De novo transcriptome analysis of weed Apera spica-venti from seven tissues and growth stages

Loose silky bentgrass (Apera spica-venti) is an important weed in Europe. It can cause up to 30% yield loses. There is little molecular information available about this species. The lack of genetic information limits the biological understanding of this important noxious weed species such as growth, genetic variation, reproduction, biosynthesis of metabolites and metabolic mechanisms of herbicide resistance. This study produced a reference transcriptome for A. spica-venti from different tissues (leaf, root, stem) and various growth stages (seed at phenological stages 05, 07, 08, 09). Seeds from six herbicide susceptible populations of Apera spica-venti originating at six different locations in Denmark, were mixed together to create a meta-population encompassing a range of genetic variations possible within the species. Each of these six populations were previously tested between 2007 and 2010 with ALS and ACCase inhibitor herbicides and found to be susceptible (Mathiassen, pers. comm). Seeds were sown under greenhouse conditions on table with an automatic watering system. Leaf, stem, and root material from individual plant were harvested once the plants were at BBCH stage 34 to 36 (stem elongation). Tissues were cut in small pieces and immediately put in liquid nitrogen and then stored at -80°C. These tissues were kept separated by individual plant. Seeds from this meta-population were sown in 9 cm petri dishes with four filter papers and grown in a climate cabinet (Termaks Series KB8000L) at night/day temperatures of 10ºC /17ºC for 10h/14h, and imbibed with 5 mL of demineralized water. Germinated seeds were harvested at stage BBCH 05 (radicle emerges from seed), 07 (coleoptile emerges from caryopsis), 08 (coleoptile elongation), and 09 (Emergence: coleoptile breaks through soil surface). A total of 0.5 g of frozen tissue from leaf, stem, and root was grounded separately using a 2010 Geno/Grinder (SPEX Sample prep, Stanmore, UK) for 45 sec at 1,500 hertz and then soaked in liquid nitrogen. A total of 0.7 g of germinated seeds were grounded using the same method but for 2 cycles of 45 seconds at 1,500 hertz and were not soaked in liquid nitrogen. The RNA mini plant kit (Qiagen, Hilsen, Germany) was used for RNA extraction following the manufacturer protocol, three wash steps were performed. RNA quality was verified using a spectrophotometer (Nanodrop 3300, Wilmington, USA) and bioanalyzer (Agilent 2100, Santa Clara, USA). Samples that showed a RIN value of 6.0 and more were treated with DNAse 1 according to the protocol (Qiagen, Hilsen, Germany) and suspended in a final volume of 14 μL. RNA quality was verified again with spectrophotometer (NanoDrop-1000 v.3.1.0) and bioanalyzer using the RNA 6000 Plant Nano program (Agilent 2100). Samples with a RIN value higher than 6 were selected for cDNA library construction. The lower than recommended (>8) RIN value threshold selected here is because of difficulties in extracting high quality RNA from germinated seed samples. Nine libraries were created; seven for each of the individual tissues/growth stage samples, and two more libraries for leaf and root respectively to be sequenced by both 5’ and 3’ end for analysis of transcript start site because we believe they have the most important number of expressed genes of interest for future study. Samples of mRNA were selected and fragmentedand transformed to 150-400 bp short insert, strand specific cDNA libraries for sequencing on Illumina HiSeq 2500 with chemistry version 3 (Eurofins MGW, Germany)

柔穗剪股颖（Loose silky bentgrass, Apera spica-venti）是欧洲地区的重要有害杂草，可导致作物最高达30%的产量损失。目前关于该物种的分子生物学信息极为匮乏，遗传信息的缺失限制了人们对这一重要有害杂草的生物学认知，包括其生长、遗传变异、繁殖、代谢物生物合成以及除草剂抗性代谢机制等多个维度。
本研究针对Apera spica-venti构建了参考转录组，采样覆盖不同组织（叶片、根、茎）以及多个生长阶段（物候期05、07、08、09的种子）。研究人员将来自丹麦6个不同地点的6个除草剂敏感种群的种子混合，构建出一个囊括该物种内全部潜在遗传变异的混合种群。上述6个种群曾于2007至2010年间经乙酰乳酸合酶（ALS）和乙酰辅酶A羧化酶（ACCase）抑制剂类除草剂测试，被证实为敏感种群（Mathiassen，私人通信）。
种子先于温室条件下播种在配备自动浇水系统的栽培台面上：当植株生长至BBCH分期34至36阶段（茎伸长阶段）时，采集单株的叶片、茎秆与根组织。将组织切成小块后立即投入液氮中速冻，随后保存于-80℃环境，所有组织均按单株分别留存。
另外将该混合种群的种子播种于铺有4层滤纸的9cm培养皿中，置于气候箱（Termaks Series KB8000L）内培养，设置昼夜温度为10℃/17℃、光照时长为10h/14h，并添加5mL去离子水进行浸种。分别在BBCH分期05（胚根突破种皮）、07（胚芽鞘突破颖果）、08（胚芽鞘伸长）以及09（出苗：胚芽鞘突破土壤表面）阶段收获萌发的种子。
称取0.5g冷冻的叶片、茎秆及根组织，分别使用2010型Geno/Grinder研磨仪（SPEX Sample Prep，英国斯坦莫尔）以1500赫兹频率研磨45秒，研磨过程中始终浸泡于液氮中。称取0.7g萌发种子，采用相同方法研磨，但需进行两轮45秒的研磨操作，且研磨过程中无需浸泡于液氮。
采用RNA微型植物提取试剂盒（Qiagen，德国希尔登），按照制造商说明书进行RNA提取，共完成3次洗涤步骤。使用分光光度计（Nanodrop 3300，美国威尔明顿）与生物分析仪（Agilent 2100，美国圣克拉拉）验证RNA质量。对于RNA完整性指数（RIN）≥6.0的样品，按照试剂盒说明书（Qiagen，德国希尔登）使用DNA酶I进行处理，最终将样品悬浮于14μL体系中。再次使用分光光度计（NanoDrop-1000 v.3.1.0）与搭载RNA 6000 Plant Nano程序的Agilent 2100生物分析仪复核RNA质量，选取RIN值大于6的样品用于cDNA文库构建。本次选取的RIN阈值低于官方推荐值（>8），原因在于从萌发种子样品中提取高质量RNA存在较大难度。
共构建9个测序文库：7个对应不同组织/生长阶段的样品，另外分别构建2个叶片与根组织的文库，采用5’端与3’端双端测序策略以分析转录起始位点——我们认为该区域包含未来研究所需的大量关键表达基因。
最终选取mRNA进行片段化，构建插入片段长度为150-400bp的链特异性cDNA文库，在Illumina HiSeq 2500平台上采用V3化学试剂进行测序（Eurofins MGW，德国）。