Cheng Acga, Ismail Ismanizan, Osman Mohamad, Hashim Habibuddin
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi 43600, Selangor Darul Ehsan, Malaysia.
Kulliyyah of Science, International Islamic University Malaysia, Jalan Istana, Bandar Indera Mahkota, Kuantan 25200, Pahang Darul Makmur, Malaysia.
Int J Mol Sci. 2012;13(5):6156-6166. doi: 10.3390/ijms13056156. Epub 2012 May 18.
The polymorphisms of Waxy (Wx) microsatellite and G-T single-nucleotide polymorphism (SNP) in the Wx gene region were analyzed using simplified techniques in fifteen rice varieties. A rapid and reliable electrophoresis method, MetaPhor agarose gel electrophoresis (MAGE), was effectively employed as an alternative to polyacrylamide gel electrophoresis (PAGE) for separating Wx microsatellite alleles. The amplified products containing the Wx microsatellite ranged from 100 to 130 bp in length. Five Wx microsatellite alleles, namely (CT)(10), (CT)(11), (CT)(16), (CT)(17), and (CT)(18) were identified. Of these, (CT)(11) and (CT)(17) were the predominant classes among the tested varieties. All varieties with an apparent amylose content higher than 24% were associated with the shorter repeat alleles; (CT)(10) and (CT)(11), while varieties with 24% or less amylose were associated with the longer repeat alleles. All varieties with intermediate and high amylose content had the sequence AGGTATA at the 5'-leader intron splice site, while varieties with low amylose content had the sequence AGTTATA. The G-T polymorphism was further verified by the PCR-AccI cleaved amplified polymorphic sequence (CAPS) method, in which only genotypes containing the AGGTATA sequence were cleaved by AccI. Hence, varieties with desirable amylose levels can be developed rapidly using the Wx microsatellite and G-T SNP, along with MAGE.
采用简化技术分析了15个水稻品种蜡质（Wx）微卫星和Wx基因区域的G-T单核苷酸多态性（SNP）。一种快速可靠的电泳方法，即MetaPhor琼脂糖凝胶电泳（MAGE），被有效地用作聚丙烯酰胺凝胶电泳（PAGE）的替代方法，用于分离Wx微卫星等位基因。包含Wx微卫星的扩增产物长度在100至130 bp之间。鉴定出5个Wx微卫星等位基因，即（CT）（10）、（CT）（11）、（CT）（16）、（CT）（17）和（CT）（18）。其中，（CT）（11）和（CT）（17）是测试品种中的主要类型。所有表观直链淀粉含量高于24%的品种都与较短的重复等位基因相关；（CT）（10）和（CT）（11），而直链淀粉含量为24%或更低的品种与较长的重复等位基因相关。所有中等和高直链淀粉含量的品种在5'-前导内含子剪接位点具有序列AGGTATA，而低直链淀粉含量的品种具有序列AGTTATA。通过PCR-AccI酶切扩增多态性序列（CAPS）方法进一步验证了G-T多态性，其中只有包含AGGTATA序列的基因型被AccI酶切。因此，利用Wx微卫星和G-T SNP以及MAGE可以快速培育出具有理想直链淀粉水平的品种。
