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Article

Development and Application of SNP-KASP Markers Based on Genes Related to Nitrogen Uptake, Assimilation and Allocation in Tea Plant (Camellia sinensis L.)

by
Kai Fan
1,*,
Jie Zhang
1,
Min Wang
2,
Wenjun Qian
1,
Litao Sun
3,
Jiazhi Shen
3,
Zhaotang Ding
1,3 and
Yu Wang
1,*
1
Tea Research Institute, Qingdao Agricultural University, Qingdao 266109, China
2
COFCO Nutrition and Health Research Institute, Beijing 102200, China
3
Tea Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, China
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(10), 2534; https://doi.org/10.3390/agronomy12102534
Submission received: 15 September 2022 / Revised: 7 October 2022 / Accepted: 14 October 2022 / Published: 17 October 2022
(This article belongs to the Special Issue Advances in Tea Agronomy: From Yield to Quality)
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Abstract

:
Nitrogen is essential for the growth and quality formation of tea plants. Excessive and inefficient use of nitrogen fertilizer leads to cost increases and ecosystem pollution. It is important to improve the nitrogen use efficiency (NUE) for tea plantation. Breeding high-NUE varieties by marker-assisted selection using NUE-associated genes is a viable approach. However, few molecular markers related to nitrogen uptake and utilization have been identified in tea plants. In this study, a total of 2554 SNP loci within NUE-related genes were identified in a database. Of the non-synonymous SNPs, 46 were successfully converted to KASP markers. These markers were deployed on 35 tea germplasms to assess their suitability and accuracy in genetic analysis. The results show that 42 markers exhibited polymorphisms and the PIC values ranged from 0.05 to 0.38. The clustering results of the phylogenetic tree was basically consistent with the phenotype, showing that tea germplasms with high nitrogen accumulation and large biomass were grouped into one cluster. Using these markers, the fingerprints of these germplasms were constructed. The preliminary association analysis showed that there were two SNPs (CsSNP07 and CsSNP11) within CsNRT2.4 (CSS0001304) that were significantly associated with nitrogen accumulation (F = 4.631, P = 0.039 and F = 3.054, p = 0.047) and one SNP (CsSNP40) within CsAAP6 (CSS0035405) that was significantly associated with biomass (F = 3.842, p = 0.032). These functional SNP-KASP markers will be valuable for the early evaluation of tea germplasms and could accelerate the breeding of high-NUE varieties.

1. Introduction

Nitrogen is an essential nutrient element for plants, especially tea plants. Tea leaves are harvested multiple times annually, accompanied by nitrogen removal [1]. To maintain growth and increase yield, a large amount of nitrogen fertilizer was applied in a tea garden [2]. However, in this case, most of the N fertilizer is not absorbed by tea plants. This N surplus leads to an increase in soil acidification, gaseous ammonia emissions in the atmosphere, and nitrate levels in water resources [3,4,5]. Selecting cultivars with high nitrogen-use efficiency (NUE) is an effective way to overcome these related problems.
Marker-assisted selection has been proven to be an effective method for NUE genetic improvement [6,7]. Numerous molecular markers have been successfully developed and applied in genetic research into tea plants, such as amplified fragment length polymorphisms (AFLPs) [8], random amplification of polymorphic DNA (RAPDs) [9], inter-simple sequence repeats (ISSRs) [10], and simple sequence repeats (SSRs) [11,12,13]. Among them, SNPs have attracted more attention from researchers for their rich polymorphisms and coverage. Previous studies have reported the development and application of SNP markers in tea genetic research. For instance, many studies have applied SNPs for genetic diversity analysis and varietal identification in different tea germplasms [14,15]. In addition, SNPs were used to construct a high-density genetic map [16,17]. More importantly, a large number of studies have identified many SNPs that are related to amino acids [18], catechins, caffeine [19], the timing of spring bud flush [20,21] and leaf shape [22], and those that could be used for MAS breeding in tea plants. However, there few SNPs related to nitrogen transport and assimilation have been identified in tea plants.
Kompetitive Allele-Specific PCR (KASP) is a closed-tube, gel-free assay based on allele-specific oligo extension and fluorescence resonance energy transfer (FRET) for signal generation. Compared with other SNP genotyping methods, it has the advantages of high accuracy, strong site adaptability, and low cost, as well as being suitable for the high-throughput genotyping of SNPs [23]. It has been widely applied in rice [24], soybean [25], oilseed rape [26], and other field crops. Thus far, there has only been one report on KASP markers in tea plants. In total, 176 tea plant accessions have been genotyped based on candidate SNPs responsible for catechin content using KASP [27].
In this study, KASP was used to genotype SNPs based on genes related to nitrogen uptake, assimilation and allocation. Then, their suitability and accuracy were assessed in different tea germplasms. Finally, correlation analyses between the markers and NUE-related traits were performed. These newly developed markers will be helpful for breeding high-NUE tea varieties via a marker-assisted selection approach.

2. Materials and Methods

2.1. Plant Materials and Sample Collection

For KASP marker development and application, a total of 35 germplasms were collected from a Nanjing superior variety tea propagation center. The 35 germplasms originated from a superior individual of the ‘Huangshanzhong’ natural hybrid progeny, which is widely planted in Shandong Province. The cutting woods contained one mature leaf and a robust bud, which were cultivated in a holed tray filled with a light substrate from November 2020 onwards. Mature leaves were collected on 17 September 2021. These samples were immediately frozen in liquid nitrogen and subsequently stored in an ultrarefrigerator for DNA extraction. For phenotypic determination, different tissues (leaves, new shoots, stems, and roots) of 3 cutting plants from each strain were harvested randomly as biological replicates. The samples were collected in triplicate and dried to a constant weight for biomass and nitrogen content analyses.

2.2. SNP Mining in Genes Related to Nitrogen Utilization and Development of KASP Primer

The SNP data were obtained from the Tea Plant Genomic Variations Database. The SNPs within genes involved in nitrogen uptake (AMT, NRT), amino acid transport (AAP) and nitrogen assimilation (GS, GOGAT and GDH) were searched for on the website (http://www.teaplant.top/teagvd, accessed on 18 June 2022). Annotation for the SNPs was conducted using SnpEff, ANNOVAR and VEP software.
To develop the KASP markers for genetic research, 46 non-synonymous SNP sites were selected. For each SNP site retained in the screening, the surrounding sequence was trimmed by 100 bp before and after the SNP, the primer design and the PCR amplification following the KASP technology manual (LGC Genomics, Beverly, MA, USA) [28]. The genotyping assays were tested in a 96-well plate and the total amplification reaction volume was 10 μL, including 4.85 μL of the template (50 ng DNA), 5 μL of 2×Kaspar mix and 0.15 μL of primer mix. PCR amplification was performed on a StepOne Plus machine, as follows: hot start at 94 °C for 15 min, followed by 10 touchdown cycles (94 °C for 20 s; touchdown 65 °C, −1 °C per cycle, for 25 s) and then 26 cycles of amplification (94 °C for10 s; 57 °C for 60 s). Fluorescence data were collected during the pre-read and post-read stages (30 °C for 1 min). Genotyping data were viewed in the form of a cluster plot using SNP viewer (LGC Limited, Leeds, UK). The SNPs and primers that were used for KASP are displayed in Table S1.

2.3. Genetic Analysis and DNA Fingerprint Construction

The values of the expected heterozygosity (He), observed heterozygosity (Ho), major allele frequency (MAF), and polymorphism information content (PIC) were calculated using PowerMarker 3.25. Nei’s genetic distances of the 35 tea germplasms based on these KASP markers were calculated using PowerMarker 3.25 [29]. A dendrogram was constructed using the neighbor-joining (NJ) algorithm, as implemented in MEGA 7.0, with bootstrap values at the default setting of 1000 replicates
According to the dimorphisms of the SNP markers, the typing data were transformed into binary coding data, as follows: a wild-type (consistent with the grape genome) table, shown as (1, 0); mutants, shown as (0, 1); a heterozygous genotype table, shown as (1, 1); and the deletion site, marked as (999, 999). Caoliaoerweima (http://cli.im/, accessed on 26 July 2022) was used to generate the 2D barcodes for different tea germplasms.

2.4. Determination of Nitrogen Accumulation and Correlation Analysis

The dried samples were weighed to determine their biomass. Total N in each tissue was determined by an elemental analyzer (Vario Max CN Analyzer, Elementar Analysensysteme GmbH, Langenselbold, Germany). The nitrogen accumulation was calculated as the sum of all tissues. Then, a correlation analysis between the phenotypic traits and the genotype was carried out using the single-locus F-test in the PowerMarker software.

3. Results

3.1. Identification of SNP in Genes Related to Nitrogen Uptake, Assimilation, and Allocation

After searching the database, a total of 2554 SNP loci were obtained in genes related to nitrogen utilization (Table 1). Based on the nucleotide substitutions, the detected SNPs were classified as transitions (Ts: G/A and C/T) and transversions (Tv: A/C, A/T, C/G, and G/T), which accounted for 70.6% (1802) and 29.4% (752), respectively, with a Ts/Tv ratio of 2.40. In transitions, the number of A/G is equivalent to the C/T type, which included 50.3% (906) and 49.7% (896), respectively (Figure 1a). In Tv, the number of A/T-type transversions is the highest, accounting for 38.4% (289), while that of the C/G type is the lowest, accounting for 17.3% (130) (Figure 1b). Most SNPs (70%) were located in introns, followed by the CDS (20%) and UTRs (10%). Among the SNPs located in the CDS, there were 217 synonymous and 282 non-synonymous SNPs (Figure 1c).

3.2. Development and Polymorphism of KASP Markers

The non-synonymous SNPs were used to develop KASP markers. A total of 46 KASP markers were successfully developed. To validate the accuracy and polymorphisms of these KASP markers, a panel of 35 tea germplasms were tested. The results show obvious distinctions between homozygous genotypes (red and blue) and the heterozygous genotype (green). The Sanger sequencing results are consistent with the KASP assay (Figure 2). There were four markers that showed no polymorphisms among the 35 tea germplasms. The polymorphism characteristics of these markers are shown in Table S2. The PIC values of these KASP markers ranged from 0.05 to 0.38, with an average of 0.27. The major allele frequencies (MAFs) ranged from 0.50 to 0.97, with an average of 0.74. Additionally, the expected heterozygosity (He) ranged from 0.06 to 0.5, with an average of 0.33.

3.3. Genetic Analysis and Construction of DNA Fingerprints

To further confirm the applicability of these KASP markers in genetic analysis, a phylogenetic tree was constructed based on the genetic distances among the 35 germplasms. As shown in Figure 3, two germplasms, L18 and L38, had the highest genetic distance, and were clearly distinguishable from other germplasms. The other germplasms could be obviously divided into four clusters, which contained the L5, L8, L13, and L7 tea germplasms, respectively. The results of the PCA also showed that L18 could be clearly distinguished from the other germplasms (Figure 3b).
Using these KASP markers, the DNA fingerprints of these germplasms were constructed (Figure 4). The results show that each germplasm had its own unique DNA fingerprint. These KASP markers could effectively distinguish between the tested materials. For easily application using a cell phone, the fingerprints of each germplasm were translated into 2D barcodes (Figure 5).

3.4. Correlation Analysis between Phenotypic Traits and SNPs

To analyze the contribution of these SNP alleles to nitrogen utilization, the biomass and nitrogen accumulation of the tested materials were determined. The results show that the data conformed to the normal distribution (Figure 6). The range of biomass of each tea seedling was 1.79~4.14 g. L20 had the largest biomass, followed by L18. The nitrogen accumulation of the 35 germplasms ranged from 33.4~74.3 mg. L18 had the largest nitrogen accumulation.
The correlation analysis showed that three SNPs were significantly correlated with nitrogen accumulation and biomass (Table S2). Among them, SNP07 and SNP11 in NRT2.4 (CSS0001304) were significantly correlated with nitrogen accumulation specifically. In SNP07, the nitrogen accumulation of genotype TT was significantly higher than that of genotype CC (Figure 7a). In SNP11, genotype GG showed a higher nitrogen accumulation than genotype AA (Figure 7b). There was only one SNP, CsSNP40, that was significantly correlated with biomass (F = 3.842, p = 0.032). The genotype AA had the highest biomass in this SNP, followed by AG, and GG had the lowest biomass.

4. Discussion

4.1. Genetic Variations in Genes Related to Nitrogen Utilization

Genome research, especially the resequencing of different tea germplasm resources, greatly accelerates the discovery of genetic variations [30,31]. A large number of SNPs/indels related to agronomic traits have been mined. In our research, we aimed to mine SNPs in crucial functional genes that are involved in nitrogen absorption, assimilation, and distribution using these massive data. The Tea Plant Genomic Variations Database (http://www.teaplant.top/teagvd, accessed on 18 June 2022) provided these massive data in our study. SNPs can be quickly obtained and filtered. Using this method, 2554 SNPs were identified in these genes. Among them, A/G and C/T transitions were the most common types, which was consistent with a previous study of tea plants [32]. Most of them (70%) were located in introns, while some of them (10%) were located in UTRs. A previous study showed that natural variations in the 5`UTR confers phosphorus acquisition diversity in soybean [33]. In tea plants, a 14 bp deletion in the upstream regulation region of the F3`5`H gene was associated with a high catechin index [34]. These results suggest that SNPs in UTRs might play an important role in tea plants and should be studied further. Regarding the function of SNPs in the CDS, the ratio of non-synonymous to synonymous SNPs was 1.29, which was lower than the results for whole genomes, ranging from 1.47 to 1.49 in different tea accessions [35]. Non-synonymous variations can usually have several functional impacts due to their altered amino acid sequence [36,37]. We speculated that the frequency of SNPs induced functional changes in the nitrogen utilization pathway, making it more conservative than other metabolic pathways. These non-synonymous SNPs were used to develop functional molecular markers.

4.2. Development and Application of SNP-KASP Markers

A high-throughput SNP genotyping platform is required for marker-assisted selection breeding. In tea plants, SNPs are more frequently converted to PCR-based dCAPS markers [21,38]. However, this is restricted to low-throughput genotyping and the special enzymes needed for specific base pair digestion. Kompetitive Allele-Specific PCR (KASP) is considered to be suitable for the high-throughput genotyping of SNPs. KASP genotyping can be performed in 96- or 384-well plates, giving it a much higher throughput than the gel-based method; more importantly, it is a closed-tube method and therefore can significantly reduce the chance of experimenters being exposed to hazardous chemicals [39]. In our research, the results of the KASP assays show that they could effectively distinguish between different genotypes, and were consistent with the Sanger sequencing. These results confirm that the KASP assays were precise and could be used for genetic analysis. The gene diversity analysis showed that the PIC values were lower than those obtained by other researchers for tea plants. This was mainly because the test materials in our study were from the same population of ‘Huangshanzhong’ and their genetic background was relatively narrow. The results of the phylogenetic tree were similar to those of the phenotype. L18, which had the highest biomass and nitrogen accumulation, had a distant genetic relationship with other germplasms. Germplasms in cluster 1 had a higher biomass and nitrogen accumulation, while those in cluster 4 had a lower biomass and nitrogen accumulation. These results suggest that these SNP-KASP markers may be valuable for genetic analysis in tea plants.

4.3. Identification of SNPs Associated with Nitrogen Utilization

In our study, F-tests were used to analyze the relationship between SNPs and phenotype traits. We found that two SNPs (CsSNP07, CsSNP11) were significantly associated with nitrogen accumulation. CsSNP07 and CsSNP11 were located at the coding sequence of high-affinity nitrate transporter 2.4 (CSS0001304). NRT2.4 is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. In N-starved NRT2.4 mutants, the nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased, suggesting that NRT2.4 is a nitrate transporter that plays a role in both the roots and shoots under N starvation [40]. In our study, the CC and GG genotypes had a high nitrogen accumulation. This suggests that these genotypes might regulate nitrate transport in the roots and shoots in order to improve nitrogen accumulation in tea plants.
CsSNP40, located at the coding sequence of amino acid permease 6 (CSS35405), was significantly associated with biomass. A previous study showed that AAP6 mutant plants had a significantly larger mean rosette width than the wild-type plants. In addition, the total amino acid concentration of the SE sap of the AAP6 mutant plants was significantly lower than that of the wild-type plants [41]. Our previous study found that the expression level of CsAAP6 was significantly correlated with the amount of 15N in mature leaves during spring shoot development [42]. In this study, the biomass of the AA genotype was prominently higher than other genotypes. This suggests that the AA genotype might increase biomass accumulation by improving nitrogen allocation. The function of SNPs should be investigated further. Moreover, these SNP-KASP markers could be used for the early evaluation of high-NUE tea germplasms.

5. Conclusions

In this study, 46 SNPs within genes involved in nitrogen uptake, assimilation, and allocation were successfully converted into KASP markers. The results of the 35 tea germplasms suggest that these KASP markers are suitable for genetic analysis and fingerprint construction in tea plants. A preliminary correlation analysis showed that two SNPs and one SNP were significantly associated with nitrogen accumulation and biomass, respectively. These SNP-KASP markers may be valuable for assessing high nitrogen use efficiency during the MAS breeding of tea varieties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12102534/s1, Table S1: Information of SNPs and primer sequences of KASP markers; Table S2: Correlation analysis of SNP-KASP markers and phenotypic traits.

Author Contributions

Conceptualization, K.F. and Z.D.; methodology, J.Z.; software, W.Q.; validation, L.S., J.S. and M.W.; formal analysis, J.Z.; investigation, K.F.; resources, Y.W.; data curation, W.Q.; writing—original draft preparation, K.F.; writing—review and editing, K.F.; visualization, M.W.; supervision, Z.D.; project administration, K.F.; funding acquisition, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Agricultural Variety Improvement Project of Shandong Province (2020LZGC010), the National Natural Science Foundation of China (32002087), the Natural Science Foundation of Shandong Province (ZR2020QC171), the Livelihood Project of Qingdao City (22-3-7-xdny-5-nsh), and the School Fund Project of Qingdao Agricultural University (1120096).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Classification and annotation of SNPs identified in genes related to nitrogen uptake, assimilation, and allocation. (a) Frequency of transitions in the SNPs, (b) frequency of transversions in the SNPs, (c) annotation of the SNPs.
Figure 1. Classification and annotation of SNPs identified in genes related to nitrogen uptake, assimilation, and allocation. (a) Frequency of transitions in the SNPs, (b) frequency of transversions in the SNPs, (c) annotation of the SNPs.
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Figure 2. KASP genotyping results confirmed by Sanger sequencing. (a) KASP genotyping result of CsSNP40, (b) Sanger sequencing of SNP site G/G, (c) Sanger sequencing of SNP site A/A.
Figure 2. KASP genotyping results confirmed by Sanger sequencing. (a) KASP genotyping result of CsSNP40, (b) Sanger sequencing of SNP site G/G, (c) Sanger sequencing of SNP site A/A.
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Figure 3. (a) Phylogenetic relationship analysis and (b) PCA based on 42 KASP markers.
Figure 3. (a) Phylogenetic relationship analysis and (b) PCA based on 42 KASP markers.
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Figure 4. Fingerprints of the 35 tea germplasms. Each line represents one marker, and each column represents one germplasm. Green represents the nucleotide in the reference genome, yellow represents the mutation, and missing data are shown in orange.
Figure 4. Fingerprints of the 35 tea germplasms. Each line represents one marker, and each column represents one germplasm. Green represents the nucleotide in the reference genome, yellow represents the mutation, and missing data are shown in orange.
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Figure 5. Two-dimensional barcodes of the 35 tea germplasms. Each 2D barcode contains the genotype information.
Figure 5. Two-dimensional barcodes of the 35 tea germplasms. Each 2D barcode contains the genotype information.
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Figure 6. Frequency distribution of (a) biomass and (b) nitrogen accumulation in 35 tea germplasms.
Figure 6. Frequency distribution of (a) biomass and (b) nitrogen accumulation in 35 tea germplasms.
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Figure 7. Phenotypic comparison of different genotypes. (a) Nitrogen accumulation of different genotypes in CsSNP07, (b) nitrogen accumulation of different genotypes in CsSNP11, (c) biomass of different genotypes in CsSNP40. * indicate significant differences (p < 0.05), ** indicate significant differences (p < 0.01).
Figure 7. Phenotypic comparison of different genotypes. (a) Nitrogen accumulation of different genotypes in CsSNP07, (b) nitrogen accumulation of different genotypes in CsSNP11, (c) biomass of different genotypes in CsSNP40. * indicate significant differences (p < 0.05), ** indicate significant differences (p < 0.01).
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Table
Table 1. Statistics of SNPs within genes related to nitrogen utilization.
Table 1. Statistics of SNPs within genes related to nitrogen utilization.
Gene NameGene IDCDSIntronUTRTotal
AMTCSS00122001713030
CSS0021429281960224
CSS0030613123015
CSS00351112011132
NRTCSS00013042220042
CSS0001748783797
CSS00356281559478
CSS00417111317711201
CSS004247223990122
GDHCSS000254312114568
CSS0021774102488266
GOGATCSS0050330576514136
CSS0050084151350150
CSS000775885969190
CSS003991331180121
GSCSS000731016243373
CSS0049154301718209
AAPCSS0000990811019
CSS003432421272573
CSS00089581112023
CSS003540598021110
CSS00132582146067
CSS0050011303953122
CSS00480492357686
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Fan, K.; Zhang, J.; Wang, M.; Qian, W.; Sun, L.; Shen, J.; Ding, Z.; Wang, Y. Development and Application of SNP-KASP Markers Based on Genes Related to Nitrogen Uptake, Assimilation and Allocation in Tea Plant (Camellia sinensis L.). Agronomy 2022, 12, 2534. https://doi.org/10.3390/agronomy12102534

AMA Style

Fan K, Zhang J, Wang M, Qian W, Sun L, Shen J, Ding Z, Wang Y. Development and Application of SNP-KASP Markers Based on Genes Related to Nitrogen Uptake, Assimilation and Allocation in Tea Plant (Camellia sinensis L.). Agronomy. 2022; 12(10):2534. https://doi.org/10.3390/agronomy12102534

Chicago/Turabian Style

Fan, Kai, Jie Zhang, Min Wang, Wenjun Qian, Litao Sun, Jiazhi Shen, Zhaotang Ding, and Yu Wang. 2022. "Development and Application of SNP-KASP Markers Based on Genes Related to Nitrogen Uptake, Assimilation and Allocation in Tea Plant (Camellia sinensis L.)" Agronomy 12, no. 10: 2534. https://doi.org/10.3390/agronomy12102534

APA Style

Fan, K., Zhang, J., Wang, M., Qian, W., Sun, L., Shen, J., Ding, Z., & Wang, Y. (2022). Development and Application of SNP-KASP Markers Based on Genes Related to Nitrogen Uptake, Assimilation and Allocation in Tea Plant (Camellia sinensis L.). Agronomy, 12(10), 2534. https://doi.org/10.3390/agronomy12102534

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