起始密码子区域相关序列多态性的开发及在苜蓿遗传多样性上的应用

Start Codon Region-related Polymorphism (SCRP): A Novel DNA Marker Technique for Assessing Genetic Diversity in Alfalfa Germplasm Collections

目前广泛使用的基于 PCR 基础的分子标记多为扩增非编码区域,或是随机基因组中扩增,在 QTL定位中得到的位点一般与目标性状基因距离较远,我们开发了一个新的基于启动子序列目的基因型分子标记技术——启动子区域相关序列多态性(SCRP) ,试图使标记能够更为准确的反映不同品种的遗传基础。它利用启动子位置保守一致序列("Kozak"序列) 作为其上游引物,利用内含子富含 "AATT"的特性,作为核心序列设计下游引物,上下游引物均为18bp,引物间通过组合配对的方式作为扩增引物对。设计了14 条上游引物和 10 条下游引物,共 140 对引物组合,对 34 个苜蓿品种进行扩增,研究了 34 个苜蓿的遗传多样性。每个 PCR 反应产生3～16个50～2 000bp 的条带,结果表明该标记简单、可靠、具有较高多态性,并且扩增区域为一种目的基因型分子标记,在种质资源研究中具有重要价值。

Mostly PCR-based molecular markers amplified non-coding regions, or the whole genome randomly, the locus is far away from the gene of targeted trait. We developed a novel marker technique called start codon region-re-lated polymorphism （SCRP） aimed for the amplification of gene start condon regions. It is based on the use of two primers of 18 nucleotides. The forward primer is designed from the targeted ＂Kozak＂ sequence; and the other prim-er, the reverse primer is an arbitrary sequence with an AT-rich core to anneal with an intron. PCR amplification is applied for the first 5 cycles with an annealing temperature of 35 ℃ , followed by 35 cycles with an annealing tempe-rature of 50 ℃. In the present study, we utilized SCRP to study genetic diversity of 34 alfalfa cultivars （varieties）. Each PCR reaction has generated as many as 3 to 16 fragments of 50 to 2 000 bp in size. The successful genetic di-versity of 34 alfalfa cuhivars by SCRP was achieved. This new technique should be useful in genotyping germplasm collections and in tagging genes govern desirable agronomic traits of crop plants.