Development Of SSR Molecular Markers In Mussaenda And Preliminary Studies On The Genetic Relationship Of Species

Mussaenda is a genus in the family Rubiaceae.This genus has about 130 species widespread in tropical and subtropical Africa,Asia and Pacific Islands,whereas 29species are from in China,distributing from southwest part to Taiwan with abundance concentrated mostly in the southwest regions.Most species appear as lianas and shrubs,but in rare cases,they can also grow as small trees.Identification of Mussaenda has been challenging because its variable morphological forms,and delimitation of Mussaenda species has been controversial.DNA barcoding methods were also inefficient as it provides low resolution in species identification.Therefore,this study aimed to develop SSR molecular markers based on transcriptomic sequencing of representative species of Mussaenda for proper molecular identification,genetic diversity and relationship studies on Mussaenda.The main results were the following:?1?64,054 and 47,928 unigenes of M.macrophylla and M.erythrophylla were obtained from transcriptome sequence,respectively.The SSR loci were analyzed using MISA.The frequency of occurrence of SSR sequences was 16.95%and 14.42%.9,703primer pairs were designed using the software Primer 3,and 243 primer pairs were screened for this study.?2?The optimized SSR-PCR reaction system condition of Mussaenda was obtained in a 20?L system containing 1.5 mmol/L Mg²⁺,0.15 mmol/L d NTPs,0.45?mol/L SSR primer,2.0 U Taq DNA polymerase and 15 ng DNA template,and the annealing temperature used was 53.4?.?3?105 out of 243 SSR primer pairs showed polymorphism and transferability,accounting for 43.21%of the synthesized primers.Among 24 species of Mussaenda,a total of 646 allelic variations were detected,with a mean of 6.15 alleles per locus.?4?Using the developed SSR molecular markers to validate genetic relationship of species,49 primer pairs amplified 319 allelic variations,with a mean of 6.51 alleles per locus.By applying UPGMA for clustering analysis,45 individuals which were divided into 3 groups showed similarity coefficient of 0.74.The results indicated that the SSR primers developed in this study can be used for the analysis of genetic relationship among species.?5?The molecular identification of Mussaenda cultivars was performed using SSR molecular markers,which directly validated that the new cultivar M.cv.nov.is a hybrid progeny of M.erythrophylla and M.macrophylla.The clustering analysis of 102individuals by 18 primers,showed that M.cv.nov.was close to M.macrophylla,and had a long genetic distance from other varieties.?6?Ten SSR primer pairs were used to analyze the genetic diversity of 11populations with 6 Mussaenda species from Yunnan and Guangxi,China,Vietnam and Japan.The results showed that there had been a genetic variability,and lower genetic diversity.SSR primers developed in this study can be used for identification of cultivars,analysis of interspecific genetic relationships,and genetic diversity of Mussaenda.