Regular Article
Identification of a Heterozygous Microsatellite Marker in Pinus pumila
2020 年 85 巻 2 号 p. 131-134
詳細
2020 年 85 巻 2 号 p. 131-134
Pinus pumila, which habits around the timber line in Japanese mountains, propagates from adventitious roots of creeping or belowground stems and from seeds that are dispersed by animals. In an attempt to detect heterozygous simple sequence repeat (SSR) alleles in the nuclear genome of P. pumila, which may be useful to identify individuals in natural populations, we performed PCR using the DNA from this species and primer sets that have been reported for P. strobus SSR (Echt et al. 1996). Aligned subclone sequences originating from RPS150 product suggested that this locus included at least two [ga(t/g)]n alleles, [gat]3[gag]3 and [gat]3[gag]1[gat]1, while the direct sequence of RPS160 product included a single SSR allele of [acag]3[gcag]1[acag]3. This is the first report of the heterozygous nuclear SSR locus in P. pumila.
Pinus pumila is a member or five-needle pines, which are assigned to subgenus Strobus. Natural flora of this species is in the Far East surrounding the Japan Sea (Goncharenko et al. 1992). P. pumila proliferates sexually (Kajimoto et al. 1998) or vegetatively (Kajimoto 1992, Araki 1998, Tani et al. 1998). The ratio of these two proliferations in natural populations has been studied by synchronization pattern of annual shoot elongation (Araki 1998) or by allozymes, random amplified polymorphic DNA and inter-simple sequence repeat (Tani et al. 1998). However, the dominance of either propagation in populations has been difficult to determine. Nuclear SSR markers are transmitted into progenies according to the Mendelian law. Then tracing SSR in a population can detect the occurrence of mating in populations (Beaulieu and Simon 1994, Tani et al. 1996, Iwasaki et al. 2013). With the development of nucleotide sequencing technology, identification of SSRs has become within the reach of plant ecologists. Nuclear SSRs consisting of two-, three- or four-base units were reported in a Strobus species, P. strobus (Echt et al. 1996, 1997). In the annealing of PCR amplification, three- or four-base unit of SSRs may generate less slippages resulting in less nucleotide-sequence errors in DNA synthesis than two-base unit of SSRs.
In order to survey heterozygous SSR loci in P. pumila, we performed PCR using genome DNA in this species and primers that are reported to amplify three- or four-base units of SSRs in P. strobus. Based on the results obtained, we discuss the possible application of these SSR markers in future studies.
Leaves of P. pumila were collected in Autumn 2018 from a shrub located at latitude 36.114373° North and longitude 137.549717° East, which is 2776 m above sea level on Mt. Norikuradake. In a point at latitude 36.122462° North and longitude 137.555346° East, where is 443 m North-northeast of the above-mentioned site, another shrub was photographed December 18, 2019.
DNA extraction, PCR amplification, and nucleotide sequencingThe genome DNA was extracted according to a previous method (Uchida et al. 2007) with a slight modification from original protocol (Murray and Thompson 1980). Using 0.17 g in fresh weight of leaves, which had been stored in −80°C, 0.22 µg of the genome DNA was extracted. This extraction yield was at the same level in another extraction. PCR was performed using genome DNA, primer sets of either RPS150 or PRS160 (Echt et al. 1996) and Ex Taq Hot Start Version (TaKaRa). PCR cycle for RPS150 amplification was as follows; one cycle of 94°C for two min, 40 or 45 cycles of 95°C for 30 s, 52°C for 30 s and 72°C for one min and one cycle of 72°C for 10 min. That of RPS160 was the same as in RPS150, except the annealing temperature of 55°C. After removing primers in amplified products using UltraClean PCR Clean-Up Kit (MO BIO), direct sequencing was performed using either forward or reverse primers that were used for PCR unless otherwise stated. In an attempt to obtain a better forward RPS150 direct sequence, a 0.25-kb PCR product was excised from the gel, purified and was subjected to direct sequencing again. To obtain individual sequences in the PCR product of RPS150, this DNA fragment was cloned into pBluescript II KS (+) using NEBuilder HiFi DNA Assembly Master Mix (NEB). Forward and reverse PCR primers for the amplification of the overlapping insert are 5′-tagggcgaattgtccatcagtgagcagtggct-3′ and 5′-gaacaaaagctgcacttgggcttcctcttccc-3′, respectively (sequences annealing to insert are underlined). Those of the overlapping vector is 5′-gaagcccaagtgcagcttttgttccctttagt-3′ and 5′-ctcactgatggacaattcgccctatagtgagt-3′, respectively (sequences annealing to vector are underlined). In order to screen positive Escherichia coli clones, colonies obtained were subject to PCR amplification using vector-specific forward and reverse primers of 5′-gatgtgctgcaaggcgattaag-3′ and 5′-agttagctcactcattaggcac-3′, respectively, followed by another PCR using insert-specific forward and reverse primers of 5′-tccatcagtgagcagtggct-3′ and 5′-cacttgggcttcctcttccc-3′, respectively. Plasmids were isolated from positive clones using GenElute Plasmid Miniprep Kit (Sigma). The independent clones were subjected to dideoxy sequencing. Sequences obtained were aligned using MAFFT version 7 online (https://mafft.cbrc.jp/alignment/software/).
Figure 1 shows the shrub of curved and dwarf stems of P. pumila in Mt. Norikuradake (Fig. 1A). From a stem of this bush, leaves were sampled for DNA extraction. On the ground, tiny seedlings were formed adjacent to distributed corns (Fig. 1B). In another place, 443 m away from this spot, densely developed creeping stems of P. pumila were observed (Fig. 1C).
Figure 2A shows PCR amplification of RPS160 locus in P. pumila genome. In the PCR reaction with forward and reverse primers added, a 0.25-kb fragment was detected, while no specific bands were formed in the negative controls of no primer addition or of only one primer addition. In most region of electropherograms in forward and reverse direct sequences, Phred-quality scores were in higher levels (data not shown). Figure 2B shows parts of forward direct sequence in RPS160 product, including a simple sequence repeat (SSR) of [acag]3[gcag]1[acag]3. The corresponding complementary sequence was obtained in high Phred-quality scores when reverse RPS160 primer was used (Fig. 2C). Since species in the genus Pinus have a common chromosome number of 2n=24 (Shibata et al. 2016), RPS160 SSR obtained in this study suggests that RPS160 locus consists of the two homozygous SSR alleles, as was reported in counterpart in P. strobus (Echt et al. 1996, 1997, GenBank accession number U60257.1).
Figure 2D shows PCR amplification when RPS150 primer set was used. A 0.25-kb fragment was amplified when forward and reverse primers were used, while the corresponding band was not detected when no primer or only one primer was added. Figure 2E indicates a part of an electropherogram showing forward direct sequence derived from PCR reaction mixture from which primers were removed. Phred-quality scores were higher in the upstream of [gat]3[gag][ga(g/t)] and drastically fell down downstream of this repeat (Fig. 2E). Clear, discreate and higher peaks of nucleotides were observed upstream of this SSR, while different colors of shorter peaks overlapped in the following. This result was reproduced in the direct sequence which derived from a 0.25-kb band excised from the gel. Then we cloned this PCR product into pBluescript KS II(+), performed nucleotide sequencing of 12 independent plasmids and aligned these sequences (Fig. 2F). Two boxes in Fig. 2F suggest two types of [ga(t/g)]n SSR alleles in RPS150 locus, [gat]3[gag]3 and [gat]3[gag]1[gat]1. This result is in contrast to the previous report that P. strobus RPS150 locus possesses a single allele having SSR of [gag]4 (Echt et al. 1996, GenBank accession number U60256.1). Though there was a study that detected single-strand conformation polymorphisms in P. pumila nuclear genome (Watano et al. 2004), there have been no reports to detect different sequences of an SSR allele. This is the first report showing heterozygous SSR sequence in this plant.
Characterization of hetero- or homozygous loci in seeds or in the different generations of seedlings is expected to reveal the dominance of sexual or vegetative proliferation in populations. Future analyses of RPS150 locus in more samples from the field may cast insight to reveal this problem (Kajimoto 1992, Tani et al. 1996, Araki 1998). Since intra-species crossing is often observed in Pinus species (Watano et al. 1996, Senjo et al. 1999, Gugerli et al. 2001, Goroshkevich 2004, Watano et al. 2004, Ito et al. 2008, Iwasaki et al. 2013), RPS150 locus can also be traced in the hybrids from P. pumila and its relatives to evaluate industrially important lines such as in pest resistance, lumber production, edible seed supply or bonsai breeding.
This work was supported in part by a grant from Institute for Cosmic Ray Research to EM, by Grant-in-Aid for JSPS KAKENHI Grant 15K00642 to KI and by Research and Investigation Promotion Fund from the Japan Securities Scholarship Foundation in 2011 (10-069) to H.U.
