and/or the genotype of the L cyA1-SNP and/or the genotype of the L cyB 1-AA and/or the genotype of the PdsB1-SNP is GG and/or the genotype of the PdsB1-SNP, namely the variety of the wheat for improving the yellow pigment content and/or the wheat more suitable for making the noodle;

(D) a screening method for breeding parent varieties of wheat with increased peroxidase activity and/or increased flour whiteness and/or reduced flour stickiness, comprising the steps of: detecting the genotype of the wheat based on PodA1-SNP, and breeding by taking the wheat with the genotype of PodA1-SNP as AA as a parent to obtain a parent variety for cultivating the wheat with improved peroxidase activity and/or improved flour whiteness and/or reduced flour viscosity;

(E) a screening method for breeding a parent variety of wheat with increased grain hardness comprises the following steps: detecting the genotype of the wheat based on PinaD1-Indel and/or PinbD1-SNP and/or PinbB2-Indel, and taking the wheat with the genotype of the PinaD1-Indel as Del and/or the genotype of the PinbD1-SNP as AA and/or the genotype of the PinbB2-Indel as Del as a parent variety for cultivating the wheat with increased grain hardness.

9. The method of claim 7 or 8, wherein: the method for detecting the genotype of each STARP marker of wheat to be detected comprises the following steps:

(1) taking genome DNA of wheat to be detected as a template, and respectively adopting the primer group in the primer group combination of any one of claims 1 to 4 to carry out PCR amplification to obtain corresponding PCR amplification products;

(2) after the step (1) is finished, respectively detecting the fluorescent signals of the PCR amplification products by using an instrument, and obtaining the genotype of each STARP marker of the wheat to be detected according to the color of the fluorescent signals;

(a1) carrying out PCR amplification on the primer pair 1, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the GluA1-Indel of the wheat to be detected is Ins; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the GluA1-Indel of the wheat to be detected is Del;

(a2) carrying out PCR amplification on the primer pair 2, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the GluA1-SNP of the wheat to be detected is CC; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the GluA1-SNP of the wheat to be detected is TT; if the fluorescent signal of the amplification product of the wheat to be detected is green, the genotype of the GluA1-SNP of the wheat to be detected is CT;

(a3) carrying out PCR amplification on the 3 by using a primer pair, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the GluD1-SNP of the wheat to be detected is CC; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the GluD1-SNP of the wheat to be detected is TT; if the fluorescent signal of the amplification product of the wheat to be detected is green, the genotype of the GluD1-SNP of the wheat to be detected is CT;

(a4) carrying out PCR amplification on the primer pair 4, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the SNP of the wheat Glu-A3e-490 to be detected is GG; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat Glu-A3e-490SNP to be detected is TT; if the fluorescence signal of the amplification product of the wheat to be detected is green, the genotype of the wheat Glu-A3e-490SNP to be detected is GT;

(a5) carrying out PCR amplification on the primer pair 5, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the wheat Glu-A3g-SNP to be detected is TT; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat Glu-A3g-SNP to be detected is AA; if the fluorescent signal of the amplification product of the wheat to be detected is green, the genotype of the wheat Glu-A3g-SNP to be detected is TA;

(a6) carrying out PCR amplification on the primer pair 6, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the wheat Glu-B3B-SNP to be detected is GG; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat Glu-B3B-SNP to be detected is AA; if the fluorescence signal of the amplification product of the wheat to be detected is green, the genotype of the wheat Glu-B3B-SNP to be detected is GA;

(a7) carrying out PCR amplification on the 7 by using a primer pair, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the Glu-B3c-SNP of the wheat to be detected is CC; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat Glu-B3c-SNP to be detected is AA; if the fluorescent signal of the amplification product of the wheat to be detected is green, the genotype of the wheat Glu-B3c-SNP to be detected is CA;

(a8) carrying out PCR amplification on the primer pair 8, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the wheat PpoA1-SNP to be detected is CC; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the PpoA1-SNP of the wheat to be detected is TT; if the fluorescence signal of the amplification product of the wheat to be detected is green, the genotype of the wheat PpoA1-SNP to be detected is CT;

(a9) carrying out PCR amplification on the wheat to be detected by adopting the primer pair 9, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the wheat PpoD1-Indel to be detected is Del; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat PpoD1-Indel to be detected is Ins;

(a10) performing PCR amplification on the primer pair 10, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the wheat PsyA1-Indel to be detected is Ins; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat to be detected PsyA1-Indel is Del;

(a11) carrying out PCR amplification on the primer pair 11, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the PsyB1-SNP of the wheat to be detected is GG; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the PsyB1-SNP of the wheat to be detected is TT; if the fluorescent signal of the amplification product of the wheat to be detected is green, the genotype of the wheat to be detected PsyB1-SNP is GT;

(a12) performing PCR amplification by using the primer pair 12, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the PsyD1-SNP of the wheat to be detected is GG; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat PsyD1-SNP to be detected is AA; if the fluorescent signal of the amplification product of the wheat to be detected is green, the genotype of the wheat to be detected PsyD1-SNP is GA;

(a13) performing PCR amplification on the primer pair 13, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the ZdsA1-Indel of the wheat to be detected is Ins; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the wheat to be detected ZDSA1-Indel is Del;

(a14) carrying out PCR amplification by using a primer pair 14, wherein the genotype of the to-be-detected wheat L oxB1-SNP is CC if the fluorescence signal of the PCR amplification product of the to-be-detected wheat is blue, the genotype of the to-be-detected wheat L oxB1-SNP is GG if the fluorescence signal of the amplification product of the to-be-detected wheat is red, and the genotype of the to-be-detected wheat L oxB1-SNP is CG if the fluorescence signal of the amplification product of the to-be-detected wheat is green;

(a15) carrying out PCR amplification by using a primer pair 15, wherein the genotype of the to-be-detected wheat L cyA1-SNP is GG if the fluorescence signal of the PCR amplification product of the to-be-detected wheat is blue, the genotype of the to-be-detected wheat L cyA1-SNP is AA if the fluorescence signal of the amplification product of the to-be-detected wheat is red, and the genotype of the to-be-detected wheat L cyA1-SNP is GA if the fluorescence signal of the amplification product of the to-be-detected wheat is green;

(a16) carrying out PCR amplification on the primer pair 16, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of L cyB1-SNP of the wheat to be detected is CC, if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of L cyB1-SNP of the wheat to be detected is GG, and if the fluorescence signal of the amplification product of the wheat to be detected is green, the genotype of L cyB1-SNP of the wheat to be detected is CG;

(a17) carrying out PCR amplification by adopting a primer pair 17, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of PdsB1-SNP of the wheat to be detected is CC; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of PdsB1-SNP of the wheat to be detected is GG; if the fluorescence signal of the amplification product of the wheat to be detected is green, the genotype of the PdsB1-SNP of the wheat to be detected is CG;

(a18) performing PCR amplification on the primer pair 18, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the PodA1-SNP of the wheat to be detected is GG; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the PodA1-SNP of the wheat to be detected is AA; if the fluorescent signal of the amplification product of the wheat to be detected is green, the genotype of the PodA1-SNP of the wheat to be detected is GA;

(a19) performing PCR amplification by using the primer pair 19, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the PinaD1-Indel of the wheat to be detected is Del; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the PinaD1-Indel of the wheat to be detected is Ins;

(a20) performing PCR amplification by using the primer pair 20, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the PinbD1-SNP of the wheat to be detected is GG; if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the PinbD1-SNP of the wheat to be detected is AA; if the fluorescence signal of the amplification product of the wheat to be detected is green, the genotype of the PinbD1-SNP of the wheat to be detected is GA;

(a21) carrying out PCR amplification on the primer pair 21, wherein if the fluorescence signal of the PCR amplification product of the wheat to be detected is blue, the genotype of the PinbB2-Indel of the wheat to be detected is Del; and if the fluorescence signal of the amplification product of the wheat to be detected is red, the genotype of the PinbB2-Indel of the wheat to be detected is Ins.

10. The method of claim 7 or 8, wherein: the method for detecting the genotype of each STARP marker of wheat to be detected comprises the following steps:

(2) taking the PCR amplification product obtained in the step (1) and sequencing;

(3) and (3) obtaining the genotype of the wheat to be detected based on the 21 STARP markers according to the sequencing result obtained in the step (2).

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a screening method for breeding parent varieties of high-quality wheat and a primer group combination used by the same.

Background

In recent years, molecular markers based on DNA sequence polymorphisms have received high attention from breeders at home and abroad. The molecular marker closely linked with the target character is used for selection, the method is not influenced by the environment, the selection result is reliable, and when favorable characters are aggregated, linkage drag is reduced and breeding process is accelerated by genetic background selection in the backcross infiltration process. The molecular marker for controlling important characters of wheat is developed by a modern molecular biology means, and has important significance for wheat breeding in China.

The molecular markers commonly used in wheat genetic breeding comprise RF L P, RAPD, AF L P, SSR, STS, CAPS, DArT, SCAR and SNP. along with the cloning of wheat genes, functional markers developed based on wheat functional genes are gradually applied in wheat, dozens of genes with important characters are cloned, corresponding functional markers thereof provide convenience for a breeder to introduce favorable genes into a new variety, the selection efficiency is improved by target selection and combining favorable allelic variation of the functional genes with interesting characters, the breeding age is shortened, the Sun and the like (2005) and the He and the like (2007) respectively develop functional markers PPO18 of polyphenol oxidase genes Ppo-A1 according to wheat PPO gene sequences, functional markers PPO16 and PPO29 of PPO-D1, the PPO mutation types detected by the markers can reflect the PPO activity value of effective varieties, the PPO activity value of the varieties can be provided for breeding wheat varieties with theoretical and methods according to the theoretical breeding of the low activity of wheat varieties (2008, 2009, WAng and the like) and the functional markers of the wheat YP-WO 2, the PCR gene is convenient for establishing a high-RT-PCR amplification test, the PCR amplification test method for wheat varieties, the PCR amplification test of the PCR amplification of the PCR gene, the PCR gene is not only for the PCR gene, the PCR gene is convenient for the PCR gene, the PCR gene is not only the PCR gene, the PCR gene is used for the PCR gene, the PCR gene is used for the PCR gene for the PCR gene for breeding of the PCR gene for breeding of the PCR gene of the.

STARP technique (Semi-thermal Asymmetric Reverse PCR)) Is one of the mainstream genotyping methods in the world at present, and carries out accurate double allele judgment on SNP and InDel on a specific site based on specific matching and artificial mismatching of a base at the tail end of a primer (a third base or a fourth base at the 3' tail end of the primer). The primers comprise 5 strips: 2 fluorescence-modified general PEA primers (Universal Priming Element-adjustablePrimers), and 3 allele-specific primers. In addition to the modifications of the fluorescent group (FAM and HEX) and the quenching group (Dabsyl), the two PEA primers in 2 general-purpose PEA primers have a 4bp difference in length, i.e., the sequence of PEA primer 1 is as follows: 5' -FAM-AGCTGGTT (SEQ ID NO:64) -Sp9-GCAACAGGAACCAGCTATGAC-3' (underlined is the modification position of Dabsyl, i.e.the quencher group modification; SP9 is PCR blocker); the PEA primer 2 has the following sequence: 5' -HEX-ACTGCTCAAGAG (SEQ ID NO:65) -Sp9-GACGCAAGTGAGCAGTATGAC-3' (underlined is the modification position of Dabsyl, i.e.the quencher group modification; SP9 is PCR blocker). The schematic structure of PEA primer 1 and PEA primer 2 is shown in FIG. 1. Thus, the STARP technique can be used for detection using fluorescence as well as polypropylene gels. In 3 specific primers, the 3 'end of 2 upstream primers is an allelic variant base (the first base at the end) and a mismatched base (the 3 rd or 4 th base at the end), the 5' end is added with universal adaptor sequences A and B, the downstream primer is a common primer, and the conventional PCR amplification and the end-point method fluorescence signal detection are carried out. The technology has high accuracy and ultra-strong flexibility, does not need expensive double-color marking probes and PCR Master Mix, has the lowest DNA sample requirement, does not need whole genome amplification, is quick and efficient, and can detect a few marks of a large number of samples at high flux.

Disclosure of Invention

The invention aims to detect the gluten strength, the polyphenol oxidase activity, the browning degree of a wheat product, the yellow pigment content, the peroxidase activity, the flour whiteness, the flour viscosity and the kernel hardness of wheat to be detected.

The primer set combination is protected firstly, and the primer set combination can comprise a primer set 1 for amplifying GluA1-Indel, a primer set 2 for amplifying GluA1-SNP, a primer set 3 for amplifying GluD1-SNP, a primer set 4 for amplifying Glu-A3 e-490-SNP, a primer set 5 for amplifying Glu-A3g-SNP, a primer set 6 for amplifying Glu-B3B-SNP, a primer set 7 for amplifying Glu-B3c-SNP, a primer set 8 for amplifying PpoA1-SNP, a primer set 9 for amplifying PpoD1-Indel, a primer set 10 for amplifying PsyA1-Indel, a primer set 11 for amplifying PsyB1-SNP, a primer set 12 for amplifying PsyD1-SNP, a primer set 1-ZDsel, a primer set 68613-Indel, a primer set 356-SNP, a primer set 3519-Pbn19-SNP, a primer set for amplifying Pibn4619-SNP, a primer set for amplifying Pibn4617-SNP, a primer set for amplifying Ppob4619-Ppob 28-SNP, Pinb4619-SNP and Pinb4619 for amplifying SNP.

GluA1-Indel and GluA1-SNP may be STARP markers developed based on the wheat Glu-A1 gene.

The GluD1-SNP may be a STARP marker developed based on the wheat Glu-D1 gene.

The Glu-A3e-490SNP and Glu-A3g-SNP may be STARP markers developed based on the wheat Glu-A3 gene.

Glu-B3B-SNP and Glu-B3c-SNP may be STARP markers developed based on the wheat Glu-B3 gene.

The PpoA1-SNP may be a STARP marker developed based on the wheat PpoA-A1 gene.

PpoD1-Indel can be a STARP marker developed based on the wheat Ppo-D1 gene.

PsyA1-Indel may be a STARP marker developed based on the wheat Psy-A1 gene.

The PsyB1-SNP may be a STARP marker developed based on the wheat Psy-B1 gene.

The PsyD1-SNP may be a STARP marker developed based on the wheat Psy-D1 gene.

ZDSA1-Indel may be a STARP marker developed based on the wheat TaZDs-A1 gene.

L oxB1-SNP can be a STARP marker developed based on the wheat Ta L ox-B1 gene.

L cyA1-SNP can be a STARP marker developed based on the wheat Ta L cy-A1 gene.

L cyB1-SNP can be a STARP marker developed based on the wheat Ta L cy-B1 gene.

The PdsB1-SNP may be a STARP marker developed based on the wheat TaPds-B1 gene.

PodA1-SNP can be a STARP marker developed based on the wheat Tapod-A1 gene.

Pina D1-Indel can be a STARP marker developed based on the wheat Pina-D1 gene.

The PinbD1-SNP can be a STARP marker developed based on the wheat Pinb-D1 gene.

PinbB2-Indel can be a STARP marker developed based on the wheat Pinb-B2 gene.

In the primer group combination, the primer group 1 can comprise a primer 1, a primer 2 and a primer 3; primer set 2 may include primer 4, primer 5, and primer 6; primer set 3 may include primer 7, primer 8, and primer 9; primer set 4 may include primer 10, primer 11, and primer 12; primer set 5 may include primer 13, primer 14, and primer 15; primer set 6 may include primer 16, primer 17, and primer 18; primer set 7 may include primer 19, primer 20, and primer 21; primer set 8 may include primer 22, primer 23, and primer 24; primer set 9 may include primer 25, primer 26, and primer 27; the primer set 10 may include a primer 28, a primer 29, and a primer 30; primer set 11 may include primer 31, primer 32, and primer 33; primer set 12 may include primer 34, primer 35, and primer 36; primer set 13 may include primer 37, primer 38, and primer 39; the primer set 14 may include a primer 40, a primer 41, and a primer 42; primer set 15 may include primer 43, primer 44, and primer 45; primer set 16 may include primer 46, primer 47, and primer 48; primer set 17 may include primer 49, primer 50, and primer 51; primer set 18 may include primer 52, primer 53, and primer 54; primer set 19 may include primer 55, primer 56, and primer 57; primer set 20 may include primer 58, primer 59, and primer 60; primer set 21 may include primer 61, primer 62, and primer 63;

the nucleotide sequence of primer 1 consists of tag sequence B and SEQ ID NO:1 from position 22 to 39 from the 5' end; the nucleotide sequence of primer 2 consists of tag sequence a and SEQ ID NO:2 from the 5' end to positions 22 to 39; the nucleotide sequence of the primer 3 is shown as SEQ ID NO: 3 is shown in the specification;

the nucleotide sequence of primer 4 consists of tag sequence B and SEQ ID NO: 4 from position 22 to 42 from the 5' end; the nucleotide sequence of primer 5 consists of tag sequence a and SEQ ID NO: 5 from the 5' end to positions 22 to 42; the nucleotide sequence of the primer 6 is shown as SEQ ID NO:6 is shown in the specification;

the nucleotide sequence of primer 7 consists of tag sequence B and SEQ ID NO: 7 from position 22 to 45 from the 5' end; the nucleotide sequence of primer 8 consists of tag sequence a and SEQ ID NO: 8 from the 5' end to positions 22 to 45; the nucleotide sequence of the primer 9 is shown as SEQ ID NO: 9 is shown in the figure;

the nucleotide sequence of primer 10 consists of tag sequence a and SEQ ID NO: 10 from the 5' end to positions 22 to 42; the nucleotide sequence of primer 11 consists of tag sequence B and SEQ ID NO: 11 from position 22 to 42 from the 5' end; the nucleotide sequence of the primer 12 is shown as SEQ ID NO: 12 is shown in the specification;

the nucleotide sequence of primer 13 consists of tag sequence B and SEQ ID NO: 13 from the 22 nd to the 44 th nucleotide sequence from the 5' end; the nucleotide sequence of primer 14 consists of tag sequence a and SEQ ID NO: 14 from the 5' end to positions 22 to 44; the nucleotide sequence of the primer 15 is shown as SEQ ID NO: 15 is shown in the figure;

the nucleotide sequence of primer 16 consists of tag sequence a and SEQ ID NO: 16 from position 22 to 46 from the 5' end; the nucleotide sequence of primer 17 consists of tag sequence B and SEQ ID NO: 17 from the 5' end to positions 22 to 46; the nucleotide sequence of the primer 18 is shown as SEQ ID NO: 18 is shown in the figure;

the nucleotide sequence of primer 19 consists of tag sequence B and SEQ ID NO: 19 from the 5' end to positions 22 to 40; the nucleotide sequence of primer 20 consists of tag sequence a and SEQ ID NO: 20 from position 22 to 40 from the 5' terminus; the nucleotide sequence of the primer 21 is shown as SEQ ID NO: 21 is shown in the figure;

the nucleotide sequence of primer 22 consists of tag sequence a and SEQ ID NO: 22 from position 22 to 40 from the 5' end; the nucleotide sequence of primer 23 consists of tag sequence B and SEQ ID NO: 23 from the 5' end to positions 22 to 40; the nucleotide sequence of the primer 24 is shown as SEQ ID NO: shown at 24;

the nucleotide sequence of primer 25 consists of tag sequence a and SEQ ID NO: 25 from the 5' end to positions 22 to 47; the nucleotide sequence of primer 26 consists of tag sequence B and SEQ ID NO: 26 from position 22 to 48 from the 5' terminus; the nucleotide sequence of the primer 27 is shown as SEQ ID NO: 27 is shown;

the nucleotide sequence of primer 28 consists of tag sequence B and SEQ ID NO: 28 from position 22 to 46 from the 5' end; the nucleotide sequence of primer 29 consists of tag sequence a and SEQ ID NO: 29 from position 22 to 46 from the 5' terminus; the nucleotide sequence of the primer 30 is shown as SEQ ID NO: 30 is shown in the figure;

the nucleotide sequence of primer 31 consists of tag sequence B and SEQ ID NO: 31 from the 5' end to positions 22 to 43; the nucleotide sequence of primer 32 consists of tag sequence a and SEQ ID NO: 32 from the 5' end to positions 22 to 43; the nucleotide sequence of the primer 33 is shown as SEQ ID NO: 33;

the nucleotide sequence of primer 34 consists of tag sequence B and SEQ ID NO: 34 from the 5' end to positions 22 to 41; the nucleotide sequence of primer 35 consists of tag sequence a and SEQ ID NO: 35 from the 5' end to positions 22 to 41; the nucleotide sequence of the primer 36 is shown as SEQ ID NO: 36 is shown;

the nucleotide sequence of primer 37 consists of tag sequence B and SEQ ID NO: 37 from the 5' end to positions 22 to 47; the nucleotide sequence of primer 38 consists of tag sequence a and SEQ ID NO: 38 from the 5' end to positions 22 to 47; the nucleotide sequence of the primer 39 is shown as SEQ ID NO: 39;

the nucleotide sequence of primer 40 consists of tag sequence a and SEQ ID NO: 40 from the 5' end to positions 22 to 39; the nucleotide sequence of primer 41 consists of tag sequence B and SEQ ID NO: 41 from the 5' end to positions 22 to 39; the nucleotide sequence of the primer 42 is shown as SEQ ID NO: 42 is shown;

the nucleotide sequence of primer 43 consists of tag sequence a and SEQ ID NO: 43 from the 5' end to positions 22 to 39; the nucleotide sequence of primer 44 consists of tag sequence B and SEQ ID NO: 44 from the 5' end to positions 22 to 39; the nucleotide sequence of primer 45 is shown in SEQ ID NO: 45 is shown;

the nucleotide sequence of primer 46 consists of tag sequence B and SEQ ID NO: 46 from the 5' end to positions 22 to 39; the nucleotide sequence of primer 47 consists of tag sequence a and SEQ ID NO: 47 from position 22 to 39 from the 5' terminus; the nucleotide sequence of the primer 48 is shown as SEQ ID NO: 48 is shown;

the nucleotide sequence of primer 49 consists of tag sequence B and SEQ ID NO: 49 from the 5' end to positions 22 to 46; the nucleotide sequence of primer 50 consists of tag sequence a and SEQ ID NO: 50 from position 22 to 46 from the 5' terminus; the nucleotide sequence of the primer 51 is shown as SEQ ID NO: 51 is shown;

the nucleotide sequence of primer 52 consists of tag sequence a and SEQ ID NO: 52 from the 5' end to positions 22 to 40; the nucleotide sequence of primer 53 consists of tag sequence B and SEQ ID NO: 53 from position 22 to 40 from the 5' end; the nucleotide sequence of the primer 54 is shown as SEQ ID NO: 54 is shown;

the nucleotide sequence of primer 55 consists of tag sequence B and SEQ ID NO: 55 from the 5' end to positions 22 to 41; the nucleotide sequence of primer 56 consists of tag sequence a and SEQ ID NO: 56 from the 5' end to positions 22 to 41; the nucleotide sequence of the primer 57 is shown as SEQ ID NO: shown as 57;

the nucleotide sequence of primer 58 consists of tag sequence B and SEQ ID NO: 58 from the 5' end to positions 22 to 42; the nucleotide sequence of primer 59 consists of tag sequence a and SEQ ID NO: 59 from the 5' end to positions 22 to 41; the nucleotide sequence of the primer 60 is shown as SEQ ID NO: 60 is shown;

the nucleotide sequence of primer 61 consists of tag sequence B and SEQ ID NO: 61 from position 22 to 46 from the 5' terminus; the nucleotide sequence of primer 62 consists of tag sequence a and SEQ ID NO: 62 from position 22 to 48 from the 5' terminus; the nucleotide sequence of the primer 63 is shown as SEQ ID NO:63, respectively.

The primer group 1 may specifically consist of a primer 1, a primer 2 and a primer 3. The primer set 2 may specifically consist of a primer 4, a primer 5 and a primer 6. The primer group 3 may specifically consist of a primer 7, a primer 8 and a primer 9. The primer set 4 may specifically consist of a primer 10, a primer 11 and a primer 12. The primer set 5 may specifically consist of a primer 13, a primer 14 and a primer 15. The primer set 6 may specifically consist of a primer 16, a primer 17 and a primer 18. The primer set 7 may specifically consist of a primer 19, a primer 20 and a primer 21. The primer set 8 may specifically consist of a primer 22, a primer 23 and a primer 24. The primer set 9 may specifically be composed of a primer 25, a primer 26 and a primer 27. The primer set 10 may specifically consist of a primer 28, a primer 29 and a primer 30. The primer set 11 may specifically be composed of a primer 31, a primer 32 and a primer 33. The primer set 12 may specifically consist of a primer 34, a primer 35 and a primer 36. The primer set 13 may specifically consist of a primer 37, a primer 38 and a primer 39. The primer set 14 may specifically be composed of a primer 40, a primer 41, and a primer 42. The primer set 15 may specifically be composed of a primer 43, a primer 44 and a primer 45. The primer set 16 may specifically consist of a primer 46, a primer 47 and a primer 48. The primer set 17 may specifically be composed of a primer 49, a primer 50 and a primer 51. The primer set 18 may specifically be composed of a primer 52, a primer 53, and a primer 54. The primer set 19 may specifically be composed of a primer 55, a primer 56, and a primer 57. The primer set 20 may specifically consist of a primer 58, a primer 59, and a primer 60. The primer group 21 may specifically be composed of a primer 61, a primer 62, and a primer 63.

The primer group combination can detect the genotype of the wheat to be detected based on 21 STARP marks, and then judge the gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of the wheat to be detected according to the genotype, so that a target parent of the wheat is screened according to breeding purposes. Specifically, the corresponding characteristics of the wheat to be detected can be judged according to the composition of the primer group combination.

The primer group 1-the primer group 21 can also comprise a primer A and/or a primer B.

The primer A sequentially comprises a DNA fragment A, a PCR blocker and a DNA fragment B from 5 'to 3'; the nucleotide sequence of the DNA fragment A is shown as SEQ ID NO: 64; the nucleotide sequence of the DNA fragment B is shown as1 st to 21 st from the 5' end of SEQ ID NO. 2.

The primer A from 5 'to 3' can be composed of a DNA fragment A, a PCR blocker and a DNA fragment B.

The 5' end of the DNA fragment A can be provided with a fluorescence labeled A. The DNA fragment B can be modified by a quenching group.

The fluorescence label A is a FAM fluorescence label. The nucleotide sequence of the tag sequence A is shown as1 st to 21 st positions from the 5' end of SEQ ID NO. 2.

The primer B sequentially comprises a DNA fragment C, a PCR blocker and a DNA fragment D from 5 'to 3'; the nucleotide sequence of the DNA fragment C is shown as SEQ ID NO. 65; the nucleotide sequence of the DNA fragment D is shown as1 st to 21 st from the 5' end of SEQ ID NO 1.

The primer B from 5 'to 3' can be composed of a DNA fragment C, a PCR blocker and a DNA fragment D.

The 5' end of the DNA fragment C can be provided with a fluorescent label B. The DNA fragment may be modified with a quencher group.

The fluorescent label B is a HEX fluorescent label. The nucleotide sequence of the tag sequence B is shown as the 1 st to 21 st positions from the 5' end of SEQ ID NO. 1.

The quenching group can be Dabsyl.

In addition to the modification of the fluorescent group (FAM and HEX) and the quenching group (Dabsyl), the primers A and B have a 4bp difference in length.

Further, the sequence of the primer A can be as follows: 5' -FAM-AGCTGGTT (SEQ ID NO:64) -Sp9-GCAACAGGAACCAGCTATGAC-3' (underlined is the modification position of Dabsyl, i.e.the quencher group modification; SP9 is PCRblocker). The sequence of the primer B is as follows: 5' -HEX-ACTGCTCAAGAG (SEQ ID NO:65) -Sp9-GACGCAAGTGAGCAGTATGAC-3' (underlined is the modification position of Dabsyl, i.e.the quencher group modification; SP9 is PCRblocker).

Further, the primer group 1 may specifically include a primer 1, a primer 2, a primer 3, a primer a, and a primer b. The primer group 2 can specifically comprise a primer 4, a primer 5, a primer 6, a primer A and a primer B. The primer group 3 specifically comprises a primer 7, a primer 8, a primer 9, a primer A and a primer B. The primer group 4 may specifically include a primer 10, a primer 11, a primer 12, a primer A, and a primer B. The primer group 5 may specifically include a primer 13, a primer 14, a primer 15, a primer A, and a primer B. The primer group 6 may specifically include a primer 16, a primer 17, a primer 18, a primer A, and a primer B. The primer group 7 may specifically include a primer 19, a primer 20, a primer 21, a primer A, and a primer B. The primer group 8 may specifically include a primer 22, a primer 23, a primer 24, a primer A, and a primer B. The primer group 9 may specifically include a primer 25, a primer 26, a primer 27, a primer A, and a primer B. The primer group 10 may specifically include a primer 28, a primer 29, a primer 30, a primer A, and a primer B. The primer group 11 may specifically include a primer 31, a primer 32, a primer 33, a primer A, and a primer B. The primer group 12 may specifically include a primer 34, a primer 35, a primer 36, a primer A, and a primer B. The primer group 13 may specifically include a primer 37, a primer 38, a primer 39, a primer A, and a primer B. The primer group 14 may specifically include a primer 40, a primer 41, a primer 42, a primer A, and a primer B. The primer set 15 may specifically include a primer 43, a primer 44, a primer 45, a primer A, and a primer B. The primer set 16 may specifically include a primer 46, a primer 47, a primer 48, a primer A, and a primer B. The primer group 17 may specifically include a primer 49, a primer 50, a primer 51, a primer A, and a primer B. The primer group 18 may specifically include a primer 52, a primer 53, a primer 54, a primer A, and a primer B. The primer group 19 may specifically include a primer 55, a primer 56, a primer 57, a primer A, and a primer B. The primer set 20 may specifically include a primer 58, a primer 59, a primer 60, a primer A, and a primer B. The primer group 21 may specifically include a primer 61, a primer 62, a primer 63, a primer A, and a primer B.

In any of the above primer set combinations, the primers of each primer set are packaged individually or each primer set is packaged individually.

The invention also protects the application of any primer group combination, which can be any one of x1) to x 6): x1) detecting the genotypes of the wheat to be detected based on the 21 STARP markers; x2) preparing a kit for detecting the genotype of the wheat to be detected based on the 21 STARP markers; x3) detecting at least one of gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of the wheat to be detected; x4) preparing a kit for detecting at least one of gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of wheat to be detected; x5) wheat breeding; x6) preparing a kit for wheat breeding.

The invention also provides a kit, which contains any one of the primer group combinations; the use of the kit may be any of x1), x3), and x 5): x1) detecting the genotypes of the wheat to be detected based on the 21 STARP markers; x3) detecting at least one of gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of the wheat to be detected; x5) wheat breeding.

In the above kit, the primers of each primer set are packaged individually.

The kit can specifically comprise any one of the primer sets or each primer set is packaged independently.

The invention also provides a method for detecting at least one of gluten strength, polyphenol oxidase activity, browning degree of wheat products, yellow pigment content, suitability for making noodles, peroxidase activity, flour whiteness, flour viscosity and kernel hardness of wheat to be detected, which comprises the following steps: respectively detecting genotypes of the wheat to be detected based on the 21 STARP markers, and then judging as follows:

the peroxidase activity of the wheat to be detected with the genotype of PodA1-SNP AA is higher than that of the wheat to be detected with the genotype of PodA1-SNP GG;

the flour whiteness of the wheat to be detected with the genotype of PodA1-SNP AA is higher than that of the wheat to be detected with the genotype of PodA1-SNP GG;

the flour viscosity of the wheat to be detected with the genotype of PodA1-SNP AA is lower than that of the wheat to be detected with the genotype of PodA1-SNP GG;

The invention also protects any one of the methods (A) - (E).

The invention provides (A) a screening method for breeding a parent variety of wheat with enhanced gluten strength, which comprises the following steps: the wheat is detected based on the genotypes of GluA1-Indel, GluA1-SNP and/or GluD1-SNP, and the wheat with the genotype of the GluA1-Indel being Del and/or the genotype of the GluA1-SNP being TT and/or the genotype of the GluD1-SNP being TT is the parent variety for cultivating the wheat with enhanced gluten strength.

The invention provides (B) a screening method for breeding parent varieties of wheat with reduced polyphenol oxidase activity and/or reduced browning degree of wheat products, which comprises the following steps: the method is characterized in that the wheat is detected based on the genotype of PpoA1-SNP and/or PpoD1-Indel, and the wheat with the genotype of 'PpoA 1-SNP being TT and/or the genotype of PpoD1-Indel being Del' is the parent variety for cultivating the wheat with reduced polyphenol oxidase activity and/or reduced browning degree of wheat products.

The invention provides a screening method for breeding parent varieties with improved yellow pigment content and/or more suitable for making noodle wheat, which comprises the following steps of detecting the genotype of wheat based on PsyA1-Indel, PsyB1-SNP, PsyD1-SNP, ZDSA1-Indel, L oxB1-SNP, L cyA1-SNP, L cyB1-SNP and/or PdsB1-SNP, and making the genotype of "PsyA 1-Indel is Del and/or PsyB1-SNP is GG and/or the genotype of PsyD1-SNP is GG and/or the genotype of ZDSA1-Indel is Del and/or the genotype of L oxB1-SNP is CC and/or the genotype of L cyA 1-387 and/or the genotype of L cyB1-SNP is GG and/or the genotype of PdsB1-SNP is" which is the parent varieties with improved yellow pigment content and/or more suitable for breeding noodle wheat.

The invention provides (D) a screening method for breeding parent varieties of wheat with improved peroxidase activity and/or improved flour whiteness and/or reduced flour stickiness, which comprises the following steps: the method is characterized in that the genotype of the wheat based on PodA1-SNP is detected, and the wheat with the genotype of PodA1-SNP AA as a parent is bred, namely the parent variety for cultivating the wheat with improved peroxidase activity and/or improved flour whiteness and/or reduced flour viscosity.

The invention provides (E) a screening method for breeding a parent variety of wheat with increased grain hardness, which comprises the following steps: detecting the genotype of the wheat based on PinaD1-Indel and/or PinbD1-SNP and/or PinbB2-Indel, and taking the wheat with the genotype of the PinaD1-Indel as Del and/or the genotype of the PinbD1-SNP as AA and/or the genotype of the PinbB2-Indel as Del as a parent variety for cultivating the wheat with increased grain hardness.

In any of the above methods, the method for detecting the genotype of each STARP marker in wheat to be detected may be as follows:

(1) taking genome DNA of wheat to be detected as a template, and respectively adopting a primer group in any one of the primer group combinations to carry out PCR amplification to obtain corresponding PCR amplification products;

In any of the above methods, the method for detecting the genotype of each STARP marker in wheat to be detected may be as follows:

(2) taking the PCR amplification product obtained in the step (1) and sequencing;

(3) and (3) obtaining the genotype of the wheat to be detected based on the 21 STARP markers according to the sequencing result obtained in the step (2).

Any of the above-mentioned methods for detecting the genotype of each STARP marker in wheat to be detected also fall within the scope of the present invention.

In the above, Ins denotes insertion mutations and Del denotes deletion mutations.

In the above, STARP markers were designed based on the difference between allelic variations of Glu-A gene, Glu-D gene, Glu-A gene, Glu-B gene, Ppo-D gene, Psy-A gene, Psy-B gene, Psy-D gene, TaZDs-A gene, Ta ox-B gene, Ta cy-A gene, Ta cy-B gene, TaPds-B gene, TaPod-A gene, Pina-D gene, Pinb-D gene and Pinb-B gene, Glu-A gene, Glu-D gene, Glu-A gene, Glu-B gene, Ppo-A gene, Ppo-D gene, Psy-A gene, Psy-B gene, Psy-D gene, TaZDs-A gene, Ta ox-B gene, Ta-A gene, Pcy-B gene, TaZDs-A gene, Taox-B gene, TanB gene, Pina-D gene, PinB-D gene, TaZDs-A gene, Taox-B gene, TanB-A gene, TanB gene, and their expression of the.

The STARP marker developed by the invention does not need enzyme digestion and electrophoresis after PCR amplification, can detect a plurality of samples at high flux, greatly improves the detection efficiency, realizes the purpose of high-flux, low-cost and quick detection, is convenient for screening parent varieties for cultivating high-quality wheat and accelerates the breeding process. The invention has important theoretical significance and economic value in auxiliary screening of wheat varieties.

Drawings

FIG. 1 is a schematic diagram of the structures of PEA primer 1 and PEA primer 2.

FIG. 2 is a diagram showing genotyping of 305 parts of wheat material detected using GluA1-Indel marker.

FIG. 3 is a genotyping chart of 305 parts of wheat material detected using GluA1-SNP marker.

FIG. 4 is a genotyping chart of 305 parts of wheat material detected using GluD1-SNP marker.

FIG. 5 is a genotyping chart of 305 parts of wheat material detected using the Glu-A3e-490SNP marker.

FIG. 6 is a diagram showing genotyping of 305 parts of wheat material using Glu-A3g-SNP marker detection.

FIG. 7 is a diagram showing genotyping of 305 parts of wheat material using Glu-B3B-SNP marker detection.

FIG. 8 is a diagram showing genotyping of 305 parts of wheat material using Glu-B3c-SNP marker detection.

FIG. 9 is a genotyping chart of 305 wheat material using PpoA1-SNP marker detection part.

FIG. 10 is a genotyping plot of 305 wheat material tested using PpoD1-Indel markers.

FIG. 11 is a genotyping plot of 305 wheat material tested using the PsyA1-Indel marker.

FIG. 12 is a genotyping chart of 305 parts of wheat material detected using the PsyB1-SNP marker.

FIG. 13 is a genotyping chart of 305 parts of wheat material detected using the PsyD1-SNP marker.

FIG. 14 is a graph of genotyping 305 parts of wheat material using the ZDSA1-Indel marker.

FIG. 15 is a diagram showing genotyping of 305 parts of wheat material using L oxB1-SNP marker detection fraction.

FIG. 16 is a diagram showing genotyping of 305 parts of wheat material using L cyA1-SNP marker detection fraction.

FIG. 17 is a diagram showing genotyping of 305 parts of wheat material using L cyB1-SNP marker detection fraction.

FIG. 18 is a genotyping chart of 305 wheat materials using PdsB1-SNP marker detection section.

FIG. 19 is a diagram showing genotyping of 305 parts of wheat material using PodA1-SNP marker detection.

FIG. 20 is a graph showing genotyping of 305 parts of wheat material using PinaD1-Indel marker.

FIG. 21 is a diagram showing genotyping of 305 parts of wheat material using the PinbD1-SNP marker detection section.

FIG. 22 is a diagram showing genotyping of 305 parts of wheat material detected using the PinbB2-Indel marker.

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 conventional biochemicals, unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The primers in the following examples were synthesized by Huada Gene (Beijing) science and technology Co.

In the following examples, the wheat varieties are provided by the national wheat improvement center, and are publicly available from the national wheat improvement center.

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