CN111926099A - SSR molecular markers based on camellia transcriptome and application of SSR molecular markers in camellia plants - Google Patents
SSR molecular markers based on camellia transcriptome and application of SSR molecular markers in camellia plants Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a group of SSR molecular markers based on a camellia transcriptome and application of the SSR molecular markers in camellia plants. 28854 pairs of SSR markers are developed in total, 13323 pairs of SSR primers are developed in total, 111 pairs of polymorphic SSR markers are further obtained, and the markers occupy 79.85 percent of effective amplification primers and are far higher than the level of the prior art; meanwhile, the PIC value range of 111 pairs of polymorphic markers in the application is 0.15-0.86, and the average value is 0.59, which is far higher than the existing level. The present application utilizes data further obtained from 27 of these polymorphism markers to reveal the true genetic relationship and population structure between 89 camellia varieties derived from camellia. In conclusion, the research result provides high-quality SSR marker resources for the genetic improvement research of camellia plants in the future.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a group of SSR molecular markers based on a camellia transcriptome and application of the SSR molecular markers in camellia plants.
Background
Camellia (genus Camellia) belongs to the family Theaceae (family Theaceae), and is widely distributed in the south and southeast Asia of China. Several varieties of camellia have important economic value. Such as camellia (c.japonica), camellia yunnanensis (c.reticulata), camellia angensis (c.saluenensis) and camellia sasanqua (c.sasanqua), are known as their attractive flowers; tea tree (c.sinensis) is well known in the world as its young leaves are used to produce tea; seeds of some species, such as camellia oleifera (c.oleifera) and camellia nandina (c.semiserrata), are used to produce high quality edible and medicinal oils.
The genus Camellia includes more than 300 species in total, and is the largest genus in the family Theaceae. Among them, ornamental camellia has been cultivated in china for over 2000 years, it has been introduced into japan over 1000 years ago, and has been introduced into europe and america in the late 70 th 19 th century. At present, more than 3000 camellia cultivars are in total in the world, and the camellia cultivar becomes one of the most popular garden ornamental flowers in many countries and regions. However, due to the relationship between natural and artificial crosses, this genus of plants has been somewhat divergent in its systematic classification.
In the traditional camellia breeding, new offspring is generated through the cross between species, and then a new high-quality variety is bred through phenotypic selection. Although the traditional breeding still plays an important role in improving the quality of camellia plants, the breeding progress is severely restricted because of the long selection time and the need of a large amount of resources for breeding new varieties. Marker Assisted Selection (MAS) can directly or indirectly convert the selection criteria from phenotype to gene, thus saving time and resources, having great potential in accelerating crop breeding, and avoiding problems in traditional plant breeding. And the molecular marker is not influenced by environmental regulation and plant growth conditions and can be detected at all stages of plant growth. Therefore, molecular marker assisted selective breeding is widely used.
Simple Sequence Repeats (SSRs) are genomic fragments consisting of consecutive repeats of 1-6 nucleotide sequence motifs flanked by unique sequences. SSR markers are widely applied to plant genetic analysis and marker-assisted selective breeding. Due to the wide distribution of SSRs throughout the genome, co-dominant inheritance and high polymorphism, SSRs have been widely used in genetic linkage map construction, genetic identification, genetic diversity analysis, and fingerprinting construction.
In recent years, with the development of high-throughput sequencing technology, people have made certain progress in the development and application of camellia SSR markers, but most of the progress is concentrated in tea trees. Wu et al developed 36 tea plant polymorphisms (ESTs) -SSR markers based on 454 sequencing data. By utilizing high-throughput Illumina RNA sequencing data, Liu et al develop 431 polymorphic SSR markers in tea trees, construct an SSR-based linkage map covering 1156.9cM, and totally 237 SSR markers are distributed in 15 linkage groups of tea trees. In another study, Ma et al developed 450 polymorphic SSR markers using tea plant transcriptome data, 406 of which were successfully added to the genetic linkage map.
Despite efforts to develop SSR based on transcriptome and genomic sequences, few presently published camellia SSR markers are available for studies such as constructing high-resolution genetic linkage maps, comparative genomic mapping, and genetics.
The SSR marker development and genetic relationship analysis based on camellia transcriptome, which is published in Panlisi and the like, published in the prior art, the university of Beijing forestry, 2019, 41(7): 111-120. This study synthesized 90 pairs of SSR primers, of which only 29 pairs were polymorphic and accounted for 39.73% of the effective amplification primers. 8 parts of camellia material were analysed for this study, with an average PIC value of 0.496. In the labeling application, the research only carries out cluster analysis on 8 materials from camellia (C. japonica) and hybrid materials thereof, and can better carry out clustering, but samples are few, and the application of the materials in the camellia plants cannot be expected.
Prior art Zhao et al genetic relationships in a sampling collection of Camellia japonica and Camellia oleifera using SSR analysis, genetics & Molecular Research,2017,16: 16019526. In the research, 138 pairs of SSR markers (polymorphic ratio is unknown) are synthesized together, and 21 pairs of SSR markers are selected from 50 parts of materials derived from two varieties of camellia (C.japonica) and camellia oleifera (C.oleifera) for analysis, and the average PIC is 0.34. In cluster analysis, although the research better separates materials derived from camellia (c. japonica) and camellia oleifera (c. oleifera), it cannot be expected how effective the application of the same in plants of the genus camellia is.
Although the prior art relates to the development and application of SSR markers, the average PIC value of the SSR markers is low, and the SSR markers cannot be widely applied to camellia plants. Therefore, there is still a need to further develop high quality SSR markers to further develop the genetic and genomics research of plants in the genus Camellia.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a group of SSR molecular markers based on a camellia transcriptome and application thereof in camellia plants, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.
The invention is realized in such a way, the SSR molecular markers are a group of SSR molecular markers based on a camellia transcriptome, and the numbers of the SSR molecular markers are CjSSR007, CjSSR008, CjSSR014, CjSSR015, CjSSR016, CjSSR021, CjSSR022, CjSSR023, CjSSR030, CjSSR034, CjSSR041, CjSSR049, CjSSR 031050, CjSSR059, CjSSR068, CjSSR073, CjSSR074, CjSSR084, CjSSR087, CjSSR094, CjSSR128, CjSSR138, CjSSR143, CjSSR145, CjSSR154 and CjSSR155, and the primer sequences of the SSR molecular markers corresponding to the numbers are shown in SEQ ID NO.1-SEQ ID NO. 54.
The SSR molecular marker is applied to phylogeny, genetic diversity analysis or linkage map construction of the camellia plants.
Further, the PCR procedure for amplification of a plant sample with the SSR molecular markers was pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension is carried out for 5min at 72 ℃.
Further, the camellia genus includes camellia sasanqua (c.sasanqua), chonglev camellia chrysantha (c.chuonggsoensis), chuanbei desmodium tea (c.rosthorniana), camellia chrysantha (c.nitidis), camellia oleifera (c.oleeiera), camellia yunnanensis (c.retnulata), camellia azalea (c.azalea), and camellia sinensis (c.sinenssis).
SSRs can be classified into genomic SSRs, which are typically developed from SSR-rich genomic libraries or random genomic sequences, and genomic SSRs, which are developed from coding regions of transcriptomes or EST sequences. Although the discovery and mining of genomic SSR markers using whole genome sequences has been successfully applied to many plant species, such as peanut, pear, sweet potato, and tea, the development of genomic SSR based on transcriptome sequences remains an important research approach. Genetic SSR is considered to be more useful for molecular marker assisted breeding than genetic SSR markers, especially when polymorphic markers are identified in breeding lines. The inventor firstly obtains a high-quality transcriptome sequence of camellia (C.japonica) by a high-throughput sequencing method, and then can identify and develop an SSR molecular marker by whole transcriptome analysis. In the present application, SSR was identified to occur at a frequency of 1/4.63kb in the leaf transcriptome of SSR Camellia japonica (C.japonica).
The effectiveness and success of SSR markers depends to a large extent on the quality of the marker. In the present application, there were 111 (64.53%) of the 172 newly designed SSR primer pairs that were polymorphic, and the polymorphism ratio was much higher than that obtained in the previous studies on Camellia plants. High polymorphism ratios may benefit greatly from stringent e-PCR screening procedures. The SSR markers developed by the application have higher PIC values, which indicates that the newly developed SSR markers are suitable for researches such as phylogeny, genetic diversity analysis and linkage map construction. In addition, the SSRs in this application have extremely high intra-generic versatility (from 92.6% to 100%, average 99.38%), higher than other camellia material studies (78.8% -90.4%; 61.6% -88.39%). In general, the SSR markers developed by the present application have high polymorphism and high metastasis. Interestingly, while the motif of the dinucleotide repeats is the most abundant type in the camellia (c. japonica) leaf transcriptome, the trinucleotide repeats are the most abundant type in our newly developed polymorphic markers, which may mean that the trinucleotide repeats may have the highest specificity in camellia (c. japonica) compared to other repeat types. Together with the high polymorphic ratio and high PIC value of the trinucleotide repeat motifs, we believe that in the research of genetic improvement in camellia plants, the development of SSR markers for trinucleotide repeat motifs may have higher polymorphism and higher efficiency.
In the present application, the NJ system analysis nicely divided all 89 materials of Camellia into 5 clusters, and the genotypic grouping and the plant taxonomic-based grouping results were consistent (FIG. 3). In addition, previous studies show that genetic distance and geographic distance of ornamental camellia populations are positively correlated. Unlike the predecessors, our studies, labeling NJ trees by flower color, showed that camellia flower color correlated closely with genetic distance of camellia population (fig. 4). Meanwhile, the result indicates that the flower color characters are relatively conservative in variation in the camellia plants.
In the genetic structure analysis of the present application, all material from camellia is well separated and clustered. The PCA analysis matched the STRUCTURE analysis when K was 7 (fig. 5, fig. 7A): all 89 materials were divided into two groups, one containing all the material of camellia (c. japonica) and the other containing the genetic material from camellia in the test material. Structural analysis by the present application showed that most clusters were clearly divided into independent subpopulations at K ═ 7, but subpopulations 3, 4(Ia, Id, Ie) and subpopulation 5(II, IV) also overlapped (fig. 7B). The structural analysis of the application provides clues for better understanding of the origin and evolution of the camellia plant and contributes to better utilization of the resources of the camellia.
In summary, the advantages and positive effects of the invention are:
the research of the application jointly develops 28,854 pairs of SSR markers, and the density of the SSR markers on the leaf transcriptome of camellia japonica (C.japonica) is divided into a pair of 4.63 kb. 13,323 pairs of SSR primers are developed in total, and 111 pairs of polymorphic SSR markers are obtained through further screening and identification, which occupy 79.85 percent of effective amplification primers and are far higher than the prior art level, so that the polymorphic SSR markers are most developed and obtained in single research carried out in camellia (C.japonica) up to now; meanwhile, the PIC value range of 111 pairs of polymorphic markers in the application is 0.15-0.86, and the average value is 0.59, which is far higher than the existing level. The present application utilizes data further obtained from 27 of these polymorphism markers to reveal the true genetic relationship and population structure between 89 camellia varieties derived from camellia. In conclusion, the research result provides high-quality SSR marker resources for the genetic improvement research of camellia plants in the future.
Drawings
FIG. 1 shows the proportions of two bases (A), three bases (B) and four bases (C) in the leaf transcriptome of camellia (C. japonica) with different SSR motifs repeated;
FIG. 2 is a PAGE electrophoresis of newly developed SSR marker validation and population analysis; (A) identification results in 10 camellia (c. japonica) materials; (B) results of population analysis in 89 materials derived from camellia; the CjSSR094, the CjSSR021, the CjSSR128 and the CjSSR166 are respectively double-base, three-base, four-base and six-base repeated motif SSR markers;
FIG. 3 is a NJ phylogenetic tree analysis showing the genetic relationship of 89 candidate materials derived from Camellia; labeling materials according to genetic background; the results show the consistency of genotypic grouping and results based on taxonomic grouping of plants;
FIG. 4 is a NJ phylogenetic tree analysis showing the genetic relationship of 89 candidate materials derived from Camellia; marking the material according to the color of the candidate material; the results show that the flower color has great correlation with the genetic distance of the camellia group;
FIG. 5 shows the results of Principal Component Analysis (PCA) of 89 camellia materials; labeling materials according to genetic background; all materials are divided into two groups, wherein materials derived from camellia (c. japonica) are divided into one group, and other materials derived from camellia are divided into the other group;
FIG. 6 shows the values of LnP (D) and the rate of change (Δ K) between two consecutive LnP (D) according to the output of the STRUCTURE software; results were obtained in 89 parts of camellia material based on 27 pairs of SSR markers;
FIG. 7 is a genetic structure analysis of 89 candidate camellia materials; labeling the abscissa as a cluster in the NJ cluster analysis; when K is 2, all 89 materials are divided into two groups: wherein materials derived from camellia (c. japonica) are divided into one group and other materials derived from camellia are divided into another group (results are consistent with PCA results); when K ═ 7, the material derived from camellia was divided into 4 groups (1-4): the first and second groups contained materials classified into sub-clusters Ib and Ic in the cluster analysis, respectively, while materials of three sub-clusters Ia, Id and Ie were assigned together to groups 3 and 4; from Yunnan camellia (C.reticulata) and hybrid seeds (cluster II) and hybrid seeds (cluster V) of the Anjiang red camellia (C.saluenensis) are divided into the 5 th group; materials derived from camellia sinensis (c.sasanqua) were divided into group 6; while materials derived from other species of camellia were classified in group 7 (fig. 7B).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.
The invention discloses a group of SSR molecular markers based on a camellia transcriptome and application of the SSR molecular markers in camellia plants. The details are shown in the following examples.
Examples
The present inventors obtained a high-quality leaf transcriptome of camellia japonica (c. japonica) earlier by an RNA sequencing technology based on Illumina platform. The sequence data provides good resources for the development of the SSR marker of the camellia. The aim of this study was: 1) developing a group of SSR molecular markers based on a camellia transcriptome; 2) determining the universality and polymorphism of the SSR markers in the camellia species; 3) the genetic relationship and genetic structure of Camellia are revealed. The research result provides important information for the genetic improvement research such as systematic classification research, genetic diversity analysis, molecular marker-assisted breeding, SSR-based genetic linkage map construction and the like of the camellia plants.
1. SSR characteristics of camellia leaf transcriptome
SSRs are very abundant in the assembled camellia leaf transcriptome. The present application identified 28,854 potential SSR sites out of 24,368 genes (motifs repeated at least 5 times more), accounting for 11.74% of 207,592 genes generated by Illumina sequencing. SSRs occur in transcriptomes at a frequency of 1 per 4.63kb (221.17 SSRs per Mb). In all repeat types, the SSR has a length of 12-279 bp, and the average length is 20.54 bp.
The incidence of different repeat types and frequencies for each motif was evaluated based on the number of repeat units, see table 1. The number of the repeat units of the SSR locus is between 5 and 18, and the repeat units exist mainly as dinucleotide repeats and trinucleotide repeats and account for 97.65 percent of all SSRs. Dinucleotide repeats account for 74.56% of the total number of SSRs, with the largest repeat units, followed by three (23.08%), four (2.04%), six (0.19%), and pentanucleotides (0.11%). Most (99.4%) of the SSR motifs have 5-10 repeat units, with over 10 repeats being rare, accounting for only 0.59%. Of the identified SSRs, AG/CT accounted for 62.19% of all dinucleotide repeat motifs, the most common type (FIG. 1A). The trinucleotide repeat motifs were predominantly AAG/CTT and AAT/ATT, accounting for 24.94% and 19.56% of SSR, respectively (FIG. 1B). Of the tetranucleotide repeats, the most common motifs were AAAT/ATTT (31.30%), followed by AAAC/GTTT (13.37%) and AAAG/CTTT (11.68%) (FIG. 1C). These results probably reflect the characteristic of rich AG/CT content of camellia transcriptome.
TABLE 1 frequencies of different SSR repeat motif types in Camellia japonica (C.japonica) transcriptome
2. Development and validation of SSR markers
Among all genes containing potential SSR sites, 12,194 (50.04%) genes can successfully design SSR primers. The application designs 13,323 pairs of SSR primers. After further electronic PCR (e-PCR) screening, a total of 8,442 primer pairs were considered to have unique amplification sites in the leaf transcriptome of Camellia japonica (C.japonica). 172 pairs of primers were selected from the 8,442 pairs of primers and synthesized (see table 2 for primer information) and used for validation in 10 camellia (c. japonica) materials (table 3), and the PAGE electrophoresis results are illustrated in fig. 2A. Of these SSR markers, 30 pairs (17.44%) amplified to give diffuse or nonspecific bands, and 3 pairs (1.74%) did not give amplified bands in all 10 materials. There were 139 pairs (80.81%) of SSR markers that gave the expected bands, of which 111 pairs (64.53%) exhibited polymorphisms. Among these polymorphic SSR markers, the number of motifs of trinucleotide repeats was the greatest (66 pairs, 59.46%), followed by dinucleotide repeats (30 pairs, 27.03%), tetranucleotide repeats (14 pairs, 12.61%) and hexanucleotide repeats (1 pair, 0.9%), and no polymorphic primers were found for pentanucleotide repeats.
Table 2 synthetic 172 pairs of primer information
Table 3 10 camellia (c. japonica) varieties for marker validation
The 111 polymorphic SSR loci identified 495 alleles (allele) in 10 camellia (C.japonica) materials, the number of alleles ranged from 1 to 12, and the average number of alleles was 4.46 per locus. The present application calculates the Polymorphism Information Content (PIC) values of these polymorphic primers, with the PIC values of these polymorphic markers ranging from 0.15 to 0.86 and the average value of 0.59 (Table 4). The PIC values of the polymorphic markers with two, three, four and six nucleotide repeats were 0.63, 0.57, 0.58 and 0.37, respectively (table 4).
TABLE 4 frequencies of different SSR motif types in polymorphic SSR markers
To verify the versatility of these polymorphic SSR markers in other species of Camellia, the present application randomly selected 27 pairs of polymorphic markers (see the polymorphic markers listed in Table 5, sequences shown in SEQ ID NO.1-SEQ ID NO.54) in 8 genera derived from Camellia, including: a total of 12 materials (named as c.sinensis, c.sanqua ' cavanza ', c.sanqua ' lova ', c.sanchi ', c.sanqua ' lova ', c.sanqua general purpose pilot, c.sanqua), chonghua camellia (c.chungdonsis), kawa desmodium (c.rosthorniana), camellia japonica (c.azalea), and tea tree (c.sinensis) were subjected to validation of their respective activities. The results show that 25 pairs of markers, except for the two pairs of cjSSR014 and cjSSR021, all amplified bands in the material tested, resulted in no band amplification in tea. The ratio of the versatility of these primers among these 8 species was 92.6% to 100%, and the average versatility was 99.38% (subject to space limitations, the results of the experiments for only 5 materials are listed in Table 5, and the results for the versatility of the other 7 materials are all 100%).
Table 527 shows the versatility of the markers in 12 parts of material derived from 8 species of Camellia
"+" indicates that PCR product was obtained; "-" indicates no amplification in PCR
3. Cluster analysis of newly developed SSR markers in plants of Camellia
In order to verify the application of the newly developed SSR marker in the plants of the genus Camellia, 89 parts of materials (Table 6) derived from the genus Camellia were selected, 27 was used to genotype the newly developed polymorphic SSR marker, and the result of PAGE electrophoresis is shown in FIG. 2B. Based on the results of the genetic distance, NJ (neighbor-joining) phylogenetic tree clustering revealed a number of distinct clusters. To simplify the description of the results, the present application divides the longest branch into 5 branches to distinguish 5 major clusters: i to V. The first cluster mainly comprises camellia (C.japonica) materials and can be divided into 5 sub-clusters: ia to Ie; camellia yunnanensis (c. reticulata) and its hybrid are mainly classified in cluster II; two materials from camellia sinensis (c.sasanqua) are grouped in cluster III; the cluster IV is all hybrid seeds from the camellia reticulata (C.saluenensis); while materials from other species of camellia are grouped into clusters V (fig. 3). It is worthwhile to note that some known materials with similar genetic backgrounds, such as: the distances in the phylogenetic tree for ` Coquetttii ` and ` Coquettinia `, ` Yudan ` and ` Yuanyang Fengguan ` and ` Grand Marshal ` and ` Grand Marshaly Variegated `arevery close (FIG. 3). These results indicate that the genotypic grouping and the results based on plant taxonomic grouping are consistent.
TABLE 6 information on 89 parts of material from Camellia in the population analysis
The NJ tree is further marked according to the flower color, and the result shows that most of the cluster II (71.4%), the sub-cluster Ia (80%) and the Id (60%) are pink materials; clusters Ib (92.3%) and Ic (69.2%) were predominantly safflower material; the cluster V mainly takes light-colored flower materials (yellow or white); interestingly, the purplish flower material is then distributed centrally in clusters IV (fig. 4). This result indicates that there is a large correlation between flower color and camellia population genetic distance.
4. Genetic structural analysis of plants of the genus Camellia
In Principal Component Analysis (PCA), two groups were identified using the first and second eigenvectors (fig. 5), with eigenvalues of the first and second axes being 5.01% and 4.56%, respectively. The results show that the material derived from camellia (c.japonica) is clearly distinguished from the varieties derived from camellia reticulata (c.reticulata), camellia sasanqua (c.sasanqua), camellia angensis (c.saluenensis) hybrid, and other species of camellia.
The application is based on a clustering algorithm of a Bayesian model, and analyzes genetic STRUCTUREs of 89 camellia materials by using STRUCTURE software. Determining the most suitable subgroup number according to the LnP (D) value output by the software and the change rate (delta K) between two continuous LnP (D). The largest Δ K peak occurs at K2 (fig. 6), where all 89 materials are divided into two groups: the first group contained all materials derived from camellia (c. japonica), while materials derived from camellia reticulata (c. reticulata), camellia sasanqua (c. sasanqua), camellia now (c. saluenensis), and other species of camellia were grouped into the second group (fig. 7A), which was highly consistent with the results of the PCA analysis. Another Δ K peak occurs at K-7 (fig. 6), where the material derived from camellia is divided into 4 groups (1-4): the first and second groups contained materials classified into sub-clusters Ib and Ic in the cluster analysis, respectively, while materials of three sub-clusters Ia, Id and Ie were assigned together to groups 3 and 4; from Yunnan camellia (C.reticulata) and hybrid seeds (cluster II) and hybrid seeds (cluster V) of the Anjiang red camellia (C.saluenensis) are divided into the 5 th group; materials derived from camellia sinensis (c.sasanqua) were divided into group 6; while materials derived from other species of camellia were classified in group 7 (fig. 7B).
The experimental methods involved in this example are as follows:
1. DNA extraction
Genomic DNA was extracted using the Plant Genomic DNA Extraction Kit of Tiangen Biochemical technology (Beijing) Ltd. The quality and concentration of the extracted DNA was determined using 1% agarose gel electrophoresis and Agilent 2100Bioanalyzer (Agilent 2100 Bioanalyzer). The DNA was then diluted to 50 ng/. mu.l using sterile ultrapure water and stored at-20 ℃ until use.
2. Primer design
Primers were designed using Primer 3 on-line tool. The parameters are set as follows: the length of the primer is between 18 and 28bp, and 20bp is the optimal primer; the product length range is 100-300 bp; the melting temperature (Tm) of the DNA is in the range of 55-65 ℃, preferably 60 ℃ and the maximum difference of the Tm is 1 ℃. All primer sequences are then compared to each other and multiple copies of the primers are deleted to ensure that there is only one SSR amplification site per primer pair. And then screening the SSR marker by using a leaf transcriptome sequence of camellia (C.japonica) as a template and adopting electronic PCR (e-PCR), thereby further ensuring that the SSR marker only has a unique amplification site. The parameters are set as follows: mismatch of 4bp, gap of 1bp, and product size of 80-2000 bp.
3. Marker identification
172 randomly selected from newly developed SSR markers were synthesized by New Biotechnology Co., Ltd of Kyoto engine, Beijing. Reagents related to PCR reaction, including buffer, MgCl2+dNTPs and Taq enzyme were purchased from Beijing Quanji Biotech Ltd. The PCR system was as follows: 2. mu.l of 10 XPCR buffer, 2. mu.l of 2.5mM dNTPs, MgCl2+1.5. mu.l primer F (5. mu.M) 1. mu.l primer R (5. mu.M), DNA 2. mu.l Taq 1. mu.l, and water to 20. mu.l. The PCR procedure was as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension is carried out for 5min at 72 ℃. The PCR products were detected electrophoretically on an 8% polyacrylamide gel. On the basis of the screening result, 27 pairs of polymorphic markers were finally selected, and 89 parts of camellia material were subjected to genotype analysis using the same PCR system program.
4. Data analysis
Based on the PCR results, a binary matrix was constructed in which the amplified products are labeled 1 and the deletions are labeled 0. Since the template used in the SSR analysis was DNA extracted from 89 plants and there were more than two alleles in each sample, individual bands were considered as a single allele. The PIC for each locus was calculated using PowerMarker 3.25 software. And constructing a dendrogram according to the genetic distance of Nei by using an NJ method, and observing and editing the dendrogram by MEGA7 software. The colony STRUCTURE was analyzed using STRUCTURE software. To determine the population number (K) of major structures in the captured data, 100,000 Markov Chain Monte Carlo (MCMC) iteration cycles were used, with a run length (run length) of 100,000, while following a Hardy-Weinberg (Hardy-Weinberg) balanced blending model. And (3) calculating ln likelihood average values when K changes from 1 to 10 according to genetic similarity, and repeating the operation for 5 times every time so as to ensure the consistency of the result.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Wuhan City college of agricultural sciences
<120> SSR molecular markers based on camellia transcriptome and application thereof in camellia plants
<160> 54
<170> SIPOSequenceListing 1.0
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tgggaaagag gtgtgtggag 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccgtcatttg tggctcccta 20
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<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cctgctcaac tttcgagggt 20
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgacgcaaaa gatagtaaaa gctca 25
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cgttttggag cttctgaggg 20
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgttgcttca cagaatgaac ca 22
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctgcgttggc actactacga 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcggctcttt tcctttccag 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggcagcttct tggagcttct 20
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggttctactg gcattcaccc a 21
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gctctctgca gtttcctcca 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tagccgagct acccagaaga 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aagcagctag taccgaagca 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tccatgccaa aatccaggga 20
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcctagcttg gatagaagtg agg 23
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gcattggctt cgtcgatcac 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tgtggggaag ccaaagttca 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
caccgaccag atgcatcctt 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ttcccagcag cagcaacata 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acagttcccc catgtttccg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gttctgggga agtgcttggt 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tccccatccc taacacctct 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cacttccacg gtgtctctcc 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tcaccaggca aggaagcaaa 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
accacctccc tttgaactca 20
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
acactcatca taacagcggg t 21
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
aggagtccac gactaccaca 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aggtcatttc acgtgggagc 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gcactctcac cctctcatgg 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aatggaagcc gacaacgaga 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgtgtgggtg tggagagatg 20
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tgcatacaag taatgtgcgg a 21
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ggcattgccg acttctctga 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
cgggaggaca agatgtctgg 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
actttctgcc ctgcttcaga 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
agcaagagga accccaatga 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tcaagcacca tcgtgttgga 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
aacccacacc caaacaccaa 20
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tgcccaattg cttcgaggaa 20
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
acccacttcc aaactcgtct 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ccatgtacga gagacaccca 20
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
accctacact acacacccct 20
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gcagcgatgt ttgtgtggaa 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
aacagttggt agagcacggg 20
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gcaagtcctt cctatcagcc t 21
<210> 46
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tgtaaagtgc ttgattacaa ttacca 26
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cttggcaagt cacaccaacg 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gagtcctcct tgaagctgca 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tcacactggt tcgacttggg 20
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cgccaccacc ttcattctct 20
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tggcggggat ttttacgact 20
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tgtccctagc agtgcttagc 20
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
acaagaacgg gttgggctag 20
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tcttccaccg gagaccatca 20
Claims (4)
1. A group of SSR molecular markers based on camellia transcriptome is characterized in that: the SSR molecular markers are respectively CjSSR007, CjSSR008, CjSSR014, CjSSR015, CjSSR016, CjSSR021, CjSSR022, CjSSR023, CjSSR030, CjSSR031, CjSSR034, CjSSR041, CjSSR049, CjSSR050, CjSSR059, CjSSR068, CjSSR073, CjSSR074, CjSSR084, Cj087, CjSSR094, CjSSR128, CjSSR138, CjSSR143, CjSSR145, CjSSR154 and CjSSR155, and the primer sequences of the SSR molecular markers corresponding to the numbers are shown in SEQ ID NO.1-SEQ ID NO. 54.
2. Use of an SSR molecular marker as claimed in claim 1 in camellia plant phylogeny, genetic diversity analysis or linkage map construction.
3. Use according to claim 2, characterized in that: the PCR program for amplifying the plant sample by using the SSR molecular marker is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension is carried out for 5min at 72 ℃.
4. Use according to claim 2, characterized in that: the camellia genus includes camellia (c.sasanqua), chonglev camellia chrysantha (c.chuonggtosensis), chuanhuoensis (c.rosthorniana), camellia chrysantha (c.nitidis), camellia oleifera (c.oleeiera), camellia yunnanensis (c.retula), camellia azalea (c.azalea) and camellia sinensis (c.sinensis).
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Cited By (1)
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| CN116970728A (en) * | 2023-07-21 | 2023-10-31 | 中国林业科学研究院亚热带林业研究所 | SSR (simple sequence repeat) marker primer, method and application for rapidly identifying tea plum variety |
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2020
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Non-Patent Citations (3)
| Title |
|---|
| Y ZHAO 等: "Genetic relationships in a germplasm collection of Camellia japonica and Camellia oleifera using SSR analysis", GENET MOL RES ., vol. 16, no. 1, pages 16019526 * |
| 张亚利 等: "SSR分子标记在山茶属观赏资源遗传多样性研究中的应用", 植物科学学报, vol. 34, no. 5, pages 755 - 764 * |
| 殷鑫 等: "山茶属叶绿体全基因组微卫星特征分析及标记开发", 分子植物育种, vol. 16, no. 20, pages 6761 - 6769 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116970728A (en) * | 2023-07-21 | 2023-10-31 | 中国林业科学研究院亚热带林业研究所 | SSR (simple sequence repeat) marker primer, method and application for rapidly identifying tea plum variety |
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