Interactions between root knot nematodes and potato. Solanum tuberosum

Plant-parasitic nematodes and especially the root-knot nematodes are ubiquitous pathogens. Eggs of root-knot nematodes (Meloidogyne javanica) were extracted from greenhouse cultures and second-stage juveniles (J2) were hatched in tap, sterile water on 30 µm sieves. Solanum tubersum cv. Desiree explants, i.e., stem including an axillary bud, were sectioned, under sterile conditions from in vitro growing seedlings and transferred to Magenta boxes containing Gamborg's media. Seedlings, 4 weeks post transferring to Gamborg's media were planted in pots containing autoclaved quartz sand. Three weeks later the plants were infected with 3000 M. javanica infective juveniles, applied to the soil in tap, sterile water. Control uninfected plants were mock-inoculated with tap, sterile water. Roots from 6 infected and 6 mock-inoculated plants were collected at a series of time points, including 5, 10 and 15 days post nematode infection/mock inoculation. Roots were observed under the microscope, and nematode feeding sites were selectively dissected, from young lateral roots. To control for the effect of tissue sectioning on gene expression, young lateral roots of the mock inoculated roots were dissected similarly to the infected roots, and collected. Dissected roots were snapped-freeze in liquid nitrogen and immediately stored in -80 C freezer. Total RNA was extracted using Qiagen RNAEasy kit. The experiment for each time point was duplicated, each duplicate derived from independent biological repeat. All RNA samples were amplified using the Ambion kit Message Amp catalog no. 1750, using as starting material 2.5 to 5 µg of total RNA. Keywords: Reference design Overall design: 13 hybs total

植物寄生线虫，尤其是根结线虫，是广泛分布的病原物。本实验从温室培养物中提取爪哇根结线虫（Meloidogyne javanica）的卵，并将卵置于30 µm筛上的无菌自来水中孵化，获得二龄幼虫（second-stage juveniles，J2）。以德西蕾品种马铃薯（Solanum tubersum cv. Desiree）的外植体——即带腋芽的茎段——为材料，从组培苗中无菌切割获取外植体，转接至含Gamborg培养基（Gamborg's media）的Magenta盒中培养。将转接至Gamborg培养基后培养4周的幼苗移栽至装有高压灭菌石英砂的花盆中。三周后，用3000头爪哇根结线虫侵染性幼虫的无菌自来水悬液接种土壤；未感染的对照植株以无菌自来水进行模拟接种。分别在接种/模拟接种后的第5、10、15天采集样本，收集6株感染植株与6株模拟接种植株的根系。在显微镜下观察根系后，从幼嫩侧根中选择性分离线虫取食位点；为控制组织切片操作对基因表达的影响，同时以相同方式分离并收集模拟接种植株的幼嫩侧根。分离得到的根系经液氮快速冷冻后，立即保存于-80℃冰箱中。总RNA采用凯杰RNAeasy提取试剂盒（Qiagen RNAEasy kit）提取。每个时间点的实验均设置两次生物学重复，每个重复样本均来自独立的生物学重复实验。所有RNA样本均以2.5~5 μg总RNA为起始材料，使用安比昂Message Amp试剂盒（货号1750，Ambion kit Message Amp catalog no. 1750）进行扩增。关键词：参考设计（Reference design）；实验设计概况：总计13次杂交实验。