一串红SRAP标记的建立与品种鉴定

Establishment of SRAP marker and cultivar identification in Salvia splendens

为建立一串红相关序列扩增多态性(sequence-related amplified polymorphism,SRAP)标记,对引物、dNTP、模板DNA、Taq酶用量及退火温度等因素进行了优化,并用建立的SRAP标记对17个一串红品种进行了鉴定。通过测试,确定了适宜一串红的SRAP-PCR反应体系,反应总体积25μL,包含PCR缓冲液(含2.0mmol·L-1 MgCl2),模板DNA 60 ng,dNTP 0.2 mmol·L-1,引物0.4μmol·L-1,Taq酶1 U。温度梯度试验结果表明,SRAP-PCR对第二阶段的退火温度变化(44～56℃)不敏感。在优化SRAP-PCR体系的基础上,采用44对引物组合对17个一串红品种进行了分析。共筛选到37个多态性引物组合。这些引物组合的多态性信息含量(PIC)为0.215～0.941,平均为0.672;所揭示的基因型数为2～17个,平均为6.76个。仅用F1/R1这一个引物组合就可以鉴别参试的17个品种,包括形态上非常相近的品种‘神州红’和‘帝王’,表明SRAP对一串红品种具有较强的鉴别能力。

The concentrations of primers,dNTP,DNA template,Taq DNA polymerase and annealing temperature were optimized in order to establish SRAP（sequence-related amplified polymorphism） marker system in Salvia splendens and 17 cultivars were identified using the SARP markers established.The suitable SRAP-PCR system was established for Salvia splendens.Totally 25 μL reaction mixture contained PCR buffer（containing 2.0 mmol·L-1 MgCl2）,DNA template 60 ng,dNTP mixture 0.2 mmol·L-1,each primer 0.4 μmol·L-1 and Taq DNA polymerase 1 U.Gradient temperature test showed that SRAP-PCR was not sensitive to the variation of annealing temperature（44-56℃） at second stage.Based on the reaction system established,17 varieties of Salvia splendens were analyzed using 44 SRAP primer combinations and 37 polymorphic primer combinations were screened in total.The PIC（polymorphism information content） value of these markers varied from 0.215 to 0.941,averaging 0.672.The genotypes revealed by each marker ranged from 2 to 17 with average of 6.76.The 17 varieties could be distinguished completely only using a single primer combination F1/R1 and even two varieties quite similar in morphology,Shenzhouhong and Diwang,could be discriminated by several primer combinations,suggesting that SRAP is powerful for variety identification.