为了搏眼球，起了一个感觉很专业的名字。其实也是通过读文章，学学大牛的科研思路。
继挪威云杉基因组2013年发表在Nature上之后，终于又有裸子植物基因组发表在顶级期刊cell上了。裸子植物基因组大，组装难度大，虽然之前发表了银杏，红豆杉，巨杉等裸子植物文章，但都没有做到顶级期刊的标准。那我们就看看这篇文章都做了哪些内容，能让它发表在cell上呢？
文章名称“The Chinese pine genome and methylome unveil key features of conifer evolution”
文章地址：https://www.sciencedirect.com/science/article/pii/S0092867421014288?
摘要：
Conifers dominate the world’s forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers’ adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.
油松基因组长达25.4G，有12条染色体。裸子植物的基因组都很大（17–35 Gb），这是由于TE扩张导致的。除了基因间的TE，油松基因内含子也包含TE。裸子植物内含子与被子植物比较长的多，之前的裸子植物文章也有研究。此文章还提到，拥有长内含子的基因表达量也很高。高水平的甲基化使TE消除速度减慢，从而导致油松基因组扩张。
组装及注释结果：
油松内含子平均长度为10,034bp，其中大于10Kb的内含子有29,883个。于是作者选了67个其它物种，比较他们基因组大小，基因，外显子，内含子长度等指标。
“We found a positive correlation between the ratio of total intron/exon length with the ge nome’s size, especially in the gymnosperm plants (Figure 2A), indicating that the genome expansion not only occurs in the intergenic region but also in the genic region.”
作者发现，基因组大小与总内含子长度/总外显子长度成正相关关系，尤其在裸子植物中。这表明基因组的扩张不止发生在基因间区，还发生在基因区。
作者真是不吝啬劳动，一下统计了67个物种，佩服。而我总想着少干活，惭愧。
We assessed the annotation completeness of P. tabuliformis using both BUSCO genome model and protein model. The result showed that BUSCO covered 84% of complete genes in the protein model in contrast to 44.5% in the genome model. We compared the gene sets that could be recognized as complete by both BUSCO protein and genome model (Pc-Gc) with the gene sets that could only be recognized by protein mode (Pc-Gm). We found that most super long genes with multi-introns were not detected under genome model but were recognized in protein model, indicating that multiple long introns are the pri�mary causes of low BUSCO genome completeness (Figure 2B).
通过BUSCO对注释的完整性进行了评估，蛋白模型的覆盖了84%的完整基因而基因组模型只覆盖了44.5%。同过比较，是因为拥有许多内含子的超大基因在基因组模型下没有检测到。（os:按照这种说法那是BUSCO这个软件自己有问题了？）
To study whether such extraordinarily long introns would disrupt transcription, we divided genes into two groups by the sizes of the first introns and found that the genes with longer first introns always had relatively higher expression levels in all eleven studied organs/tissues than those with shorter introns (Figure S2B).
为了验证超长内含子是否会阻断转录，作者根据首个内含子长度将基因分成两组，结果显示拥有长的首个内含子的基因表达量在所有11个研究的器官/组织中相对高。
（简书设置文字颜色的方法，需要用到插入公式 \color{red}{你要写的文字} ）
To gain insight into the gene-expression recognition mechanism of small exons from super-long introns in conifers, we manually checked the RNA-junction and DNA methylation patterns of the 10 long genes. Large amounts of RNA-junction data confirm that small exons can be accurately identified and transcribed in a huge DNA that was thousands of times longer than exons (Figure S2E). It is noteworthy that almost all CG and CHG sites in long introns were methylated, whereas exon regions were marked by low methylation levels, especially for the CHG context (Figure S2F), indicating that DNA methylation was probably involved in the accurate exon recognition from super-long introns.
作者为了探究从超长内含子中识别小外显子的基因表达机制，查看了10个长基因的RNA连接和DNA甲基化模式。长内含子的CG和CHG几乎全被甲基化，而外显子为低甲基化。
