Sun X, Mu Q, Jiang D, Wang C, Wang X C, Fang J G
College of Horticulture, Nanjing Agricultural University, Nanjing, P.R. China.
Genet Mol Res. 2012 Aug 6;11(3):2071-80. doi: 10.4238/2012.August.6.11.
We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.
我们通过增加随机扩增多态性DNA（RAPD）引物的长度并严格筛选PCR退火温度，对RAPD技术进行了优化，以区分中国农业科学院果树研究所的60个甜橙品种。一种名为品种鉴定图（CID）的新方法被用于提高RAPD标记在品种鉴定中的效率。首先根据54个RAPD随机11聚体引物的扩增产物和扩增出的多态性条带，从其中筛选出13条有效引物；然后用这些引物对所有60个品种进行PCR扩增。所有品种通过13条引物的DNA指纹图谱中出现的多态性条带被人工且完全区分开；随后构建了60个甜橙品种的CID。该CID根据多态性条带将所有品种相互区分开；相应的引物被标记在甜橙CID的正确位置上。CID策略有助于利用DNA标记鉴定果实品种。这种甜橙品种的CID对于品种权保护以及苗圃行业幼苗的早期鉴定将非常有用。
