CN104561348A - Specific PCR molecular markers for detecting rice high-grain weight alleles - Google Patents
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Abstract
The invention provides eight specific PCR markers for detecting high thousand seed weight alleles of miyang 46 on rice grain weight QTL (quantitative trait loci) qTGW1.2a and qTGW1.2b, wherein seven specific PCR markers for detecting qTGW1.2a are respectively Wn32837, Wn32886, Wn32893, Wn33185, Wn33186, Wn33304b and Wn33252; and one specific PCR marker for detecting the qTGW1.2b is Wn34526. The specific PCR markers are characterized by being applied to PCR primers. The rice DNA is identified by the eight markers; whether a to-be-tested material is transferred into the high thousand seed weight alleles of the rice variety miyang 46 on two target loci and has the capacity of improving the rice grain weight can be respectively detected; and the overall detection process is simple to operate and high in accuracy.
Description
Technical field
The present invention relates to the detection technique field in rice breeding, particularly detect rice grain weight QTL
qTGW1.2awith
qTGW1.2ballelic 8 specific PCRs mark of upper Milyang 46 height thousand seed weight.
Background technology
At present, the rate of rise of world population, higher than increases in grain production speed, is short of food and becomes global safety problem.Paddy rice, as the staple food of half population in the world, is improved its output and is significant to solution Food Security.The volume increase of paddy rice, after experienced by of short stemization and heterosis utilization, is in slow growth state always, and within nearly 30 years, China's new rice variety output year increase rate is only 1-2%, and wherein some types is even lower than 1%.Slowly increase production the stage at this, thousand seed weight has played vital effect to output increased.Therefore, in the Breeding Process of new rice variety, the height of thousand seed weight is one of focus of paying close attention to of breeding men.The material of the kind that the cultivation of high thousand seed weight kind often need improve and high thousand seed weight carries out hybridizing and obtaining, measure comparatively accurate after the mensuration of thousand seed weight need wait seed fully matured, and sample to be tested quantity is huge in Breeding Process, hinders breeding process and strengthen breeding cost.Along with the fast development of molecule marker, by molecular marker assisted selection, utilize suitable molecule marker can play the importing of allelotrope in acceptor kind of synergism to thousand seed weight in rice at whole growth periods qualification, thus from offspring, select the rice varieties of high thousand seed weight.At present, Molecular Marker Assisted Selection Technology at Rice Resistance characteristic of disease, the widespread use of the proterties such as insect-resistance and salt tolerant.
Applicant navigates to the QTL(Wang L-L that 3 control paddy rice thousand seed weight early stage on paddy rice the 1st karyomit(e), et al. Dissection of
qTGW1.2to three QTLs for grain weight and grain size in rice (Oryza sativa L.). Euphytica, DOI 10.1007/s10681-014-1237-7).In these 3 QTL intervals, the allelotrope deriving from rice varieties Milyang 46 all has the effect improving thousand seed weight, positioning precision then with
qTGW1.2awith
qTGW1.2bfor height.The present invention on this basis, according to the heavy sequencing result of rice varieties Milyang 46 and precious Shan 97, for
qTGW1.2awith
qTGW1.2bcarry out sequence alignment between location, design sequence mark (STS) mark, and select can the allelic mark of specificity identification Milyang 46.Molecule marker provided by the invention is to rice grain weight QTL
qTGW1.2awith
qTGW1.2bthe allelic detection of upper Milyang 46 synergy has general using value, can be widely used in
qTGW1.2awith
qTGW1.2bin the allelic transformation research of upper Milyang 46 synergy.
Summary of the invention
The technical problem that the present invention solves there is provided detection
qTGW1.2awith
qTGW1.2bthe allelic specific molecular marker of upper Milyang 46 height thousand seed weight 8, wherein, detects
qTGW1.2a7, detect
qTGW1.2b1.
The present invention is by the following technical solutions: according to the heavy sequencing result of Milyang 46 and precious Shan 97, right
qTGW1.2awith
qTGW1.2bbetween location and the sequence in close linkage interval compare, according to insertion and deletion condition, at this section design, 12 STS marks, through laboratory qualification checking, filter out the mark that 8 have polymorphism in two kinds.
For detecting
qTGW1.2aupper Milyang 46 height thousand seed weight is allelic is marked with 7, respectively called after Wn32837, Wn32886, Wn32893, Wn33185, Wn33186, Wn33304b and Wn33252, and concrete sequence is as follows:
The upstream primer sequence of Wn32837 is 5'-CGTCCTGCGTTTCGACATGACT-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:1;
The downstream primer sequence of Wn32837 is 5'-TATTTCTACCACTCCAAGCACCA-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:2;
The upstream primer sequence of Wn32886 is 5'-CGTGTTACTAACCCAGTCAACTCAGG-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:3;
The downstream primer sequence of Wn32886 is 5'-CAATAGTAGCAGCTAAATTGGCGTTC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:4;
The upstream primer sequence of Wn32893 is 5'-TTCCGATTTTGTTTTCTTGGTCCC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:5;
The downstream primer sequence of Wn32893 is 5'-GCCGCACATTTATTACCTTAATCCTG-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:6;
The upstream primer sequence of Wn33185 is 5'-GGAGGATAACGCAAGCAAACTTGA-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:7;
The downstream primer sequence of Wn33185 is 5'-CCATAGCTTGTAAAAGACAGTGCC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:8;
The upstream primer sequence of Wn33186 is 5'-AGTTAGTGCAGCAATCTACGTCCT-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:9;
The downstream primer sequence of Wn33186 is 5'-AGCTCCTCCAGTGACGATACGG-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:10;
The upstream primer sequence of Wn33304b is 5'-AAGCAACATAACTTATTCTACTC-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:11;
The downstream primer sequence of Wn33304b is 5'-ACGACATCCACTTCGCACC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:12;
The upstream primer sequence of Wn33252 is 5'-GCATGTATCAAAGATTCGATGAGA-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:13;
The downstream primer sequence of Wn33252 is 5'-TGAAAACTCATGGCTACGCT-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:14.
For detecting
qTGW1.2bupper Milyang 46 height thousand seed weight is allelic is marked with 1, called after Wn34526, and concrete sequence is as follows:
The upstream primer sequence of Wn34526 is 5'-TCATCATCTGTTCGTGGGTT-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:15;
The downstream primer sequence of Wn34526 is 5'-TTGAAATATAAACTCTATACAATCTGCT-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:16.
The PCR amplification system of 8 STS marks is consistent.Amplification system is, 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, each 3.3 ng/ μ l of upstream and downstream primer,
taqarchaeal dna polymerase 2.0 unit, masterplate DNA 50 ng; Other is consistent except annealing temperature for PCR reaction conditions, denaturation temperature 94 DEG C 2 minutes; Denaturation temperature 94 DEG C 45 seconds, annealing temperature 50-55 DEG C (Wn33815, Wn33252 and Wn34526 are 50 DEG C, and all the other 5 marks are all 55 DEG C) 45 seconds, 72 DEG C 1 minute, 30 circulations; 72 DEG C 8 minutes.
Accompanying drawing explanation
Fig. 1 STS marks the detected result of Wn32886.
M: molecular weight contrasts; P1: precious Shan 97; P2: Milyang 46; 1 ~ 16: testing sample
Fig. 2 two overlaps the thousand seed weight distribution of near isogenic line two places plantation.
A.2014 Lingshui, spring in year Hainan
qTGW1.2a
B.2014 Lingshui, spring in year Hainan
qTGW1.2b
C.2014 summer in year Fuyang, Zhejiang
qTGW1.2a
D.2014 summer in year Fuyang, Zhejiang
qTGW1.2b .
Embodiment
Explain the present invention further below in conjunction with embodiment, but embodiment does not limit in any form to the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Experiment material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
embodiment
1
paddy rice thousand seed weight
qTL
qTGW1.2a
with
qTGW1.2b
upper close sun
46
the exploitation of specific molecular marker
Utilize sequence alignment program DNASTAR MegAlign module (Lasergene) to the precious Shan 97 of the parent of QTL target group and Milyang 46 at thousand seed weight QTL
qTGW1.2awith
qTGW1.2bbase sequence between location is compared, the insertion shown in comparison according to 2 kinds or deletion segment, and application primer-design software Oligo 7.0 develops 12 and is respectively used to detect
qTGW1.2awith
qTGW1.2bthe STS mark of synergy allelotrope Milyang 46 specific fragment.Whether that identifies that these develop by following step marks whether to there is specificity between precious Shan 97 and Milyang 46, and can accurately identify in segregating population and carry Milyang 46 allelotrope.The present invention marks Wn32886 for STS and introduces detection method in detail:
1. DNA Trace bio-element
(1) single-strain seed of precious Shan 97, Milyang 46 and 16 random chooses in segregating population is placed in respectively the culture dish of label in advance, 30 DEG C germinate 7 days.
(2) clip each culture dish seedling leaves 2 ~ 3 cm, is cut into the fragment that 0.5 cm is long, puts into 2.0 mL centrifuge tubes.
(3) add 450 μ l DNA extraction liquid and a steel ball, application organizes beveller to grind.
(4) add 450 μ l chloroform extraction liquid, cover tightly lid, mixing of turning upside down.
(5) 11,000 rpm extremely clear phase-splittings in centrifugal 2 minutes.Aspirate supernatant 400 μ l, proceeds in 1.5 new ml centrifuge tubes, abandons rifle head
(6) add the dehydrated alcohol of 800 μ l precoolings, cover tightly lid, mixing of turning upside down.Place 30 minutes for-20 DEG C.
(7) 11,000 rpm invest bottom centrifuge tube to precipitation in centrifugal 3 minutes, abandon supernatant liquor.
(8) precipitate 2 times by 70% washing with alcohol, 1.5 ml centrifuge tubes are inverted on paper, seasoning.
(9) 1/10 × TE buffer solution precipitation of 100 μ l is added.
(10) get 2 μ l and carry out pcr amplification.
2. pcr amplification and detection
Amplification system is, 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, each 3.3 ng/ μ l of upstream and downstream primer,
taqarchaeal dna polymerase 2.0 unit, masterplate DNA 50 ng; Other is consistent except annealing temperature for PCR reaction conditions, denaturation temperature 94 DEG C 2 minutes; Denaturation temperature 94 DEG C 45 seconds, annealing temperature 50-55 DEG C (Wn33815, Wn33252 and Wn34526 are 50 DEG C, and all the other 4 marks are all 55 DEG C) 45 seconds, 72 DEG C 1 minute, 30 circulations; 72 DEG C 8 minutes.
Get 2 μ l PCR primer and be splined on 6% non-denaturing polyacrylamide gel (PAGE); Connect electrode, electrophoresis 3 hours under 100V constant voltage, powered-down; Take off gel, the colour developing of silver dye.
As shown in Figure 1, on Wn32886 seat, precious Shan 97 and Milyang 46 show polymorphism, in segregating population, sample 3,4,7,8,10,11,12 and 14 is homozygous in Milyang 46,13 and 16 in heterozygous, and all the other are individual homozygous in precious Shan 97, illustrates that this STS marks and can control paddy rice thousand seed weight QTL as detecting Milyang 46
qTGW1.2aspecific mark.Select 8 by the method finishing screen and have specific molecule marker, wherein, 7 control paddy rice thousand seed weight QTL for detecting Milyang 46
qTGW1.2a, 1 is detected Milyang 46 and controls paddy rice thousand seed weight QTL
qTGW1.2b.
embodiment
2
the close sun of application specific molecular markers for identification
46
the allelic checking of high thousand seed weight
1 near isogenic line builds
Adopt the molecule marker that embodiment 1 is set up, hand over screening the Gao Dai colony of precious Shan 97/ // precious Shan 97//precious Shan 97/ Milyang 46 of combination individual from Indica Xian, establish 2 cover near isogenic line colonies, exist respectively
qTGW1.2awith
qTGW1.2binterval separation, consistent between all the other background areas.1st is enclosed within
qTGW1.2abe separated, containing precious Shan 97 type and each 39 of Milyang 46 type near isogenic line; 2nd is enclosed within
qTGW1.2bbe separated, containing precious Shan 97 type and each 42 of Milyang 46 type near isogenic line.
2 phenotypic evaluation
Spring-2014 winter in 2013 and summer in 2014 are respectively at Lingshui county of Hainan Province and China Paddy Rice Inst of Fuyang City of Zhejiang Province test base plantation near isogenic line, each strain is by randomized block design plantation, 2 repetitions are set, each strain kind 8 individual plants, get middle 5 strains and measure thousand seed weight after maturation.
3 results and analysis
Statistic analysis result shows,
qTGW1.2ainterval, compared with precious Shan 97 type strain, carries the allelic strain of Milyang 46 type and on average can improve thousand seed weight 0.52 g in Lingshui, on average can improve 0.44 g in Fuyang; ?
qTGW1.2binterval, compared with precious Shan 97 type strain, carries the allelic strain of Milyang 46 type and on average can improve thousand seed weight 0.30 g in Lingshui, on average can improve 0.31 g(Fig. 2 in Fuyang).Two places test all confirms that the allelic importing of Milyang 46 can improve thousand seed weight, and effect-size two ground level is consistent.
Embodiment 2 confirms that molecule marker provided by the invention is to rice grain weight QTL
qTGW1.2awith
qTGW1.2bthe allelic detecting reliability of middle Milyang 46 synergy is high, can be applied to thousand seed weight QTL
qTGW1.2awith
qTGW1.2bin the transformation research of middle synergy allelotrope Milyang 46.
<110> China Paddy Rice Inst
The allelic specific PCR molecular markers of <120> detection control rice grain weight
<160> 14
<170> PatentIn 3.3
<210> 1
<211> 22
<212> DNA
<213> paddy rice
<400> 1
cgtcctgcgt ttcgacatga ct
<210> 2
<211> 23
<212> DNA
<213> paddy rice
<400> 2
tatttctacc actccaagca cca
<210> 3
<211> 26
<212> DNA
<213> paddy rice
<400> 3
cgtgttacta acccagtcaa ctcagg
<210> 4
<211> 26
<212> DNA
<213> paddy rice
<400> 4
caatagtagc agctaaattg gcgttc
<210> 5
<211> 24
<212> DNA
<213> paddy rice
<400> 5
ttccgatttt gttttcttgg tccc
<210> 6
<211> 26
<212> DNA
<213> paddy rice
<400> 6
gccgcacatt tattacctta atcctg
<210> 7
<211> 24
<212> DNA
<213> paddy rice
<400> 7
ggaggataac gcaagcaaac ttga
<210> 8
<211> 24
<212> DNA
<213> paddy rice
<400> 8
ccatagcttg taaaagacag tgcc
<210> 9
<211> 24
<212> DNA
<213> paddy rice
<400> 9
agttagtgca gcaatctacg tcct
<210> 10
<211> 22
<212> DNA
<213> paddy rice
<400> 10
agctcctcca gtgacgatac gg
<210> 11
<211> 23
<212> DNA
<213> paddy rice
<400> 11
aagcaacata acttattcta ctc
<210> 12
<211> 19
<212> DNA
<213> paddy rice
<400> 12
acgacatcca cttcgcacc
<210> 13
<211> 24
<212> DNA
<213> paddy rice
<400> 13
gcatgtatca aagattcgat gaga
<210> 14
<211> 20
<212> DNA
<213> paddy rice
<400> 14
tgaaaactca tggctacgct
<210> 15
<211> 20
<212> DNA
<213> paddy rice
<400> 15
tcatcatctg ttcgtgggtt
<210> 16
<211> 28
<212> DNA
<213> paddy rice
<400> 16
ttgaaatata aactctatac aatctgct
Claims (2)
1. detect rice grain weight QTL
qTGW1.2aallelic 7 specific PCRs mark of upper Milyang 46 height thousand seed weight, called after Wn32837, Wn32886, Wn32893, Wn33185, Wn33186, Wn33304b and Wn33252 respectively, concrete sequence is as follows:
The upstream primer sequence of Wn32837 is 5'-CGTCCTGCGTTTCGACATGACT-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:1;
The downstream primer sequence of Wn32837 is 5'-TATTTCTACCACTCCAAGCACCA-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:2;
The upstream primer sequence of Wn32886 is 5'-CGTGTTACTAACCCAGTCAACTCAGG-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:3;
The downstream primer sequence of Wn32886 is 5'-CAATAGTAGCAGCTAAATTGGCGTTC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:4;
The upstream primer sequence of Wn32893 is 5'-TTCCGATTTTGTTTTCTTGGTCCC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:5;
The downstream primer sequence of Wn32893 is 5'-GCCGCACATTTATTACCTTAATCCTG-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO:6;
The upstream primer sequence of Wn33185 is 5'-GGAGGATAACGCAAGCAAACTTGA-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:7;
The downstream primer sequence of Wn33185 is 5'-CCATAGCTTGTAAAAGACAGTGCC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:8;
The upstream primer sequence of Wn33186 is 5'-AGTTAGTGCAGCAATCTACGTCCT-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:9;
The downstream primer sequence of Wn33186 is 5'-AGCTCCTCCAGTGACGATACGG-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:10;
The upstream primer sequence of Wn33304b is 5'-AAGCAACATAACTTATTCTACTC-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:11;
The downstream primer sequence of Wn33304b is 5'-ACGACATCCACTTCGCACC-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:12;
The upstream primer sequence of Wn33252 is 5'-GCATGTATCAAAGATTCGATGAGA-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:13;
The downstream primer sequence of Wn33252 is 5'-TGAAAACTCATGGCTACGCT-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:14.
2. detect rice grain weight QTL
qTGW1.2ballelic 1 specific PCR mark of upper Milyang 46 height thousand seed weight, called after Wn34526, concrete sequence is as follows:
The upstream primer sequence of Wn34526 is 5'-TCATCATCTGTTCGTGGGTT-3', and described nucleotides sequence is classified as the shown nucleotide sequence of SEQ ID NO:15;
The downstream primer sequence of Wn34526 is 5'-TTGAAATATAAACTCTATACAATCTGCT-3', and described nucleotides sequence is classified as the nucleotide sequence shown in SEQ IDNO:16.
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| CN107760794A (en) * | 2017-10-24 | 2018-03-06 | 中国水稻研究所 | Detect the specific PCR molecular markers of the weak response to temperature allele of qHd1 of rice varieties treasure Shan 97 |
| CN108060259A (en) * | 2018-01-24 | 2018-05-22 | 中国水稻研究所 | Detect the specific PCR molecular markers of high grain weight allele on rice grain weight QTLqGW35.5 |
| CN108103230A (en) * | 2018-01-24 | 2018-06-01 | 中国水稻研究所 | Detect the specific PCR molecular markers of elongated grain allele on rice grain shape QTLqGL35.1 |
| CN108179221A (en) * | 2018-02-28 | 2018-06-19 | 中国水稻研究所 | Detect the specific molecular marker of high mass of 1000 kernel allele on rice mass of 1000 kernel QTL qTGW10.2a |
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| CN108060259A (en) * | 2018-01-24 | 2018-05-22 | 中国水稻研究所 | Detect the specific PCR molecular markers of high grain weight allele on rice grain weight QTLqGW35.5 |
| CN108103230A (en) * | 2018-01-24 | 2018-06-01 | 中国水稻研究所 | Detect the specific PCR molecular markers of elongated grain allele on rice grain shape QTLqGL35.1 |
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| CN108060259B (en) * | 2018-01-24 | 2021-03-23 | 中国水稻研究所 | Specific PCR molecular marker for detecting high grain weight allele on rice grain weight QTLqGW35.5 |
| CN108179221A (en) * | 2018-02-28 | 2018-06-19 | 中国水稻研究所 | Detect the specific molecular marker of high mass of 1000 kernel allele on rice mass of 1000 kernel QTL qTGW10.2a |
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