摘要
AFL P分子标记技术将对玉米品种指纹文库的构建发挥巨大作用 ,因此对玉米 AFL P指纹图谱的引物选择研究具有重要意义。通过研究发现 :1用 Mse 和 Eco R 酶切后 ,酶切位点后第一个碱基 T占的比例高达 30 %左右 ,其次 A占 2 7%左右 ,G、C占的比例较少 ,为 2 1%左右。第二个碱基 T占 2 7%左右 ,A、G、C分别占 2 4 %左右较均匀。因此以 TT选择引物时扩增的谱带较多。 2不同引物扩增的谱带的多态性不同 ,多态性强的引物组合可分为两类。其一为多态性主要由一端引物产生的 ;其二为多态性由两端共同决定的。对多态性贡献较大的两个引物组合可能产生更高的多态性。 3随引物选择碱基的增加 ,扩增的谱带数有所降低。同时增加一个选择碱基 ,在 M端增加一个选择碱基较在 E端增加一个选择碱基 ,谱带数目降低较多。 4在多态性较高的 2 +2引物组合中 ,增加第 3个选择碱基 ,可选出多态性更强的引物组合。 5选择多态性强的引物组合可按如下程序 :第一步选择 2～ 4组亲缘关系相近的材料 6～ 12个。第二步 ,将两端碱基相同的 2 +2引物组合 (16对 )全部选择扩增 ,以筛选有多态性的 2 +2引物组合。第三步 ,重组有多态的 2+2引物 ,以筛选多态性较强的 2 +2引物。第四步 ,将多态性较强的 2 +2引物一端分别增加第 3个选择碱基 ,?
AFLP mark technique will play an important role in constructing cultivar fingerprint database of maize, so selecting suitable primers for AFLP fingerprints of maize is very important. Study results showed as follows: ①The percentages of first bases after digested by Mse Ⅰ and Eco RⅠ were about 30% for T 27% for A and 21% for G and G respectly. The percentages of second bases were about 27% for T, 24% for A, G and C respectly. So, the amplification bands were much more while to use primers with TT selective bases. ②Different primers brought about different polymorphism. High polymorphic primer combinations could be divided into two kinds, one of which mainly brought into polymorphism by one end primer, another one of which did it by two end primers. Much more polymorphism could be produced by recombinations of primers, which contributed much to polymorphism. ③The bands decreased with adding selective bases. The bands decreased much more while adding one selective base at M end than at E end. ④Highly polymorphic primer combinations could be selected from the primers added third selective base to 2+2 primers that could bring about high polymorphism. ⑤Selecting highly polymorphic primer combinations might follow this procedure: first. selecting 6～12 materials about 2～4 groups which have close genetic relations; secondly, selecting polymorphic 2+2 primers through selective amplification with all same 2+2 primers at two ends; thirdly, recombinating polymorphic 2+2 primers to choose highly polymorphic 2+2 primer combinations; then, selecting much more polymorphic 2+3 or 3+2 primer combinations by adding one selective base at end of 2+2 primers. M GA E GAG and M GAA E GA were selected by above mentioned program, Which would produce more bands. high polymorphism and high quality bands, and could be used as standard primers of maize AFLP fingerprints. ⑥Fingerprints of hybrids contained all bands of double parents.
出处
《作物学报》
CAS
CSCD
北大核心
2002年第2期221-226,共6页
Acta Agronomica Sinica
基金
国家九 .五攻关课题资助 (96-0 14 -0 1-0 6)
