CN116769962A - Method for distinguishing olive, olea aculeata and filial generation thereof - Google Patents
Method for distinguishing olive, olea aculeata and filial generation thereof Download PDFInfo
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- CN116769962A CN116769962A CN202311034961.XA CN202311034961A CN116769962A CN 116769962 A CN116769962 A CN 116769962A CN 202311034961 A CN202311034961 A CN 202311034961A CN 116769962 A CN116769962 A CN 116769962A
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- 240000007817 Olea europaea Species 0.000 title claims abstract description 54
- 241000795633 Olea <sea slug> Species 0.000 title claims abstract description 48
- 241000256852 Aculeata Species 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000012163 sequencing technique Methods 0.000 claims abstract description 45
- 239000012634 fragment Substances 0.000 claims abstract description 30
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 8
- 238000010899 nucleation Methods 0.000 claims description 22
- 235000002725 Olea europaea Nutrition 0.000 claims description 11
- 230000002902 bimodal effect Effects 0.000 claims description 4
- 230000035772 mutation Effects 0.000 description 6
- 238000002864 sequence alignment Methods 0.000 description 4
- 244000016119 Elaeagnus latifolia Species 0.000 description 2
- 235000001456 Elaeagnus latifolia Nutrition 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 235000004598 Cordia boissieri Nutrition 0.000 description 1
- 235000009692 Crataegus pubescens Nutrition 0.000 description 1
- 244000217406 Crataegus pubescens Species 0.000 description 1
- 244000147058 Derris elliptica Species 0.000 description 1
- 235000017643 Elaeagnus angustifolia Nutrition 0.000 description 1
- 235000007630 Elaeagnus umbellata var parvifolia Nutrition 0.000 description 1
- 235000001444 Elaeocarpus serratus Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 235000015248 Forestiera neo mexicana Nutrition 0.000 description 1
- 235000016128 Halesia carolina Nutrition 0.000 description 1
- 241000207836 Olea <angiosperm> Species 0.000 description 1
- 235000002852 Olea africana Nutrition 0.000 description 1
- 235000018136 Olea europaea var sylvestris Nutrition 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 235000006801 Ximenia americana Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- NBDQGOCGYHMDSJ-NRFPMOEYSA-M sodium;(e,3r,5s)-7-[2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl]-3,5-dihydroxyhept-6-enoate Chemical compound [Na+].[O-]C(=O)C[C@H](O)C[C@H](O)\C=C\C1=C(C2CC2)N=C2C=CC=CC2=C1C1=CC=C(F)C=C1 NBDQGOCGYHMDSJ-NRFPMOEYSA-M 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- General Health & Medical Sciences (AREA)
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- Developmental Biology & Embryology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for identifying and distinguishing olive, olea aculeata and filial generation thereof, belonging to the technical field of crop variety identification. The method disclosed by the invention utilizes the upstream primer shown in SEQ ID NO. 1 and the downstream primer shown in SEQ ID NO. 2 to respectively amplify and sequence fragments in the genome of the olive, the olea aculeata and the filial generation thereof, and distinguishes the olive, the olea aculeata and the filial generation thereof through the distinguishing of the sequencing result, so that the olive, the olea aculeata and the filial generation thereof can be distinguished and distinguished rapidly and reliably, and the cost is lower.
Description
Technical Field
The invention belongs to the technical field of crop variety identification, in particular to a technical method for identifying olive seedlings, and particularly relates to a method for identifying and distinguishing olive, olea aculeata and filial generation thereof.
Background
Fructus Oleae EuropaeaeOlea europaea L.) is an important woody oil tree species, widely distributed on coasts of the mediterranean, and a large number of introduction and cultivation are started in the year 60 of the 20 th century in China. Along with the continuous improvement of the living standard of people in China, the demand for olive oil is increased year by year. The method drives the continuous development of the olive planting industry in China, and the demand of seedlings is vigorous.
After the original midsea area is introduced into China, a plurality of inadaptation characteristics exist, and in order to realize high yield of the olives in China, the genetic component of the wild olives subspecies distributed in China, namely, the oleaster aculeata, is required to be hybridized to obtain a new variety with better adaptability. Because olive is wind-borne pollinating, artificial pollination is inefficient and also difficult to preserve in air the pollutes of pollen. In the new variety breeding process, hybrid offspring identification is currently a great difficulty. Meanwhile, the olive seedling market also has the possibility of mixing the olea aculeata seedlings into olive variety seedlings for sale. Therefore, there is a need to develop a reliable, rapid, accurate PCR primer for identifying whether the sown seedlings are seedlings of the hybrid offspring of Olea europaea.
At present, a technology suitable for identifying the filial generation of olive-olea aculeata is not proposed in China.
Disclosure of Invention
In view of one or more of the problems of the prior art, one aspect of the present invention provides a method for identifying and differentiating olea europaea, olea aculeata and filial generation thereof, comprising amplifying and sequencing 1040bp long fragments in the genome of olea europaea, olea aculeata and filial generation thereof respectively using an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, and identifying and differentiating olea europaea, olea aculeata and filial generation thereof according to sequencing results; wherein:
the 1040bp long fragment from the olive genome was sequenced as T at position 285 and/or 884 and/or as C at position 508;
a1040 bp long fragment from the genome of Olea acutifolia had a sequencing result of C at position 285 and/or 884 and/or a sequencing result of T at position 508;
the sequencing of 1040bp long fragments from the genomes of the filial generation of olive and olea acutifolia at 285 th, and/or 508 th, and/or 884 th was CT bimodal.
In another aspect, the present invention provides a method of identifying the relationship between a sown seedling and its parent and olive and olea aculeata, comprising the steps of:
s1: extracting DNA of a leaf sample of the seeding seedling;
s2: amplifying the DNA obtained in the step S1 by using an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2 to obtain an amplified fragment with 1040bp length;
s3: sequencing the amplified fragment obtained in the step S2, and judging the result according to the sequencing result, wherein the judgment standard is as follows:
if the sequencing result of the amplified fragment at 285 th and/or 884 th is T and/or the sequencing result at 508 th is C, indicating that the parents of the seeding seedlings are olea europaea;
if the sequencing result of the amplified fragment at 285 th and/or 884 th is C and/or the sequencing result at 508 th is T, indicating that the parents of the seeding seedlings are olea acutifolia;
if the sequencing result of the amplified fragment at 285 th, and/or 508 th and/or 884 th is CT bimodal, the seeding seedling is the hybrid offspring of olive-olea acutifolia.
The method for distinguishing the olive, the olea aculeata and the filial generation thereof based on the technical scheme comprises the steps of respectively amplifying an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2 to obtain 1040bp long fragments in the genome of the olive, the olea aculeata and the filial generation thereof and sequencing, and distinguishing the olive, the olea aculeata and the filial generation thereof according to the sequencing result, wherein the sequencing result of the 1040bp long fragments contains 3 reliable sites which can be used for distinguishing the olive, the olea aculeata and the filial generation thereof, and the method can be used for distinguishing the olive, the olea aculeata and the filial generation thereof quickly and reliably, and the PCR and sequencing cost for distinguishing the olive, the olea aculeata and the filial generation thereof can be only tens of elements, so that the method has low cost.
In addition, because the leaves for seeding are diploid, half of DNA is from female parent and the other half is from male parent, the invention can also provide a method for identifying the relationship between the (olive) seeding and the male parent thereof and the olea europaea and the olea acutifolia, namely a method for identifying the (olive) seeding and the varieties of the male parent thereof based on the upstream primer shown in SEQ ID NO. 1 and the downstream primer shown in SEQ ID NO. 2.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
FIG. 1 shows the sequence alignment of 249-329bp in 1040bp long fragments derived from olive and olea acutifolia genomes, respectively.
FIG. 2 shows the sequence alignment of 475-545bp in 1040bp long fragments derived from the olive and olea aculeata genomes, respectively.
FIG. 3 shows the sequence alignment of 848-928bp in 1040bp long fragments derived from olive and olea aculeata genomes, respectively.
In FIGS. 1-3, the first 22 sequences were from Olea acutifolia and the last 22 sequences were from Olea europaea.
Description of the embodiments
When the inventors studied the genome information of 22 olive varieties (Lucques, empeltre, thiaki, kalamon, cornicabra, hojiblanca, coratina, pendolino, farga, rogani, adramittini, megaritiki, arbosana, yuntai, ogliarola, manzanilla, salonenque, picholine, picual, moraiolo, nikita, chalkidikis, respectively) and 22 olea acutifolia samples (the genome sequence information about the above olive and olea acutifolia obtained from the applicant laboratory) by bioinformatics method, it was unexpectedly found that there was a sequence in the genome of olive and olea acutifolia that can reliably distinguish the two from the filial progeny of the two, characterized in that 3 mutation sites were present in the sequence, and as shown in the following boxes of fig. 1-3, the olive, olea acutifolia and olea acutifolia-olea acutifolia can be clearly separated by using the sequencing results of these 3 mutation sites alone or in combination (only the sequence information of olive and olea acutifolia are listed in the figure), specifically: the sequencing result of the 3 sites of the sequence from olive is T, C and T in sequence, the sequencing result of the 3 sites of the sequence from olea aculeata is C, T and C in sequence, and the sequencing result of the 3 sites of the sequence from olive-olea aculeata filial generation of the sequence from olive-olea aculeata is C/T (the sequencing peak diagram shows CT double peaks and represents merger base Y), C/T and C/T in sequence. Based on the sequence, the inventors purposefully designed an upstream primer and a downstream primer for amplifying a sequence containing 3 sites, the primer sequence information is shown in the following table 1, the fragment with the length of 1040bp can be effectively amplified from the genome of olive, olea aculeata and the hybrid progeny of olive-olea aculeata (for example, the fragment with the length of 1040bp derived from olea aculeata is shown as SEQ ID NO: 3), and the sequencing result of the fragment contains the 3 mutation site information, so that olive, olea aculeata and the hybrid progeny of olive-olea aculeata can be reliably distinguished. Because the leaves of the seeding seedling are diploid, one half of DNA comes from female parent and the other half comes from male parent, the DNA extracted from the leaves of the seeding seedling can reflect the genetic information of the male parent of the seeding seedling, and the relationship between the seeding seedling and the male parent thereof and the olive and the olea acutifolia can be identified by the DNA, namely, whether the varieties of the seeding seedling and the male parent thereof are the olive, the olea acutifolia or the hybrid offspring of the olive and the olea acutifolia.
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present invention. The following description is, therefore, to be taken in an illustrative and not a limiting sense.
The present invention will be described in detail with reference to examples.
Example 1: method for identifying relationship between seeding seedling and father and mother thereof and olea europaea and olea aculeata
1. Obtaining DNA of a seeding seedling leaf sample: collecting two leaves of a single plant to be identified, drying the leaves by silica gel, and then cutting leaf samples (1 cm multiplied by 1 cm) to extract DNA;
2. and (3) PCR amplification: amplifying the PCR products by using a conventional PCR instrument and method, wherein the primers are listed in the table 1, and the specific amplification procedure is shown in the table 2 below;
3. sequencing: sending the PCR product obtained in the step 2 to a sequencing company, and sequencing by adopting a sanger method to obtain a sequencing result;
4. sequence alignment and identification: 1-3, identifying the sown seedlings and parent and female varieties thereof according to the sequence information of 3 mutation sites marked in the frame, wherein the identification standards are as follows:
if the sequencing result of the PCR amplified fragment at 285 th and/or 884 th positions is T and/or the sequencing result at 508 th position is C, the parents of the seeding seedlings are olea europaea;
if the sequencing result of the PCR amplified fragment at 285 th and/or 884 th positions is C and/or the sequencing result at 508 th position is T, the parents of the seeding seedlings are olea acutifolia;
if the sequencing result of the PCR amplified fragment at 285 th position and/or 508 th position and/or 884 th position is CT double peak, the seeding seedling is the hybrid offspring of olive-olea aculeata.
10 seeding samples (samples 1-10) collected in the olive field and varieties of the father and the mother were identified by using steps 1-4 in the above method, and the results are shown in the following table 3.
Compared with the prior art, the invention has the following advantages.
1. The cost is low, and the PCR and sequencing costs can be completed by tens of yuan.
2. The method has high speed, and can be completed in one week from acquisition to acquisition of the identification result.
3. The result is reliable, one PCR reaction obtains 1040bp sequence, 3 reliable mutation sites which can be used for accurate identification are contained, and the 3 mutation sites can be mutually verified.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. A method for distinguishing olive, olea aculeata and filial generation thereof comprises the steps of respectively amplifying an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2 to obtain 1040bp long fragments in the genome of olive, olea aculeata and filial generation thereof, sequencing, and distinguishing olive, olea aculeata and filial generation thereof according to sequencing results; wherein:
the 1040bp long fragment from the olive genome was sequenced as T at position 285 and/or 884 and/or as C at position 508;
a1040 bp long fragment from the genome of Olea acutifolia had a sequencing result of C at position 285 and/or 884 and/or a sequencing result of T at position 508;
the sequencing of 1040bp long fragments from the genomes of the filial generation of olive and olea acutifolia at 285 th, and/or 508 th, and/or 884 th was CT bimodal.
2. A method of identifying the relationship between a sown seedling and its parent and olea europaea and olea aculeata, comprising the steps of:
s1: extracting DNA of a leaf sample of the seeding seedling;
s2: amplifying the DNA obtained in the step S1 by using an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2 to obtain an amplified fragment with 1040bp length;
s3: sequencing the amplified fragment obtained in the step S2, and judging the result according to the sequencing result, wherein the judgment standard is as follows:
if the sequencing result of the amplified fragment at 285 th and/or 884 th is T and/or the sequencing result at 508 th is C, indicating that the parents of the seeding seedlings are olea europaea;
if the sequencing result of the amplified fragment at 285 th and/or 884 th is C and/or the sequencing result at 508 th is T, indicating that the parents of the seeding seedlings are olea acutifolia;
if the sequencing result of the amplified fragment at 285 th, and/or 508 th and/or 884 th is CT bimodal, the seeding seedling is the hybrid offspring of olive-olea acutifolia.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311034961.XA CN116769962B (en) | 2023-08-17 | 2023-08-17 | Method for distinguishing olive, olea aculeata and filial generation thereof |
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| CN202311034961.XA CN116769962B (en) | 2023-08-17 | 2023-08-17 | Method for distinguishing olive, olea aculeata and filial generation thereof |
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| CN116769962A true CN116769962A (en) | 2023-09-19 |
| CN116769962B CN116769962B (en) | 2023-10-17 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109554501A (en) * | 2018-12-30 | 2019-04-02 | 中国林业科学研究院林业研究所 | Amplimer, screening technique and the identification method of olive cultivar identification based on SNP site |
| CN116072229A (en) * | 2022-09-16 | 2023-05-05 | 中国林业科学研究院林业研究所 | Germplasm resource database for olive variety identification and construction method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109554501A (en) * | 2018-12-30 | 2019-04-02 | 中国林业科学研究院林业研究所 | Amplimer, screening technique and the identification method of olive cultivar identification based on SNP site |
| CN116072229A (en) * | 2022-09-16 | 2023-05-05 | 中国林业科学研究院林业研究所 | Germplasm resource database for olive variety identification and construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHRISTOS BAZAKOS ET AL.: "Whole genome scanning of a Mediterranean basin hotspot collection provides new insights into olive tree biodiversity and biology", THE PLANT JOURNAL, pages 1 - 17 * |
| MASOUD SHEIDAI ET AL.: "Evaluation of genetic diversity in cultivated (O. europaea subsp. europaea L. ssp. europea var. europaea) and wild olives (Olea cuspidata Wall) using genome size and RAPD markers", THE NUCLEUS, vol. 57, pages 215 - 222 * |
| 潘莉 等: "油橄榄×尖叶木樨榄种间杂交种真实性的SSR鉴定", 分子植物育种, vol. 17, no. 7, pages 2279 - 2284 * |
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