Genetic Diversity of the Critically Endangered Ferula Sinkiangensis K.M.Shen (Apiaceae) and the Implications for Conservation
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Turkish Journal of Botany Turk J Bot (2020) 44: 145-152 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1906-2 Genetic diversity of the critically endangered Ferula sinkiangensis K.M.Shen (Apiaceae) and the implications for conservation 1,2 1 1,4 1,2 1,2,3, Wenjun LI , Zhihao SU , Lei YANG , Qiumei CAO , Ying FENG * 1 CAS Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Urumqi, P.R. China 2 The Specimen Museum of Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, P.R. China 3 University of Chinese Academy of Sciences, Beijing, P.R. China 4 College of Life Sciences, Shihezi University, Xinjiang, P.R. China Received: 03.06.2019 Accepted/Published Online: 31.12.2019 Final Version: 17.03.2020 Abstract: Research on the genetic diversity and structures of endangered plant species with small population sizes is a prerequisite for developing reasonable conservation strategies. Ferula sinkiangensis K.M.Shen is a critically endangered herbaceous perennial and monocarpic medical plant growing in semiarid steppe ecosystems with a small population size within the Yili Valley in Xinjiang, China. Unfortunately, the lack of genetic information has limited our ability to initiate effective conservation strategies for the species. In this study, 14 polymorphic microsatellite primers of F. sinkiangensis were developed and characterized using Illumina HiSeq paired-end reads from genomic DNA. Primers were used to assess the genetic diversity and structure of the species in the Yili Valley. A total of 92 alleles were detected across 14 microsatellite loci from 65 specimens of F. sinkiangensis. Moderate to high levels of genetic diversity (He = 0.446, I = 0.821, PPB = 92.86%) were found within the species. Our results contradict the general hypothesis that rare and endangered species with small population sizes and narrow ranges of distribution exhibit low levels of genetic diversity. The moderate to high levels of genetic diversity within the species can be explained by the mating system, life history traits, and human activities in recent years. Several reasonable conservation and reintroduction strategies are proposed, including maintenance of the plant’s natural habitats, seeds or seedlings collection for germplasm storage, and artificial breeding of the species. Key words: Ferula sinkiangensis, genetic assessment, microsatellite, small population size, medicinal plant 1. Introduction al., 2006; Dong et al., 2007), and human factors (Qiao et The genetic diversity of a population or species is the al., 2010; Wu et al., 2015). In particular, anthropogenic foundation of evolutionary change (Darwin, 1859) and activities, including overexploitation of natural resources, provides a genetic basis for adaptation to environmental habitat degradation, land reclamation, and overgrazing, are change (Markert et al., 2010). The genetic analysis helps responsible for reductions in population size (Chiang et al., us to understand the genetic variation within and between 2006; Luan et al., 2006; Qiao et al., 2010; Wu et al., 2015). individuals and populations. This information is useful For rare and endangered species with small population to identify management units for conservation efforts sizes and narrow ranges of distribution, it has been and to detect population-level effects of habitat loss, hypothesized that a relatively low level of genetic diversity fragmentation, and isolation (Palsbøll et al., 2007; Allendorf reduces species fitness (Young et al., 1996; Markert et al., et al., 2013). For rare and endangered species, genetic 2010) because species with low genetic diversity are more diversity assessment within and between populations is susceptible to genetic drift, founder effect, and inbreeding an essential step for formulating appropriate management depression (Nei et al., 1975; Hamrick et al., 1992; Hamrick strategies for natural populations (Frankham et al., 2002; and Godt, 1996; Frankham, 1997; Nybom, 2004). However, Palsbøll et al., 2007; Gordon et al., 2012; Allendorf et al., some endangered species (Zawko et al., 2001; Wu et al., 2013; Ottewell et al., 2015; Frankham et al., 2017). 2015; Feng et al., 2019) and some common species with In general, the genetic diversity of plant populations small population size (Luan et al., 2006; Schou et al., 2017) is determined by population size, mating system, genetic have moderate to high genetic diversity. drift, gene flow, and evolutionary and life history (Loveless The genus Ferula L. (Apiaceae) consists of more than and Hamrick, 1984; Hamrick and Godt, 1996; Leimu et 170 perennial herbaceous taxa, mainly distributed in the * Correspondence: [email protected] 145 This work is licensed under a Creative Commons Attribution 4.0 International License. LI et al. / Turk J Bot Mediterranean region, Central Asia, and northwest China 2. Materials and methods (Pimenov and Leonov, 1993). In this genus, many species 2.1. Plant sampling are used as medical resources (Shen, 1987; Mahendra In 2017–2018, a total of 65 specimens of F. sinkiangensis and Bisht, 2012). In China, there are 26 Ferula spp., of were collected from Yili Valley. Within the valley, 61 which seven are endemic (She and Watson, 2005). Ferula specimens were sampled from the Yining locality (YN, sinkiangensis K.M.Shen is an herbaceous perennial and 43°44′N, 82°06′E) and 4 specimens were sampled from the monocarpic plant growing in semiarid steppe ecosystems, Nileke locality (NLK, 43°41′N, 82°18′E; just 4 specimens and the species is endemic to Xinjiang, China (She and were found in 2017 and were lost in 2018 at this locality). Watson, 2005). This species grows for about 7 years before All fresh plant samples were dried in silica gel and then flowering and fruiting (Shen, 1987). F. sinkiangensis was stored at –20 °C until DNA extraction. reported from Yining, Nileke, and Manashi counties in 2.2. DNA extraction, libraries, and isolation of microsatel- Xinjiang, and it has been widely used as a medicinal herb lite loci in China (Shen et al., 1975). Owing to its narrow range of All 65 genomic DNA samples were extracted using the distribution and important medicinal value, F. sinkiangensis Plant Genomic DNA Isolation Kit (Tiangen, Beijing, China) has been listed as a critically endangered (CR) species following the manufacturer’s instructions. DNA sequencing (Wang and Xie, 2004; Qin et al., 2017) and an important libraries were constructed from one sample from the YN wild medicinal plant used to treat stomach disorders locality according to the method used by Li et al. (2019). in Xinjiang District for centuries (Zhang et al., 2019). After screening the raw sequences and removing redundant Despite these listings, the distribution of F. sinkiangensis sequences, the remaining sequences were assembled using is further shrinking, likely due to global climate change, PANDAseq version 2.9. Then the microsatellite loci were overexploitation, habitat degradation, land reclamation, detected by 4 software programs (MISA, http://pgrc.ipk- and overgrazing in recent years. At the same time, lack gatersleben.de/misa/misa.html; MREP, http://mreps.univ- of enough conservation awareness from the government mlv.fr/; SSRIT, http://archive.gramene.org/db/markers/ and local people and insufficient basic research limit the ssrtool; and TRF, http://code.google.com/p/highssr/). successful conservation of the species although a small Primer3 was used to design primers for the sequences nature reserve has been established in Yining county. A detected by the 4 software programs simultaneously (Rozen recent report suggested that F. sinkiangensis has lost its and Skaletsky, 2000). ground in Manashi county, and the population size in 2.3. Amplification and sequencing Yining county has been reduced to 1/20th of its size in the Fifty pairs of primers were randomly selected to amplify 15 1980s (Huang et al., 2012). samples from the YN locality. The amplification used the With the availability of diverse molecular markers, such method of Schuelke (2000), based on the use of a forward as allozyme, amplified fragment length polymorphism SSR-specific primer with the 13M universal primer sequence (AFLP), random amplified polymorphic DNA (RAPD), (TGTAAAACGACGGCCAGT) at the 5’ end labeled with inter-simple sequence repeat (ISSR), single nucleotide 6-FAM. For DNA amplification, the SimpliAmp Thermal polymorphisms (SNP) and simple sequence repeat Cycler (Thermo Fisher Scientific, Waltham, MA, USA) was (SSR) analysis, the genetic diversity of several rare and used with initial denaturation at 95 °C for 5 min followed by endangered species has been widely studied (Hamrick and 10 cycles at 95 °C for 30 s (denaturation), at 58 °C for 30 s Godt, 1989; Nybom, 2004). The best molecular markers (annealing), and at 72 °C for 30 s (extension), then followed suited for detecting genetic variability should be relatively by 32 cycles at 95 °C for 30 s (denaturation), at 55 °C for 30 s easy to implement, inexpensive, and polymorphic enough. (annealing), and at 72 °C for 30 s (extension), with 5 min of As a codominant and polymorphism genetic marker, final extension at 72 °C. The PCR reactions were run with 20 microsatellites (SSRs) have been proven to be a reliable µL of reaction mixture containing 30 ng of template DNA, technique, capable of detecting the genetic diversity and 0.5 mM dNTPs, 2 µL of 10X PCR buffer (TransGen, Biotech), structures of rare and endangered plants (Yang et al., 10 pmol of each primer, and 0.5 U of Taq DNA polymerase. 2015; Nisar et al., 2017; Bilgen et al., 2019). In order to The PCR products were checked on a 1.0% agarose gel. The implement reasonable conservation strategies and promote primer set that had a successful amplification was used for effective management practices, the genetic diversity ofF. all collected samples. The PCR products were genotyped on sinkiangensis was researched. The aims of this study were an ABI3730 automatic sequencer using mixed molecular to develop and characterize microsatellite markers for F.