PDF(Pubmed)
Abstract：
Cassava Brown Streak Disease (CBSD), which is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), represents one of the most devastating threats to cassava production in Africa, including in Rwanda where a dramatic epidemic in 2014 dropped cassava yield from 3.3 million to 900,000 tonnes (1). Studying viral genetic diversity at the genome level is essential in disease management, as it can provide valuable information on the origin and dynamics of epidemic events. To fill the current lack of genome-based diversity studies of UCBSV, we performed a nationwide survey of cassava ipomovirus genomic sequences in Rwanda by high-throughput sequencing (HTS) of pools of plants sampled from 130 cassava fields in thirteen cassava-producing districts, spanning seven agro-ecological zones with contrasting climatic conditions and different cassava cultivars. HTS allowed the assembly of a nearly complete consensus genome of UCBSV in twelve districts. The phylogenetic analysis revealed high homology between UCBSV genome sequences, with a maximum of 0.8 per cent divergence between genomes at the nucleotide level. An in-depth investigation based on Single Nucleotide Polymorphisms (SNPs) was conducted to explore the genome diversity beyond the consensus sequences. First, to ensure the validity of the result, a panel of SNPs was confirmed by independent reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Furthermore, the combination of fixation index (FST) calculation and Principal Component Analysis (PCA) based on SNP patterns identified three different UCBSV haplotypes geographically clustered. The haplotype 2 (H2) was restricted to the central regions, where the NAROCAS 1 cultivar is predominantly farmed. RT-PCR and Sanger sequencing of individual NAROCAS1 plants confirmed their association with H2. Haplotype 1 was widely spread, with a 100 per cent occurrence in the Eastern region, while Haplotype 3 was only found in the Western region. These haplotypes\' associations with specific cultivars or regions would need further confirmation. Our results prove that a much more complex picture of genetic diversity can be deciphered beyond the consensus sequences, with practical implications on virus epidemiology, evolution, and disease management. Our methodology proposes a high-resolution analysis of genome diversity beyond the consensus between and within samples. It can be used at various scales, from individual plants to pooled samples of virus-infected plants. Our findings also showed how subtle genetic differences could be informative on the potential impact of agricultural practices, as the presence and frequency of a virus haplotype could be correlated with the dissemination and adoption of improved cultivars.
摘要：
木薯棕色条纹病（CBSD），由木薯褐条病毒（CBSV）和乌干达木薯褐条病毒（UCBSV）引起，代表了非洲木薯生产最具破坏性的威胁之一，包括在卢旺达，2014年的一次严重流行将木薯产量从330万吨降至90万吨（1）。在基因组水平上研究病毒遗传多样性对疾病管理至关重要。因为它可以提供有关流行病事件的起源和动态的有价值的信息。为了填补目前缺乏基于基因组的UCBSV多样性研究，我们通过高通量测序（HTS）从13个木薯产区的130个木薯田采样的植物池，对卢旺达的木薯类病毒基因组序列进行了全国调查，跨越七个农业生态区，具有不同的气候条件和不同的木薯品种。HTS允许在12个地区组装几乎完整的UCBSV共有基因组。系统发育分析揭示了UCBSV基因组序列之间的高度同源性,在核苷酸水平上，基因组之间的差异最大为0.8％。进行了基于单核苷酸多态性（SNP）的深入研究，以探索共有序列之外的基因组多样性。首先,为了确保结果的有效性，通过独立逆转录聚合酶链反应(RT-PCR)和Sanger测序确认了一组SNP.此外，基于SNP模式的固定指数（FST）计算和主成分分析（PCA）的组合确定了三种不同的UCBSV单倍型。单倍型2（H2）被限制在中心区域，NAROCAS1品种主要种植。单个NAROCAS1植物的RT-PCR和Sanger测序证实了它们与H2的关联。单倍型1分布广泛，在东部地区100%发生，而单倍型3仅在西部地区发现。这些单倍型与特定品种或地区的关联需要进一步确认。我们的结果证明，在共有序列之外，可以破译更复杂的遗传多样性图景，对病毒流行病学有实际影响，进化,和疾病管理。我们的方法提出了对基因组多样性的高分辨率分析，超出了样品之间和内部的共识。它可以用于各种规模，从单个植物到病毒感染植物的汇集样本。我们的发现还表明，微妙的遗传差异如何对农业实践的潜在影响提供信息，因为病毒单倍型的存在和频率可能与改良品种的传播和采用相关。
