1 Crop Breeding and Applied Biotechnology - 23(1): e439023112, 2023 Genetic diversity of Rhododendron henanense subsp. lingbaoense revealed by whole-genome resequencing

: Rhododendron henanense subsp. lingbaoense Fang (Rhl) is a sub-species endemic to China with important ornamental value. Recently, the Rhl population has been shrinking as a result of its limited distribution range, poor natural regeneration, and rising human exploitation. In this study, high-quality single nucleotide polymorphisms (SNPs) identified through whole-genome re - sequencing were examined in 50 individuals from four populations of Rhl with a depth of ~34× to evaluate genome-level diversity and population structure. Population structure profiling, principal component analysis, and phylogenetic analysis clustered the Rhl samples into two groups corresponding to their geographical distributions. Analysis of SNPs indicated that Rhl populations have high genetic diversity and little genetic differentiation. Additionally, demographic history indicated that all four populations of Rhl have experienced long-term population decline. The above findings showed that we should take action to protect this rare species.


INTRODUCTION
Rhododendron is the collective name for Rhododendron (Ericaceae) plants, and of its flowers are among the ten most recognizable flowers in China.In addition to its high ornamental value, rhododendron also serves the purposes of ecological conservation, medical application, and scientific research (Li et al. 2018, Zhang et al. 2021).Rhododendron contains more than 1,000 species and has a global distribution.However, some unmanaged wild species with small populations have gone extinct or are on the verge of extinction due to narrow habitats or severe anthropogenic interference (Ma et al. 2014, Liu et al. 2020).Rhododendron henanense lingbaoense Fang (Rhl) is a perennial evergreen plant up to 3-6 m tall that belongs to subgen.Hymenanthes and subsect.Campylocarpa (Fang 1983).The main difference between Rhl and closely related species of Rhododendron is the white corolla without spots.Among the numerous Rhododendron taxa, Rhl has received less scientific attention.This may be because it is endemic to China, naturally distributed in the alpine region at altitudes above 2000 m on the border of Henan and Shaanxi provinces.Rhl has significant landscape usage value as a result of its extremely large blooms and clustered growth, as well as scientific research value and natural heritage value as an endemic species (Weng et al. 2012).The population of Rhl has X Zhou et al. recently been declining due to its constrained geographic range, its weak natural regeneration potential, and growing human exploitation (Han et al. 2008, Ma et al. 2017, Zhou et al. 2022).As a result, it is essential to properly assess the natural resources of Rhl and take emergency conservation measures.
The core objective of species conservation is to protect their genetic diversity and genetic structure as much as possible (Hendricks et al. 2017).Studying the genetic variation of the target population can not only provide a basis for the development of artificial recovery techniques for wild populations of the species but also provide important information for the development of conservation strategies (Barbosa et al. 2018).With advances in high-throughput sequencing technology, population genomics can now support genetic analysis at a genomic scale in natural populations with thousands of genetic markers rather than a few genetic loci (Hohenlohe et al. 2010).Single nucleotide polymorphisms (SNPs) are the most abundant and universal sequence variations in all genomes, which makes them excellent markers for genetic studies (Wang et al. 2015).By using next-generation sequencing (NGS) platforms to resequence the genomes of representative individuals from different groups within a species, we can quickly and efficiently obtain genomic information from different groups and obtain a large number of SNP loci by comparing them with existing genomes, thus exploring the genetic, evolutionary and biological properties of living organisms at the genomic scale (Wang and Zhang 2011).
In this study, SNP loci identified by genome resequencing were used to determine the genetic diversity, population structure, and demographic history of Rhl.We believe that the results presented here will be useful for the management and conservation of Rhl in the future and will also serve as an example for population genetic studies employing NGS for the preservation of species with restricted distributions.

Plant sampling and DNA extraction
Rhl often grows in moist, slightly acidic (pH 6.2~7) mountain brown soil rich in organic matter.It is intolerant to high temperatures, with an optimum growth temperature of 10-25 ℃ (Han et al. 2008).Common companion species are Betula platyphylla Suk. and Acer davidii Franch.Rhl is mainly distributed in three populations (CA for Chang'an, LY for Laoya, and NC for Nanchuang) in the Xiaoqinling National Nature Reserve (lat 34° 25' 12" N, long 110° 28' 45" E, alt 2200 m asl) in Henan Province.While conducting fieldwork, we found another population showing characteristics consistent with those of Rhl in the Taibai Mountains (TB, lat 34° 00' 73" N, long 107° 48' 86" E, alt 2700 m asl) of Shaanxi Province, China.Therefore, we sampled the TB population as an outgroup for our study.Due to limitations imposed by field conditions, a total of 50 individuals from the four locations (CA 13,LY 18,NC 8,and TB 11) covering major habitats were sampled.Individuals from each population were chosen at random and spaced approximately 100 m apart.Fresh leaves were collected in self-sealing bags with dried silica gel and preserved until DNA extraction, and all plant materials were kept in the Plant Diversity Research Laboratory (PDRL) of Luoyang Normal University.
Genomic DNA was isolated using a modification of the CTAB method (Allen et al. 2006).The concentration and quality of DNA were measured using a NanoDrop™ 2000 spectrophotometer (IMPLEN, CA, USA) and the Qubit® DNA Assay Kit Fluorometer (Life Tech, CA, USA).DNA integrity was evaluated on a 1% (w/v) agarose gel.

Library construction and genome resequencing
The extracted DNA was enzymatically disrupted using the MGIEasy Enzymatic PCR-Free DNA Library Preparation Kit (MGI Tech, China), and the fragments (450~600 bp) were purified using two-step magnetic bead selection.The fragmented DNA was end-repaired, splice ligated, and then purified.The purified product was denatured using a PCR instrument to obtain a single-stranded product, which was then cyclized.Then, enzymatic digestion and purification were performed.The quality-checked products were subjected to DNBSEQ-T7 (MGI Tech, China) sequencing (Jeon et al. 2021).

Population structure and population genetics analysis
After filtering, high-quality SNPs were employed in the population genetics analysis.A phylogenetic tree is a diagram representing evolutionary relationships among organisms derived from a common ancestral form.We constructed neighbor-joining (NJ) trees for 50 individuals using FastTreeMP software (version 2.1.11SSE3) with 200 bootstrap values (Price et al. 2010).To examine population stratification, structure analysis was carried out by Admixture software (v1.3.0) with K values from 1 to 4 (Alexander et al. 2009).Principal component analysis (PCA) was performed with the EIG-6.1.4program package (Patterson et al. 2006).Nucleotide diversity (Pi) and fixation statistic (Fst) analyses were applied to estimate the degree of variation among groups and to explain population differentiation based on the variance between the two groups, respectively.Both Pi and Fst were calculated using the "population" command in Stacks software (version 2.54) (Catchen et al. 2013).

Identification of SNPs in Rhl germplasms
In this study, the genomes of 50 Rhl individuals were resequenced to explore genomic diversity with a mean sequencing depth of ~ 34× and 95% coverage.In total, ~ 114 G bp of sequences or 7.60 billion reads were generated.Our sequencing data showed high Phred quality (Q20 > 96%, Q30 > 88%), with a stable GC content ranging from 40.91% to 42.01%.A total of 316,555,560 SNPs were obtained in the 50 individuals by comparison with the reference genome.After stringent alignment and filtering, 249 million high-quality SNPs were discovered and used for subsequent analysis.The SNP data have been deposited in the National Genomics Data Center (NGDC) under accession number GVM000370.

Population genetic structure of Rhl germplasms
Using the high-quality SNPs, a phylogenetic relationship analysis of 50 Rhl individuals was conducted to explore the degree of admixture in the populations.It is clear from Figure 1a that the 50 individuals of Rhl were divided into Shaanxi and Henan populations when K=2.When K=3 and 4, two different subpopulations emerged in the Shaanxi population, and there was no significant genetic structure between populations.In addition, at the junction of different populations, the genetic structures were intermixed with each other.Therefore, the optimal K value is two, at which the four populations can be divided into two groups based on geographic distance: Henan and Shaanxi.Moreover, the NJ tree was reconstructed.The samples were clearly divided between two branches, where the red section showed individuals from the Shaanxi TB population and the blue section showed individuals from Henan Province (Figure 2).This result is consistent with the genetic structure revealed by admixture.PCA was further performed based on the high-quality SNPs to study the genetic background similarity and clustering relationships among the 50 samples.As shown in Figure 3a, PC1 and PC2 accurately divided the individuals into two groups in Henan and Shaanxi, which is fully consistent with the geographical information of the source of Rhl.PC1 and PC2 explained 2.14% and 1.57% of the total variation, respectively.To further analyze the relationship between the Rhl materials in different populations, the expected heterozygosity (He), observed heterozygosity (Ho), and nucleotide diversity (Pi) of each population were quantified.Analysis of SNP datasets for all populations revealed high genetic diversity (Pi>0.3648,He >0.3412) in Rhl (Table 1).The results of Fst analysis indicated little genetic differentiation among these populations (Fst = 0.0110-0.0240)(Table 2).
It is widely recognized that characterizing population structure and genetic diversity is essential for the effective conservation of threatened species.Compared to widespread plant species, narrowly distributed species may have lower genetic diversity due to genetic drift and inbreeding depression (Gibson et al. 2008).However, resequencing indicated that the nucleotide diversity (0.3648-0.4017) of Rhl is higher than that of other endangered plant species, such as Geodorum densiflorum (0.0359), Rhododendron meddianum (0.0741), and Pinus bungeana (0.2427-0.2842) (Tian et al. 2022, Roy et al. 2016, Zhang et al. 2021, respectively).Additionally, Rhl has higher genetic diversity than other widely distributed species, e.g., Physalis philadelphica (0.127-0.206) and Phaseolus vulgaris (0.31) (Delfini et al. 2021, Alcalá-Gómez et al. 2022, respectively).These findings are largely consistent with the hypothesis that some rare and endangered species can maintain high levels of genetic diversity even at small population sizes (Zhao et al. 2012, Wu et al. 2015, Stone et al. 2019).
The genetic diversity of a species is generally influenced by its breeding system, life span, seed dispersal mechanism, distribution range, and evolutionary history.Additionally, outcrossing organisms typically exhibit higher genetic variety than selfing species (Nybom 2004, Zhang et al. 2021).Previous research revealed that Rhododendron requires pollinators and produces adhesive pollen, suggesting that relies mostly on outcrossing (Ng and Corlett 2000, Huang et al. 2017, Li et al. 2018).The high genetic diversity of the four populations of Rhl might also be derived from their ancestral populations, as observed for other threatened Rhododendron plants, e.g., R. protistum var.gigantum and R. meddianum (Wu et al. 2015, Zhang et al. 2021).
The population genetic structure, PCA results, and NJ trees demonstrated that populations (LY, NC, and CA) located in Henan Province were clustered together, while population TB from Shaanxi Province was separated.This is due to the geographical proximity of the three populations in Henan Province, whereas the TB population in Shaanxi Province is distant from them.The results of the population genetic analysis were consistent with the geographical distributions of the populations.Fst is a classical measure of population genetic differentiation and population genetic outcome (Holsinger and Weir 2009).Fst values range from 0-1, with a maximum value of 1 indicating complete differentiation between two populations and a minimum value of 0 indicating no differentiation between populations.The lowest peak was discovered to be at K = 1 according to the cross-validation errors of K values (Figure 1b).In light of this finding and the Fst values (0.0110-0.0240), it is likely that the four populations of Rhl used in this study all descended from the same ancestral population.

Demographic history of Rhl populations
MSMC2 software was used to assess changes in the effective population size of Rhl over time, and the results are shown in Figure 3b.Long-term shrinkage was observed in all four populations, and the trends in effective population size were  generally consistent.After a long period of bottleneck effects, the LY population expanded significantly approximately 700,000 years ago.By approximately 400,000 years ago, the LY population had undergone another significant contraction, after which its changes remained largely consistent with those of other populations.
The key to conserving a species is preserving its genetic diversity (Rauch and Bar-Yam 2005).Our study on the genetic analysis of Rhl has significant conservation implications for this rare species.All populations of Rhl maintain a high level of genetic diversity, so it is important to enhance the restoration and conservation of its native habitat.In wild populations of Rhl, seedlings and saplings are rarely found, and its habitat is disturbed and destroyed by mining and other forms of anthropogenic interference.Therefore, it is extremely urgent to establish conservation plots to protect its natural habitat.In addition, the results of the population history dynamics study revealed long-term population decline in all four Rhl populations accompanied by Pleistocene climatic oscillations.Therefore, seed collection from all four Rhl populations should be conducted as soon as possible so that they can be used for ex situ conservation and storage of the germplasm.

ACKNOWLEDGMENTS
This study was funded by the National Natural Science Foundation of China (31870697).

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Figure 1 .
Figure 1.Genetic structure analysis of four populations (CA, LY, NC, and TB) of R. henanense subsp.lingbaoense based on SNPs.Population assignment by admixture analysis for K = 1-4 (a).The cross-validation (CV) error rate of K value (b).

Figure 2 .
Figure 2. Neighbor-joining (NJ) phylogenetic tree of 50 R. henanense subsp.lingbaoense samples.The red section indicates individuals from the Shaanxi TB population, and the blue section indicates individuals from Henan Province.

Figure 3 .
Figure 3. Principal component analysis (PCA) of all 50 R. henanense subsp.lingbaoense individuals, with the proportion of the variance explained being 2.14% for PC1 and 1.57% for PC2 (a).The effective population size of four R. henanense subsp.lingbaoense populations estimated using MSMC2 software (b).

Table 1 .
Genetic diversity analysis of four R. henanense subsp.lingbaoense populations performed by using Stacks software Poly Loci, percentage of sites found to be polymorphic; Num Indv, mean number of individuals per locus in this population.P, average frequency of major allele; Ho, average observed heterozygosity; He, average expected heterozygosity; Pi, mean nucleotide diversity; Fis, average Wright's inbreeding coefficient.

Table 2 .
Genetic differentiation among populations of R. henanense subsp.lingbaoense based on SNPs XZhou et al.