Characterization of genome-wide genetic variations between two varieties of tea plant (Camellia sinensis) and development of InDel markers for genetic research

- Characterization of genome-wide genetic variations between two varieties of tea plant ( Camellia sinensis ) and development of InDel markers for genetic research.
- Shuchazao ’ and Camellia sinensis var.
- assamica ‘ Yunkang 10.
- A total of 48 InDel markers that yielded polymorphic and unambiguous fragments were developed when screening six tea cultivars.
- These markers were further deployed on 46 tea cultivars for transferability and genetic diversity analysis, exhibiting information with an average 4.02 of the number of alleles (Na) and 0.457 of polymorphism information content (PIC).
- Conclusion: The identified genome-wide genetic variations and newly-developed InDel markers will provide a valuable resource for tea plant genetic and genomic studies, especially the SNPs/InDels within catechin/caffeine biosynthesis-related genes, which may serve as pivotal candidates for elucidating the molecular mechanism governing catechin/caffeine biosynthesis..
- The tea plant ( Camellia sinensis (L.) O.
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Full list of author information is available at the end of the article.
- With the rapid development of the high-throughput sequencing approaches, the third- generation single nucleotide polymorphism (SNP) and insertion/deletion (InDel) markers are gradually be- coming the most widely used molecular markers, demonstrating a promising future in plant genetic and breeding research..
- SNPs are the most abundant genetic variations in most plant species, and the exploitation of SNP markers in single-copy regions is considerably easier than use of the other DNA markers [14–16].
- InDel markers have prac- tical value for those laboratories with limited resources, which also showed reliable transferability between dis- tinct populations .
- Several stud- ies have also reported the development and application of SNP/InDel markers in tea plant genetic studies.
- The completion of the two reference genome sequences is a notable advance for genetic and genomic studies and a basis for this study.
- The tea plant whole genome CSA.
- ‘Yunkang 10’ was first reported based on the Illumina.
- A number of polymorphic and stable InDel markers were developed, providing informative molecu- lar markers for genetic and genomic studies.
- The cat- echin and caffeine contents of the two tea cultivars were detected, and SNPs/InDels within catechin/caffeine biosynthesis-related genes were characterized.
- The iden- tified genome-wide genetic variations and newly devel- oped InDel markers provide valuable resources for tea plant genetic and genomic studies, and the identification of SNPs/InDels within catechin/caffeine biosynthesis- related genes can serve as important candidate loci for functional analysis..
- The completion of the two reference genome sequences.
- Shuchazao ’ and.
- Yunkang 10.
- b Distribution of the length of InDels identified between the two tea cultivars.
- It is obvious that mononucleotide InDels is the most abundant type, accounting for of the total number.
- (243,749) of the total InDels.
- Location and functional annotation of SNPs and InDels The annotation of the ‘Shuchazao’ reference genome was used to uncover the distribution of SNPs and InDels.
- According to the gene structure of the reference genome, the overwhelming number of SNPs (94%) was identified in intergenic re- gions, while only of SNPs were located in genic regions (Fig.
- Similarly, a small pro- portion of InDels were located in the genic regions, which accounted for only of the total num- ber (Fig.
- Remarkably, 3406 InDels were located in the CDs region, which can be regarded as the preference for developing InDel markers..
- 3 Annotation of SNPs and InDels identified between ‘ Shuchazao ’ and ‘ Yunkang 10.
- Validation and polymorphism of newly-developed InDel markers.
- To validate the InDels and develop polymorphic InDel markers, we selected 100 InDel markers that were distributed on different scaffolds.
- To determine the reliability and polymorphisms of the primers, six tea cultivars were selected for testing their amplified fragments using Frag- ment Analyzer™ 96.
- Of the total primer sets tested, 48 primer pairs were successfully amplified with unambigu- ous bands and length polymorphisms among the six tea cultivars, 19 primer sets generated non-polymorphic or empty amplifications, and 33 primer pairs yielded non- specific amplification or ambiguous bands..
- Consequently, the 48 primer sets were regarded as ele- gant InDel markers and used for further analysis..
- To test cross-cultivars/subspecies transferability, the 48 InDel markers were conducted on a panel of 46 tea cultivars belonging to section Thea of genus Camellia .
- The detailed information of the 46 tea cultivars is listed in Additional file 4: Table S1.
- The results of 18 InDel markers testing on various tea cultivars are shown in Fig.
- The amplified results of the remaining 30 markers were also demonstrated (Additional file 3: Figure S3).
- For the newly developed markers, 20, 25 and 3 InDel markers generated high polymorphism, moderate polymorphism, and low polymorphism in the 46 tea cultivars, respect- ively.
- 4 Exhibition of transferability and polymorphism detected by 18 out of 48 InDel markers among 46 tea cultivars.
- Table 1 Characteristics of 48 newly developed InDel markers.
- within the ranges detected in the donor tea cultivar, im- plying that the amplified fragments were derived from the same loci and that the primer binding sites of the al- leles were highly conserved among distinct tea cultivars/.
- These results showed that these newly developed InDel markers are informative and possess good transferability among vari- ous tea subspecies/cultivars..
- Population structure and genetic relationship analysis Population structure analysis was performed on the 46 tea cultivars using Structure 2.3.3 software based on 48 newly-developed InDel markers.
- To further confirm the applicability of the developed InDel markers for classification, we constructed a phylo- genetic tree based on their genetic distances (Fig.
- Tea cultivars belonging to Camellia sinensis var.
- both ‘Shuchazao’ and ‘Yunkang 10’ based on HPLC ana-.
- Table 1 Characteristics of 48 newly developed InDel markers (Continued).
- ‘Shuchazao’ and ‘Yunkang 10’..
- Identification of genetic variations in tea plant whole genome.
- The recent release of the ‘Shuchazao’ and ‘Yunkang 10’.
- This advance may enable researchers to study numerous agronomic traits associated with the perennial tea trees with a complete set of tools, including identifi- cation and development of SNP/InDel markers.
- Never- theless, genome-wide identification and development of SNP/InDel markers are still in infancy, especially genetic variations related to important agronomical traits.
- By mapping the clean reads of ‘Yunkang 10’ to the reference genome assembly ‘Shuchazao’, we comprehensively.
- 5 Population structure and phylogenetic relationship analysis based on 48 InDel markers.
- a Estimation of the optimal group number through Δ K, the number of K was set from 2 to 9.
- b Q-plot of the population structure when K = 2.
- It was shown that only minimal SNPs and InDels were distributed in the CDs region, which can be ex- plained by the fact that the CDs region only accounted for a small proportion of the whole genome sequences and had relatively higher conservation compared with other regions.
- a Detection of catechin content of the bud and leaf of both ‘ Shuchaza ’ and ‘ Yunkang 10.
- Nevertheless, genetic variations at UTRs may also play important roles, such as modifi- cation of regulatory elements affecting the interaction of the UTRs with proteins and miRNAs [37].
- Development and application of InDel markers.
- In fact, InDel markers are also PCR-based markers and are similarly affected by genomic complex- ity.
- Through a series of screenings, we developed a final of 48 polymorphic and stable InDel markers with 5–20 bp in length based on the genomic assembled sequences (Table 1).
- The length of fragments of the alleles amplified across tea cultivars was consist- ent with the expected sizes of the products, implying that the primer binding sites of the alleles were highly conserved.
- The large proportion of InDel markers dis- played a moderate PIC value (0.25 <.
- It is obvious that the PIC values of most InDel markers were lower than the PIC of the majority SSR markers supporting that the InDel markers are stable and bi-allelic throughout the genome.
- Therefore, these newly developed InDel markers are suitable for germplasm identification and conservation, genetic diversity analysis, population struc- ture and phylogenetic relationship analysis.
- To test the reliabil- ity and practicability of the newly-developed InDel markers, population structure and phylogenetic.
- Detection of catechin content of the bud and leaf of both ‘ Shuchaza ’ and ‘ Yunkang 10.
- These results indi- cate that the population structure analysis and phylogen- etic tree reflect the relationships of the 46 tea cultivars, demonstrating the high reliability of these InDel markers for genetic analysis..
- Unsurprisingly, a large number of SNPs and InDels were identified and some of them were located in the CDs (Table 2).
- Remarkably, a number of.
- Comparison of the whole genome sequences between.
- ‘Yunkang 10’ and ‘Shuchazao’ revealed a large amount of.
- Based on these InDel loci, a total of 48 novel InDel markers with moderate polymorphism and high stability were developed.
- ‘Shuchazao’, and a number of SNPs and InDels were identified within genes related to the catechin/caffeine biosynthesis pathways..
- three clonal tea cultivars (‘Liubaoxiye’, ‘Lingyun 2’ and ‘Zihong’) were collected from the Tea Germplasm Repository of the Tea Research Institute of Guangxi Province with permis- sion.
- Then take the intersection of the two results and use the GATK software to filter according to the following parameters:.
- Validation and development of InDel markers.
- To develop suitable InDel markers for genetic research, the InDel lengths ≥5 and ≤ 20 bp were used as candidate.
- The number of alleles ( Na.
- Estimation of the sub- groups and the best K value was performed according to a previous study [50]..
- Nei’s genetic distances of the 46 tea cultivars based on 48 InDel markers were calculated using PowerMarker.
- Pairwise gap deletion mode was employed to guarantee that the divergent domains could contribute to the topology of the tree [52]..
- Flowchart diagram for identifying genome-wide genetic variations between ‘ Shuchazao ’ and ‘ Yunkang 10 ’ and functional annotation..
- Functional categorization of the genes containing genetic variations within the CDs region.
- Exhibition of transferability and polymorphism detected by the remaining 30 InDel markers among 46 tea cultivars..
- Primer sequences of 48 newly developed InDel markers..
- Na: Number of alleles.
- The funding bodies had no role in the design of the study, collection, analysis, and interpretation of data, and in writing the manuscript..
- Most of the important data generated or analyzed during this study are included in the article and its supplementary information files.
- Morphological studies on the origin of the tea plant V, a proposal of one place of origin by cluster analysis.
- Thea — a section of beveragial tea trees of the genus Camellia..
- sinensis provides insights into the evolution of the tea genome and tea quality.
- Development of InDel markers for Brassica rapa based on whole-genome re-sequencing.
- Development of novel InDel markers and genetic diversity in Chenopodium quinoa through whole-genome re-sequencing.
- Genetic analysis and gene mapping of the orange flower trait in Chinese cabbage (Brassica rapa L.
- Clonal tea cultivars in China.
- Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds.

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