Development And Application Of G-SSR Markers Based On Genome Survey Sequencing Of Napier Grass (Pennisetum Purpureum)

Pennisetum species,which belongs to Poaceae,is an important annual or perennial forage grass in tropical,subtropical and temperate regions.It has strong adaptability and is able to grow under various environmental conditions.Despite of its economic importance,the inheritance information of Pennisetum has remained largely unknown,and the limited number of molecular markers also become the barrier of its further research.To obtain a more accurate genomic information,genomic survey sequencing based on the next-generation sequencing(NGS)platform was conducted on elephant grass,which is known as a famous Pennisetum species,in this experiment.Besides,a large number of Genomic-SSR(G-SSR)markers were developed based on the obtained sequence data.Then genetic diversity analysis and fingerprint analysis were carried out on 24 species of Pennisetum using developed molecular markers.At last,the genetic integrity of the artificially aged seed was studied,and the optimum sample size and the germination rate criterion for regeneration were determined.The main research results are as follows:(1)Survey sequencing of elephant grass and development of its G-SSR markers.Illumina platform based on NGS technology was used to de novo sequence the whole genome of elephant grass.According to the sequencing results and k-mer analysis,the genome size of elephant grass was 2.02 Gb,the proportion of repetitive elements was71.36% and the heterozygosity was 1.02%.A total of 14.46% of the repetitive elements were annotated,in which the retroelements accounting for the most(9.36%),followed by DNA transposons accounting for 3.66%.A total of 83,796 G-SSR markers were developed.Verification showed that the success rate of amplification was 100%,the polymorphism ratio was 93.3%,the polymorphic information content(PIC)was 0.2052 to 0.4170,the effective multiplex ratio(EMR)was 6.85,and the marker index(MI)was 3.49.The reference genome provided in this experiment enriched the inheritance resources of Pennisetum,and the high-efficiency molecular markers developed in this study would play an important role in the further research.(2)Fingerprint construction of 24 Pennisetum species based on G-SSR markers.Twenty pairs of SSR primers were selected,and 176 clear polymorphic bands were amplified based on 24 species of Pennisetum,which stood for a percentage of polymorphic band at 88.44%.The average value of H and I were 0.3131 and 0.4690,respectively.24 species were classified into three groups after cluster analysis.7 pairs of primers with higher polymorphism were selected for further study.2 combinations that could distinguish24 species of Penniseum,in which two primers were used,were selected to successfully construct two DNA fingerprints.The fingerprints constructed in this study provided a theoretical basis for the identification and development of Pennisetum species in the future.(3)The study of effect of seed aging on genetic integrity based on G-SSR markers.The SSR markers and single plant sampling method were used to analyze the genetic integrity of seeds of “Douniu” pearl millet.Six groups of different sample size were set(15,30,45,60,75,90 individuals per group).The result showed that four polymorphism indexes(PPB,Ne,H and I)increased steadily with the increase of the amount of sample size.PPB of the group with sample size at 45 had no significant difference with ones of the groups with sample size above 45,while Ne,H and I of the group with sample size at 60 had no significant different with ones of the groups above 60.Therefore,the optimal sample size was set at 60.Then the seeds were artificially aged using high-temperature and high-humidity method,and five different germination rate gradients were selected for further research.The result showed that H and I decreased gradually with the decrease of germination rate.The polymorphic indexes were significantly different from the ones of control group when the germination rate decreased to 68.23%,which was determined as the germination rate criterion for regeneration.The loss of genetic integrity of the germplasm could be reduced or avoided by monitoring the germination rate of germplasm and regenerating the germplasm timely.The results in this study provided a reference for protecting the genetic diversity.