多态性分子标记是图位克隆和分子标记技术的基础,为了开发水稻品种间有多态性的分子标记,本研究利用转录组测序技术寻找水稻粳型亚种拉木加和水晶3号两品种间有多态性的InDel标记并进行实验验证,以期为水稻品种间分子标记开发提供参考。研究结果发现通过测序原始序列比对参考基因组后对得到的InDel以测序深度大于2 (DP>2)、等位基因频率等于100%(AF1=1)进行过滤后得到的多态性InDel可信度较高,而通过转录组组装序列比对比较两品种间的InDel假阳性率比较高。利用该方法两个品种转录组数据共发现多态性InDel 279个,随机选取其中24个InDel进行验证,阳性率为91%。本研究利用转录组测序技术开发水稻亚种内品种间InDel标记效率较高,可用于基因图位克隆、品种指纹图谱构建等。
Polymorphic molecular marker is the basis for map-based gene cloning and molecular marker techniques. To provide guidance for development of polymorphic markers between cultivars in rice, transcriptome sequencing technique was used to find and verify polymorphic InDels between japonica rice cultivars Lamujia and Shuijing No.3. The results showed that the InDels found by raw clean reads mapped to reference genome and filtered by criteria of sequencing depth greater than 2 (DP>2) and allele frequency equal to 1 00%(AF1 = 1) was more relia- ble. While, the false positive rate of InDels found by assembled transcriptome sequences was relatively high. In total, 279 polymorphic InDels were found by this method. 24 InDels were randomly chosen for verification and the positive rate was 91%. InDel finding by transcriptome sequencing method used in this study was efficient for polymorphic molecular marker between cultivars of same rice subspecies, which could be used for map-based gene cloning and molecular finger-print construction of cultivars.
Molecular Plant Breeding