Orquera-Tornakian Gabriela K, Garrido Patricia, Kronmiller Brent, Hunger Robert, Tyler Brett M, Garzon Carla D, Marek Stephen M
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, United States.
Centro de Investigación de Alimentos, CIAL, Facultad de Ciencias de la Ingeniería e Industrias, Universidad Tecnológica Equinoccial, EC171029 Quito, Ecuador.
J Microbiol Methods. 2017 Aug;139:113-122. doi: 10.1016/j.mimet.2017.04.011. Epub 2017 Apr 27.
Switchgrass (Panicum virgatum L.) can be severely affected by rust disease. Recently switchgrass rust caused by P. emaculata (now confirmed to be Puccinia novopanici) has received most of the attention by the research community because this pathogen is responsible for reducing the biomass production and biofuel feedstock quality of switchgrass. Microsatellite markers found in the literature were either not informative (no allele frequency) or showed few polymorphisms in the target populations, therefore additional markers are needed for future studies of the genetic variation and population structure of P. novopanici. This study reports the development and characterization of novel simple sequence repeat (SSR) markers from a Puccinia emaculata s.l. microsatellite-enriched library and expressed sequence tags (ESTs). Microsatellites were evaluated for polymorphisms on P. emaculata s.l. urediniospores collected in Iowa (IA), Mississippi (MS), Oklahoma (OK), South Dakota (SD) and Virginia (VA). Puccinia novopanici single spore whole genome amplifications were used as templates to validate the SSR reactions protocol and to assess a preliminary population genetics statistics of the pathogen. Eighteen microsatellite markers were polymorphic (average PIC=0.72) on individual urediniospores, with an average of 8.3 alleles per locus (range 3 to 17). Of the 49 SSRs loci initially identified in P. emaculata s.l., 18 were transferable to P. striiformis f. sp. tritici, 23 to P. triticina, 20 to P. sorghi and 31 to P. andropogonis. Thus, these markers could be useful for DNA fingerprinting and population structure analysis for population genetics, epidemiology and ecological studies of P. novopanici and potentially other related Puccinia species.
柳枝稷（Panicum virgatum L.）极易受到锈病的影响。最近，由黄斑柄锈菌（现确认是新柳枝稷柄锈菌Puccinia novopanici）引起的柳枝稷锈病受到了研究界的广泛关注，因为这种病原菌会导致柳枝稷生物量产量下降以及生物燃料原料质量降低。文献中发现的微卫星标记要么信息量不足（没有等位基因频率），要么在目标群体中显示出很少的多态性，因此，未来对新柳枝稷柄锈菌的遗传变异和群体结构研究需要更多的标记。本研究报告了从黄斑柄锈菌复合种（Puccinia emaculata s.l.）的微卫星富集文库和表达序列标签（ESTs）中开发新型简单序列重复（SSR）标记并对其进行特征分析的过程。对从爱荷华州（IA）、密西西比州（MS）、俄克拉荷马州（OK）、南达科他州（SD）和弗吉尼亚州（VA）采集的黄斑柄锈菌复合种夏孢子上的微卫星进行了多态性评估。使用新柳枝稷柄锈菌单孢子全基因组扩增产物作为模板来验证SSR反应方案，并评估该病原菌的初步群体遗传学统计数据。18个微卫星标记在单个夏孢子上具有多态性（平均PIC = 0.72），每个位点平均有8.3个等位基因（范围为3至17）。在最初在黄斑柄锈菌复合种中鉴定出的49个SSR位点中，18个可转移至条形柄锈菌小麦专化型（P. striiformis f. sp. tritici），23个可转移至小麦叶锈菌（P. triticina），20个可转移至高粱柄锈菌（P. sorghi），31个可转移至须芒草柄锈菌（P. andropogonis）。因此，这些标记可用于新柳枝稷柄锈菌以及潜在的其他相关柄锈菌物种的群体遗传学、流行病学和生态学研究中的DNA指纹分析和群体结构分析。
