Seagrist John F, Su Shih-Heng, Krysan Patrick J
Department of Horticulture, University of Wisconsin-Madison, Madison, WI, United States of America.
Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, United States of America.
PeerJ. 2018 Jul 3;6:e5076. doi: 10.7717/peerj.5076. eCollection 2018.
We previously described the identification of a chromosomal deletion in that resulted in the elimination of genomic DNA between two T-DNA insertions located ca. 25 kilobases apart on chromosome IV. The mechanism responsible for this deletion appears to have been recombination between the closely spaced T-DNA elements located in trans in a parent plant. In our original study, we observed one such deletion event after screening ca. 2,000 seedlings using a polymerase chain reaction (PCR) assay. Because a method for precisely deleting a selected region of the genome would have significant value as a research tool, we were interested in determining the frequency with which this type of T-DNA-directed deletion occurs. To do this we designed a genetic screen that would allow us to phenotypically screen for deletions caused by recombination between T-DNA inserts. This screen involved crossing T-DNA single-mutant lines in order to produce F1 plants in which the two T-DNA insertions flanked a () locus present in the genome. Loss-of-function mutations of genes cause a distinctive developmental phenotype that can be easily scored visually in young seedlings. We used T-DNA lines flanking , , , and for this study. Recombination between the T-DNAs in an F1 parent should result in deletion of the gene located between the T-DNAs. Because the deletion would be heterozygous in the F2 generation, we screened the F3 progeny of pooled F2 individuals to search for the loss-of-function phenotype. Using this strategy we were able to evaluate a total of 28,314 meioses for evidence of deletions caused by recombination between the T-DNA inserts. No seedlings displaying the phenotype were recovered, suggesting that deletions caused by recombination between T-DNA inserts are relatively rare events and may not be a useful tools for genome engineering in .
我们之前描述了在[具体物种]中鉴定出的一个染色体缺失，该缺失导致位于第四条染色体上相距约25千碱基的两个T-DNA插入之间的基因组DNA被消除。导致这种缺失的机制似乎是亲本植物中反式排列的紧密间隔的T-DNA元件之间的重组。在我们最初的研究中，使用聚合酶链反应（PCR）检测筛选了约2000株幼苗后，我们观察到了一个这样的缺失事件。由于一种精确删除[具体物种]基因组选定区域的方法作为研究工具将具有重要价值，我们有兴趣确定这种T-DNA定向缺失发生的频率。为此，我们设计了一个遗传筛选，使我们能够从表型上筛选由T-DNA插入之间的重组引起的缺失。这个筛选包括杂交T-DNA单突变系，以产生F1植株，其中两个T-DNA插入位于基因组中一个[具体基因]位点的两侧。[具体基因]基因的功能丧失突变会导致一种独特的发育表型，在幼苗中可以很容易地通过视觉进行评分。我们在这项研究中使用了位于[具体基因1]、[具体基因2]、[具体基因3]和[具体基因4]两侧的T-DNA系。F1亲本中T-DNA之间的重组应导致位于T-DNA之间的[具体基因]基因缺失。由于这种缺失在F2代中是杂合的，我们筛选了合并的F2个体的F3后代，以寻找[具体基因]功能丧失表型。使用这种策略，我们总共评估了28314次减数分裂，以寻找由T-DNA插入之间的重组引起的缺失证据。没有回收显示[具体基因]表型的幼苗，这表明由T-DNA插入之间的重组引起的缺失是相对罕见的事件，可能不是[具体物种]基因组工程的有用工具。
