Settles A Mark, Holding David R, Tan Bao Cai, Latshaw Susan P, Liu Juan, Suzuki Masaharu, Li Li, O'Brien Brent A, Fajardo Diego S, Wroclawska Ewa, Tseung Chi-Wah, Lai Jinsheng, Hunter Charles T, Avigne Wayne T, Baier John, Messing Joachim, Hannah L Curtis, Koch Karen E, Becraft Philip W, Larkins Brian A, McCarty Donald R
Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA.
BMC Genomics. 2007 May 9;8:116. doi: 10.1186/1471-2164-8-116.
Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.
Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.
We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.
基因敲除是功能基因组学的关键资源。在拟南芥中，通过扩增和测序插入突变体侧翼的基因组DNA生成了全面的敲除文库。这些侧翼序列标签（FSTs）将每个突变体定位到基因组内的特定位点。在玉米中，已利用DNA转座子生成了FSTs。转座元件可产生不稳定的插入，难以分析其简单的敲除表型。转座子还可产生体细胞插入，这些插入在后代中无法分离。
通过位点特异性PCR检测了106个UniformMu FSTs的转座子插入位点的遗传情况。我们证实89%的FSTs为生殖系转座子插入。在未得到证实的11%的插入位点中，我们未发现体细胞插入的证据。相反，由于基因组序列不完整或质量低，该插入位点子集在位点特异性引物设计上存在错误。位点特异性PCR分析鉴定出一个与种子突变表型共分离的6-磷酸葡萄糖酸脱氢酶基因的敲除。与该敲除相关的突变表型产生了关于质体定位的氧化戊糖磷酸途径在籽粒灌浆过程中作用的新假说。
我们表明，来自UniformMu群体的FSTs可鉴定玉米中稳定的生殖系插入位点。此外，我们表明这些序列索引突变可很容易地用于反向遗传学分析。我们从这些数据得出结论，目前来自UniformMu的1882个非冗余插入位点的文库为反向遗传学提供了全基因组资源。
