Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project.
Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project.
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DOI：
10.1186/1471-2164-4-40
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发表时间：
2003-09-30
期刊：
影响因子：
4.4
通讯作者：
Heinz RA
中科院分区：
文献类型：
--
作者：
Fernández P;Paniego N;Lew S;Hopp HE;Heinz RA
消减杂交方法是用于鉴定给定组织中差异调节基因的有价值的工具，避免了代表相同表达基因的克隆的冗余测序，最大化了低丰度转录本的检测，从而影响了旨在特异性鉴定用于育种目的的有用基因的小规模cDNA测序项目的效率和成本效益。这项工作的目的是评估替代战略的高通量测序项目，用于识别新的基因差异表达的向日葵作为一个来源的器官特异性遗传标记，可以在功能上与重要性状。在两个发育阶段（R1和R4）的叶、茎、根和花芽中产生了差异器官特异性EST。使用不同来源的RNA作为用于构建差异文库的测试者和驱动者cDNA作为检测稀有或低丰度转录物的工具进行了评估。器官特异性范围从75%到100%的非冗余序列在不同的cDNA文库。序列冗余根据每种情况下使用的靶和驱动cDNA而变化。R4花cDNA文库是冗余度较小的文库，具有62%的独特序列。在总共919个被编辑和注释的序列中，318个是非冗余序列。与公共数据库中的序列比较显示，60%的非冗余序列与已知序列具有显著的相似性。预测的新基因的数量在不同的cDNA文库中变化，从R4花中的56%到R1花蕾文库中的16%。与公共数据库中的向日葵EST序列比较发现，197个非冗余序列（60%）与先前报道的向日葵EST序列没有表现出显著的相似性。这种方法有助于成功地分离出大量新报道的序列，这些序列与重要农艺性状和关键调控和生理基因的反应有关。抑制消减杂交技术的应用不仅使得差异表达序列的分离具有成本效益，而且当与主要测序项目相比时，其还允许从相对少量的分析序列中鉴定向日葵中的新序列。
Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important agronomic traits and key regulatory and physiological genes. The application of suppressed subtracted hybridization technology not only enabled the cost effective isolation of differentially expressed sequences but it also allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences when compared to major sequencing projects.
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影响因子：
11.6
作者：
LOTAN, T;ORI, N;FLUHR, R
通讯作者：
FLUHR, R
影响因子：
7.4
作者：
MAUCH, F;MAUCHMANI, B;BOLLER, T
通讯作者：
BOLLER, T
影响因子：
7.3
作者：
Gago, GM;Almoguera, C;Chan, RL
通讯作者：
Chan, RL
影响因子：
5.4
作者：
Cato, SA;Gardner, RC;Richardson, TE
通讯作者：
Richardson, TE
DOI：
10.1007/bf00272349
发表时间：
1992-07-01
期刊：
MOLECULAR & GENERAL GENETICS
影响因子：
--
作者：
GU, Q;KAWATA, EE;CHEUNG, AY
通讯作者：
CHEUNG, AY