Molecular Genetics Department, Center for Research in Agricultural Genomics CRAG, CSIC-IRTA-UAB, Jordi Girona 18-26, 08034 Barcelona, Spain.
BMC Plant Biol. 2010 Nov 12;10:246. doi: 10.1186/1471-2229-10-246.
Cucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has a high intra-specific genetic variation, morphologic diversity and a small genome size (454 Mb), which make it suitable for a great variety of molecular and genetic studies. A number of genetic and genomic resources have already been developed, such as several genetic maps, BAC genomic libraries, a BAC-based physical map and EST collections. Sequence information would be invaluable to complete the picture of the melon genomic landscape, furthering our understanding of this species' evolution from its relatives and providing an important genetic tool. However, to this day there is little sequence data available, only a few melon genes and genomic regions are deposited in public databases. The development of massively parallel sequencing methods allows envisaging new strategies to obtain long fragments of genomic sequence at higher speed and lower cost than previous Sanger-based methods.
In order to gain insight into the structure of a significant portion of the melon genome we set out to perform massive sequencing of pools of BAC clones. For this, a set of 57 BAC clones from a double haploid line was sequenced in two pools with the 454 system using both shotgun and paired-end approaches. The final assembly consists of an estimated 95% of the actual size of the melon BAC clones, with most likely complete sequences for 50 of the BACs, and a total sequence coverage of 39x. The accuracy of the assembly was assessed by comparing the previously available Sanger sequence of one of the BACs against its 454 sequence, and the polymorphisms found involved only 1.7 differences every 10,000 bp that were localized in 15 homopolymeric regions and two dinucleotide tandem repeats. Overall, the study provides approximately 6.7 Mb or 1.5% of the melon genome. The analysis of this new data has allowed us to gain further insight into characteristics of the melon genome such as gene density, average protein length, or microsatellite and transposon content. The annotation of the BAC sequences revealed a high degree of collinearity and protein sequence identity between melon and its close relative Cucumis sativus (cucumber). Transposon content analysis of the syntenic regions suggests that transposition activity after the split of both cucurbit species has been low in cucumber but very high in melon.
The results presented here show that the strategy followed, which combines shotgun and BAC-end sequencing together with anchored marker information, is an excellent method for sequencing specific genomic regions, especially from relatively compact genomes such as that of melon. However, in agreement with other results, this map-based, BAC approach is confirmed to be an expensive way of sequencing a whole plant genome. Our results also provide a partial description of the melon genome's structure. Namely, our analysis shows that the melon genome is highly collinear with the smaller one of cucumber, the size difference being mainly due to the expansion of intergenic regions and proliferation of transposable elements.
甜瓜属于葫芦科，其在园艺作物中的经济重要性仅次于茄科。甜瓜具有高度的种内遗传变异、形态多样性和较小的基因组大小（454Mb），这使其适合进行各种分子和遗传研究。已经开发了许多遗传和基因组资源，例如几个遗传图谱、BAC 基因组文库、基于 BAC 的物理图谱和 EST 集。序列信息对于完成甜瓜基因组图谱的绘制将是非常有价值的，这将有助于我们进一步了解该物种与其亲缘物种的进化，并为其提供重要的遗传工具。然而，时至今日，可用的序列数据仍然很少，只有少数甜瓜基因和基因组区域被存入公共数据库。大规模平行测序方法的发展使得人们可以设想新的策略，以比以前基于 Sanger 的方法更快、更低成本地获得基因组序列的长片段。
为了深入了解甜瓜基因组的重要部分的结构，我们着手对 BAC 克隆池进行大规模测序。为此，我们使用 454 系统对来自双单倍体系的 57 个 BAC 克隆进行了两个池的测序，采用了鸟枪法和配对末端法。最终组装估计包含甜瓜 BAC 克隆实际大小的 95%，其中 50 个 BAC 很可能具有完整的序列，总序列覆盖率为 39x。通过将一个 BAC 的先前可用的 Sanger 序列与 454 序列进行比较，评估了组装的准确性，发现的多态性仅涉及每 10000bp 中有 1.7 个差异，这些差异定位于 15 个同聚区域和两个二核苷酸串联重复。总体而言，该研究提供了大约 6.7Mb 或甜瓜基因组的 1.5%。对这些新数据的分析使我们能够进一步了解甜瓜基因组的特征，如基因密度、平均蛋白质长度或微卫星和转座子含量。BAC 序列的注释显示，甜瓜与其近缘种黄瓜（黄瓜）之间存在高度的共线性和蛋白质序列同一性。对同线性区域的转座子含量分析表明，在两种葫芦科物种分化后，黄瓜中转座子的活性很低，但在甜瓜中转座子的活性非常高。
这里呈现的结果表明，所采用的策略，结合鸟枪法和 BAC 末端测序以及锚定标记信息，是一种极好的方法，可用于对特定基因组区域进行测序，特别是对像甜瓜这样相对紧凑的基因组。然而，与其他结果一致，这种基于图谱的 BAC 方法被证实是一种昂贵的测序整个植物基因组的方法。我们的结果还提供了甜瓜基因组结构的部分描述。即，我们的分析表明，甜瓜基因组与较小的黄瓜基因组高度共线性，大小差异主要是由于基因间区域的扩展和转座因子的增殖所致。
