A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.)
A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.)
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DOI：
10.1111/j.1365-313x.2004.02190.x
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发表时间：
2004-10-01
期刊：
影响因子：
7.2
通讯作者：
Waugh, R
中科院分区：
文献类型：
--
作者：
Caldwell, DG;McCallum, N;Waugh, R
以大麦（Hordeum vulgare L.）CV.已经开发了光学来促进这种作物的正向和反向遗传。分别收获约20000株M-2植物的叶材料和种子，冷冻干燥并存档。从20和30 mM EMS处理的9216株植物中分离DNA，并组装成1152个8株植物库。为了促进基于PCR的突变扫描，已经采用了一种方法，该方法组合了使用Cel核酸酶（Cel I）切割异源双链体、切割后嵌入染料标记和随后在转基因WAVE-HS上检测切割产物。通过筛选两个目标基因中的诱导突变来评估群体，并通过序列分析验证诱导突变。为了增强筛选过程，评估来自每个M-2植物的12-16个M-3后代的可见表型，并将数据输入网络可访问的数据库（http：//bioinf.scri.sari.ac.uk/bioing/bioing.html）。
Two large-scale ethylmethanesulfonate (EMS) mutant populations from barley (Hordeum vulgare L.) cv. Optic have been developed to promote both forward and reverse genetics in this crop. Leaf material and seed from approximately 20 000 M-2 plants were individually harvested, freeze-dried and archived. DNA was isolated from 9216 plants from the 20 and 30 mM EMS treatments and assembled into 1152 eight-plant pools. To facilitate PCR-based mutation scanning an approach has been employed that combines cleavage of heteroduplexes using the Cel nuclease (Cel I), post-cleavage intercalating dye labeling and the subsequent detection of cleaved products on a Transgenomic WAVE-HS. The populations were evaluated by screening for induced mutations in two genes of interest and the induced mutations were validated by sequence analysis. To enhance the screening process, 12-16 M-3 progeny from each of the M-2 plants were assessed for visible phenotypes and the data entered into a web accessible database (http://bioinf.scri.sari.ac.uk/distilling/distilling.html).