Development of polymorphic microsatellite markers for Fagus pashanica (Fagaceae) using next-generation sequencing

ABSTRACT

Fagus pashanica is an endangered and endemic tree species in China. To understand its genetic diversity and structure for effective conservation, we used next-generation sequencing data to develop a set of microsatellite markers. Twenty-three of the 68 designed loci were successfully amplified. Fifteen polymorphic loci with clear peaks were selected for further analyses in three F. pashanica populations sampled from Nanjiang, Wangcang and Pingwu counties in Sichuan Province, China. The number of alleles per locus ranged from two to 11. The levels of observed and expected heterozygosity ranged from 0.033–0.852 and 0.033–0.787, respectively. All 23 loci were also successfully amplified in F. longipetiolata and F. lucida, and 19 were successfully amplified in F. engleriana. These microsatellite markers will be useful for population genetic studies of F. pashanica and other Fagus species.

MAIN

Beeches (Fagus L., Fagaceae) are deciduous broadleaved trees that dominate certain temperate forests in the Northern Hemisphere (Denk, 2003). They are broadly distributed in three isolated regions: East Asia, Europe and West Asia, and North America (Fang and Lechowicz, 2006). There are four Fagus species in China (Huang et al., 1999): F. engleriana, F. longipetiolata, F. lucida and F. hayatae. All of these species mainly occur in the moist subtropical mountains in South China, between 700 m and 2,500 m altitude (Cao et al., 1995; Li and Li, 2008).

Fagus pashanica C. C. Yang is an endangered and endemic species in China that has been identified as a subspecies of F. hayatae (Huang et al., 1999; Ji et al., 2002). However, recent molecular phylogenetic analyses in Fagus suggested that F. pashanica should be a distinct species from F. hayatae in Taiwan Island (Li et al., 2003; Yang, 2020). Fagus pashanica has a more restricted and fragmented habitat than other Fagus species in China, and is distributed in isolated high mountains between 1,300 m and 1,900 m altitude in Sichuan, Hubei and Zhejiang provinces (Li and Li, 2008). Human activities and climate change have accelerated its habitat fragmentation and population decline. To effectively manage and conserve this species, we need to understand its genetic diversity and structure so that genetic conservation strategies (e.g., genetic rescue) can then be applied (Bell et al., 2019; Hoffmann et al., 2021).

Previous studies on F. pashanica are mainly about taxonomy, population characteristics and geographical distribution. Two used allozyme markers to study genetic diversity of several F. pashanica populations (Li et al., 1999; Li and Li, 2005). Because of the disadvantage of allozyme markers and the limitation of sample size, these reports have revealed little about the genetic diversity and structure of F. pashanica. Further studies with adequate sampling and sensitive molecular markers are required.

Microsatellites, also known as simple sequence repeats or short tandem repeats, are polymorphic, codominant markers that have been widely used in population genetic studies. With technical developments such as next-generation sequencing and multiplex PCR, the costs of identifying and genotyping large numbers of microsatellite loci in non-model species have been reduced greatly (Guichoux et al., 2011). In this study we identified microsatellite markers for F. pashanica based on Illumina sequencing genome data. We then used these markers to assess the genetic diversity of three F. pashanica populations and evaluated their cross-species application to F. longipetiolata, F. lucida and F. engleriana.

We used the genome sequencing data of 74 F. pashanica individuals from Yang (2020). These data were generated by an Illumina HiSeq 2500 with 150-bp paired-end sequencing. A total of 27,764,466 clean reads were used for de novo assembly using SOAPdenovo V2.04 (Luo et al., 2012). The scaffolds generated by de novo assembly were then used to detect microsatellite loci using QDD V3.1.2 (Meglécz et al., 2014) with default parameters. Primers were designed using QDD, taking into consideration the optimal PCR product size (100–200 bp), primer annealing temperature (57–63 ℃) and primer length (18–27 nucleotides).

To evaluate the microsatellite markers, samples of F. pashanica were collected from three natural populations from Nanjiang (NJ, n = 27), Wangcang (WC, n = 30) and Pingwu (PW, n = 28) counties in Sichuan Province. To evaluate the cross-species applicability of these markers, samples of three other Fagus species were collected: 30 individuals from one natural population of each species. The locality information of the sampled populations is detailed in Supplementary Table S1. Individuals were randomly selected and were at least 10 m apart from each other in one site. Fresh and healthy leaves of each individual were collected in a paper bag and dried with silica gel. Genomic DNA of all sampled individuals was extracted from the dried leaf tissues using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) and stored at −30 ℃.

Microsatellite locus validation had three steps. First, we used seven individuals from population WC of F. pashanica to test the success of amplification for the designed primer pairs. The 15-μl PCR reaction mixture contained 9.2 μl of deionized water, 1.5 μl of 10× buffer (Mg²⁺-free), 0.9 μl of MgCl₂ (25 mM), 1.2 μl of dNTP mixture (2.5 mM each), 0.5 μl of each primer (10 μM), 0.2 μl of Taq polymerase (5 U/μl, Takara Bio, Dalian, China) and 1 μl of DNA. Touchdown PCRs were performed under the following conditions: an initial denaturation of 4 min at 94 ℃; 10 cycles of 94 ℃ for 45 s, 63 ℃ for 45 s with a decrease of 1.3 ℃ per cycle, 72 ℃ for 40 s; 20 further cycles of 94 ℃ for 45 s, 50 ℃ for 45 s, 72 ℃ for 40 s; and a final extension at 72 ℃ for 8 min. The success of amplification was determined by electrophoresis using 1.5% agarose gels. Second, the forward primers of the successfully amplified primer pairs in all seven WC individuals were labeled with a fluorescent dye. DNA from all individuals collected from the three F. pashanica populations and three other Fagus species populations was used for PCR reactions using the above conditions. PCR products were scanned with an ABI 3730xl DNA Analyzer (Applied Biosystems) at Tsingke Biotechnology (Beijing, China), and genotyped by GENEMAPPER V4.0 (Applied Biosystems). For each Fagus species, microsatellite loci that could be successfully amplified in more than 90% of individuals and had clear peaks, suitable fragment lengths and polymorphism were further analyzed. Third, population genetic analyses were conducted. The number of alleles (N_A), Shannon’s information index (I), observed heterozygosity (H_O) and expected heterozygosity (H_E) were estimated using GenAlEx version 6.5 (Peakall and Smouse, 2012). Pairwise F_ST values were calculated using GenAlEx. Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium between microsatellite loci were tested using GENEPOP4.7.2 (Rousset, 2008). Frequencies of null alleles were estimated by Micro-Checker V2.2.3 (Van Oosterhout et al., 2004).

Twenty-three of the 68 primer pairs randomly selected from the designed primer pairs using QDD successfully amplified the target loci in the first step (Table 1). For F. pashanica, 15 primer pairs were finally selected for population genetic screening. All these loci had > 95% PCR amplification success rate (Table 2). The N_A per locus ranged from two to 11. The I values were from 0.085 to 1.688. The levels of H_O and H_E ranged from 0.033 to 0.852 and 0.033 to 0.787. Significant deviations from HWE (P < 0.05) were detected in five loci: F45 in population NJ; F66 and F68 in population WC; and F11, F34 and F45 in population PW. Potential inbreeding and genetic drift may have led to the deviations from HWE in small populations of F. pashanica. No significant linkage disequilibrium or null alleles were observed in any of the 15 loci. Among the three F. pashanica populations, NJ had the lowest mean value of N_A (3.9), but the highest mean values of I (0.889), H_O (0.459) and H_E (0.485) (Table 2). The pairwise F_ST values suggested moderate differentiation between populations NJ and WC (0.069), and between populations NJ and PW (0.084), but low differentiation between populations WC and PW (0.023). The transferability of the 23 primer pairs was tested across 90 individuals of F. lucida, F. longipetiolata and F. engleriana (Table 3). All the primer pairs successfully amplified the target loci, except loci F19, F43, F59 and F66 in F. engleriana. Amplifications of these four loci may have failed because F. engleriana belongs to another subgenus. These loci performed differently in population genetic analyses for different Fagus species (Table 3).

Table 1. Characteristics of 23 microsatellite loci of Fagus pashanica

Locus	Primer sequences (5′–3′)	Repeat motif	GenBank accession no.
F5	F: GCAACTTGGATTATGTTCTTCCAG	(AAT)8	OR020943
	R: TGTCCGTTAATAAAGAACTCGGG
F8	F: CCAGCAACTCTCGTTAACTAGC	(AAT)8	OR020944
	R: AACGAGATGCGCGTCTCTAC
F10	F: TGCCAAACAAGCGACAAACA	(AAC)8	OR020945
	R: AGCATGTGCAGAAGTTGTGG
F11	F: ACTCTCACCAGCTTTCAGGC	(AGG)7	OR020946
	R: ACACCTACACGTTATATCACACGA
F14	F: TGCCGCACTTAACCTCACAT	(ATC)7	OR020947
	R: CTCACAGCATAAAGATCTTATACCG
F16	F: GGGTGTTAGCTTCCTAATTTCCC	(AC)16	OR020948
	R: CAACAGGATTGAGTGAGATGCC
F18	F: GCATCACAGGTCTGTCCACA	(AG)13	OR020949
	R: GAGCAACATAGCATGTGGTTGA
F19	F: CTTGTATATTGCTTGGCTACTAGC	(AG)12	OR020950
	R: TGATGGAAAGTTGAGCCACC
F20	F: CTCAAACCCGACCTAACCCG	(AG)12	OR020951
	R: TCGTAACCGATACCAACCACA
F24	F: CAAGAAATGCAGAATAGGAGAGAGA	(AG)11	OR020952
	R: ATGGCTCACTGCTTCGTGAA
F27	F: ACGTCGATTTCTTTAACGGCT	(AG)10	OR020953
	R: CAATTTCCACACTCCGATGACC
F31	F: TCCAAACACCGAATAGCCCTC	(AG)9	OR020954
	R: TGAGAGGCGTTGTGAGGTTC
F34	F: AGGTGACTTGTGACGTGAATGA	(AG)9	OR020955
	R: AGAAAGAGCCATCCCTTTCCT
F37	F: ACTGAACACTTTCTAGAGCTTCGA	(AG)9	OR020956
	R: TGCAGTCAAACAAGACAACCA
F43	F: AACCCAGAGTTGCTAGTTGC	(AG)8	OR020957
	R: ACTTCCAAAGTTCGAAGTGCT
F45	F: TGAACATGTAGCTAGCCCGC	(AC)8	OR020958
	R: ATGCCTACTCATTCTGTGCGA
F47	F: GCCACGGGATGCTGAAAGAT	(AC)8	OR020959
	R: TGACGAGGTGGTGTTTGGG
F58	F: AGGCACGCCCACATACAATT	(AG)7	OR020960
	R: CAAGTCTACTTCATGATTCCCTCT
F59	F: ACTCGCCCATTGGTTAACCT	(AT)7	OR020961
	R: TAAGAGCTGCTGCCACAACA
F62	F: TCCCTTCGAGCTTGTTATGCA	(AG)7	OR020962
	R: ACTGAGTCTGAGTAGAATCCCAT
F64	F: GCCTCCTATTGGCCATCTTGA	(AG)7	OR020963
	R: CCTCTGCTACCTCCTCCCAT
F66	F: CACATGTGGAGATAAAGAGTCCA	(AG)7	OR020964
	R: ACCTGCTCTCGATTCTTTCCA
F68	F: ACCAGATGCCCTTCCATTGA	(AG)5	OR020965
	R: TGTTGCGGTGGTGGTTTAGT

Bold loci were successfully validated in three populations of F. pashanica.

Table 2. Genetic diversity statistics of 15 polymorphic microsatellite loci in three populations of Fagus pashanica

Locus	Population												Total (n = 85)		Mean			PCR amplification rate (%)
	NJ (n = 27)				WC (n = 30)				PW (n = 28)
	NA	I	HO	HE	NA	I	HO	HE	NA	I	HO	HE	NA	Size range (bp)	I	HO	HE
F8	4	1.302	0.630	0.711	3	0.992	0.567	0.593	4	1.103	0.500	0.622	5	164–176	1.132	0.565	0.642	100
F10	4	0.565	0.333	0.288	4	0.586	0.333	0.310	5	0.645	0.286	0.288	6	110–125	0.599	0.317	0.295	100
F11	5	1.030	0.407	0.501	4	0.844	0.367	0.451	5a	0.698	0.286	0.337	5	135–147	0.858	0.353	0.430	100
F16	6	1.193	0.667	0.606	10	1.525	0.700	0.656	8	1.093	0.500	0.489	11	160–184	1.270	0.622	0.584	100
F18	5	1.266	0.593	0.679	5	1.357	0.667	0.681	6	1.382	0.714	0.692	6	165–177	1.335	0.658	0.684	100
F19	2	0.593	0.240	0.403	2	0.287	0.100	0.153	2	0.652	0.429	0.459	2	151–157	0.511	0.256	0.338	98
F20	6	1.505	0.852	0.748	7	1.688	0.833	0.787	5	1.330	0.750	0.696	7	129–141	1.508	0.812	0.744	100
F24	5	0.669	0.296	0.298	4	0.748	0.433	0.387	5	0.870	0.393	0.448	6	171–181	0.762	0.374	0.378	100
F34	3	0.625	0.407	0.341	3	0.394	0.200	0.185	2a	0.090	0.036	0.035	4	189–195	0.370	0.214	0.187	100
F37	3	1.011	0.667	0.604	3	1.020	0.667	0.613	3	0.931	0.500	0.576	3	121–125	0.987	0.611	0.598	100
F45	4a	1.142	0.538	0.639	5	1.054	0.567	0.547	5a	1.018	0.429	0.505	6	164–176	1.072	0.511	0.564	99
F47	3	0.768	0.481	0.514	4	0.740	0.467	0.414	4	0.613	0.321	0.328	5	163–175	0.707	0.423	0.418	100
F62	3	0.730	0.333	0.426	4	0.748	0.300	0.387	2	0.441	0.250	0.270	4	155–163	0.639	0.294	0.361	100
F66	2	0.349	0.148	0.198	3a	0.563	0.300	0.309	3	0.346	0.179	0.165	4	126–142	0.419	0.209	0.224	100
F68	3	0.592	0.296	0.316	2a	0.085	0.033	0.033	2	0.154	0.071	0.069	3	109–129	0.277	0.134	0.139	100
Mean	3.9	0.889	0.459	0.485	4.5	0.842	0.436	0.434	4.1	0.758	0.367	0.399

N_A = number of alleles per locus; I = Shannon’s information index; H_O = observed heterozygosity; H_E = expected heterozygosity; n = number of individuals.

^a  Significant deviation from Hardy–Weinberg equilibrium (P < 0.05).

Table 3. Transferability of the 23 microsatellite markers developed from Fagus pashanica in F. longipetiolata, F. lucida and F. engleriana

	F. longipetiolata (n = 30)						F. lucida (n = 30)						F. engleriana (n = 30)
Locus	NA	Size range (bp)	I	HO	HE	PCR amplification rate (%)	NA	Size range (bp)	I	HO	HE	PCR amplification rate (%)	NA	Size range (bp)	I	HO	HE	PCR amplification rate (%)
F5	3	118–124	0.886	0.600	0.555	100	4	121–133	1.289	0.767	0.704	100	4c	118–130	1.079	0.567	0.613	100
F8	5	164–182	1.192	0.533	0.644	100	3abc	164–176	1.033	0.345	0.627	100	1	164	0.000	0.000	0.000	100
F10	4	111–126	0.781	0.633	0.477	100	4c	111–123	1.139	0.800	0.640	100	1	108	0.000	0.000	0.000	100
F11	4	135–147	1.220	0.867	0.683	100	6a	135–150	1.557	0.655	0.756	100	6	135–153	1.443	0.821	0.718	94
F14	3	157–163	0.755	0.500	0.505	100	3	154–163	0.485	0.267	0.240	100	3a	154–163	0.343	0.100	0.156	100
F16	5	160–168	1.170	0.567	0.578	100	6	160–172	1.307	0.667	0.648	100	5	160–168	1.170	0.567	0.578	100
F18	10	167–195	1.848	0.724	0.769	100	12	157–201	2.123	0.967	0.854	100	4c	157–179	1.110	0.667	0.610	100
F19	4	147–153	1.016	0.700	0.596	100	3abc	149–153	1.004	0.333	0.602	100	0	NA				0
F20	9c	125–151	1.843	0.759	0.810	100	7	123–143	1.688	0.867	0.798	100	7	117–143	1.511	0.800	0.697	100
F24	4c	172–178	0.967	0.667	0.537	100	4	172–192	0.472	0.233	0.214	100	7	172–200	1.456	0.690	0.707	100
F27	2	165–167	0.666	0.433	0.473	100	3	165–169	0.703	0.367	0.384	100	3c	164–168	0.843	0.533	0.483	100
F31	7	126–144	1.565	0.833	0.743	100	10	128–154	2.016	0.967	0.848	100	3	126–138	0.370	0.200	0.183	100
F34	4	185–191	0.818	0.433	0.512	100	7	187–207	1.461	0.900	0.713	100	5	189–203	0.869	0.667	0.508	100
F37	3	120–126	0.282	0.067	0.126	100	5c	120–128	1.391	0.900	0.731	100	6	151–171	1.461	0.733	0.720	100
F43	3	116–140	0.703	0.533	0.455	100	2a	136–138	0.085	0.033	0.033	100	0	NA				0
F45	5	162–174	1.133	0.533	0.621	100	4c	164–172	0.986	0.633	0.535	100	3	166–174	0.505	0.333	0.283	100
F47	4c	160–170	0.847	0.400	0.463	100	4c	166–192	1.085	0.700	0.593	100	3	160–164	0.230	0.100	0.096	100
F58	2ab	137–139	0.500	0.000	0.320	100	7a	107–141	1.565	0.667	0.735	100	11	139–177	1.561	0.800	0.659	100
F59	7	171–189	1.458	0.733	0.688	100	8c	173–235	1.652	0.833	0.781	100	0	NA				0
F62	2abc	155–157	0.451	0.133	0.278	100	3ac	155–159	1.038	0.533	0.629	100	2c	159–161	0.543	0.400	0.358	100
F64	3	166–176	0.703	0.600	0.455	100	4	172–178	0.922	0.500	0.501	100	1	166	0.000	0.000	0.000	100
F66	5c	134–144	1.279	0.607	0.686	94	3b	140–150	0.716	0.167	0.438	100	0	NA				0
F68	2	107–127	0.085	0.033	0.033	100	1	127	0.000	0.000	0.000	94	1	107	0.000	0.000	0.000	90

N_A = number of alleles per locus; I = Shannon’s information index; H_O = observed heterozygosity; H_E = expected heterozygosity; n = number of individuals; NA = unsuccessful PCR amplification.

Bold loci were successfully validated in three populations of F. pashanica.

^a  Significant deviation from Hardy–Weinberg equilibrium (P < 0.05).

^b  Null allele present.

^c  Significant linkage disequilibrium (P < 0.01).

In this study, we developed 23 microsatellite markers for F. pashanica using next-generation sequencing data and successfully validated 15 of them in three populations of F. pashanica. These markers will be useful for future genetic diversity and structure studies of F. pashanica, which are essential to effectively manage and conserve this species.

ACKNOWLEDGMENTS

This work was supported by the National Key R&D Program of China (2017YFA0605100) and the State Key Laboratory of Earth Surface Processes and Resource Ecology (2022-TS-04).

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