Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials， and carry out the study on the genetic differentiation and structure. MethodGenomic DNA of 47 I. indigotica germplasm materials were extracted by kit extraction method. Two chloroplast DNA （cp DNA） sequences and five inter-simple sequence repeat （ISSR） primers were used for amplification and sequencing. Chromas， Mega 7.0， DanSP5， and GenALEx were used to calibrate， splice， and analyze the sequence characteristics. PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure， and NTSYS was used to obtain the unweighted pair-group method with arithmetic means（UPGMA） clustering tree plot of 47 I. indigotica germplasm materials. ResultA total of 129 samples from 47 I. indigotica germplasm materials were successfully amplified and sequenced. The length of 2 cp DNA sequences after spliced was 1 412 bp， and there were 377 polymorphic variation loci， and 36 haplotypes. Fu and Li's D* test was significant （P<0.01）. The values of Pi， HS， and HT based on cp DNA were 0.119 89， 0.787， and 0.891， respectively. The genetic differentiation coefficients of gene differentiation coefficient（Gst）， nucleotide differentiation coefficient（Nst）， and fixation index（Fst） were 0.117， 0.468， and 0.488， respectively， and the gene flow （Nm） was 0.615. The mean values of PPB， Shannon information diversity index（I）， Nei's genetic diversity index（H）， and Gst based on ISSR were 78.85%， 0.334 8， 0.218 6， and 0.754 4， respectively， and the Nm value was 0.162 8. ConclusionI. indigotica has high genetic diversity and abundant haplotypes at the species level， with abundant haplotypes. Genetic differentiation among different germplasm materials is obvious， and gene exchange is not frequent. Genetic variation mainly exists among populations. The population has accumulated various low-frequency gene mutations recently， suggesting that it has experienced significant regional expansion in the history.