Simple Sequence Repeat Markers Distinguish among Morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5′ end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49°C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.

松壳色二孢（Sphaeropsis sapinea）是松属（Pinus spp.）的真菌内生菌（fungal endophyte），可在宿主树木遭受生物或非生物胁迫诱发易感后引发病害。该真菌自然分布范围内已报道存在4种形态型（morphotype），而在南半球的外来松树上仅发现1种形态型。本研究旨在开发可同时应用于分类学与种群研究的稳定多态性分子标记。采用携有微卫星（microsatellite）序列且5'端带有简并锚定序列的分子间短串联重复序列（Inter-short-sequence-repeat）引物，靶向扩增松壳色二孢分离株中富集微卫星的基因组区域。以49℃退火温度进行PCR扩增，可获得包含5至10条条带的扩增图谱。对上述条带进行克隆与测序后，设计出了锚定富集微卫星区域两侧的新型短序列重复（SSR）引物对。针对11个多态性SSR标记，我们对40株代表不同形态型的松壳色二孢分离株，以及2株近缘物种葡萄座腔菌（Botryosphaeria obtusa）分离株开展了扩增验证。推测的I型形态型被证实与葡萄座腔菌（Botryosphaeria obtusa）完全一致。除此之外，该标记组可清晰区分剩余的3种形态型，且结果显示C型形态型与A型形态型的亲缘关系较B型更近。B型形态型的遗传多样性最高，且分离株可依据其地理来源进一步聚类分组。对每个位点的不同等位基因进行测序分析后发现，多态性最高的标记其微卫星序列内部存在突变。