Abstract
Platycladus orientalis is one of the main species used in afforestation projects in the arid mountains of north and northwest China, meaning that the species has high ecological and economic value. Studying its genetic diversity and obtaining a core germplasm base and genetic fingerprint data are important for the screening, development, and utilization of the species. This can provide the core materials for the preservation and evaluation and mining of germplasm resources and can provide superior gene resources for breeding programs. In this study, the genetic diversity among 104 P. orientalis germplasm resources was examined using simple sequence repeat (SSR) markers, and a core germplasm containing 31 accessions was constructed that represents the most genetic diversity of P. orientalis accessions. Each of 20 pairs of primers showed polymorphism, and 117 alleles were identified. The average number of alleles at each locus was six, and the mean effective allele number was 2.607. The average Shannon’s information index was 0.983, and the average polymorphism information content was 0.445. There is thus a significant amount of genetic variation within P. orientalis germplasm, yielding a rich genetic diversity. The constructed core germplasm accounted for 30% of the original germplasm. There was no significant difference in genetic diversity between the core germplasm and the original germplasm resources, indicating that the obtained core germplasm resources could fully represent the original germplasm. Using 17 SSR primers with high polymorphism, the DNA fingerprints of 104 P. orientalis germplasm resources were constructed. The results showed that 98 had specific DNA fingerprints. The results of this study provide a valuable basis for the collection, preservation, and utilization of P. orientalis germplasm resources, and the methods adopted in this study have important reference value for the construction of core germplasm of other perennial woody plants.
Platycladus orientalis, belonging to the genus Platycladus (Cupressaceae), is a single-species genus native to China that is currently cultivated throughout the world (Li et al. 2016a). P. orientalis is one of the conifer species with the widest natural distribution in China; as such, it has high ecological and economic value due to several adaptive advantages. It is an important component of the boreal forests of China and one of the main afforestation trees used in the arid mountains of north and northwest China. It can withstand extremely high temperatures of 45 °C and extremely low temperatures of −35 °C. In addition, it can tolerate poor soils, and grow well on alkaline soil, slightly alkaline soil, and acidic soil, as well as on sunny and dry mountain slopes.
A national collection of P. orientalis germplasm resources has been preliminarily established, and these germplasm resources provide data for the genetic improvement and innovative utilization of P. orientalis. The rich germplasm resources make the realization of breeding targets feasible, but the large amount of germplasm resources necessary results in difficulties in conservation and evaluation. Therefore, it is important to construct a core germplasm and make full use of the genetic diversity of existing germplasm resources. The utilization of breeding resources in China is still in the initial stages, and the genetic background of breeding resources is still unclear. Therefore, the following are urgently needed: the development of genetic markers, genetic evaluation of species resources, identification of relatives, and clonal allocation of P. orientalis.
Frankel and Brown (1984) first proposed the concept of a core germplasm defined as the minimum number of germplasm resources in a sample that can represent the genetic diversity of the entire sample. Further in-depth mining of genetic core resources can effectively improve the utility and efficiency of such collection efforts. Most tree species are perennial woody plants, and the conservation methods are primarily in situ and off-site preservation, measures that cover a large area and incur substantial management costs. The proposal of the core germplasm opens up a new way to solve the problem that the scale of germplasm resources is too large to manage and use. In recent years, the construction of a core germplasm has become the focus of research on forest trees (Belaj et al. 2012; Liu et al. 2015). Research has summarized the development of the core germplasm of 146 species of plants in the past 10 years (Gu et al. 2023), and it can be seen that the research on core germplasm resources is mainly focused on economic crops, fruit trees, and forages. Compared with crops, the establishment of a forest core germplasm started relatively late, and the tree species involved are limited, especially in terms of endemic afforestation trees. At present, there are mainly reports on the establishment of forest core germplasm resources, such as Cunninghamia lanceolata (Wu et al. 2023), Robinia pseudoacacia (Guo et al. 2022), Pinus massoniana (Yang et al. 2021), Populus tomentosa (Mao et al. 2020), Catalpa bungei (Fang et al. 2017), Schima superba (Yang et al. 2017), Ginkgo biloba (Wang et al. 2016), and Populus nigra (Zeng et al. 2014), among others. The construction of a core germplasm provides a way for effective preservation and in-depth evaluation of genetic resources. At present, the construction of a core germplasm is mainly based on morphological and molecular marker data. For perennial and tall woody plants, it takes a long time to obtain phenotypic data. However, molecular markers based on DNA polymorphism are unaffected by plant growth period and are more suitable for the construction of a forest core germplasm than morphological markers.
The application of DNA fingerprinting has provided new methods for accurate identification of plant cultivars. By using a combination of multiple microsatellite (or SSR) loci in the genome of a species with a rapid evolutionary rate, a DNA profile specific to an individual can be obtained, and the ability to identify individuals is comparable to that of human fingerprints. The genetic data can also be converted into digital codes, similar to human identification numbers. For this reason, the data are referred to as a DNA fingerprint or a molecular identity (ID) card. DNA fingerprinting technology has been widely applied in the identification of plant cultivars. The method reveals the variation in the genetic material that is unaffected by the external environment or human disturbance. The results of molecular testing of plant cultivars as an auxiliary means of cultivar identification were recognized by the International Union for the Protection of New Varieties of Plants (UPOV) in 2005.
In this study, SSR molecular markers were used to examine the genetic diversity of 104 P. orientalis plants. The goals of this study were to construct a core germplasm for P. orientalis and to preliminarily verify the representativeness of the core germplasm resources for the conservation of genetic diversity and the effective utilization of core materials. Such a collection can provide a reliable basis for breeding, genetic improvement, and identification of new cultivars as well as theoretical guidance for subsequent production and afforestation.
Materials and Methods
Materials.
A total of 104 accessions of P. orientalis were obtained from the Forest Germplasm Resource Nursery of the Institute of Forestry and Pomology (FGRN-IFP, Beijing, China), the National Base for Improved Species of Platycladus orientalis (NBISPO, Jiaxian, Henan, China), the Beijing West Mountains Experimental Tree Farm (BWMETF, Beijing, China), the Zhuifeng Mountain of Miyun (ZFM, Beijing, China), the Qinglong Mountain of Pinggu (QLM, Beijing, China), and the Fahai Temple of Shijingshan (FHT, Beijing, China). After the leaves of P. orientalis were collected, genomic DNA of P. orientalis was extracted by an improved cetyltrimethylammonium bromide (CTAB) method (Jin 2020).
SSR primers and polymerase chain reaction amplification.
A total of 6075 SSR primer sequences were developed based on transcriptome sequencing analysis of P. orientalis adventitious roots. Three hundred SSR primer pairs were selected, and 11 polymorphic SSR primer pairs were obtained (P9, P64, P74, P84, P85, P89, P97, P105, P133, P139, and P166). In addition, published SSR sequences were downloaded and screened from the literature of Cupressaceae (Jin et al. 2016; Zhu and Lou 2012), and nine polymorphic SSR primers were obtained (PLOC1, PLOC2, PLOC8, PLOC20, PO53, PO57, PO171, PO182, and POA10). These 20 pairs of SSR primers were used to evaluate the genetic diversity of 104 P. orientalis germplasm resources and to construct a core germplasm and DNA fingerprints. Primer sequence information is presented in Table 1. The reaction system for polymerase chain reaction (PCR) amplification comprised 20 µL: 14.8 µL distilled deionized water (ddH2O), 0.4 µL deoxyribonucleoside triphosphate (dNTP), 2 µL buffer, 0.3 µL forward primer (20 µM), 0.3 µL reverse primer (20 µM), 2 µL genomic DNA (50 ng·μL−1), and 0.2 µL ExTaq DNA polymerase (Takara, Dalian, China). The PCR amplification reaction program was as follows: predenaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, renaturation at 54 °C for 35 s, and elongation at 72 °C for 40 s, followed by a final extension at 72 °C for 3 min.
Simple sequence repeat primer names and sequences used for polymerase chain reaction amplification analysis in Platycladus orientalis.
Capillary electrophoresis.
Formamide was mixed with a molecular weight internal standard at a volume ratio of 100:1, and 15 μL was added to the upper sample along with 1 μL of the 10-fold diluted PCR product. The Fragment (Plant) analysis software in Genemarker V2.20 (SoftGenetics LLC, State College, PA, USA) was used to analyze the raw data obtained by a DNA analyzer (ABI 3730XL; Thermo Fisher Scientific, Waltham, MA, USA). The position of the molecular weight internal standard in each lane was compared with the position of the peak of each sample to obtain the fragment size.
Data analysis.
The number of alleles (Na), the number of effective alleles (Ne), Shannon’s information index (I), observed heterozygosity (Ho), and expected heterozygosity (He) were calculated by POPGENE version 1.32 software (Yeh et al. 1999). The polymorphism information content (PIC) was calculated using statistical software (Power Stats V12. xls; Promega Biotech, Madison, WI, USA). NTSYSpc2.1 software (Rohlf 2000) was used to calculate the similarity coefficient and genetic distance among accessions, and the unweighted pair group method with arithmetic mean (UPGMA) was used for cluster analysis. The R language package Genetic Subsetter (Beukelaer et al. 2012; Graebner et al. 2016) was used to construct the core germplasm. The sizes of allelic fragments obtained with different primer pairs for each sample were then coded in Arabic numerals in order from the smallest to the largest. If Na exceeded nine by one or more digits, it was represented by a single digit number with “*”, such as “0*” for the 10th allele and “1*” for the 11th allele so that the number of digits in the DNA barcodes of all samples was the same.
Results
Genetic diversity analysis.
A total of 117 allelic variants were detected for the 20 SSR loci (Table 2), with Na per locus ranging from two (P85, P105, and P166) to 15 (P84), with an average of six alleles. The highest Ne was 8.375 (P84), the lowest was 1.090 (P85), and the average Ne was 2.607. The highest value of I was 2.314 (P84), the lowest was 0.178 (P85), and the average was 0.983. The highest value of Ho was 0.817 (P84), and the lowest was 0.000 (PLO2), with an average of 0.363. The highest He was 0.885 (P84), the lowest was 0.083 (P85), and the average was 0.486. The proportion of polymorphism was 100%. The highest PIC was 0.869 (P84), the lowest was 0.079 (P85), and the average PIC was 0.445.
Genetic diversity of the germplasm resources of Platycladus orientalis.
Cluster analysis.
To study the genetic relationships of the 104 accessions of P. orientalis, the genetic distances were calculated and a phylogenetic tree was constructed (Fig. 1). The phylogenetic tree comprised seven significant clades, A, B, C, D, E, F, and G. Group A contained two accessions, PLO13 and PLO104. Group B contained only the PLO1 accession. Group C contained 12 accessions (PLO7, PLO102, PLO39, PLO2, PLO18, PLO26, PLO96, PLO95, PLO36, PLO71, PLO38, and PLO45). Group D contained two accessions, PLO11 and PLO46. Group E contained PLO29, PLO6, and PLO10. Group F contained five accessions, PLO89, PLO98, PLO99, PLO65, and PLO100. Group G contained 79 accessions.
Genetic relationship clustering of 104 Platycladus orientalis accessions.
Citation: J. Amer. Soc. Hort. Sci. 149, 3; 10.21273/JASHS05356-23
Construction and evaluation of a core germplasm.
Based on the clustering of 104 P. orientalis accessions, the R package Genetic Subsetter was used to rank the germplasm data of P. orientalis, and 30% of the core germplasm was constructed according to the original germplasm. Table 3 shows that compared with the original germplasm, Na in the initially constructed core germplasm was slightly less, and Ne, the observed and He, I, and the PIC values were slightly higher. The retention rates of alleles, effective alleles, and I of the core germplasm were 88.89%, 112.21%, and 110.54%, respectively. Statistical software (IBM SPSS Statistics version 22.0; IBM Corp., Armonk, NY, USA) was used to perform a t test on the genetic diversity parameters of the constructed core and original germplasms, and the results (Table 3) showed that the genetic diversity parameters of the core germplasm were not significantly different from those of the original germplasm (P < 0.05). Therefore, 30% of the original data was selected as the core germplasm in this project, as this maintained the genetic diversity of the original sample while ensuring that the less used germplasm represented the maximum genetic diversity of the original germplasm. Principal coordinate analysis (PCoA) was used to compare and analyze the constructed core germplasm to further confirm its representativeness. The results showed that the P. orientalis core germplasm was evenly distributed within the principal coordinate map of the entire germplasm resource, indicating that the constructed P. orientalis core germplasm was well representative (Fig. 2). Therefore, the core germplasm of P. orientalis constructed in this study would fully represent the genetic diversity of the original sample.
Comparison of genetic diversity between 31 core germplasm and 104 original germplasm accessions of Platycladus orientalis.
The principal coordinate map showing 31 core germplasm evenly distributing within 104 primary germplasm resources in Platycladus orientalis.
Citation: J. Amer. Soc. Hort. Sci. 149, 3; 10.21273/JASHS05356-23
The 31 core germplasm samples were from accessions 1, 2, 3, 4, 6, 10, 13, 14, 15 (Liye), 18 (Dieye), 20, 21, 24, 29, 32, 33, 34, 35, 36, 39, 40, 43, 46, 64, 80, 82, 89, 94, 97, 100, and 104.
DNA fingerprinting construction.
Among the 20 SSR primer pairs, 17 could be used as core primers. The exceptions were P85, 97, and 166 that were less polymorphic. The 17 core primer pairs had a total of 34 digits, and the 17 primer pairs amplified DNA barcodes that consisted of 34 digits. The sizes of the genotype fragments obtained by the 17 pairs of primers for each cultivar were directly encoded (Table 4) to construct DNA fingerprints for each of the 104 P. orientalis accessions. The results showed that 98 of the 104 accessions had specific DNA fingerprints. “Liye” had the same fingerprint as Jiaxian 1745 and Xishan 2-1; Xishan 1-1 had the same fingerprint as Xishan 1-2; Xishan 3-1 had the same fingerprint as Xishan 3-2; Xishan 6-1 had the same fingerprint as Xishan 6-2; and Xishan 8-1 had the same fingerprint as Xishan 8-2 (Table 5).
Encoded standard for alleles obtained by 17 core simple sequence repeat primers in Platycladus orientalis.
DNA fingerprint codes of 104 Platycladus orientalis cultivars.
Discussion
Development and evaluation of molecular markers for P. orientalis.
P. orientalis is an important tree species for ecological afforestation as well as a common species for landscaping in China. In recent years, it has been widely used in afforestation projects in barren areas, and the species plays a significant role in afforestation of desolate mountains. The development of molecular markers for P. orientalis can assist the genetic improvement of trees, accelerate the breeding process, and be used to explore the characteristics of genetic variation at the genome level.
SSR markers are widely used in genetic and breeding research for assessing the distribution of genetic variation, analyzing mating systems, and determining parent-child relationships (El-Kassaby and Barclay 1992; Shimono et al. 2011; Wang et al. 2010). Coniferous genomes are extremely large, ranging in size from 4.067 Gb of Microcachrys tetragona to 35.084 Gb of Pinus gerardiana (Burleigh et al. 2012; Neale and Wheeler 2019). At the same time, a large number of repetitive sequences have been found in the conifer genome (De La Torre et al. 2014). Therefore, it is difficult to identify gene loci that segregate in a Mendelian manner due to their large, complex, and many repetitive sequences. For species with large and unsequenced genomes such as P. orientalis, SSR markers extracted from transcriptome databases have clear advantages over other strategies. For species with unknown genome sequences, SSR markers extracted from transcriptome databases are cost-effective and efficient. Moreover, due to the strong conservation of transcriptome sequences among species, SSR markers developed based on transcriptome databases can be used in closely related species, providing tools for biological evolution and phylogenetic studies.
In recent years, with the rapid development of molecular biology, SSR has been widely used in P. orientalis. Zhu and Lou (2012, 2013) successfully developed nine pairs of SSR markers with high polymorphism and applied them in the genetic diversity analysis of P. orientalis. Jin et al. (2016) developed 27 polymorphism expressed sequence tag SSR (EST-SSR) markers, and later used 10 EST-SSR markers to evaluate genetic variation in breeding populations. Ding et al. (2017) developed 129 EST-SSR markers based on the transcriptome sequencing of Fokienia hodginsii, among which 10 polymorphic loci could be amplified in P. orientalis. Huang et al. (2018) developed 26 chloroplast SSR (cpSSRs) that were polymorphic in different genera of Cupressaceae (Platycladus, Sabina, Juniperus, and Cupressus), and a total of 192 P. orientalis samples were genotyped using 10 nuclear SSRs (nSSRs) and 10 cpSSRs. Cui et al. (2021a, 2021b) analyzed the genetic diversity and population structure of an ancient P. orientalis population in the middle reaches of the Yellow River based on cpSSRs and nSSRs. In this study, the genetic diversity among 104 P. orientalis germplasm resources was examined using SSR markers, and 104 P. orientalis germplasm resources were clustered into seven major groups. It was found that the clustering results were not completely consistent with the geographical area, indicating significant genetic differences among samples from the same area. This discrepancy may be attributed to the presence of resource nurseries at some sampling sites, where the origin of germplasm resources may vary, which leads to a change in the genetic relationship of the germplasm resources in the same sampling site. In addition, it can be speculated that mixed sowing occurred during the cultivation of P. orientalis, which increased the gene exchange of germplasm resources in different geographical environments, thus causing confusion in the genetic relationship of germplasm resources in different habitat conditions. The numbers of Na for 20 pairs of SSR markers was six, and the average PIC value was 0.445, indicating that the selected SSR polymorphism primers in this study could be effectively applied to subsequent studies on the genetic relationships of P. orientalis.
Construction of a core germplasm for P. orientalis.
The markers that have been used to construct core germplasm collections include morphological characteristics, ecological adaptability, physiological and biochemical characteristics, and molecular genetic markers (Falk 1990). For perennial trees, it is difficult to collect and conserve resources. Phenotypic data require many years of observation and are affected by environmental factors. Molecular markers are based on DNA polymorphisms, traits that generally have no biological function and are unaffected by the plant growth period or the external environment. Molecular markers can truly reflect the genetic diversity of tree species and are more suitable for constructing a core germplasm and for evaluating genetic diversity than morphological markers (Liu et al. 2014). In particular, SSR markers have the characteristics of codominant inheritance, full genome coverage, high polymorphism, and good repeatability, factors that provide more abundant allele information than other molecular markers. Therefore, the construction of a core germplasm based on SSR data has significant advantages over other markers and thus has been widely used in the construction of a forest core germplasm. Therefore, in this study, 20 pairs of polymorphic SSR primers were used to analyze the genetic diversity of 104 P. orientalis accessions, and 31 core accessions of P. orientalis were effectively obtained.
The sample size in the construction of a core germplasm is an important factor concerning whether the core germplasm is effective (Yang et al. 2011). The key to constructing a forest core germplasm is to maintain the genetic diversity of the original sample with the minimum amount of germplasm. Due to the characteristics of the species, the sampling ratio of the core germplasm should be based on the genetic diversity and genetic structure of the plants under study. If the original sample is small but has a high level of genetic diversity, the sampled proportion will be greater. Conversely, the sampling proportion may be appropriately reduced. If the sampling ratio is too high, there may be genetically redundant germplasm in the core germplasm bank. If the sampling ratio is too low, rare alleles will be lost. Therefore, a reasonable sampling ratio is particularly important. The constructed core germplasm resources of different plants generally accounted for 5% to 40% of the original germplasm (Escribano et al. 2008; Wang et al. 2007). Wang et al. (2016) analyzed the genetic diversity of 180 Ginkgo biloba trees and obtained a core germplasm comprising 63 accessions, accounting for 35% of the original sample. Using SSR markers, Liang et al. (2014) obtained a core germplasm of Malus domestica, comprising 55 accessions, accounting for 13.2% of the original sample. Wang et al. (2014) used EST-SSR markers to establish a core germplasm of Litchi chinensis accounting for 22.9% of the original material. All these results suggest that the sampling ratio should depend on the quantity and genetic diversity of the germplasm resources. According to Frankel and Brown (1984), the core germplasm should represent at least 70% of the genetic diversity of the original germplasm. In this study, a total of 104 accessions of P. orientalis germplasm were collected, and the core germplasm was constructed using random sampling. When the sampling ratio was 20%, it did not meet the requirement of representing at least 70% of the genetic diversity of the original germplasm. However, with a sampling ratio of 30%, the constructed core germplasm could maximally represent the genetic diversity of the whole P. orientalis germplasm resource. This may be because the genetic differentiation of the germplasm resources involved in this study is high, and a large sampling proportion is needed to represent the genetic diversity of the original germplasm.
In the process of constructing a core germplasm, selecting genetic parameters that can reflect species characteristics to test the genetic integrity and validity of the constructed core germplasm is one of the key points. The genetic diversity parameters of a core germplasm based on molecular marker data usually include Na, the PIC, I, Ne, Ho, and He. In this study, these genetic diversity parameters were used to evaluate the validity of the core germplasm, and t tests and PCoA analysis were used to verify and confirm the representativeness of the core germplasm. The results of the t tests and PCoA analysis showed that the obtained core germplasm had rich genetic diversity and was evenly distributed in the original germplasm resources, indicating that the constructed core germplasm was reliable, effective, and representative.
Although the constructed core germplasm of P. orientalis could represent the genetic diversity of the entire sample, the remaining germplasm may contain traits that breeders need but do not occur in the core germplasm (Cipriani et al. 2010). The representative degree of genetic diversity of the germplasm resources in the gene bank is a basic indicator of the effectiveness of the core germplasm. However, the material in gene banks often does not fully represent the full genetic diversity of a species. The most effective way to solve this problem is to allow the size and content of the core germplasm to change over time. Therefore, to preserve the genetic diversity of P. orientalis, it will be necessary to continuously add the newly collected germplasm of selected trees to the core germplasm of P. orientalis. This study is the first attempt to construct a core germplasm of P. orientalis, and because of the limitations of the number of markers and the lack of phenotypic characteristic data, the results should be supplemented and improved in future studies.
Construction of a DNA fingerprint of P. orientalis.
A DNA fingerprint database is an important tool for tree germplasm identification, conservation, and management. Identification of germplasm resources by DNA molecular markers is an accurate and effective method that has been widely used in germplasm research (Li et al. 2016b; Tang et al. 2015). Using molecular marker data to analyze the genetic diversity of P. orientalis germplasm resources and construct a germplasm fingerprint database has important guiding significance for the utilization and conservation of P. orientalis germplasm resources.
Most of the P. orientalis germplasm resources in this study were obtained from different resource nurseries or woodlands. Some of the accessions were selected from the same half-sib family or full-sib family. Because of the long period of germplasm collection and cultivation, the lineage relationships of some materials were unclear, and thus, it was necessary to carry out germplasm identification. Molecular markers are important technical means for rapid identification of tree germplasm, as they are unaffected by environmental conditions. Among these, SSRs are codominant markers and are ideal for identifying germplasm resources (Zhang et al. 2012). In this study, 17 highly polymorphic SSR primer pairs were used to obtain DNA fingerprints of 104 accessions of P. orientalis. The results showed that 98 of the 104 accessions had specific DNA fingerprints. Limited primers can be applied only to a certain number of sample populations, and as the number of germplasm resources increases, more suitable primers need to be added for identification (Heckenberger et al. 2002). In future work, morphological information should be combined with molecular data to form a more complete fingerprint of P. orientalis. Breeders could thus carry out more targeted research with the help of molecular identity information, greatly shortening the time required for germplasm identification.
Conclusion
Based on the SSR marker data, a core germplasm of P. orientalis was constructed that contained 31 accessions, accounting for 30% of the original sample. The retention rates of alleles, effective alleles, and I were 88.89%, 112.21%, and 110.54%, respectively. These results indicate that the core germplasm established in this study would be effective. Seventeen highly polymorphic SSR primer pairs were used to obtain the DNA fingerprints of 104 accessions of P. orientalis. The results showed that 98 accessions had specific DNA fingerprints. In addition, the method adopted in this study has important reference value for the construction of core germplasm resources for other species of perennial woody plants.
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