CN102586435A - Method for identifying cytoplasm type of cytoplasmic-genic male sterile rice by detecting mitochondrial DNA - Google Patents
Method for identifying cytoplasm type of cytoplasmic-genic male sterile rice by detecting mitochondrial DNA Download PDFInfo
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- CN102586435A CN102586435A CN2012100345417A CN201210034541A CN102586435A CN 102586435 A CN102586435 A CN 102586435A CN 2012100345417 A CN2012100345417 A CN 2012100345417A CN 201210034541 A CN201210034541 A CN 201210034541A CN 102586435 A CN102586435 A CN 102586435A
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Abstract
The invention discloses a method for identifying cytoplasm type of cytoplasmic-genic male sterile rice by detecting mitochondrial DNA. The method comprises the following steps of: extracting the mitochondrial DNA from rice leaves serving as materials; performing polymerase chain reaction (PCR) amplification by using mitochondrial primers mt1, mt6, mt7 and mt11, and cloning and sequencing amplification products; and comparing with molecular fingerprints of the mitochondrial DNA of the cytoplasmic-genic male sterile rice to easily and efficiently identify the cytoplasm type of the cytoplasmic-genic male sterile rice.
Description
Technical field
It is the method for male sterile rice cytoplasm type that this invention relates to a kind of evaluation three.
Background technology
1966, the BT type (with India's rice variety Chinsuran Boro II is the tenuigenin donor, does the nuclear donor seed selection with japonica rice variety and forms) three that the long friend of Japanese scholar's new city has bred by tenuigenin and the common control of nucleus was a sterile line.1970, Yuan Longping, Li Bihu etc. found a strain male sterile plant in the wild-rice of Hainan, cultivated into " open country is lost " type sterile line through nucleus substitution.Become the red lotus type sterile line with the red awns wild-rice in Hainan with rice variety Lian Tang selection cross morning subsequently.K type, ridge type, seal water type etc. have been bred again.Because these tenuigenin do not have the tenuigenin mark property except that outside the Pass having with male sterile, can't distinguish through its tenuigenin of General Biology character pair.
Summary of the invention
The object of the present invention is to provide a kind of evaluation three is the method for male sterile rice cytoplasm type, through being that the pcr amplification product of male sterile rice Mitochondrial DNA carries out cloning and sequencing and can identify that three is the cytoplasm type of male sterile rice to three.
Detection line mitochondrial DNA of the present invention identifies that three is the method for male sterile rice cytoplasm type, may further comprise the steps:
(1) Mitochondrial DNA in the extraction rice leaf to be measured;
(2) Mitochondrial DNA that with plastosome primer mt1, mt6, mt7, mt11 step (1) is extracted carries out pcr amplification;
(3) the gained pcr amplification product is carried out cloning and sequencing;
(4) be the contrast of male sterile rice Mitochondrial DNA molecular fingerprint with gained base sequence and three, confirm rice cytoplasmic type to be measured.
Detection line mitochondrial DNA of the present invention identifies that three is the method for male sterile rice cytoplasm type; Identifying that three only need carry out cloning and sequencing to the pcr amplification product of plastosome primer mt1, mt6, mt7, mt11 when being the male sterile rice cytoplasm type, comparing with the Mitochondrial DNA molecular fingerprint again that can to identify three simply, efficiently be the cytoplasm type of male sterile rice.
Embodiment
The present invention is contrast with 32 portions of typical long-grained nonglutinous rices and Typical Japonica tenuigenin; 17 three to different sources is that the sterile line mtdna sequence is analyzed; Find that dissimilar sterile cytoplasms have characteristic base or base sequence, set up Yebai, red lotus type and three kinds of Mitochondrial DNA molecular fingerprints of BT type with the extension increasing sequence difference of plastosome primer mt1, mt6, mt7, mt11.
Fine with conventional rice Japan is contrast, is that the pcr amplification product of the Mitochondrial DNA of sterile line carries out cloning and sequencing to V20A etc. three, and its sequence results is seen table 1.
Table 1
Annotate: this sequence of "-" expression disappearance.
Plastosome primer wherein
Mt1: forward sequence 5-tcttcttcggacttgatgcac-3, reverse sequence 5-gcgcccttgaaatcatctta-3;
Mt6: forward sequence 5-ggtaagcggaccgtggaa-3, reverse sequence 5-gacgcgaccggacttca-3;
Mt7: forward sequence 5-tgccctgccttcacctt-3, reverse sequence 5-ttcgcacttcagaaaatcg-3;
Mt11: forward sequence 5-ccttaaccccgctttca-3, reverse sequence 5-gcagttctcactctgtccg-3.
Above-mentioned three is in the male sterile rice Mitochondrial DNA molecular fingerprint
Yebai cytoplasmic: mt1 extension increasing sequence 1223-729 position is that I-494 fragment, mt6 extension increasing sequence 104-113 position are that I-9 fragment, mt7 extension increasing sequence 115-123 position are that I-8 fragment, mt11 section extension increasing sequence 104-95 position are the II-9 fragment;
HL cytoplasm: mt1 extension increasing sequence 1223-729 position is that I-494 fragment, mt6 extension increasing sequence 104-113 position lack, mt7 extension increasing sequence 115-123 position lacks, mt11 section extension increasing sequence 104-95 position is the II-9 fragment;
BT type tenuigenin: mt1 extension increasing sequence 1223-729 position disappearance, mt6 extension increasing sequence 104-113 position are that I-9 fragment, mt7 extension increasing sequence 115-123 position are that I-8 fragment, mt11 amplify two kinds of sequences; The II-9 fragment that both a kind of sequence 104-95 position is, another kind of sequence deletion II-9 fragment.
Said I-494 sheet represents is inserted fragment sequence:
tttgttgtgg?tttttggcgt?gggttttttc?ccaacgaaaa?acgaattaga?acttccatga
caaaaccaaa?tgctagttgc?ctacagtgtg?gcaactgcta?attaaaataa?tttagaatgt
atataaagta?gggactagcg?tccccatgaa?cgaccagatc?aagatagatc?agatttaatg
taagaatctg?ctcctctata?gagtccccag?gaaaaaccat?atttggaatt?atatcctgga
gtgcgagtcc?ctccggcaat?tgggcattac?ctgaagtttg?ctcaaaaagc?atttttagct
tatattcaag?cttttgagag?agcatttctc?ttttcaatgc?gagggaatgg?tctctaagac
cccaccccgc?tttataagcg?aaaaaaatgg?caaccgaaac?aatgattccg?gcgcctacat
atgaaatgaa?ttggttatcc?ataaatgcag?aatgttttat?tcttgttccg?cttcttgctc
caagcagaaa?gacttctttg/gatctc；
Said I-9 sheet represents is inserted fragment sequence: ccctcctga;
Said I-8 sheet represents is inserted fragment sequence: cgttgcgc;
Said II-9 sheet represents is inserted fragment sequence: aggtttctc
Detected result is: V20A, T98A, K17A are the Yebai male sterile rice, and the safe A in Guangdong is the red lotus type male sterile rice, and T1A, 6A, 199A are BT type male sterile rice.
SEQUENCE?LISTING
< 110>Hunan Normal University
< 120>the detection line mitochondrial DNA identifies that three is the method for male sterile rice cytoplasm type
<160> 2
<170> PatentIn?version?3.3
<210> 1
<211> 500
<212> DNA
<213> I-494(1)
<400> 1
tttgttgtgg?tttttggcgt?gggttttttc?ccaacgaaaa?acgaattaga?acttccatga 60
caaaaccaaa?tgctagttgc?ctacagtgtg?gcaactgcta?attaaaataa?tttagaatgt 120
atataaagta?gggactagcg?tccccatgaa?cgaccagatc?aagatagatc?agatttaatg 180
taagaatctg?ctcctctata?gagtccccag?gaaaaaccat?atttggaatt?atatcctgga 240
gtgcgagtcc?ctccggcaat?tgggcattac?ctgaagtttg?ctcaaaaagc?atttttagct 300
tatattcaag?cttttgagag?agcatttctc?ttttcaatgc?gagggaatgg?tctctaagac 360
cccaccccgc?tttataagcg?aaaaaaatgg?caaccgaaac?aatgattccg?gcgcctacat 420
atgaaatgaa?ttggttatcc?ataaatgcag?aatgttttat?tcttgttccg?cttcttgctc 480
caagcagaaa?gacttctttg 500
<210> 2
<211> 500
<212> DNA
<213> I-494(2)
<400> 2
tttgttgtgg?tttttggcgt?gggttttttc?ccaacgaaaa?acgaattaga?acttccatga 60
caaaaccaaa?tgctagttgc?ctacagtgtg?gcaactgcta?attaaaataa?tttagaatgt 120
atataaagta?gggactagcg?tccccatgaa?cgaccagatc?aagatagatc?agatttaatg 180
taagaatctg?ctcctctata?gagtccccag?gaaaaaccat?atttggaatt?atatcctgga 240
gtgcgagtcc?ctccggcaat?tgggcattac?ctgaagtttg?ctcaaaaagc?atttttagct 300
tatattcaag?cttttgagag?agcatttctc?ttttcaatgc?gagggaatgg?tctctaagac 360
cccaccccgc?tttataagcg?aaaaaaatgg?caaccgaaac?aatgattccg?gcgcctacat 420
atgaaatgaa?ttggttatcc?ataaatgcag?aatgttttat?tcttgttccg?cttcttgctc 480
caagcagaaa?gactgatctc 500
Claims (3)
1. a detection line mitochondrial DNA identifies that three is the method for male sterile rice cytoplasm type, it is characterized in that may further comprise the steps:
Extract the Mitochondrial DNA in the rice leaf to be measured;
The Mitochondrial DNA that step (1) is extracted with plastosome primer mt1, mt6, mt7, mt11 carries out pcr amplification;
The gained pcr amplification product is carried out cloning and sequencing;
With gained base sequence and three is the contrast of male sterile rice Mitochondrial DNA molecular fingerprint, confirms rice cytoplasmic type to be measured.
2. identify that according to the said detection line mitochondrial DNA of claim 1 three is the method for male sterile rice cytoplasm type; It is characterized in that: said plastosome primer mt1: forward sequence 5-tcttcttcggacttgatgcac-3, reverse sequence 5-gcgcccttgaaatcatctta-3; Mt6: forward sequence 5-ggtaagcggaccgtggaa-3, reverse sequence 5-gacgcgaccggacttca-3; Mt7: forward sequence 5-tgccctgccttcacctt-3, reverse sequence 5-ttcgcacttcagaaaatcg-3; Mt11: forward sequence 5-ccttaaccccgctttca-3, reverse sequence 5-gcagttctcactctgtccg-3.
3. identify that according to claim 1 or 2 said detection line mitochondrial DNAs three is the method for male sterile rice cytoplasm type, it is characterized in that said three is male sterile rice Mitochondrial DNA molecular fingerprint:
Yebai cytoplasmic: mt1 extension increasing sequence 1223-729 position is that I-494 fragment, mt6 extension increasing sequence 104-113 position are that I-9 fragment, mt7 extension increasing sequence 115-123 position are that I-8 fragment, mt11 section extension increasing sequence 104-95 position are the II-9 fragment;
HL cytoplasm: mt1 extension increasing sequence 1223-729 position is that I-494 fragment, mt6 extension increasing sequence 104-113 position lack, mt7 extension increasing sequence 115-123 position lacks, mt11 section extension increasing sequence 104-95 position is the II-9 fragment;
BT type tenuigenin: mt1 extension increasing sequence 1223-729 position disappearance, mt6 extension increasing sequence 104-113 position are that I-9 fragment, mt7 extension increasing sequence 115-123 position are that I-8 fragment, mt11 amplify two kinds of sequences; The II-9 fragment that both a kind of sequence 104-95 position is; Another kind of sequence deletion II-9 fragment, said I-494 sheet represents is inserted fragment sequence:
tttgttgtgg?tttttggcgt?gggttttttc?ccaacgaaaa?acgaattaga?acttccatga
caaaaccaaa?tgctagttgc?ctacagtgtg?gcaactgcta?attaaaataa?tttagaatgt
atataaagta?gggactagcg?tccccatgaa?cgaccagatc?aagatagatc?agatttaatg
taagaatctg?ctcctctata?gagtccccag?gaaaaaccat?atttggaatt?atatcctgga
gtgcgagtcc?ctccggcaat?tgggcattac?ctgaagtttg?ctcaaaaagc?atttttagct
tatattcaag?cttttgagag?agcatttctc?ttttcaatgc?gagggaatgg?tctctaagac
cccaccccgc?tttataagcg?aaaaaaatgg?caaccgaaac?aatgattccg?gcgcctacat
atgaaatgaa?ttggttatcc?ataaatgcag?aatgttttat?tcttgttccg?cttcttgctc
caagcagaaa?gacttctttg/gatctc；
Said I-9 sheet represents is inserted fragment sequence: ccctcctga;
Said I-8 sheet represents is inserted fragment sequence: cgttgcgc;
Said II-9 sheet represents is inserted fragment sequence: aggtttctc.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630347A (en) * | 2015-01-07 | 2015-05-20 | 湖南省蔬菜研究所 | Method for identifying chili cytoplasmic male sterile line by detecting mitochondria DNA |
| CN111621552A (en) * | 2019-06-13 | 2020-09-04 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425276A (en) * | 2002-12-31 | 2003-06-25 | 扬州大学 | Broad sprectum wide affinity restoring line selective breeding technology for rice |
| CN1740337A (en) * | 2001-03-28 | 2006-03-01 | 科学与工业研究委员会 | DNA marker for estimating seeds purity and method for applying DNA sequence to estimate seeds purity |
| CN1831138A (en) * | 2005-11-21 | 2006-09-13 | 安徽省农业科学院水稻研究所 | Molecular marking supplementary breeding method for multi-purpose restoring series of three-series hybrid rice |
| CN101347095A (en) * | 2008-09-05 | 2009-01-21 | 扬州大学 | Honglian type method for breeding japonica hybrid rice |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1740337A (en) * | 2001-03-28 | 2006-03-01 | 科学与工业研究委员会 | DNA marker for estimating seeds purity and method for applying DNA sequence to estimate seeds purity |
| CN1425276A (en) * | 2002-12-31 | 2003-06-25 | 扬州大学 | Broad sprectum wide affinity restoring line selective breeding technology for rice |
| CN1831138A (en) * | 2005-11-21 | 2006-09-13 | 安徽省农业科学院水稻研究所 | Molecular marking supplementary breeding method for multi-purpose restoring series of three-series hybrid rice |
| CN101347095A (en) * | 2008-09-05 | 2009-01-21 | 扬州大学 | Honglian type method for breeding japonica hybrid rice |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630347A (en) * | 2015-01-07 | 2015-05-20 | 湖南省蔬菜研究所 | Method for identifying chili cytoplasmic male sterile line by detecting mitochondria DNA |
| CN104630347B (en) * | 2015-01-07 | 2018-12-25 | 湖南省蔬菜研究所 | A method of detection mitochondrial DNA identifies capsicum cytoplasmic male sterile line |
| CN111621552A (en) * | 2019-06-13 | 2020-09-04 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
| CN111621552B (en) * | 2019-06-13 | 2022-05-06 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
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