重要食药同源植物余甘子转录组微卫星特征分析

为全面了解余甘子转录组SSR位点的分布特征和变异规律,本研究利用Illumina Hiseq 4000平台对余甘子叶片转录组进行测序,通过MISA软件对获得的Unigenes进行SSR位点搜索和统计分析。结果发现9 538条包含SSR位点的Unigenes,共检测到9 991个SSR位点,平均每5.49 kB出现1个SSR。单碱基和二碱基为余甘子转录组SSR主要重复类型,分别占SSR总数的42.3%和30.79%。位于基因编码区的SSR位点共有1 731个,出现频率为0.039 SSRs/kB,优势重复类型为三碱基重复。余甘子转录组SSR中共有169种重复基元,其中所占比例最高的是A/T（42.10%）,其次是AG/CT（22.91%）和AAG/CTT（5.02%）。SSR各基元的重复次数波动于4~75次,且多数集中于4~20次。重复片段长度≥ 20 bp的SSR占21.20%,且SSR发生频率与片段长度呈显著负相关（P<0.01）,相关系数为-0.561。本研究获得的余甘子转录组SSR位点出现频率较高、分布密度较大、低级重复基元较多,重复次数较高、长片段较多,大多数SSR位点的多态性潜能较高,用于余甘子遗传多样性分析的潜力较大,为下一步余甘子转录组SSR标记的大规模开发和群体遗传学研究提供了重要的数据信息,进而为余甘子野生资源的保护和合理开发利用提供了参考依据。     

Genome-Wide Characterization and Linkage Mapping of Simple Sequence Repeats in Mei (Prunus mume Sieb. et Zucc.)

Because of its popularity as an ornamental plant in East Asia, mei (Prunus mume Sieb. et Zucc.) has received increasing attention in genetic and genomic research with the recent shotgun sequencing of its genome. Here, we performed the genome-wide characterization of simple sequence repeats (SSRs) in the mei genome and detected a total of 188,149 SSRs occurring at a frequency of 794 SSR/Mb. Mononucleotide repeats were the most common type of SSR in genomic regions, followed by di- and tetranucleotide repeats. Most of the SSRs in coding sequences (CDS) were composed of tri- or hexanucleotide repeat motifs, but mononucleotide repeats were always the most common in intergenic regions. Genome-wide comparison of SSR patterns among the mei, strawberry (Fragaria vesca), and apple (Malus×domestica) genomes showed mei to have the highest density of SSRs, slightly higher than that of strawberry (608 SSR/Mb) and almost twice as high as that of apple (398 SSR/Mb). Mononucleotide repeats were the dominant SSR motifs in the three Rosaceae species. Using 144 SSR markers, we constructed a 670 cM-long linkage map of mei delimited into eight linkage groups (LGs), with an average marker distance of 5 cM. Seventy one scaffolds covering about 27.9% of the assembled mei genome were anchored to the genetic map, depending on which the macro-colinearity between the mei genome and Prunus T×E reference map was identified. The framework map of mei constructed provides a first step into subsequent high-resolution genetic mapping and marker-assisted selection for this ornamental species.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

李白盾蚧转录组分析及SSR位点开发

李白盾蚧Pseudaulacaspis prunicola (Maskell)寄主范围广泛,是一种重要的入侵害虫。本研究利用高通量测序平台（Illumina NovaSeq 6000）对李白盾蚧进行转录组测序、de novo从头组装及功能注释,在此基础上筛选其微卫星（SSR）位点,并挖掘微卫星引物。研究共获得李白盾蚧转录组60 296条转录本,24 967条单基因（unigenes）序列。通过GO数据库注释,将所有unigenes的功能分为生物学进程、细胞组分和分子功能三大类41个亚类功能区。KOG数据库注释结果显示,5 085条unigenes归到25个基因家族,注释到一般功能预测的数目最多。KEGG代谢通路富集分析显示6 668条unigenes注释到280个代谢通路,其中注释到内质网中蛋白质加工的数目最多。利用MISA软件共搜索到微卫星位点18 193个,分布在9 043条unigenes中,占总unigenes数量的36.22%,平均每2.29 kb出现一个SSR位点。其中主要重复类型为单核苷酸重复,占SSR位点总数的72.03%,其次为三核苷酸重复（15.90%）和二核苷酸重复（8.48%）。单核苷酸重复主要为A/T（71.16%）,二核苷酸重复主要为AG/CT（5.20%）。基于Primer Primer 3软件设计出12 538对李白盾蚧SSR引物,从中随机挑选50对引物进行PCR验证,共29对引物可以稳定扩增出目的片段。本研究成功组装了李白盾蚧转录组数据,并基于转录组数据成功筛选出其微卫星位点,为未来该虫的种群遗传学以及入侵生物学研究提供了数据支撑。     

Rapid microsatellite identification from Illumina paired-end genomic sequencing in two birds and a snake

Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct "Seq-to-SSR" approach has an advantage over enrichment-based strategies in that it does not require a priori selection of particular motifs, or prior knowledge of genomic SSR content. It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake (the Burmese python, Python molurus bivittatus), and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes (Gunnison Sage-grouse, Centrocercus minimus, and Clark's Nutcracker, Nucifraga columbiana), which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as $10 per sample--a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable.     

基于短序列测序数据的四倍体拟南芥转录组研究

为了促进对四倍体拟南芥(A.suecica)的研究,阐明多倍体植物在染色体加倍过程中遗传物质的变化,从而在分子层面上解释多倍体植物的环境适应和进化机制,描述了一套基于第二代测序技术的转录组短序列组装和生物信息学分析方法.通过对23 000 000条来至于Illumina测序平台的序列数据进行SOAPdenovo组装,以...