CN102864245A

CN102864245A - Specific PCR (polymerase chain reaction) molecular markers for detecting high-silicon content allele qHUS6 of rice variety Miyang 46

Info

Publication number: CN102864245A
Application number: CN2012103928323A
Authority: CN
Inventors: 应杰政; 樊叶杨; 庄洁云; 朱玉君
Original assignee: China National Rice Research Institute
Current assignee: China National Rice Research Institute
Priority date: 2012-10-16
Filing date: 2012-10-16
Publication date: 2013-01-09
Anticipated expiration: 2032-10-16
Also published as: CN102864245B

Abstract

The invention provides 16 specific PCR (polymerase chain reaction) molecular markers for detecting high-silicon content allele qHUS6 of rice variety Miyang 46. The 16 specific PCR molecular markers are respectively PCR primers of Si2925A, Si2925B, Si2926A, Si2926B, Si2927A, Si2927B, Si2927C1, Si2927C2, Si2927C3, Si2933A, Si2933B, Si2933C, Si2936, Si2939, Si2940 and Si2946. The 16 markers are utilized to identify DNA extracted at a rice seedling stage, whether the high-silicon content allele qHUS6 of the rice variety Miyang 46 is shifted into a to-be-measured material can be detected, namely whether the capability of improving content of silicon in the rice is provided can be detected, and operation is simple and accuracy is high in the whole detection process.

Description

Detect the specific PCR molecular markers of rice varieties Milyang 46 high silicon content allelotrope qHUS6

Technical field

The invention belongs to the rice breeding technology field, particularly detect rice varieties Milyang 46 high silicon content allelotrope QHUS6Specific PCR molecular markers.

Background technology

Paddy rice is typically to inhale the silicon crop, and silicon has vital role in the rice growth process.High silicon rice varieties can effectively alleviate various biologies in process of growth and abiotic association compels, and improves the resistance to rice blast and banded sclerotial blight, improves lodging tolerance and drought resistance.The cultivation of high silicon rice varieties is generally by using high silicon rice material, adopt selection cross to obtain, and silicon in paddy rice particularly the content in the clever shell must after the ripe results of paddy rice, could detect, can't select by traditional field Phenotypic Observation.

Along with the fast development of molecule marker, by molecular marker assisted selection, can detect the gene of controlling silicone content by one-phase in office, identify whether the allelotrope to silicon accumulation synergy has imported the acceptor kind, the high silicon rice varieties of seed selection.

Carrying out of molecular marker assisted selection breeding need possess three conditions:

(1) donor parents carry effect clearly, Fine Mapping or clone's target gene;

(2) developed with the target gene close linkage and detected easy mark;

(3) linked marker is polymorphic between donor and receptor parent.

Early-stage Study shows, cloned control Rice Silica content at paddy rice the 6th karyomit(e) QHUS6Gene, its synergy allelotrope derives from the rice varieties Milyang 46.

The present invention is right on this basis QHUS6Checking order in gene and close linkage interval thereof, according to sequencing result design gene function and order mark (STS) mark, but and selects the allelic mark of specificity identification Milyang 46.Molecule marker provided by the invention is to high silicon content allelotrope in the Milyang 46 QHUS6Detection have general using value, can be widely used in Milyang 46 high silicon content allelotrope QHUS6Transformation research in.

Summary of the invention

The technical problem that the present invention solves has provided 16 and has detected rice varieties Milyang 46 high silicon content allelotrope QHUS6Specific molecular marker.

The present invention is by the following technical solutions:

According to early stage QHUS6Clone result, gene and close linkage interval thereof are checked order, sequencing result and the japonica rice variety Nipponbare that announced and the genome sequence of rice variety 93-11 are compared, according to insertion and the deletion condition of target sequence, designed 53 with QHUS6Closely linked STS mark.These 53 STS marks for detection of the parent of target group Zhenshan 97B and Milyang 46, are filtered out 16 marks that detect specific fragment in the rice varieties Milyang 46.These 16 marks can be used for identifying that whether detected materials exists QHUS6Carry the allelotrope of rice varieties Milyang 46 control Rice Silica content on the seat.

Detect rice varieties Milyang 46 high silicon content allelotrope QHUS6Specific PCR molecular markers, comprising the primer of 16 specific marker Si2925A, Si2925B, Si2926A, Si2926B, Si2927A, Si2927B, Si2927C1, Si2927C2, Si2927C3, Si2933A, Si2933B, Si2933C, Si2936, Si2939, Si2940, Si2946, each is a pair of:

The upstream primer sequence of Si2925A is 5'-TTGGCTAGCTTAACCTTCC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:1 as; The downstream primer sequence of Si2925A is 5'-GGCCATGTCAAATTAATAACCTC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:2 as;

The upstream primer sequence of Si2925B is 5'-CATCTGTGTCACTGTTTGGCT-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:3 as; The downstream primer sequence of Si2925B is 5'-GGCCATGTCAAATTAATAACCTC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:4 as;

The upstream primer sequence of Si2926A is 5'-TACGGATCATATCTTGGATGC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:5 as; The downstream primer sequence of Si2926A is 5'-TAACATATCTGATCGGTGCCTA-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:6 as;

The upstream primer sequence of Si2926B is 5'-TACGGATCATATCTTGGATGC-3', described nucleotides sequence classify as SEQ ID NO:7 shown in nucleotide sequence; The downstream primer sequence of Si2926B is 5'-ACCAGGAATATCGGTGACCA-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:8 as;

The upstream primer sequence of Si2927A is 5'-AGACCTGCCTAGCTGT-3', described nucleotides sequence classify as SEQ ID NO:9 shown in nucleotide sequence; The downstream primer sequence of Si2927A is 5'-AAAATGTTTGGCTAGCTTATGAGA-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:10 as;

The upstream primer sequence of Si2927B is 5'-CATGCAGGCTCTTAAGTGT-3', described nucleotides sequence classify as SEQ ID NO:11 shown in nucleotide sequence; The downstream primer sequence of Si2927B is 5'-AAAATGTTTGGCTAGCTTATGAGA-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:12 as;

The upstream primer sequence of Si2927C1 is 5'-AACATGAGAAAATAACTTGCACAT-3', described nucleotides sequence classify as SEQ ID NO:13 shown in nucleotide sequence; The downstream primer sequence of Si2927C1 is 5'-TCATTCTGATAGCTTTGTCCC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:14 as;

The upstream primer sequence of Si2927C2 is 5'-AACATGAGAAAATAACTTGCACAT-3', described nucleotides sequence classify as SEQ ID NO:15 shown in nucleotide sequence; The downstream primer sequence of Si2927C2 is 5'-CCGTCATCGCCCGTTC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:16 as;

The upstream primer sequence of Si2927C3 is 5'-AACTTGGGACAAAGCTATCAGA-3', described nucleotides sequence classify as SEQ ID NO:17 shown in nucleotide sequence; The downstream primer sequence of Si2927C3 is 5'-CCGTCATCGCCCGTTC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:18 as;

The upstream primer sequence of Si2933A is 5'-AGACGCTCATATTCAACAC-3', described nucleotides sequence classify as SEQ ID NO:19 shown in nucleotide sequence; The downstream primer sequence of Si2933A is 5'-TAAAGCGCAATCAGACT-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:20 as;

Si2933B the upstream primer sequence be 5'-AGTACTACATACCCCTATCCAC-3', described nucleotides sequence classify as SEQ ID NO:21 shown in nucleotide sequence; The downstream primer sequence of Si2933B is 5'-AATATATGTATAGACCACGCGCACT-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:22 as;

The upstream primer sequence of Si2933C is 5'-GAAAATATATACTAGCAATCCTCGCAAA-3', described nucleotides sequence classify as SEQ ID NO:23 shown in nucleotide sequence; The downstream primer sequence of Si2933C is 5'-CATAGGCTTGCATCCAGACTC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:24 as;

The upstream primer sequence of Si2936 is 5'-AAACTTAAAGAAATTTGACTATGAAA-3', described nucleotides sequence classify as SEQ ID NO:25 shown in nucleotide sequence; The downstream primer sequence of Si2936 is 5'-AACAAGATCTGATCAACTTCAC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:26 as;

The upstream primer sequence of Si2939 is 5'-GGTAAATTGTCTTTCCATACTATTG-3', described nucleotides sequence classify as SEQ ID NO:27 shown in nucleotide sequence; The downstream primer sequence of Si2939 is 5'-AATTATTTGGAGGAGCGTATC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:28 as;

The upstream primer sequence of Si2940 is 5'-ACACTCATTTCAAGATTCAACTT-3', described nucleotides sequence classify as SEQ ID NO:29 shown in nucleotide sequence; The downstream primer sequence of Si2940 is 5'-GCCAATATAAACAAGGTTACTACTC-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:30 as;

The upstream primer sequence of Si2946 is 5'-ATGCCAATTGAGTTTCGTTCAC-3', described nucleotides sequence classify as SEQ ID NO:31 shown in nucleotide sequence; The downstream primer sequence of Si2946 is 5'-CATGACATCGACGCGAAGT-3', and described nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:32 as.

The pcr amplification of 16 marks all adopts following system: Tris-HCL(pH 8.8) 33.5 mM, (NH ₄) ₂SO ₄8.0 mM, MgCl ₂1.5 mM, TWEEN-20 0.05%, dNTPs 0.2 mM, and each 3.3 ng/ μ l of upstream and downstream primer, TaqArchaeal dna polymerase 2.0 units, masterplate DNA 50 ng.Other is consistent except annealing temperature for the PCR reaction conditions, and design parameter is: 94 ℃ 2 minutes; 45 seconds, 72 ℃ of 94 ℃ of 45 seconds, 50-60 ℃ (Si2940 is 60 ℃, and Si2939 and Si2946 are 55 ℃, and all the other 13 marks are all 50 ℃) 1 minute, 30 circulations; 72 ℃ 8 minutes.

Four, description of drawings

Fig. 1 is that Si2926A is to the detected result figure of 192 parting materials.

Wherein, M: molecular weight marker; ZS: Zhenshan 97B; MY: Milyang 46; S1, S2, S3, S4, S8, S9, S13, S15, S17, S18, S19, S20, S21, S27, S29, S32, S33, S34, S36, S40 are that 20 of filtering out are the homozygous individual plant of Zhenshan 97B; S5, S6, S7, S10, S11, S12, S14, S16, S22, S23, S24, S25, S26, S28, S30, S31, S35, S37, S38, S39 are that 20 of filtering out are the homozygous individual plant of Milyang 46.

Fig. 2 is the silicone content detected result figure of 40 homozygous strains.

Wherein,, ordinate zou is the silicon percentage composition, X-coordinate is strain; The silicone content of 20 homozygous strains of Zhenshan 97B (S1, S2, S3, S4, S8, S9, S13, S15, S17, S18, S19, S20, S21, S27, S29, S32, S33, S34, S36, S40) (the unfilled histogram graph representation of silicone content) significantly ( P＜0.01) is lower than 20 homozygous strains of Milyang 46 (S5, S6, S7, S10, S11, S12, S14, S16, S22, S23, S24, S25, S26, S28, S30, S31, S35, S37, S38, S39) (histogram graph representation that silicone content is filled with black).

Five, embodiment

1, Milyang 46 high silicon content allelotrope QHUS6The screening of specific PCR molecular markers

According to the clone result in early stage, right QHUS6And the close linkage interval checks order, and sequencing result and the warm and fine rice variety 93-11 sequence of japonica rice variety Japan of having announced are compared, and analyzes insertion and disappearance between Japanese warm and fine 93-11 sequence, designed 53 QHUS6Functional label and closely linked STS mark are used the following step detection and location parent of colony Zhenshan 97B and Milyang 46, filter out 16 marks that detect specific fragment at Milyang 46.

(1) DNA extraction: the seed of Zhenshan 97B and Milyang 46 is placed respectively the in advance culture dish of label, and 30 ℃ germinateed 7 days, adopted simplified method to extract DNA from seedling.

(2) pcr amplification: adopt 20 μ l reaction systems, reaction conditions increases at the PCR instrument as mentioned above.

(3) PCR product electrophoresis and colour developing: add sample loading buffer in the amplified production, every sample is got 2 μ l and is splined on 6% non-denaturing polyacrylamide gel (PAGE); Connect electrode, electrophoresis is 4 hours under the 100V constant voltage, powered-down; Take off gel, silver dyes colour developing.

As shown in Figure 1: the mark that filters out can screen from segregating population and carry QHUS6Not homoallelic individual plant, wherein, S1, S2, S3, S4, S8, S9, S13, S15, S17, S18, S19, S20, S21, S27, S29, S32, S33, S34, S36, that S40 shows as Zhenshan 97B is homozygous, and S5, S6, S7, S10, S11, S12, S14, S16, S22, S23, S24, S25, S26, S28, S30, S31, S35, S37, S38, that S39 shows as Milyang 46 is homozygous.

2, the specific PCR mark is to Milyang 46 high silicon content allelotrope QHUS6The checking of identification result

Gather in the crops 40 seeds of being selected on the individual plant, planted with strain at China Paddy Rice Inst's Experimental Base in 2010, establish 2 repetitions.Each repeats each strain and gets the grain of middle 5 strains in 55 ℃-60 ℃ bakings 48 hours after ripe, through ridge paddy machine to grain husking, and husk placed 55 ℃ of-60 ℃ of drying and processings 24 hours, then with whirlwind abrasive dust instrument husk is polished, and the powder sample that grinds is positioned over 60 ℃ of oven for drying to constant weight, adopt at last molybdenum blue colorimetric method to measure the silicone content of each sample, unit is %.Every sample surveys twice, averages.

Show from Fig. 1, Fig. 2 result, genotype identification and silicone content detected result are in full accord, the silicone content of 20 homozygous strains of Zhenshan 97B (the unfilled histogram graph representation of silicone content) remarkable ( P＜0.01) is lower than 20 strains (histogram graph representation that silicone content is filled with black) that Milyang 46 is homozygous.The result shows, whether molecule marker provided by the invention effectively test material carries the high silicon content allelotrope of Milyang 46 QHUS6

SEQUENCE LISTING

＜110〉China Paddy Rice Inst

＜120〉specific PCR molecular markers of detection rice varieties Milyang 46 high silicon content allelotrope qHUS6

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Claims

1. detect rice varieties Milyang 46 high silicon content allelotrope QHUS6Specific PCR molecular markers, comprising the primer of 16 specific marker Si2925A, Si2925B, Si2926A, Si2926B, Si2927A, Si2927B, Si2927C1, Si2927C2, Si2927C3, Si2933A, Si2933B, Si2933C, Si2936, Si2939, Si2940, Si2946, each is a pair of: