CN111088385A - Method for identifying sex of female and male heteroplant in early growth stage - Google Patents
Method for identifying sex of female and male heteroplant in early growth stage Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 241000196324 Embryophyta Species 0.000 claims description 38
- 108020004414 DNA Proteins 0.000 claims description 11
- 239000004922 lacquer Substances 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 230000010196 hermaphroditism Effects 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 244000044283 Toxicodendron succedaneum Species 0.000 claims 2
- 238000009396 hybridization Methods 0.000 abstract description 2
- 244000223760 Cinnamomum zeylanicum Species 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 235000017803 cinnamon Nutrition 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 230000020509 sex determination Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 241001533085 Aquilaria sinensis Species 0.000 description 2
- 108091092878 Microsatellite Proteins 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 230000000366 juvenile effect Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 241000763620 Aquilaria yunnanensis Species 0.000 description 1
- 244000002917 Excoecaria cochinchinensis Species 0.000 description 1
- 240000003152 Rhus chinensis Species 0.000 description 1
- 235000014220 Rhus chinensis Nutrition 0.000 description 1
- 102100023706 Steroid receptor RNA activator 1 Human genes 0.000 description 1
- 101710187693 Steroid receptor RNA activator 1 Proteins 0.000 description 1
- 241000895647 Varroa Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000035425 carbon utilization Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
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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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- 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
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
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Abstract
The invention discloses a sex identification method for early growth stage of a male and female heteroplant, which can directly identify the sex of the naturally-growing male and female heteroplant, thereby avoiding the sex identification of offspring generated by experimental hybridization of a plurality of adult male plants and female plants, long identification period and complicated method. The sex specificity markers M1 and M2 of the screened heterogynic plants are used for identifying the sex of the heterogynic plant population growing in the early stage under the natural environment, and the method has the characteristics of simplicity, accuracy and high efficiency.
Description
Technical Field
The invention belongs to the field of plant identification, and particularly relates to a method for identifying the sex of a male and female heterotrophic plant in the early growth stage.
Background
Over 15000 species of hermaphrodite plants are grown worldwide. In the juvenile period of most of the male and female heteroplants, the sex of the plants cannot be accurately identified through morphological characters, and great obstruction is brought to the forestry and agricultural production and application of the plants. Currently, sex identification of the young stage of hermaphroditic plants is performed by using technologies such as Amplified Fragment Length Polymorphism (AFLP), Random Amplified DNA Polymorphism (RAPD), Inter-Simple Sequence repeat (ISSR), Sequence Amplified region (scarr), Related Sequence Amplified Polymorphism (SRAP), and Simple repeat Sequence repeat (SSR) to perform sex-specific molecular marking on plants. However, these methods are based on PCR techniques and require extremely large numbers of primers or primer pairs to screen for markers associated with sex, which is time consuming, laborious and inefficient.
RAD-seq is a new technology based on genome restriction enzyme cutting and high-throughput sequencing, and can complete sequencing of tens of thousands or even hundreds of thousands of genome sites in a short time. Since many hermaphrodite plants are woody plants, with long generation cycles or low seed numbers, experimental crossing is impractical in many woody plants. Even for annual plants, the construction of mapping populations takes a significant amount of manpower, material and time, which limits the application of the RAD-seq technique to hermaphroditic plant identification.
The library created by ddRAD-seq is highly informative. However, no report has been made on the existence of partial information for the sex identification of the hermaphrodite plants. In light of the above problems, it is an urgent problem to search for information useful for identifying the sex of a plant of a sexually-emasculated plant.
Disclosure of Invention
The invention aims to simply, quickly, accurately and efficiently identify the sex of a male and female plant in the juvenile period by utilizing a genome restriction enzyme digestion and high-throughput sequencing technology and using an improved double-enzyme digestion RAD-seq (ddRAD-seq) method. Unless otherwise specified, the following reagents are commercially available.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, sex-specific ddRAD loci M1 and M2 are presented. According to an embodiment of the invention, the nucleotide sequences of the loci M1 and M2 are as follows:
M1：
GGAAAATTTCCATGTGATTCAGATCTTGAGAAATATTTAGATCACATGGTTCATATTCCTGTTAGCGAACTCCAGTATTAGATCACATG；
M2：
CGTGGCAATACAATAACAAAAACAGAAAATAATAACAACAGCAATACAGTTTTTACAATAAATCGTTGGGAATTGCTCATATACTAGCCGAC。
in a second aspect of the invention, a primer set for amplifying the sex-specific ddRAD loci M1 and M2 as described above is provided. According to an embodiment of the present invention, the nucleotide sequence of the primer set is as follows:
M1
M1-F：GCATTGATAGTGTGGATCATGC；
M1-R：CCATGTGATTCAGATCTTGAGAA；
M2
M2-F：CGCATACGTACGCCACTTAG；
M2-R：CGCAAGCAGCCTAAGGGATCAA。
in a third aspect of the invention, a kit for detecting sex identification of a heterothermaphrodite plant in an early growth stage is provided, which comprises the primer set.
In a fourth aspect of the present invention, there is provided a vector comprising the sex-specific ddRAD loci M1 and M2 as described above.
In the fifth aspect of the invention, the application of the sex-specific ddRAD locus and the primer group in the preparation of the sex determination kit for the early growth stage of the hermaphroditic plants is provided.
In the sixth aspect of the invention, a sex identification method for the female and male heterozygote plants in the early growth stage is provided. According to an embodiment of the invention, the method comprises the steps of:
1) extracting the genome DNA of the sample;
2) PCR amplification was performed using the extracted genomic DNA as a template, using the aforementioned primer sets M1-F, M1-R, M2-F and M2-R;
3) analyzing the amplified fragments to identify the sex of the sample.
According to an embodiment of the invention, the hermaphrodite plant is a plant of the genus sumac.
According to an embodiment of the invention, the plant of the genus seawood is a seawood.
According to an embodiment of the invention, the early stage sex identification is a pre-anthesis sex identification of the heterogynic plant.
According to an embodiment of the present invention, the sample sex determination method of step 3): if the M1 primer amplifies 3 bands in the sample, the sample is male; if 2 bands are amplified in a sample, the sample is female.
According to an embodiment of the present invention, the sample sex determination method of step 3): if the M2 primer amplifies 1 strip in the sample, the sample is male; if no band is amplified in the sample, the sample is female.
The invention has the beneficial effects that:
the invention can directly carry out sex identification on the female and male plants which naturally grow, thereby avoiding the sex identification of offspring generated by experimental hybridization of a plurality of adult male plants and female plants, having long identification period and complicated method. The sex specificity marker of the screened heterogynic plant can be used for identifying the sex of the heterogynic plant population growing in the early stage under the natural environment, and has the characteristics of simplicity, accuracy and high efficiency.
Detailed Description
The technical solution of the present invention is further described and illustrated by the following specific examples.
Example 1
The invention selects the nucleotide sequences of ddRAD loci (M1 and M2), M1:
GGAAAATTTCCATGTGATTCAGATCTTGAGAAATATTTAGATCACATGGTTCATATTCCTGTTAGCGAACTCCAGTATTAGATCACATG(SEQ ID NO.1)；
nucleotide sequence of M2:
CGTGGCAATACAATAACAAAAACAGAAAATAATAACAACAGCAATACAGTTTTTACAATAAATCGTTGGGAATTGCTCATATACTAGCCGAC(SEQ ID NO.2)。
designing PCR primers aiming at M1 and M2 respectively according to the nucleic acid sequence; the invention screens out primers with high sensitivity and strong specificity respectively through a large amount of screens, and the sequences are as follows:
M1：
M1-F：GCATTGATAGTGTGGATCATGC(SEQ ID NO.3)；
M1-R：CCATGTGATTCAGATCTTGAGAA(SEQ ID NO.4)；
M2：
M2-F：CGCATACGTACGCCACTTAG(SEQ ID NO.5)；
M2-R：CGCAAGCAGCCTAAGGGATCAA(SEQ ID NO.6)。
example 3
A vector comprising the nucleotide sequences of the ddRAD loci M1 and M2 described in example 1.
Comparative example 1
A method for identifying the sex of the gynoecial heterotrophy plant, namely the red-back cinnamon (Excoecaria cochinchinensis) in the early growth stage comprises the following steps:
cross-species amplification was performed on male and female allogenic red-dorsal cinnamon using the primers of M1 and M2 loci of the present invention, respectively.
As a result: m1 amplified a band in the red-backed cinnamon, but did not amplify a male specific band, and the amplification results between the female and the male were not obvious. M2 did not amplify a male-specific band in the red cinnamon. The M1 and M2 have higher specificity.
Comparative example 2
A method for identifying the sex of the heterotrophic plant of Aquilaria yunnanensis (Excoecia acerifolia) in the early growth stage comprises the following steps:
the primers M1 and M2 of the invention are used for cross-species amplification on male and female allogenic Yunnan native agilawood respectively.
As a result: m1 amplified a band in Aquilaria sinensis, Yunnan, but did not amplify a male-specific band. M2 did not amplify a male-specific band in Aquilaria sinensis. The M1 and M2 have higher specificity.
Verification experiments were performed below for the M1 and M2 loci of the invention.
1. PCR validation of sex-specific ddRAD loci M1, M2
The method comprises the following steps: the PCR procedure was: initial denaturation at 94 ℃ for 4 min, followed by 30 denaturation cycles (45 s at 94 ℃), annealing (56 and 40s at 62 ℃) and extension (25 s at 72 ℃).
As a result: through PCR verification, the M1 primer is used for amplification, and detection shows that M1 can amplify 3 bands in 20 male strains of the lacquer, but only 2 bands in 20 female strains of the lacquer. M2 amplified 1 band in the male strain of Lacquer, but not in the female strain of Lacquer.
It is understood that the female strain-specific markers M1 and M2 of the invention are ideal sex-specific markers for PCR-based sex determination. Among them, M1 was more effective and could be the first choice for identifying the sex of the varroa jalapa plants, since male and female PCR products with different banding patterns could be obtained at the M1 locus.
2. Seedling sex identification verification
The M1 screened by the invention respectively identifies the sex of 100 seeds of the lacquer and 100 young seedlings of the lacquer with the seedling age of 1 month.
The results show that the seed male-female ratio of the sea-lacquer plant is 49: 51, the male-female ratio of the sea lacquer seedlings is 45: 55, gender ratio did not deviate significantly from 1: 1, the gene locus M1 has high accuracy, and can identify the sex of the hermaphrodite plants of the lacca.
SEQUENCE LISTING
<110> Guangzhou Pont Garden shares GmbH,
Zhongshan university, Guangzhou Jieli Biotech Co., Ltd
<120> method for identifying sex of female and male heteroplant in early growth stage
<130>
<160>6
<170>PatentIn version 3.5
<210>1
<211>89
<212>DNA
<213> Artificial sequence
<400>1
ggaaaatttc catgtgattc agatcttgag aaatatttag atcacatggt tcatattcct 60
gttagcgaac tccagtatta gatcacatg 89
<210>2
<211>92
<212>DNA
<213> Artificial sequence
<400>2
cgtggcaata caataacaaa aacagaaaat aataacaaca gcaatacagt ttttacaata 60
aatcgttggg aattgctcat atactagccg ac 92
<210>3
<211>22
<212>DNA
<213> Artificial sequence
<400>3
gcattgatag tgtggatcat gc 22
<210>4
<211>23
<212>DNA
<213> Artificial sequence
<400>4
ccatgtgatt cagatcttga gaa 23
<210>5
<211>20
<212>DNA
<213> Artificial sequence
<400>5
cgcatacgta cgccacttag 20
<210>6
<211>22
<212>DNA
<213> Artificial sequence
<400>6
cgcaagcagc ctaagggatc aa 22
Claims (10)
1. Sex-specific ddRAD loci M1 and M2, characterized in that the nucleotide sequences of said loci M1 and M2 are as follows:
M1：
GGAAAATTTCCATGTGATTCAGATCTTGAGAAATATTTAGATCACATGGTTCATATTCCTGTTAGCGAACTCCAGTATTAGATCACATG；
M2：
CGTGGCAATACAATAACAAAAACAGAAAATAATAACAACAGCAATACAGTTTTTACAATAAATCGTTGGGAATTGCTCATATACTAGCCGAC。
2. a primer set for amplifying the sex-specific ddRAD loci M1 and M2 of claim 1, wherein the nucleotide sequences of the primer set are:
M1
M1-F：GCATTGATAGTGTGGATCATGC；
M1-R：CCATGTGATTCAGATCTTGAGAA；
M2
M2-F：CGCATACGTACGCCACTTAG；
M2-R：CGCAAGCAGCCTAAGGGATCAA。
3. a kit for detecting sex identification of a heterothermaphroditic plant in early growth stage, which is characterized by comprising the primer group in claim 2.
4. A vector comprising the sex-specific ddRAD loci M1 and M2 of claim 1.
5. The use of the sex-specific ddRAD locus and the primer set as claimed in any one of claims 1 to 2 in the preparation of a kit for identifying the sex of a heterothermaphroditic plant in the early growth stage.
6. A method for identifying the sex of a male and female heteroplant in the early growth stage is characterized by comprising the following steps:
1) extracting the genome DNA of the sample;
2) performing PCR amplification using the primer sets M1-F, M1-R, M2-F and M2-R of claim 2, using the extracted genomic DNA as a template;
3) analyzing the PCR amplified band to identify the sex of the sample.
7. The method according to claim 6, wherein the hermaphrodite plant is a plant of the genus Lacquertree.
8. The method of claim 7, wherein said plant of the genus Lacquertree is Lacquer.
9. The method of claim 6, wherein the step 3) of sexing the sample comprises: if 3 bands are amplified in the sample by using the M1 primer, the sample is male; if 2 bands are amplified in a sample, the sample is female.
10. The method of claim 6, wherein the step 3) of sexing the sample comprises: if the M2 primer amplifies 1 strip in the sample, the sample is male; if no band is amplified in the sample, the sample is female.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115211321A (en) * | 2022-08-22 | 2022-10-21 | 中国林业科学研究院林业研究所 | Early identification method for sex of male and female poplar hybrid progeny |
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| CN115211321B (en) * | 2022-08-22 | 2024-02-20 | 中国林业科学研究院林业研究所 | Early identification method for male and female sex of poplar hybrid progeny |
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