基于叶绿体DNA的籼-粳稻谷(米)SYBR-Green实时荧光PCR鉴定方法的建立

Identification Method of Hsien and Keng Rice by SYBR-Green Fluorescent-PCR Detection for Chloroplast DNA

亚洲栽培稻分为籼稻(hsien或indica)和粳稻(keng或japonica)两个亚种。基于籼-粳稻叶绿体DNA(cpDNA)的PstⅠ-12片段ORF100区域的序列差异,即籼稻cpDNA的ORF100区域缺失69 bp的碱基片段,建立籼-粳稻亚种鉴定方法。本文选择典型的籼稻谷(米)样品(台籼11号)和粳稻谷(米)(东北珍珠大米)进行叶绿体DNA(cpDNA)PstⅠ-12片段的ORF100区域DNA的SYBR-Green实时荧光PCR扩增和测序,在籼-粳稻2个PCR产物序列比对的基础上,设计正向引物4条(F1-F4)、反向引物3条(R1-R3),组合成12对引物进行优化筛选,选出F4R1引物对。在粳稻样品中,检出cpDNA PCR产物片段长度为204 bp(非缺失型),荧光PCR扩增熔解温度Tm值为78.00;在籼稻样品中,检出cpDNA PCR产物片段长度为135 bp(cpDNA缺失型),荧光PCR扩增熔解温度Tm值为76.50。建立了籼-粳稻亚种SYBR-Green实时荧光PCR鉴定方法。基于上述实验结果,选择国内多个省份的典型籼/粳稻品种与籼/粳型杂交稻组合共538个,对该籼粳稻米亚种鉴定方法进行验证。在177个粳稻及粳型杂交组合样品中,cpDNA荧光PCR熔解温度Tm值为78.00的样品170个,与粳稻符合率为96.05%,Tm值为76.50的7个样品,不符合率为3.95%;在361个籼稻及籼型杂交稻样品中,Tm值为76.50的样品331个,与籼稻符合率为91.69%,Tm值为78.00的30个样品,不符合率为8.31%。本方法只检测一个遗传稳定的细胞质叶绿体DNA位点,用于籼-粳稻米的亚种鉴定,简便高效,准确率高。

Asia cultivated rice(Oryza sativa L.)is divided into two subspecies,namely hsien rice(also known as Oryza sativa L.subs.indica rice)and keng rice(also known as Oryza sativa L.subs.japonica rice).Based on the sequence difference of ORF100 region within the PstⅠ-12 fragments of chloroplast DNA(cpDNA)of hsien-keng rice,it was discovered that there was a 69 bps deletion in the ORF100 region of cpDNA in hsien(indica)rice.Typical hsien rice samples(Taixian 11)and keng rice(Northeast Pearl Rice)were used to amplify and sequence for ORF100 region nucleotides within the cpDNA PstⅠ-12 fragment by SYBR-Green realtime PCR.Based on the comparison of two PCR product sequences of hsien and keng rice,four forward primers(F1-F4)and three reverse primers(R1-R3)were designed,and 12 pairs of primers were synthesized,a SYBR-Green real-time PCR method was developed for the identification of hsien-keng subspecies.In hsien subspecies,the length of cpDNA fragment was 204 bp(non-deletion type),and the melting temperature Tm value was 78.00,while in keng subspecies,and the length of cpDNA fragment was 135 bp(cpDNA deletion type),and the melting temperature Tm value was 76.50 in hsien subspecies.Furthermore,538 typical hsien-keng rice varieties and hsien-keng hybrids combinations from several provinces in China were selected to validate above identification method of hsienkeng subspecies rice based on chloroplast DNA SYBR-Green real-time fluorescent PCR detection.Among 177 samples of keng rices including keng type hybrids combinations,170 samples with a Tm value of 78.00 were detected by SYBR-Green real-time PCR for cpDNA.The coincidence rate was 96.05%and the non-coincidence rate was 3.95%.Among the 361 samples of hsien rices including hsien type hybrids combinations,331 samples with a Tm value of 76.50 were detected by SYBR-Green real-time fluorescence PCR for cpDNA.The coincidence rate was 91.69%and the non-coincidence rate was 8.31%.This method is simple,efficient and accurate for the identification of typical hsien-keng subspecies rice by detecting only one deletion site of chloroplast DNA in the cytoplasm.