CN116814837B - SSR (simple sequence repeat) marker primer, method and application for rapidly identifying varieties of camellia yunnanensis - Google Patents
SSR (simple sequence repeat) marker primer, method and application for rapidly identifying varieties of camellia yunnanensis Download PDFInfo
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Abstract
The invention relates to a method for rapidly identifying varieties of camellia yunnanensis, and provides 3 pairs of SSR marker primers SSR512, SSR568 and SSR623 which are independently developed and are applied to the identification of varieties of camellia yunnanensis. Specifically, different varieties are identified according to different SSR genotypes of different Yunnan camellia varieties of the same primer. The 3 pairs of SSR primers obtained by the invention can completely distinguish 10 varieties of Yunnan camellia one by one, and the identification efficiency is high. Compared with the traditional experience identification method, the method has high accuracy and high repeatability. Compared with a qualitative or quantitative comprehensive judging method for observed data, the method can save time and cost. Therefore, the method has wide application prospect for identifying the varieties of the Yunnan camellia, and provides technical support and scientific basis for effectively identifying the varieties of the Yunnan camellia and further developing and utilizing the varieties of the Yunnan camellia.
Description
Technical Field
The invention relates to a Yunnan camellia variety identification technology, establishes an SSR marker primer for rapidly identifying the Yunnan camellia variety, a method and application thereof, and belongs to the technical field of biology.
Background
The Yunnan camellia (Camellia reticulata) is a camellia plant of the camellia family (Theaceae), and compared with camellia (C.japonica) and camellia (C.sasanqua) variety groups, the Yunnan camellia variety has larger flower diameter (more than 20 cm), bright and colorful flower color and higher ornamental value, is a rare flower in the world, and is widely applied at home and abroad. The Yunnan camellia forms a large number of varieties with rich colors and various postures in the long-term cultivation and application history process, becomes an important flower variety for potted plants and landscaping, and plays a very important role in flower production.
Morphological differences and variations among individuals in the Yunnan camellia species are common in nature, and the influence of natural hybridization and artificial breeding increases the diversity and complexity of the variations and increases the difficulty of morphological classification. Meanwhile, with the wide collection and continuous accumulated exchange of the Yunnan camellia germplasm resources, a large number of homonymous foreign matters and homonymous foreign matters appear, and great inconvenience and confusion are brought to production and research, so that the research on the Yunnan camellia varieties by utilizing the latest molecular marking technology is necessary. The rapid, efficient and accurate Yunnan camellia variety identification technology is established, and the technology support and scientific basis are hopeful to be provided for variety communication, research, popularization and application.
Identification of the varieties of the Yunnan camellia, which is usually judged from the appearance form by a professional technician according to experience, often causes subjective misjudgment; qualitative or quantitative comprehensive determination of observed data typically requires the observation of data collected for one or more growing seasons or for a particular period of growth and development, lacks timeliness and accuracy, and further, is susceptible to culture management and environmental conditions. Therefore, it is difficult to effectively identify and select the varieties of the Yunnan camellia with similar morphological characters. The identification is performed by using the DNA molecular marker, has the characteristics of being immediate, efficient, visual and accurate, can be used for identifying similar varieties more quickly and accurately, can also reveal genetic variation occurring and existing in germplasm, and provides scientific basis for sustainable utilization of Yunnan camellia resources.
SSR markers, also known as microsatellites DNA (Microsatellite DNA), are inherited in a Mendelian manner and are co-dominant compared to other molecular markers; the number is rich, and the polymorphism is high; multiallelic genes have large information quantity; meanwhile, the method has the characteristics of good repeatability, strong reliability and the like, and is widely applied to the aspects of germplasm resource identification, genetic diversity, genetic structure analysis, genetic map construction and the like. At present, an SSR molecular identification technology is mainly applied to crops and various cash crops, and related reports on the identification of the varieties of the Yunnan camellia by utilizing an SSR molecular marking technology are not yet seen.
According to the invention, 3 pairs of SSR marker primers are independently developed, 10 varieties of the camellia yunnanensis are rapidly identified through high-throughput sequencing and SSR genotyping technologies, and technical support is provided for the identification and further development and utilization of the varieties of the camellia yunnanensis.
Disclosure of Invention
The invention provides 3 pairs of SSR marker primers which are independently developed and designed, and combines high-throughput sequencing and SSR genotyping technologies through SSRseq TM technologies, so that rapid, accurate and efficient identification of the camellia yunnanensis varieties is realized, and technical support and scientific basis are provided for efficient identification of the camellia yunnanensis varieties.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The camellia yunnanensis variety SSR marker primer is developed autonomously, and the primer is specifically as follows:
Primer number: SSR512, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2;
Primer number: SSR568, the nucleotide sequence of the upstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4;
Primer number: the nucleotide sequence of the SSR623 and the nucleotide sequence of the upstream primer are shown as SEQ ID No.5 and SEQ ID No.6 respectively.
The SSR marker primer is applied to the identification of the varieties of the Yunnan camellia, and the varieties of the Yunnan camellia are rapidly identified by SSRseq TM technology. The method for identifying the varieties of the Yunnan camellia comprises the following steps:
DNA extraction: DNA in fresh leaves of the Yunnan camellia variety is extracted by using a DNA extraction kit.
DNA quality assessment: the integrity of the nucleic acid was checked by agarose gel electrophoresis, the band should be clear, and no degradation and contamination of the nucleic acid occurred. The purity of the DNA was evaluated by NanoDrop2000, the concentration of DNA was not less than 20 ng/. Mu.L, and the content was not less than (200+n.times.30) ng (n is the panel number).
And (3) PCR amplification: single or multiple PCR reaction amplification is carried out on 10 Yunnan camellia varieties by using SSR512, SSR568 and SSR623 primer loci.
High throughput sequencing: all PCR products were pooled and libraries were prepared and sequenced on a IlluminaHiSeq2500 (PE 150 bp) platform. The raw readings were subjected to a series of analyses including quality control using FastQC, merging reads with FLASH, and constructing a reference alignment using Blastn.
SSR genotyping: the number of SSR alleles was calculated by aligning reads with the sequence data. SSR motifs and repeat numbers are listed after two-step correction, including slip adjustment and amplification efficiency adjustment.
And (3) identifying varieties of Yunnan camellia: according to the SSR genotyping of different Yunnan camellia varieties of the same primer, different varieties can be effectively identified.
The beneficial effects obtained by the invention are as follows:
SSR primers used for identifying the SSR molecular markers of the camellia yunnanensis varieties are independently developed and designed for the laboratory, and the primer information is shown in Table 1.
TABLE 1 SSR primers for Yunnan camellia varieties
According to the SSR genotyping of different Yunnan camellia varieties of the same primer, different varieties can be effectively identified. The 48 pairs of primers independently developed in the laboratory are utilized to carry out genotyping identification on 168 camellia yunnanensis varieties, and 3 pairs of SSR primers can be screened to respectively distinguish 10 camellia yunnanensis varieties one by one.
The invention utilizes 3 pairs of autonomously developed SSR primers to carry out high-throughput sequencing and SSR genotyping on the DNA of the camellia varieties, and can effectively identify 10 camellia varieties. 1. The SSR mark parting map obtained by the invention has strong specificity and high polymorphism. 2. The 3 pairs of SSR primers independently developed can completely distinguish 10 varieties of the Yunnan camellia at one time, and the identification efficiency is high; 3 pairs of SSR primers can be independently used for identification, and can also be used for simultaneously carrying out multiple PCR reactions, so that a high polymorphism parting map can be obtained by a small number of amplification cycles, the reaction time is greatly shortened, and the rapid identification of the Yunnan camellia variety can be realized. 3. The data obtained by the method is based on bioinformatics analysis, does not need to manually judge genotype, has high efficiency in flow and accurate typing, reduces artificial errors, and has high accuracy and strong repeatability compared with the traditional experience identification method. 4. Compared with a qualitative or quantitative comprehensive judging method for observed data, the method can save time and cost. Therefore, the method has wide application prospect in identifying the varieties of the Yunnan camellia.
Drawings
Fig. 1:10 Yunnan camellia variety primer SSR512 genotyping diagrams.
Fig. 2:10 camellia yunnanensis variety primer SSR568 genotyping charts.
Fig. 3:10 Yunnan camellia variety primer SSR623 genotyping diagrams.
Detailed Description
Example 1
And 10 camellia varieties can be identified at one time according to SSR genotyping results by utilizing autonomous development SSR primers to carry out high-throughput sequencing and SSR genotyping on the 10 camellia varieties. An SSR primer for rapidly identifying a Yunnan camellia variety and an application method thereof are specifically carried out according to the following steps:
Sampling: collecting fresh leaves of Yunnan camellia varieties. The serial numbers of the 10 Yunnan camellia varieties are respectively: 10: chrysanthemum petals, 11: smart peak, 103: xiao Gong powder, 108: early peony, 111: drunk and lovely red, 113: red Jin Ling, 118: chrysanthemum morifolium ramat, 119: folium Daturae Metelis, 121: brocade red, 128: zixi.
DNA extraction: DNA in fresh leaves of the Yunnan camellia variety is extracted by using a DNA extraction kit.
DNA quality assessment: the integrity of the nucleic acid was checked by agarose gel electrophoresis, the band should be clear, and no degradation and contamination of the nucleic acid occurred. The purity of the DNA was evaluated by NanoDrop2000, the concentration of DNA was not less than 20 ng/. Mu.L, and the content was not less than (200+n.times.30) ng (n is the panel number).
And (3) PCR amplification: 10 varieties of Yunnan camellia are amplified by multiplex PCR reaction with SSR512, SSR568 and SSR623 primer loci. Reaction system (25 μl): the template DNA contained 100ng in 1.5. Mu.L, 2X TSINGKE MASTER Mix in 12.5. Mu.L, 10mmol/L primer in 2. Mu.L and ultrapure water in 9. Mu.L. PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 30s,11 cycles; extending at 72 ℃ for 9min; preserving at 4 ℃.
High throughput sequencing: all PCR products were pooled and libraries were prepared and sequenced on a IlluminaHiSeq2500 (PE 150 bp) platform. The raw readings were subjected to a series of analyses including quality control using FastQC, merging reads with FLASH, and constructing a reference alignment using Blastn.
SSR genotyping: the number of SSR alleles was calculated by aligning reads with the sequence data. SSR motifs and repeat numbers are listed after two-step correction, including slip adjustment and amplification efficiency adjustment.
And (3) identifying varieties of Yunnan camellia: different varieties can be effectively identified according to different SSR genotypes of different Yunnan camellia varieties of the same primer, and the results are shown in Table 2.
TABLE 2 SSR genotyping results for 10 Yunnan camellia varieties
| Sample/primer | SSR512 | SSR 568 | SSR 623 |
| 10 | 5/6/7/7/9/10 | 8/8/8/8/8/9 | 8/8/8/12/16/17 |
| 11 | 3/5/5/7/7/10 | 7/7/7/7/14/18 | 8/8/8/9/13/14 |
| 103 | 3/5/5/6/6/7 | 7/7/7/7/9/10 | 7/7/8/8/8/12 |
| 108 | 3/5/5/6/6/10 | 4/7/7/9/9/14 | 8/8/8/8/11/13 |
| 111 | 5/5/7/7/7/10 | 8/9/9/14/17/18 | 8/9/9/9/10/10 |
| 113 | 5/5/7/10/10/10 | 4/7/7/7/7/9 | 8/8/8/9/9/14 |
| 118 | 3/5/6/6/7/10 | 5/8/9/9/10/14 | 8/8/9/9/9/14 |
| 119 | 3/3/6/7/10/10 | 4/9/9/10/11/13 | 8/8/9/10/14/14 |
| 121 | 6/7/8/8/8/10 | 7/8/8/8/8/8 | 8/8/10/10/10/14 |
| 128 | 3/3/3/5/5/5 | 7/7/9/9/11/14 | 8/8/9/9/9/12 |
Example 2
Sampling: collecting fresh leaves of Yunnan camellia varieties. The serial numbers of the 10 Yunnan camellia varieties are respectively: 10: chrysanthemum petals, 11: smart peak, 103: xiao Gong powder, 108: early peony, 111: drunk and lovely red, 113: red Jin Ling, 118: chrysanthemum morifolium ramat, 119: folium Daturae Metelis, 121: brocade red, 128: zixi.
DNA extraction: DNA in fresh leaves of the Yunnan camellia variety is extracted by using a DNA extraction kit.
DNA quality assessment: the integrity of the nucleic acid was checked by agarose gel electrophoresis, the band should be clear, and no degradation and contamination of the nucleic acid occurred. The purity of the DNA was evaluated by NanoDrop2000, the concentration of DNA was not less than 20 ng/. Mu.L, and the content was not less than (200+n.times.30) ng (n is the panel number).
And (3) PCR amplification: and carrying out PCR amplification on 10 Yunnan camellia varieties by using SSR512 primer loci. Reaction system (25 μl): the template DNA contained 100ng in 1.5. Mu.L, 2X TSINGKE MASTER Mix in 12.5. Mu.L, 10mmol/L primer in 2. Mu.L and ultrapure water in 9. Mu.L. PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 30s,11 cycles; extending at 72 ℃ for 9min; preserving at 4 ℃.
High throughput sequencing: all PCR products were pooled and libraries were prepared and sequenced on a IlluminaHiSeq2500 (PE 150 bp) platform. The raw readings were subjected to a series of analyses including quality control using FastQC, merging reads with FLASH, and constructing a reference alignment using Blastn.
SSR genotyping: the number of SSR alleles was calculated by aligning reads with the sequence data. SSR motifs and repeat numbers are listed after two-step correction, including slip adjustment and amplification efficiency adjustment.
And (3) identifying varieties of Yunnan camellia: according to the SSR genotyping results of the primer SSR512 on different varieties of the Yunnan camellia, different varieties can be effectively identified, the results are shown in the figure 1 and the table 3, the genotyping results of 10 varieties of the Yunnan camellia 10, 11, 103, 108, 111, 113, 118, 119, 121 and 128 are respectively ：5/6/7/7/9/10、3/5/5/7/7/10、3/5/5/6/6/7、3/5/5/6/6/10、5/5/7/7/7/10、5/5/7/10/10/10、3/5/6/6/7/10、3/3/6/7/10/10、6/7/8/8/8/10、3/3/3/5/5/5.10 varieties of the primer SSR512, the genotyping results are different, and 10 varieties of the Yunnan camellia can be identified once by using the primer SSR512, so that the identification efficiency is high.
TABLE 3 SSR512 genotyping results for 10 Yunnan camellia variety primers
| Sample/primer | SSR512 |
| 10 | 5/6/7/7/9/10 |
| 11 | 3/5/5/7/7/10 |
| 103 | 3/5/5/6/6/7 |
| 108 | 3/5/5/6/6/10 |
| 111 | 5/5/7/7/7/10 |
| 113 | 5/5/7/10/10/10 |
| 118 | 3/5/6/6/7/10 |
| 119 | 3/3/6/7/10/10 |
| 121 | 6/7/8/8/8/10 |
| 128 | 3/3/3/5/5/5 |
Example 3
Sampling: collecting fresh leaves of Yunnan camellia varieties. The serial numbers of the 10 Yunnan camellia varieties are respectively: 10: chrysanthemum petals, 11: smart peak, 103: xiao Gong powder, 108: early peony, 111: drunk and lovely red, 113: red Jin Ling, 118: chrysanthemum morifolium ramat, 119: folium Daturae Metelis, 121: brocade red, 128: zixi.
DNA extraction: DNA in fresh leaves of the Yunnan camellia variety is extracted by using a DNA extraction kit.
DNA quality assessment: the integrity of the nucleic acid was checked by agarose gel electrophoresis, the band should be clear, and no degradation and contamination of the nucleic acid occurred. The purity of the DNA was evaluated by NanoDrop2000, the concentration of DNA was not less than 20 ng/. Mu.L, and the content was not less than (200+n.times.30) ng (n is the panel number).
And (3) PCR amplification: and carrying out PCR amplification on 10 Yunnan camellia varieties by using SSR568 primer loci. Reaction system (25 μl): the template DNA contained 100ng in 1.5. Mu.L, 2X TSINGKE MASTER Mix in 12.5. Mu.L, 10mmol/L primer in 2. Mu.L and ultrapure water in 9. Mu.L. PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 30s,11 cycles; extending at 72 ℃ for 9min; preserving at 4 ℃.
High throughput sequencing: all PCR products were pooled and libraries were prepared and sequenced on a IlluminaHiSeq2500 (PE 150 bp) platform. The raw readings were subjected to a series of analyses including quality control using FastQC, merging reads with FLASH, and constructing a reference alignment using Blastn.
SSR genotyping: the number of SSR alleles was calculated by aligning reads with the sequence data. SSR motifs and repeat numbers are listed after two-step correction, including slip adjustment and amplification efficiency adjustment.
And (3) identifying varieties of Yunnan camellia: according to the SSR genotyping results of the primer SSR568 on different varieties of the Yunnan camellia, different varieties can be effectively identified, the results are shown in fig. 2 and table 4, the genotyping results of 10 varieties of the Yunnan camellia 10, 11, 103, 108, 111, 113, 118, 119, 121 and 128 are ：8/8/8/8/8/9、7/7/7/7/14/18、7/7/7/7/9/10、4/7/7/9/9/14、8/9/9/14/17/18、4/7/7/7/7/9、5/8/9/9/10/14、4/9/9/10/11/13、7/8/8/8/8/8、7/7/9/9/11/14.10 varieties of the primer SSR568, the genotyping results are different, and 10 varieties of the Yunnan camellia can be identified once by using the primer SSR568, so that the identification efficiency is high.
TABLE 4 SSR568 genotyping results for 10 Yunnan camellia variety primers
| Sample/primer | SSR 568 |
| 10 | 8/8/8/8/8/9 |
| 11 | 7/7/7/7/14/18 |
| 103 | 7/7/7/7/9/10 |
| 108 | 4/7/7/9/9/14 |
| 111 | 8/9/9/14/17/18 |
| 113 | 4/7/7/7/7/9 |
| 118 | 5/8/9/9/10/14 |
| 119 | 4/9/9/10/11/13 |
| 121 | 7/8/8/8/8/8 |
| 128 | 7/7/9/9/11/14 |
Example 4
Sampling: collecting fresh leaves of Yunnan camellia varieties. The serial numbers of the 10 Yunnan camellia varieties are respectively: 10: chrysanthemum petals, 11: smart peak, 103: xiao Gong powder, 108: early peony, 111: drunk and lovely red, 113: red Jin Ling, 118: chrysanthemum morifolium ramat, 119: folium Daturae Metelis, 121: brocade red, 128: zixi.
DNA extraction: DNA in fresh leaves of the Yunnan camellia variety is extracted by using a DNA extraction kit.
DNA quality assessment: the integrity of the nucleic acid was checked by agarose gel electrophoresis, the band should be clear, and no degradation and contamination of the nucleic acid occurred. The purity of the DNA was evaluated by NanoDrop2000, the concentration of DNA was not less than 20 ng/. Mu.L, and the content was not less than (200+n.times.30) ng (n is the panel number).
And (3) PCR amplification: and carrying out PCR amplification on 10 Yunnan camellia varieties by using SSR623 primer loci. Reaction system (25 μl): the template DNA contained 100ng in 1.5. Mu.L, 2X TSINGKE MASTER Mix in 12.5. Mu.L, 10mmol/L primer in 2. Mu.L and ultrapure water in 9. Mu.L. PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 30s,11 cycles; extending at 72 ℃ for 9min; preserving at 4 ℃.
High throughput sequencing: all PCR products were pooled and libraries were prepared and sequenced on a IlluminaHiSeq2500 (PE 150 bp) platform. The raw readings were subjected to a series of analyses including quality control using FastQC, merging reads with FLASH, and constructing a reference alignment using Blastn.
SSR genotyping: the number of SSR alleles was calculated by aligning reads with the sequence data. SSR motifs and repeat numbers are listed after two-step correction, including slip adjustment and amplification efficiency adjustment.
And (3) identifying varieties of Yunnan camellia: according to the SSR genotyping of the primer SSR623 on different varieties of the Yunnan camellia, different varieties can be effectively identified, the results are shown in the figure 3 and the table 5, the genotyping results of 10 varieties of the Yunnan camellia 10, 11, 103, 108, 111, 113, 118, 119, 121 and 128 are ：8/8/8/12/16/17、8/8/8/9/13/14、7/7/8/8/8/12、8/8/8/8/11/13、8/9/9/9/10/10、8/8/8/9/9/14、8/8/9/9/9/14、8/8/9/10/14/14、8/8/10/10/10/14、8/8/9/9/9/12.10 varieties of the Yunnan camellia, the genotyping results of the primer SSR623 of the Yunnan camellia are different, 10 varieties of the Yunnan camellia can be identified by using the primer SSR623 once, and the identification efficiency is high.
TABLE 5 results of SSR623 genotyping of 10 Yunnan camellia variety primers
| Sample/primer | SSR 623 |
| 10 | 8/8/8/12/16/17 |
| 11 | 8/8/8/9/13/14 |
| 103 | 7/7/8/8/8/12 |
| 108 | 8/8/8/8/11/13 |
| 111 | 8/9/9/9/10/10 |
| 113 | 8/8/8/9/9/14 |
| 118 | 8/8/9/9/9/14 |
| 119 | 8/8/9/10/14/14 |
| 121 | 8/8/10/10/10/14 |
| 128 | 8/8/9/9/9/12 |
Claims (3)
1. The camellia yunnanensis variety SSR marker primer is characterized in that: the 3 pairs of primers are all autonomously developed primers, and the primers are specifically as follows:
Primer number: SSR512, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2;
Primer number: SSR568, the nucleotide sequence of the upstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4;
Primer number: the nucleotide sequence of the SSR623 and the nucleotide sequence of the upstream primer are shown as SEQ ID No.5 and SEQ ID No.6 respectively.
2. A method for identifying a variety of camellia yunnanensis, which is characterized in that: the method for rapidly identifying the varieties of the Yunnan camellia by using SSRseq TM technology comprises the following steps:
DNA extraction: extracting DNA in fresh leaves of the Yunnan camellia variety by using a DNA extraction kit;
DNA quality assessment: checking the integrity of nucleic acid by agarose gel electrophoresis, wherein the band is clear, no nucleic acid degradation and no nucleic acid pollution are caused, and the purity of deoxyribonucleic acid is evaluated by using a spectrophotometer, wherein the DNA concentration is more than or equal to 20 ng/mu L, and the content is more than or equal to (200+n x 30) ng (n is the detection number);
and (3) PCR amplification: carrying out single or multiple PCR reaction amplification on 10 varieties of Yunnan camellia by using SSR512, SSR568 and SSR623 primer loci;
High throughput sequencing: all PCR products were pooled and library prepared, sequenced on a IlluminaHiSeq2500 (PE 150 bp) platform, and the original reads were subjected to a series of analyses, including quality control using FastQC, merging reads with FLASH, and construction of a reference alignment using Blastn;
SSR genotyping: calculating the number of SSR alleles by comparing reads with sequence data, and listing SSR motifs and repetition numbers after two-step correction, including slippage adjustment and amplification efficiency adjustment;
And (3) identifying varieties of Yunnan camellia: according to the SSR genotyping difference of different Yunnan camellia varieties of the same primer, different varieties of chrysanthemum petals, smart peaks, xiao Gong powder, early peony, buddleia red, red Jin Ling, siraitia, carmine cassia leaves and brocade red Zixi are effectively identified.
3. The use of an SSR marker primer for the variety of camellia in rapid identification of varieties of camellia in chrysanthemum, lingfeng, xiao Gong powder, early peony, buddleia red, red Jin Ling, siraitia, carmine, broccoli red and Zixi according to claim 1.
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| WO2003085133A2 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics | Novel fissr-pcr primers and method of genotyping diverse genomes of plant and animal systems including rice varieties |
| CN111944917A (en) * | 2019-05-16 | 2020-11-17 | 南京农业大学 | Method for developing camellia plant SSR primers based on transcriptome sequencing |
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| WO2003085133A2 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics | Novel fissr-pcr primers and method of genotyping diverse genomes of plant and animal systems including rice varieties |
| CN111944917A (en) * | 2019-05-16 | 2020-11-17 | 南京农业大学 | Method for developing camellia plant SSR primers based on transcriptome sequencing |
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| 茶树EST-SSRs在山茶科植物中的通用性研究;程小毛;陈自兰;王华;;安徽农业科学;20110501(第13期);7596-7598 * |
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