González Víctor M, Aventín Núria, Centeno Emilio, Puigdomènech Pere
Molecular Genetics Department, Center for Research in Agricultural Genomics CRAG (CSIC-IRTA-UAB-UB), Campus UAB, Edifici CRAG, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain.
BMC Genomics. 2014 Dec 17;15(1):1131. doi: 10.1186/1471-2164-15-1131.
Plant NBS-LRR -resistance genes tend to be found in clusters, which have been shown to be hot spots of genome variability. In melon, half of the 81 predicted NBS-LRR genes group in nine clusters, and a 1 Mb region on linkage group V contains the highest density of R-genes and presence/absence gene polymorphisms found in the melon genome. This region is known to contain the locus of Vat, an agronomically important gene that confers resistance to aphids. However, the presence of duplications makes the sequencing and annotation of R-gene clusters difficult, usually resulting in multi-gapped sequences with higher than average errors.
A 1-Mb sequence that contains the largest NBS-LRR gene cluster found in melon was improved using a strategy that combines Illumina paired-end mapping and PCR-based gap closing. Unknown sequence was decreased by 70% while about 3,000 SNPs and small indels were corrected. As a result, the annotations of 18 of a total of 23 NBS-LRR genes found in this region were modified, including additional coding sequences, amino acid changes, correction of splicing boundaries, or fussion of ORFs in common transcription units. A phylogeny analysis of the R-genes and their comparison with syntenic sequences in other cucurbits point to a pattern of local gene amplifications since the diversification of cucurbits from other families, and through speciation within the family. A candidate Vat gene is proposed based on the sequence similarity between a reported Vat gene from a Korean melon cultivar and a sequence fragment previously absent in the unrefined sequence.
A sequence refinement strategy allowed substantial improvement of a 1 Mb fragment of the melon genome and the re-annotation of the largest cluster of NBS-LRR gene homologues found in melon. Analysis of the cluster revealed that resistance genes have been produced by sequence duplication in adjacent genome locations since the divergence of cucurbits from other close families, and through the process of speciation within the family a candidate Vat gene was also identified using sequence previously unavailable, which demonstrates the advantages of genome assembly refinements when analyzing complex regions such as those containing clusters of highly similar genes.
植物NBS-LRR抗性基因往往成簇存在，这些基因簇已被证明是基因组变异的热点区域。在甜瓜中，81个预测的NBS-LRR基因中有一半归为9个基因簇，五号连锁群上一个1兆碱基区域包含甜瓜基因组中最高密度的R基因以及存在/缺失基因多态性。已知该区域包含Vat基因座，Vat是一个在农业上具有重要意义的基因，可赋予对蚜虫的抗性。然而，重复序列的存在使得R基因簇的测序和注释变得困难，通常会导致具有高于平均错误率的多缺口序列。
采用结合Illumina双末端映射和基于PCR的缺口封闭策略，对甜瓜中发现的包含最大NBS-LRR基因簇的1兆碱基序列进行了改进。未知序列减少了70%，同时约3000个单核苷酸多态性（SNP）和小插入缺失得到了校正。结果，该区域总共23个NBS-LRR基因中的18个基因的注释得到了修改，包括额外的编码序列、氨基酸变化、剪接边界校正或共同转录单元中开放阅读框的融合。对R基因的系统发育分析以及它们与其他葫芦科植物的同线序列的比较表明，自葫芦科与其他科分化以来，以及通过科内物种形成，存在局部基因扩增模式。基于韩国甜瓜品种报道的Vat基因与未精炼序列中先前不存在的序列片段之间的序列相似性，提出了一个候选Vat基因。
一种序列优化策略使甜瓜基因组的1兆碱基片段得到了实质性改进，并对甜瓜中发现的最大NBS-LRR基因同源物簇进行了重新注释。对该基因簇的分析表明，自葫芦科与其他近缘科分化以来，抗性基因是由相邻基因组位置的序列重复产生的，并且通过科内物种形成过程，还利用先前不可用的序列鉴定了一个候选Vat基因，这证明了在分析包含高度相似基因簇的复杂区域时，基因组组装优化的优势。
