**1. Introduction**

*Pinus koraiensis* (Sieb. et Zucc), commonly known as Korean pine, is a perennial evergreen tree in the Pinaceae family with five needles per fascicle [1–3]. It is an ancient and valued forest tree in East Asia, and natural forests of this species have undergone long-term succession and are described as tertiary forest [4]. Compared with other *Pinus* species, *P. koraiensis* is long-lived and is a dominant species in mixed conifer and broadleaved forest [5,6]. Currently, *P. koraiensis* is distributed mainly in cool-temperate regions in northeast China, the Russian Far East, the Korea peninsula (note that information is not available from North Korea due to limited access) and Honshu, Japan. It typically occurs in mild regions with more than 70% humidity and at altitudes from 600 m to 1500 m [7,8]. However, in China, it only grows from the Changbai Mountains to Xiaoxinganling Mountains in northeast China, mainly on slopes and rolling hills and in river valleys [9]. Nearly half of the

**Citation:** Li, X.; Zhao, M.; Xu, Y.; Li, Y.; Tigabu, M.; Zhao, X. Genetic Diversity and Population Differentiation of *Pinus koraiensis* in China. *Horticulturae* **2021**, *7*, 104. https://doi.org/10.3390/ horticulturae7050104

Academic Editors: Rosario Paolo Mauro, Carlo Nicoletto and Leo Sabatino

Received: 6 April 2021 Accepted: 6 May 2021 Published: 9 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

germplasm resources of *P. koraiensis* in the world are found in Xiaoxinganling Mountains in Yichun city, China, where the largest and most undisturbed primeval forest remaining in Asia and a natural climax community of *P. koraiensis* exists [10].

*P. koraiensis* has high economic, ornamental and ecological values in East Asia. Timber of *P. koraiensis* is widely used for architecture, bridges, furniture and ships because of the light, soft, fine structure and straight texture of the wood and its strong corrosion resistance [11]. Furthermore, it produces edible nuts that are nutritious and distinctly flavored, containing abundant unsaturated fatty acids, vitamins and minerals [12]. It also has high medicinal value, able to lower cholesterol levels and allay ultraviolet injury and tiredness [13]. Natural *P. koraiensis* forest absorbs large amounts of carbon dioxide and contributes to climate change regulation [14]. Therefore, it is a prominent conifer tree species of grea<sup>t</sup> value for the maintenance and protection of the environment in East Asia.

Genetic improvement of *P. koraiensis* began in the 1960s, which then developed slowly due to a lack of systematic breeding strategies and objectives [8]. In the early stages of selective breeding, large numbers of superior trees or natural populations were selected from natural forest to establish primary seed orchards, mainly through phenotype selection [15–17]. Earlier studies have mainly focused on propagation technology [18], provenance division [19], progeny determination [20] and selection of improved varieties [21], while studies of molecular plant breeding, including studies of genetic diversity, genomic selection and construction of genetic maps, are lacking [22]. Existing natural forests of this species have grea<sup>t</sup> significance for the conservation of breeding materials, the development of gene resources and the study of population genetic diversity [23]. However, in the past few decades, with the increasing demand for wood and cones of *P. koraiensis* as well as increasing wildfire, the area of natural *P. koraiensis* forest has decreased extensively [24]. Thus, to protect existing natural forests under the background of illegal logging and unpredictable biotic stress, such as white pine blister rust diseases, the collection and evaluation of germplasm resources of *P. koraiensis* are urgently needed.

Genetic diversity and population structure are key parameters of population genetics research. Analyses of genetic variation among and within populations can guide the formulation of conservation strategies. The use of molecular markers identified from wholegenome, chloroplast genome and transcriptome analysis is a primary method of revealing genetic diversity and population structure. Many DNA molecular markers are codominant and highly polymorphic, and many have been identified in the genome and transcriptome, unlike morphological and biochemical markers [25–27]. Simple sequence repeats (SSRs) are considered powerful and advantageous molecular tools due to their low cost, easy detection by polymerase chain reaction (PCR), high polymorphism, and codominance. Thus, they can be used for genetic diversity analysis, genome-wide association analysis, core collections and genetic linkage map construction in many plants and animals [28–30]. Furthermore, multiple EST-SSR markers can easily be developed from microsatellite loci of public transcriptome data. At present, there are few reports of analyses of genetic diversity in *P. koraiensis* based on DNA molecular markers; studies to date have employed random amplified polymorphic DNA (RAPD) analysis [31], single primer amplification reaction (SPAR) [32], intersimple sequence repeat (ISSR) analysis [33,34] and expressed sequence tag-simple sequence repeat (EST-SSR) analysis [35]. All these studies have identified high levels of genetic diversity in *P. koraiensis*, with the greatest levels of genetic differentiation occurring within populations. However, those previous studies focused on a limited number of populations, few molecular markers and population size. Thus, a systematic and comprehensive population genetic study, involving widespread germplasm collection and abundant polymorphic markers developed from high-throughput sequencing, is necessary to study the genetic relationships and diversity of *P. koraiensis* populations.

In this study, germplasm resources from 480 individuals of 16 natural populations of *P. koraiensis* were collected within the species' main distribution area in northeastern China, and analyzed for genetic diversity using 15 EST-SSRs. This study is the first comprehensive study evaluating the genetic diversity and population structure of *P. koraiensis* in China using large samples and wider distribution as well as a sufficient number of molecular markers. The aims of the study were to (1) investigate genetic variation using polymorphic EST-SSRs, (2) evaluate the genetic diversity and structure of natural populations, (3) conduct a comprehensive, range-wide genetic diversity study of *P. koraiensis* in China, and (4) propose a protection conservation strategy. The hypothesis of the study was that high genetic diversity could be detected within populations and significant genetic differentiation could exist among populations due to restricted natural distribution of the species and low to moderate degree of gene flow between populations. Thus, the results will provide insights into the conservation of this species and lay a foundation for further studies of marker-assisted selection (MAS) and genomic selection (GS) in *P. koraiensis* for genetic improvement.
