The Study of Genetic Diversity and Structure of Extremely Endangered Manglietia longipedunculata and Endangered Manglietia insignis

doi:10.21203/rs.3.rs-3555362/v1

Download PDF

Article

The Study of Genetic Diversity and Structure of Extremely Endangered Manglietia longipedunculata and Endangered Manglietia insignis

https://doi.org/10.21203/rs.3.rs-3555362/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

To strengthen the protection of Manglietia insignis and Manglietia longipedunculata germplasm resources, the endangered mechanism and scientific protection strategies of these two species were investigated. A novel, effective, and flexible DNA sequencing library construction approach-Hyper-seq was used in this study to develop large-scale genomic single nucleotide polymorphism (SNP) and to evaluate the genetic diversity and population structure of M. insignis and M. longipedunculata. Genetic diversity analysis demonstrated that the genetic diversity of M. insignis, which expected heterozygosity (H_e) was 0.1435 and nucleotide diversity (π) was 0.1541.M. longipedunculata, lower than in M. longipedunculata (H_e= 0.1793, π = 0.1916) with narrow distribution. The genetic diversity of the two ex-situ conservation populations (H_e=0.1797, 0.1990, π = 0.1894, 0.2121 ) was higher than the wild populations (H_e=0.1591, π = 0.1733), suggesting that the artificial ex-situ conservation population can effectively protect its genetic diversity and the genetic breeding in future studies. The mean inbreeding coefficient (F_IS) in the M. longipedunculata and M. insignis populations were − 0.0141 and − 0.0176, respectively, indicating that the two populations were largely outcrossing in the natural state. The frequency of rare alleles in M. insignis (Tajima's D = 0.3436 > 0) and M. longipedunculata (Tajima's D = 0.2119 > 0) populations was low, which may be under balanced selection pressure or experienced population shrinkage. A high degree of genetic differentiation between the two species was detected, but moderate among the populations of the same species. The results of cluster analysis demonstrated a obvious separation between the two species with all individuals clustering according to species differences. It is worth noting that individuals 7, 8, and 9 in the ML-CW M. insignis population first clustered with M. longipedunculata. Furthermore, these individuals also contained a small genetic component from M. longipedunculata. Principal component and genetic relationship analysis also demonstrated that some M. insignis individuals exhibited an evident genetic relationship with M. longipedunculata. These populations can therefore be preferentially selected in the further cross-breeding experiments of the two species. The genetic diversity and structure of M. insignis and M. longipedunculata were compared in this study. Not only were the genetic characteristics of the two species explored for the first time, but the genetic relationship between the two species was also preliminarily explored. This study provides a scientific basis for the development of future protection strategies at the molecular level.

Biological sciences/Genetics

Biological sciences/Molecular biology

Biological sciences/Plant sciences

Manglietia insignis is an evergreen tree widely distributed throughout Guizhou, Guangxi, Yunnan, and other provinces in China. This species is commonly found growing in broad-leaved forests in the form of individual plants or groups. However, pure forest or wild community with Manglietia rosea as the dominant population has not been found to date ¹. Manglietia insignis trees are straight, tall, and exhibit rapid growth. The wood has the advantages of fine texture, luster, corrosion resistance, and easy processing. In addition, its flowers have a great aroma and bright color ² and its roots, leaves, flowers, and fruits are commonly used for medicinal purposes ³. Manglietia insignis has, therefore, become a popular garden tree species. However, due to its high ornamental value and economic value, M. insignis has become a major target of deforestation due to its popularity. Combined with the poor regeneration ability of its natural populations, the number of wild populations of M. insignis has been decreasing annually. This species has become rare and endangered and has been listed as a national third-class protected plant in China ⁴. Similar to M. insignis, Manglietia longipedunculata is an evergreen tree in the Family Magnoliaceae. Manglietia longipedunculata was first discovered in 2003 by Professor Zeng Qingwen (Chinese Academy of Sciences) and his research team in Nankunshan Nature Reserve, Huizhou City, Guangdong Province, China ⁵. To date, only 11 wild plants have been identified in the Nankunshan Nature Reserve. No other populations of M. longipedunculata have been found in other parts of the world ⁶. This species is now listed in the red list of Magnoliaceae plants. Due to the lack of pollinators, the asynchronous development of pistil and stamen, and the extremely short stigma receptivity period, the propagation of this species is extremely challenging. In its natural state, M. longipedunculata only blooms and does not bear fruit. Therefore, to protect this rare species and its population size, researchers have rapidly performed a series of in-situ and ex-situ protection measures including artificial pollination and grafting ⁷. At present, M. longipedunculata has been successfully introduced to the Nankunshan Botanical Garden in Huizhou City and Dongguan Botanical Garden in Dongguan City (China).

Due to the limitation of population size, M. insignis research has been limited to resource investigation ^8,9, seedling breeding ^10,11, and medicinal ingredients ^12,13, all requiring further investigation. In addition to some research on resource investigation ¹⁴ and seedling breeding⁸, there are few reports on M. longipedunculata at home and abroad. Therefore, to better implement effective protection strategies for this species, it is imperative to expand M. longipedunculata population size. Current research on the two species of Manglietia is still in its infancy. Research on the genetic diversity and structure of M. insignis and M. longipedunculata throughout the world is lacking. Investigating genetic diversity is therefore of great significance to the protection of species resources, specifically in rare and endangered plants. This study has aimed to effectively identify the environmental adaptability and evolutionary potential of this species, provide a scientific and effective basis for the formulation of protection measures for related species and the screening of core germplasm resources, and provide a solid material basis for resource utilization ¹⁵. Genetic diversity of species elucidation began from the initial simple phenotypic identification to the cell identification but now has progressively advanced to molecular markers. Compared with the previous marker techniques, molecular markers are highly polymorphic, fast, highly efficient, and are not affected by the developmental stage of the species or the external environment ¹⁶. With the continuous progression of molecular biology research, molecular marker technology has been developed to the third generation represented by single nucleotide polymorphisms (SNPs). SNPs are widely distributed within the genome, which commonly exhibits high polymorphisms, high genetic stability, and easy automatic detection, becoming the best option for population genetic analysis and genomics¹⁷. SNP-based molecular marker technology has been widely used in genetic diversity studies of many species ^18–20. In addition, we used Hyper-seq method, which is a novel, effective, and flexible marker-assisted selection and genotyping approach. Compared with the traditional whole-genome sequencing ( WGS ), Hyper-seq approach has a certain gene region enrichment effect. This approach can flexibly adjust the marker density by using different Hyper-seq primers based on the needs of different species and projects. And without additional enzyme digestion and ligation joints, through a special PCR method, a large number of samples can be built at the same time, and a large amount of genotype big data can be produced ²¹.

Theoretically, the genetic diversity of widely distributed species is higher than that of small and narrowly distributed species ²². Due to the high probability of inbreeding, unstable population structure, and genetic drift, small populations of plants will exhibit accelerated loss of genetic diversity. However, some recent studies have identified that the genetic diversity of small-scale populations with fewer individuals is higher than that of large-scale populations ^23,24. In addition, M. insignis is a relatively primitive species, occupying an important position in the study of the classification, distribution, and flora of the adjacent areas ²⁵. Therefore, this study investigated the genetic diversity of the widely distributed species M. insignis and the extremely small population of M. longipedunculata. The genetic diversity and structure differences were compared between these two species, to identify the cause for the decrease in the population size and the genetic relationship between the populations and present recommendations for the protection and restoration of the population. Furthermore, excellent germplasm resources were screened out to provide a theoretical scientific and material basis for future introduction and cultivation of this species.

SNP calling based on the reference genome. A total of 430,379 raw reads were generated from 22 M. insignis and 36 M. longipedunculata samples. After filtering, 6,596 high-quality SNP loci were identified and evenly distributed on the 19 chromosomes (Fig. 1).

Genetic diversity. The genetic diversity of M. insignis and M. longipedunculata was calculated using 430,379 SNPs (Tables 1 and 2 and Fig. 2). Among the five populations, ML-NKC H_O was the largest, followed by ML-DG and ML-NKW, and the smallest was in MI-CW. The H_E of ML-NKC was the largest, followed by ML-DG and ML-NKW, and the smallest was in MI-CW. The ranking of the H_E in each population was consistent with the H_O. The average H_O value of the two populations of M. insignis was 0.1649, which was higher than that of H_E. The average H_O value of the three populations of M. longipedunculata was 0.2024, which was also higher than that of H_E. The mean value of π (0.1916) in the three populations of M. longipedunculata was greater than in M. insignis. The genetic diversity was evaluated using H_E and π as follows: M. longipedunculata (ML-NKC > ML-DG > ML-NKW) > M. insignis (MI-YZL > MI-CW). Between the three M. longipedunculata populations, the genetic diversity of the two ex-situ populations was higher than that of the wild population. These results demonstrate the significance of ex-situ conservation for maintaining and improving the genetic diversity of M. longipedunculata.

The F_IS coefficient was used to assess the inbreeding level within the populations. Among the two M. insignis populations, the F_IS coefficient of one population (MI-CW, F_IS=0.0226) was greater than 0, while the other (MI-YZL, F_IS=-0.0507) was less than 0. Among the three M. longipedunculata populations, only ML-DG (F_IS=0.0080) F_IS was greater than 0, while the other two populations (ML-NKC and ML-NKW) were less than 0. This suggests that selfing or inbreeding between individuals may be occurring in the MI-CW population of M. insignis and the ML-DG population of M. longipedunculata, demonstrating the loss of heterozygosity phenomenon. The results of Tajima’s D calculation of M. insignis and M. longipedunculata were positive at 0.3039–0.3833 and 0.0440–0.3513, respectively (Fig. 2). These results suggest that the rare allele frequency in these two populations was low and may be under balanced selection pressure or experiencing population shrinkage. The genetic differentiation (F_st) of the population was preliminarily established according to the genetic differentiation coefficient between the populations (Table 2). The genetic distance between M. insignis and M. longipedunculata populations was large, as was the degree of differentiation. The F_st of the M. insignis MI-CW population and M. longipedunculata populations was lower than that of MI-YZL and M. longipedunculata populations, suggesting that M. insignis MI-CW population was closer to M. longipedunculata than the MI-YZL population.

Table 1

Genetic diversity of five *M. insignis* and *M. longipedunculata* populations.
Pop ID	Private SNP	H_O	H_E	π	F_IS	Tajima’s D
MI-CW	119	0.1485	0.1421	0.1554	0.0226	0.3039
MI-YZL	365	0.1812	0.1426	0.1528	-0.0507	0.3833
Mean value		0.1649	0.1424	0.1541	-0.0141	0.3436
ML-DG	18	0.1890	0.1797	0.1894	0.0080	0.2404
ML-NKC	30	0.2385	0.1990	0.2121	-0.0495	0.3513
ML-NKW	23	0.1826	0.1591	0.1733	-0.0112	0.0440
Mean value		0.2024	0.1793	0.1916	-0.0176	0.2119

Table 2

Coefficient of genetic differentiation among *M. insignis* and *M. longipedunculata* populations.
	MI-YZL	ML-DG	ML-NKC	ML-NKW
MI-CW	0.141	0.168	0.170	0.189
MI-YZL		0.234	0.236	0.251
ML-DG			0.051	0.065
ML-NKC				0.073

Population structure. The most direct manifestation of genetic diversity is the level of genetic variation. However, a single individual of a species has a limited lifespan and can be susceptible to external factors. A continuous population composed of individuals is therefore a unit of genetic evolution. The specific distribution pattern of these types of populations in nature is the genetic structure of the population. The mixed analysis results demonstrated that K = 2 was the best clustering result among all the samples of M. insignis and M. longipedunculata (Fig. 3). When K = 2, the CV error value was the lowest, and the two species were obviously separated (Fig. 4). Since the two M. longipedunculata ex-situ populations were derived from wild populations (ML-NKW), some individuals were not separated according to population differences but exhibited some individual confounding phenomena. All individuals within the two populations were separated according to population differences. It is worth noting that some individuals (ML-CW-2,3,7,8,9) in the M. insignis ML-CW population were mixed, suggesting that a small number of genetic components of M. longipedunculata were mixed in the MI-CW M. insignis population. Furthermore, the M. insignis ML-CW-7, 8, and 9 individuals were the first to be clustered with M. longipedunculata in the phylogenetic tree. This demonstrates that M. longipedunculata may have a certain relationship with M. insignis in its genetic evolution.

When K = 3, all M. longipedunculata individuals still clustered into one group (Fig. 4). While the individuals of the two populations were clustered into one group after the separation of M. longipedunculata and M. insignis, two of the populations did not show a common genetic component. Two individuals (ML-CW-7 and 8) from the M. insignis ML-CW population were also mixed with M. longipedunculata genetic components. When K = 4, most M. longipedunculata individuals were divided into three categories according to different populations (Fig. 4). However, a small number of individuals was separated from the population and clustered with other populations. A few M. longipedunculata individuals (ML-NKC-5, 6, and ML-NKW-4) exhibited same genetic components of other M. longipedunculata populations, but all same genetic components within the population. All M. insignis individuals clustered into one group and individuals (ML-CW-2, 3, 7, 8, and 9) in the ML-CW population were mixed with M. longipedunculata. Furthermore, ML-CW-7 and 8 individuals contained mixed genetic information from the three M. longipedunculata populations.

Principal component analysis (PCA) and genetic relationship analysis. According to the two-dimensional PCA diagram (Fig. 5), the contribution rates of the first and the second principal components were 31.15 and 7.34%, respectively. The position distance in the two-dimensional map demonstrated the distance of the genetic relationship between the germplasm resources of the M. insignis and M. longipedunculata populations. The results showed that M. longipedunculata and M. insignis were obviously separated (Fig. 5). The two M. insignis populations exhibited considerable separation, with the MI-YZL in the first quadrant and the MI-CW in the third and fourth quadrants, likely due to the geographical distance between the two populations. The three M. longipedunculata populations exhibited a small degree of separation and close genetic distance, and all individuals were mixed, which may be related to all individuals originating from the same population. Furthermore, PCA results demonstrated that some ML-CW individuals tended to move closer to M. longipedunculata, suggesting the presence of a genetic evolutionary relationship between the two populations, consistent with the genetic structure analysis results.

The genetic relationship analysis of all M. insignis and M. longipedunculata individuals demonstrated that M. insignis was largely divided into two categories according to the genetic relationship (Fig. 6). Most individuals in the MI-CW population were clustered into one group, as were all individuals in the MI-YZL population. In addition, the MI-CW-7, 8, and 9 individuals in the MI-CW population exhibited considerable separation from most of the MI-CW individuals, suggesting significant differences in the genetic relationship between the two populations. Furthermore, the three MI-CW-7, 8, and 9 individuals demonstrated an evident genetic relationship. In the M. longipedunculata genetic relationship analysis, most individuals of the three populations were generally divided into three categories according to the differences in populations. Some individuals however were mixed into other populations. Overall, the genetic distance between all individuals of the three M. longipedunculata populations was not significantly different. The genetic differentiation was not obvious when compared with M. insignis. This was consistent with the PCA results.

Comparison of genetic diversity between M. insignis and M. longipedunculata. Genetic diversity is the basis of species adaptability and evolutionary potential ²⁶. Generally, higher genetic diversity of the species results in greater adaptability to the environment, decreased influence of the external environment, and increased evolutionary potential. On the contrary, the evolutionary potential is reduced with lower genetic diversity of the species, increasing the vulnerability to environmental factors²⁷. In this study, a large-scale genomic analysis of SNP loci was performed for the first time to evaluate the genetic diversity of M. insignis and M. longipedunculata. The results showed that the genetic diversity of M. insignis (π = 0.1541) was lower than that of M. longipedunculata (π = 0.1916). On one hand, this phenomenon may be due to M. insignis ecology and habitat has undergone great changes in recent years despite having accumulated rich genetic variation over the long-term evolution process. In addition, due to excessive anthropogenic deforestation and natural disasters, its distribution has become fragmented with the individual numbers continually decreasing, leading to a decrease in the genetic diversity of its population. On the other hand, due to the continuous habitat fragmentation of M. insignis, its original wide and continuous distribution has been broken up. This reduces the species adaptability, causing some populations to completely disappear, increasing the inbreeding rate in the residual population, and resulting in the disappearance of some alleles, which in turn leads to the continuous reduction of M. insignis genetic diversity. In addition, this study only collected samples from two wild populations of M. insignis. The low sampling rate or one-sidedness may also cause the low results. Although M. longipedunculata population was small, high genetic diversity was still maintained. A previous study demonstrated that the high genetic diversity of small populations is dependent on frequent gene flow ²⁸. Similar phenomena have been reported in extremely small populations of Camellia dongxingensis ²⁹, Chinese wild apricot ³⁰, and other species.

Many studies have found that the ability of some rare and endemic species to maintain high genetic diversity is closely related to the evolutionary history of species, the maintenance of genetic diversity of glacial refuge population, the regional characteristics of distribution area, and the ecological habits of species including the mating system ³¹. Therefore, the potential effects of these factors on the genetic diversity of M. insignis and M. longipedunculata were comprehensively analyzed in the current study. The Tajima's D values of the five populations of these two species were positive, suggesting that the frequency of rare alleles in these two species was low and the existing populations may be under balanced selection pressure or have already experienced population shrinkage. The inbreeding (F_IS>0) was detected in the M. insignis MI-CW population, while in the M. longipedunculata population, the wild population (ML-NKW) specifically was dominated by outcrossing (F_IS<0). In the case of limited pollinators, selfing is one of the most effective strategies for species to maintain or increase population size ³². Although the outcrossing-based mating system of M. longipedunculata limits the rapid population expansion, it ensures the high genetic diversity of the population and reduces the risk of species extinction due to any future changes in the habitat. Despite the small size of the wild M. longipedunculata population, selfing was not detected. Therefore, it is likely that in natural habitat, there may be self-incompatibility and partial asexual reproduction in M. longipedunculata, eventually leading to heterozygous and homozygous deletions within the population ^{33, 34}. However, further research is required via experimental analyses such as artificial pollination.

When the raw habitat of species is destroyed, the measures of ex-situ migration and protection are called ‘ex-situ conservation’ ³⁵. However, due to the lack of complete sampling, unclear resources, and repeated sampling during the implementation of the ex-situ conservation plan, the isolated populations established by the ex-situ conservation often have the risk of loss or reduction in genetic diversity ³⁶. The genetic diversity and population structure information of ex-situ populations is one of the most important reference indices to evaluate the success of ex-situ conservation. If the genetic diversity of the ex-situ population is higher than or generally consistent with the genetic diversity of the wild population, the protection measures are considered successful ³⁷. The genetic diversity (π = 0.1894, 0.2121) in this study of the two ex-situ conservation populations of M. longipedunculata was higher than that of its wild population (π = 0.1733). Therefore, the two artificial ex-situ conservation populations of ML-DG and ML-NKC can effectively protect the genetic diversity of M. longipedunculata species and fully represent the genetic diversity of its wild populations. The ex-situ conservation of M. longipedunculata individuals largely originated from the seeds collected from the sexual reproduction of wild populations (ML-NKW). In addition, the mating strategy of the wild population based on outcrossing ensures frequent gene exchange. This may be one of the reasons why the genetic diversity of ex-situ conservation populations was higher than in the wild populations. Therefore, the ex-situ population can be used as the key application material in future genetic breeding work. In this study, the average H_e of M. insignis and M. longipedunculata was 0.1424 and 0.1793, respectively. This value was significantly lower than in other Magnoliaceae species, such as Manglietia conifera (H_e = 0.66) 38, Kmeria septentrionalis (H_e = 0.69) 39, indicating that the genetic diversity of these two Manglietia species was low and the genetic basis was narrow, which may be related to the small natural distribution range of these two plant species.

Comparison of genetic structure and differentiation between M. insignis and M. longipedunculata. The investigation of the genetic structure of species is very important for the protection of species and the formulation of protection strategies^{40, 41}. The M. longipedunculata and M. insignis clustering map demonstrated a few individual confounding phenomena between the two populations. However, due to the geographical distance between the two populations, there may be two reasons for this phenomenon. On the one hand, a population of M. insignis was located near M. longipedunculata and there is gene exchange between the two groups. On the other hand, combined with phylogenetic tree clustering analysis, M. longipedunculata may be derived from the genetic differentiation of some individuals in M. insignis. This hypothesis is also supported by PCA and genetic relationship analysis between individuals. These two analyses demonstrate that some individuals in the MI-CW population of M. insignis tend to gradually approach M. longipedunculata. Therefore, in future breeding studies, these species with close genetic relationships can be first selected to improve the success rate of the study.

In addition, the F_st was also used to measure the distance of genetic relationships or the degree of genetic differentiation ⁴². The genetic differentiation coefficients of the two populations of M. insignis and the three populations of M. longipedunculata were different, and the value of MI-CW was lower, suggesting that the population was closely related to M. longipedunculata. Previous studies have demonstrated an absence of differentiation among subgroups in populations with F_st values between 0 and 0.05. If the Fst value was 0.05 to 0.15, it was moderately differentiated. High differentiation was considered if the F_st value was between 0.15 and 0.25 ⁴³. The results of this study showed that there was moderate differentiation between the two populations (F_st =0.141), similar to a high degree of differentiation. This may be related to the geographical distance between the two populations. The three M. longipedunculata populations (F_st =0.051, 0.065, 0.073) also belonged to moderate differentiation, with almost no differentiation between the two ex-situ conservation populations. The degree of genetic differentiation between the wild and ex-situ conservation populations was higher than that of the two ex-situ conservation populations. The degree of differentiation between ex-situ conservation and wild populations and between ex-situ conservation populations will gradually increase ³⁵. Furthermore, the adaptability of ex-situ conservation species to the native habitat will gradually be lost, eventually delaying the subsequent wild regression experiments of the species ⁴⁴. Therefore, to avoid this putative risk, an increase in the gene exchange between the ex-situ conservation and the wild populations is necessary via artificial pollination, systematic updates of the ex-situ conservation population gene diversity, and finally ensuring that the ex-situ population maintains the adaptability to the native environment.

Protection recommendations. The results of this study demonstrated that the genetic diversity of M. insignis was low. In addition to ensuring that the population was able to self, the number of individuals was also an important factor due to the continuous reduction of anthropogenic activities. It is, therefore, necessary to improve the investigation of the plant resources and appropriately protect the native environment of the large-scale population and the ex-situ conservation of the small-scale population. The protection of species does not only provide protection for a single group but for different groups or different genotype groups. In regard to multi-population species, the screening of excellent germplasm resources should not only improve the protection and management efficiency of germplasm resources but also reduce costs ⁴⁵. However, the genetic information of only two M. insignis populations was investigated. The sampling range in future studies should be expanded to accurately screen out excellent germplasm resources. In addition, a one-way mixing phenomenon was identified between the two species. These two species should avoid proximity in ex-situ conservation to ensure the purity of genetic information for the species. Due to the geographical distance between the two species, this gene mixing phenomenon can not only explain the possible evolutionary relationship between the two species but also more importantly, shows that there may be an M. longipedunculata population in Caowang Mountain, Leye County, Baise City (China). Therefore, the species investigation in this area should be accelerated and the corresponding protection should be rapidly established. The inability to rapidly expand the population is due to the self-pollination mating strategy. Therefore, the primary task should be to protect the wild seedlings in situ or ex-situ and expand the population size based on maintaining the excellent characteristics of the female parent via tissue culture, cuttings, and other asexual reproduction methods, to provide a solid material basis for future introduction and breeding.

Two ex-situ conservation populations with high genetic diversity can therefore be utilized for breeding improved varieties. Although the ex-situ conservation duration of M. longipedunculata was relatively short, the degree of differentiation between M. longipedunculata and wild populations has gradually increased. Therefore, to ensure the adaptability of the species to the native habitat and the success of the subsequent introduction and regression experiments, it is necessary to identify the genetic diversity of the ex-situ conservation population and increase the gene exchange between the ex-situ and the wild populations in time via artificial pollination and other breeding methods.

In this study, Hyper-seq method was used to sequence a total of 58 samples from two wild populations of M. insignis as well as one wild population and two ex-situ conservation populations of M. longipedunculata. A total of 6,596 high-quality SNP loci were detected. The genetic diversity, population genetic structure, and phylogenetic tree of the two populations were constructed. The results showed that the genetic diversity of the widely distributed M. insignis was lower than that of the narrowly distributed M. longipedunculata. This may be related to the significant anthropogenic destruction of M. insignis habitat. In addition, although the M. longipedunculata population was small, high genetic diversity was still maintained. The results of this study demonstrate that the individuals were primarily outcrossing, which may be the reason for this phenomenon. The genetic diversity of the two ex-situ conservation populations was higher than that of their wild populations, suggesting that the ex-situ conservation can effectively protect the genetic diversity of M. longipedunculata and this population can be used for future breeding. In addition, the results of the population genetic structure of the two populations demonstrated mixed genetic components between the two populations. This material could be preferred for future cross-breeding testing. Since only two populations of M. insignis were selected for genetic diversity investigation, the results were not comprehensive enough. Therefore, the sampling range should be expanded in the future to screen out high-quality germplasm resources and accelerate the protection of these species.

Plant materials. Leaf samples, which are all healthy and mature were collected from 22 M. insignis individuals from two populations and 36 M. longipedunculata individuals from three populations (Fig. 7 and Table 3). Five leaves were collected from each plant. Among the three populations of M. longipedunculata, both ML-DG and ML-NKC were ex-situ conservation populations, originating from ML-NKW. Due to the small size of some populations, the total number of samples with the sampling interval was balanced in order to collect as many possible representative samples. The interval between plant individuals sampled was more than ten meters. The samples were all collected from healthy and mature individuals. Fresh leaf samples were collected and quickly dried in discolored silica gel. The materials used in this study were collected and identified by associate researcher Tang Jianmin (Guangxi Botanical Research Institute, China). One sample of each population was preserved in the Herbarium of Guangxi Botanical Research Institute (China). The plant materials collected in this study are in compliance with Chinese and international guidelines and laws, including the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. One plant sample was collected from each population of M. insignis and M. longipedunculata., and all of them were preserved in the South China Botanical Garden, Guangdong, China. The specimen numbers were MI-CW-20221024, MI-YZL-20221025, ML-DG-20221105, ML-NKC-20221106, ML-NKW-20221107, respectively.

Table 3. Sampling information of M. insignis and M. longipedunculata.

Population	Location	Type	Latitude	Longitude	No. of samples
MI-CW	Caowang Mountain, Leye County, Guangxi, China	Wild population	24°43'46ʺN	106°21'21ʺE	9
MI-YZL	Yinzhulao Mountain, Ziyuan County, Guangxi, China	Wild population	26°15'19ʺN	110°33'19ʺE	13
ML-DG	Dongguan Botanical Garden, Guangdong, China	Ex-situ population	22°51'16ʺN	113°47'26ʺE	14
ML-NKC	Nankunshan Botanical Garden, Huizhou, Guangdong, China	Ex-situ population	23°38'40ʺN	113°52'47ʺE	12
ML-NKW	Nankun Mountain, Huizhou County, Guangdong, China	Wild population	23°36'60ʺN	113°50'60ʺE	10

Hyper-seq library construction and sequencing. E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, USA) was used to extract DNA from 22 M. insignis and 36 M. longipedunculata leaf samples according to the manufacturer’s instructions. The DNA quality was assessed using 1% agarose gel electrophoresis and a Nanodrop 2000 (Thermo Fisher, USA) spectrophotometer. The extracted DNA was quantified using Qubit 3.0 (Thermo Fisher, USA) to ensure that each sample met the following criteria: total mass > 3 μg, concentration > 30 ng/μL, and OD₂₆₀/OD₂₈₀= 1.80-1.20. The barcode adaptor was then ligated and the fragment size selection and PCR amplification were performed to obtain a Hyper-seq library ²¹. The constructed library was sent for 150-bp paired-end sequencing on the Illumina Novaseq 6000 platform (Illumina, San Diego, USA).

SNP calling. FastQC ⁴⁶ was used to evaluate the quality of the original data. Trimmomatic v0.36 (set to the default parameters) ⁴⁷ was used to filter the adaptor sequences, cleavage sites (the first six base pairs of the reads), and low-quality reads (Q20 < 20, length < 36 bp) from the raw sequence data ⁴⁸. The clean reads were then mapped to the Magnolia hypoleuca (Magnoliaceae) reference genome ⁴⁹ using BWA v0.7.12 ⁵⁰. The rm-pcr-duplicates parameter of the gstacks module in the Stacks v.2.59 software ⁵¹ was used to remove the PCR repeats and perform SNP calling. Vcftools v0.1.11 ⁵² was used to filter SNPs, removing sites with a population loss rate of more than 40%, maf > 0.05, and a sequencing depth of less than 3.

Population genetics analysis. Based on the selected high-quality SNP data, Stacks v2.59 was used to calculate genetic diversity parameters, including observed heterozygosity (H_O), expected heterozygosity (H_E), nucleotide diversity (π), and inbreeding coefficient (F_IS) ⁵³. The Tajima's D value of the site was calculated using Vcftools, with the sliding window size of the operation set to 500 kb.

Phylogenetic and population structure analyses. A phylogenetic tree between individuals was constructed for 22 M. insignis and 36 M. longipedunculata using the maximum likelihood (ML) method in IQtree v2.0 ⁵⁴, with M. insignis set as the outgroup. Bayesian clustering was performed on all individuals using Admixture v1.3.0 ⁵⁵ with clustering values (K) set from 1 to 9. The optimal K value was determined based on the minimum cross-validation (CV) error.

Principal component analysis (PCA) and genetic relationship analysis. PCA and and kinship analysis among individuals were performed using MingPCACluster, a new simple and efficient software for population VCF file ⁵⁶.

Acknowledgments

This work was supported by the Special Funds for Natural Resource Affairs in Guangdong Province in 2023, the National Key Research and Development Program (No.2022YFF1300700), Chinese Academy of Sciences ‘Light of West China’ Program (2022) and Guangxi Forestry Science and Technology Promotion Demonstration Project (2023LYKJ03 and [2022]GT23). This publication is partially based on work at Guangxi Key Laboratory of Plant Functional Substances and Resources Sustainable Utilization of Guangxi Institute of Botany.

Author Contributions

Conceptualization, X.W.; methodology, X.L.; validation, Q.M.; formal analysis, Y.Y. and X.L.; investigation, L.W., S.L., Q.F., W.Z. (sampling), L.Z. and J.T. (sequencing), X.L. and L.P. (data analysis); writing—original draft preparation, Y, Y.; writing—review and editing, Y, Y.; visualization, X.W. and J, T; supervision, J.T.; funding acquisition, X.W. All co-authors have reviewed and approved the final version of the manuscript for publication. All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests

Correspondence and requests for materials should be addressed to J.T.

Data Availability

Raw sequencing data are available at NCBI with the SRA accession number of PRJNA1037033 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA1037033?reviewer=pjnht5rev90an34aft2ojnjfti).

Gao, Z. Q. Manglietia insignis and its cultivation techniques. Northern Horticulture.1, 160-161 (2007).
Li, M., Ma, H.C. & Li, F.X. Study of Photosynthesis Characteristics of Manglietia insignis at Seedling Stage. Journal of West China Forestry Science. 1, 42-45 (2004).
Peng, C. S. Breeding technology of improved varieties of Manglietia insignis. Forest By-Product and Specialty in China. 1, 52-54 (2022).
Chen, Y. J., Deng, L. X., Chen J. Y.; Chen, Z. P. & Tan, H. M. A Study on the Annual Growth Dynamics of Manglietia insignis Seedlings from Different Provenances. Chinese Agricultural Science Bulletin. 29, 8-14 (2013).
Zeng, Q. W. & Law, Y. Manglietia longipedunculata (Magnoliaceae), a new species from Guangdong, China. Annales Botanici Fennici. 41 (2004).
Xie, C., Lin F., Liu, D. M., Wen, X. Y., & Zhong, W.C. Conservation of Rare and Endangered Species Manglietia longipedunculata (Magnoliaceae). Proceedings of the second international symposium on the family Magnoliaceae. Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PR China Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China; Key Laboratory.
Ren, H. et al. The use of grafted seedlings increases the success of conservation translocations of Manglietia longipedunculata (Magnoliaceae), a Critically Endangered tree. Oryx. 50, 437-445. DOI: 10.1017/S0030605315000423 (2015).
Zhu H. Vegetation geography of evergreen broad-leaved forests in Yunnan, southwestern China. Chinese Journal of Plant Ecology. 45, 224-241 (2021).
Zou T.C. et al. Species diversity conservation and utilization of Guizhou rare and endangered spermatophyte. Guihaia. 41, 1699-1717 (2021).
Yu, Y. P., Liang, D. L., Liu K. C., Wang, L. H. & Huang, R. F. Effects of Chilling Stress on Physiological Characteristics of Manglietia florida Seedlings. Northern Horticulture. 1, 69-71 (2013).
Guan, L. W., Kuang, D. L. Yang, W. L. & Liu, Z. Y. Effects of Cutting Roots during Different Periods on Transplanting Survival in Manglietia insignis. Southwest China Journal of Agricultural Sciences. 24, 707-711 (2011).
Shang, S. Z. et al. Two new neolignans from Manglietia insignis. Natural Products and Bioprospecting. 2, 227-230. DOI: 10.1007/s13659-012-0063-7 (2012).
Shang S. Z. et al. Bioactive phenolics and terpenoids from Manglietia insignis. Fitoterapia. 84, 58-63. DOI: 10.1016/j.fitote.2012.10.010 (2013).
Zhang Y. K. Investigation and Evaluation of Wild Ornamental Plants in Nankun Mountain Nature Reserve in Guangdong Province. Ph.D. Thesis, South China Agricultural University, Guangdong, China, 2016.
Eduardo, C., Marie Stéphanie, S., Paul, G. & Fernández P. J. A. Genetic structure in peripheral Western European populations of the endangered species Cochlearia pyrenaica (Brassicaceae). Plant Systematics and Evolution. 297, 75-85. DOI: 10.1007/s00606-011-0500-9 (2011).
Liu, J. X., Shan, J. P. & Wang, P. Genetic Diversity Analysis of Sunflowers Resources Based on SNP Markers. Institute of Crops. Molecular Plant Breeding. 1, 1-18 (2023).
Wang, X., Lu, B., Shao, L., Ali, A. & Sun, F. Genome-wide SNPs reveal fine-scale population structure of Laodelphax striatellus in China using double-digest restriction site-associated DNA sequencing Genomics. 114, 110329-110329. DOI: 10.22541/au.159141074.44080801 (2020).
Phoebe, M. et al. Genetic Diversity and Population Structure of Sorghum (Sorghum bicolor (L.) Moench) Landraces Using DArTseq-Derived Single-Nucleotide Polymorphism (SNP) Markers. Journal of molecular evolution. 91, 552-561. DOI: 10.1007/S00239-023-10108-1 (2023).
Liu, C. G. et al. Genetic Diversity of Tea Plant (Camellia sinensis (L.) Kuntze) Germplasm Resources in Wuyi Mountain of China Based on Single Nucleotide Polymorphism (SNP) Markers. Horticulturae. 8, 932. DOI: 10.3390/HORTICULTURAE8100932 (2022)
Yukio, N., Hiroaki, T., Sayoko, N., Naofumi, H., Atsushi, J. N. & Shinji, F. Genetic diversity of loquat (Eriobotrya japonica) revealed using RAD-Seq SNP markers. Scientific Reports. 12, 10200 (2022).
Zou, M., Xia, Z. Hyper-seq: A novel, effective, and flexible marker-assisted selection and genotyping approach. Innovation. 3, 100254 (2022). DOI: 10.1016/J.XINN.2022.100254.
Kaijund, K. & Jaaska, V. No loss of genetic diversity in small and isolated populations of Medicago sativa subsp. Falcata. Biochemical Systematics and Ecology. 38, 510-520. DOI: 10.1016/j.bse.2010.05.007 (2010).
Li W. J.; Su, Z. H. & Yang, L. Genetic diversity of the critically endangered Ferula sinkiangensis K. M. Shen (Apiaceae) and the implications for conservation. Turkish Journal of Botany. 44, 145-152. DOI: 10.3906/bot-1906-2 (2020).
Schou, M. F., Loeschche, V., Bechsgaard, J., Christian, S. & Torsten, N. K. Unexpected high genetic diversity in small populations suggests maintenance by associative overdominance, Molecular Ecology. 26, 6510-6523. DOI: 10.1111/mec.14262 (2017).
Wang, C. M., Zhuang, Z. Y., Li, W. D. & Li, W. B. Study on Seedling Reproduction Technique of Manglietia insignis(Wall.). Modern Agricultural Science and Technology. 6, 193-195 (2010).
Du, L. M., Yang, L. W., Lin, X. J., Huang, G. N., Li, X. B. & Guo, X. Genetic Diversity Analysis of Ficus hirta Vahl Based on SNP Molecular Marker. Molecular Plant Breeding 21, 5709-5719 (2023).
Nybom, H. Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Mol. Ecol. 13, 1143-1155. DOI: 10.1111/j.1365-294X.2004.02141.x (2004).
Cai, M., Wen, Y., Uchiyama, K., Onuma, Y. & Tsumura, Y. Population genetic diversity and structure of ancient tree populations of Cryptomeria japonica var. Sinensis based on RAD-seq data. Forests. 11, 1192. DOI: 10.3390/f11111192 (2020).
Tang, J. M. et al. Genetic diversity and mating system of camellia tunghinensis Chang extremely small population. Fresenius Environmental Bulletin. 29, 6508-6517 (2020).
Wang, Z. et al. High-level genetic diversity and complex population structure of Siberian apricot (Prunus sibirica L.) in China as revealed by nuclear SSR markers. PloS one. 9, e87381. DOI: 10.1371/journal.pone.0087381 (2014).
Eduardo, S. L., Márcia, G., Camila, M. Z., & Fernanda B. Genetic diversity and population structure of Vriesea reitzii (Bromeliaceae), a species from the Southern Brazilian Highlands. Genetics and Molecular Biology. 41, 308－317. DOI: 10.1590/1678-4685-GMB-2017-0062 (2018).
Kalisz, S., Vogler, D. W. & Hanley, K. M. Context-dependent autonomous self-fertilization yields reproductive assurance and mixed mating. Nature. 430, 884–887. DOI: 10.1038/nature02776 (2004).
Stoeckel, S., Grange, J., Fernández-Manjarres, J. F., Bilger, I. & Mariette, S. Heterozygote excess in a self-incompatible and partially clonal forest tree species: Prunus avium L. Mol Ecol. 15, 2109-2118. DOI: 10.1111/j.1365-294X.2006.02926.x (2006).
Wang, B., Yan, L.L., Zhang, Y. & Song, L.H. Genetic diversity analysis of four wild Jujube populations in Ningxia based on SNP molecular markers. Journal of Anhui Agricultural University. 49, 432-437 (2022).
Cibrian-Jaramillo, A. et al. What is the Conservation Value of a Plant in a Botanic Garden? Using Indicators to Improve Management of Ex Situ Collections. The Botanical Review. 79, 559–577. DOI: 10.1007/s12229-013-9120-0 (2013).
Wei, X. Z. & Jiang, M. X. Meta-analysis of genetic representativeness of plant populations under ex-situ conservation in contrast to wild source populations. Conservation Biology. 35, 12-23. DOI: 10.1111/cobi.13617 (2021).
Su, Z. H., Bryce, R. B., Zhuo, L., & Jiang, X. L. Divergent population genetic structure of the endangered Helianthemum (Cistaceae) and its implication to conservation in northwestern China. Frontiers in Plant Science. 7, 2010. DOI: 10.3389/fpls.2016.02010 (2017).
Wen, S. N., Zhong, C. L., Jiang, Q. B., Zhang, Y., Chen, Z. & Chen, Y. Genetic Diversity Analysis of Manglietia conifera Dandy Germplasm by SSR Markers. Molecular Plant Breeding. 15, 3788-3797 (2017).
Lin, Y. F. The population genetic structure and gene flow of Kmeria septentrionalis assessed by microsatellite markers. Ph.D. Thesis, Guangxi Normal University, Ph.D. Thesis, Guilin, China, 2012.
Pan, W., Sun, J., Yuan, Q., Zhang, L., Deng, K. & Li, Y. Analysis of genetic diversity and structure in different provenances of liriodendron by RAD-seq technique. Scientia Silvae Sinicae. 58, 74-81 (2022).
Zhu, X. L. et al. Genome-wide diversity evaluation and core germplasm extraction in ex situ conservation: A case of golden Camellia tunghinensis. Evolutionary Applications. 16, 1519–1530. DOI: 10.1111/eva.13584 (2023).
Holsinger, K. E. & Weir, B. S. Genetics in geographically structured populations: Defining, estimating and interpreting. Nature Reviews Genetics. 10, 639-650 (2009).
Wright, S. Evolution and the genetics of populations. A treatise in four volumes. Volume 4. Variability within and among natural populations. Journal of Biosocial Science. 4, 253-256 (1972).
Enßlin, A., Sandner, T. M. & Matthies, D. Consequences of ex-situ cultivation of plants: Genetic diversity, fitness and adaptation of the monocarpic Cynoglossum officinale L. in botanic gardens. Biological Conservation. 144, 272–278. DOI: 10.1016/j.biocon.2010.09.001 (2011).
Miao, L., Wang, S., Zou, M., Li, J., Kong, L. & Yv, X. Review of the studies on Core collection for horticultural crops. Journal of Plant Genetic Resources. 17, 791–800 (2016).
Andrews, S. Fastqc: A quality control tool for high throughput sequence data. 2010.
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 30, 2114–2120. DOI: 10.1093/bioinformatics/btu170 (2014).
Bolger, A. M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 30, 2114–2120. DOI: 10.1093/bioinformatics/btu170 (2014).
Zhou, L. J. et al. The genome of Magnolia hypoleuca provides a new insight into cold tolerance and the evolutionary position of magnoliids. Frontiers in Plant Science. 14, 1108701-1108701. DOI: 10.3389/FPLS.2023.1108701 (2023).
Li, H. & Durbin, R. Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics. 25, 1754-1760. DOI: 10.1093/bioinformatics/btp324 (2009).
Rochette, N. C., Rivera-Col´on, A.G., Catchen, J.M. Stacks 2: analytical methods for paired-end sequencing improve RADseq-based population genomics. Mol.Ecol. 28, 4737–4754. DOI: 10.1111/mec.15253 (2019).
Danecek, P. et al. The Variant Call Format and VCFtools. Bioinformatics 27, 2156–2158. DOI: 10.1093/bioinformatics/btr330 (2011).
Rochette, N. C., Rivera-Colón, A. G. & Catchen, J. M. Stacks 2: Analytical methods for paired-end sequencing improve RADseqbased population genomics. Molecular Ecology. 28, 4737-4754. DOI: 10.1111/mec.15253 (2019).
Minh, B.Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534. DOI: 10.1093/molbev/msaa015 (2020).
Alexander, D. H., Novembre, J. & Lange, K. Fast Model-Based Estimation of Ancestry in Unrelated Individuals. Genome Res. 19, 1655–1664. DOI: 10.1161/01.ATV.0000137190.63214.c5 (2009).
Zhu, X. L., Zou, R., Tang, J. M., Deng, L. L. & Wei, X. Genetic diversity variation during the natural regeneration of Vatica guangxiensis, an endangered tree species with extremely small populations. Global Ecology and Conservation 1, 42. DOI: 10.1016/J.GECCO. 2023. E02400 (2023).

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

The Study of Genetic Diversity and Structure of Extremely Endangered Manglietia longipedunculata and Endangered Manglietia insignis

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Conclusions

Methods

Declarations

References

Additional Declarations

Status:

Version 1