Molecular Sex Identification In Herpetospermum Pedunculosum(Ser.) C.B.Clarke Via RAPD And SCAR Markers

Objective Herpetospermum pedunculosum(Ser.)C.B.Clarke is a diecious annual herb vegetating in Ganzi and Aba areas in Sichuan.It is of great clinical value for heat-clearing and detoxifying,and nourishing liver etc.,and its efficacy on hepatitis B has been verified by the modern medicine.As a diecious herb whose seeds can serve as medicinal materials,the values of female Herpetospermum pedunculosum(Ser.)C.B.Clarke are greater in practices.However,it is difficult to differentiate the male and female plants by means of the morphological and physiological methods before its blooming.In view of this,the molecular marker technology with RAPD and SCAR are used to study the gender-related molecular marker of Herpetospermum pedunculosum(Ser.)C.B.Clarke.Method DNA of mature Herpetospermum pedunculosum(Ser.)C.B.Clarke with a given gender is used as a template,and orthogonal design is used for PCR optimization of the RAPD system of Herpetospermum pedunculosum(Ser.)C.B.Clarke.Then primer with clean background and clear band without tail are selected,and primer that fail to amplify Herpetospermum pedunculosum(Ser.)C.B.Clarke or has poor amplification are deleted.Mixed samples from the sample pool of male and female Herpetospermum pedunculosum(Ser.)C.B.Clarke are chosen respectively as templates to further screen the efficient primer,and primer that can amplify with differentiated fragments are selected from the sample pool.Single sample from the sample pool of male and female Herpetospermum pedunculosum(Ser.)C.B.Clarke is used to verify the primer that has variance,then to find out the gender difference-related specific fragments for molecular marker RAPD.Prime5 software is utilized to design SCAR primer so as to verify the effectiveness of molecular marker RAPD-SCAR Result(1)The orthogonal experiment has verified the RAPD-PCR system of 20μl:10×buffer 2μl,RAPD primer of 0.80μl with a concentration of 1μmol/L,DNA of 2μl template with a concentration of 1.25～20ng/μl,Taq DNA polymerase of 4μl with a concentration of5U/μl.(2)The obtained optimal reaction system of RAPD-PCR is used to screen 312 RAPD primer,and has confirmed S94 and S15 can stably amplify the specific band that is related to the gender of Herpetospermum pedunculosum(Ser.)C.B.Clarke.(3)Band sequence amplified by S94 has been obtained by sequencing,based on which SCAR primer is designed.A 423 bp specific band can be amplified stably from a single female plant(transformation of SCAR primer of S15 has failed,but transformation of SCAR marker of S94 is successful).Conclusion One SCAR molecular marker related to female has been obtained to fast and precisely identify the genders of Herpetospermum pedunculosum(Ser.)C.B.Clarke,which can be applied for the identification of plants with unknown genders and the testing for batch production,thus providing a foundation in gender test for Herpetospermum pedunculosum(Ser.)C.B.Clarke in the early stage.