Development And Application Of Retrotransposon-based SSAP Molecular Marker In Moso Bamboo

Moso bamboo(Phyllostachys edulis),ecologically and economically important non-timber forest resource,is widely distributed with large planting area,particularly in Asia.Retrotransposons(Class I,RNA transposon)account for a large proportion of Moso bamboo genome and are widely distributed in all the chromosomes.It regulates the gene expression and changes the genome size,which is an important factor causing the genomic diversity of Moso bamboo.In the present study,five pairs of SSAP(Sequence Specific Amplified Polymorphism)molecular markers were developed based on the insertion site of LTR retrotransposon and used to analyze the genetic diversity of different Moso bamboo populations.The SSAP analysis provided the reliable technical and theoretical support for the development and utilization of retrotransposons markers for Moso bamboo genetic diversity studies.1、A total of 7731 full-length long terminal repeat(LTR)-retrotransposons were identified in Moso bamboo genome by LTRharvest software.Then 35 full-length LTR-retrotransposons were screened,and finally five SSAP molecular markers were developed.2、Five SSAP molecular markers were applied to analyze the genetic diversity of wild-type Moso bamboo and its 15 forms.A total of 83 polymorphic loci were amplified,with an average polymorphic loci ratio of 98.81%.Compared with previous studies on the genetic diversity of Moso bamboo species,it was found that SSAP molecular markers exhibited a higher polymorphism than other DNA molecular markers.3、Three SSAP molecular markers,such as hic19 G,hic26G and hic34 G with high polymorphism(PIC>0.3)were further selected to analyze the genetic diversity of Moso bamboo seedling materials.(1)Wild-type Moso bamboo half-sib seedlings had an average polymorphic loci ratio of 96.67%,an average polymorphic information content(PIC)of 0.37,Nei’s index(H)of 0.251 6,and Shannon’s index(I)of 0.399 7.Moso bamboo Kikko-chiku seedlings had an average polymorphic loci ratio of 93.02%,PIC of 0.45,Nei’s genetic diversity of 0.173 7,and Shannon information index of 0.267 5.Statistical analysis showed the significant differences between populations(P<0.05),and AMOVA analysis showed polymorphism was mainly from intraspecific variation(79%).(2)Heterozygous seedling of Moso bamboos had an average polymorphic loci ratio of 100%,an average polymorphic information content(PIC)of 0.41,Nei’s index(H)of 0.275 2,and Shannon’s index(I)of 0.430 6.Statistical analysis showed significant differences between the two populations(P<0.05),and polymorphism was mainly from intraspecific variation(81%).4、The SSAP molecular markers(hic19G,hic26 G,and hic34G)were also used to analyze the genetic diversity of flowering and non-flowering Moso bamboos.The results showed that the two populations had relatively approximate average polymorphic loci ratios(87.93% and 87.27%),PIC(0.44 and 0.46),Nei’s genetic diversity(0.234 4 and 0.225 4),and Shannon information index(0.365 8and 0.348 1).There was no huge genetic difference between the two populations.It was verified by the AMOVA analysis that showed the difference between the two populations was not significant(P<0.5).5、Focused on the identifying the transposition activity of PHRE1,a kind of retrotransposon,we analyzed the insertion site polymorphism of PHRE1 in Arabidopsis transgenic using these SSAP markers.Our analysis showed that polymorphic bands were identified in Arabidopsis transgenic grown under heat stress(42℃).The PHRE1 transposition was found in Arabidopsis through illumina sequencing,indicating that SSAP technology could be used to identify the retrotransposon transposition activities.Further,we analyzed the genomic diversity of cold-stressed,heat-stressed and untreated half-sib Moso bamboo seedlings using these markers.The results revealed that the polymorphism rates of cold-stressed and untreated wild-type Moso bamboo half-sib seedlings were 98.33% and 95.83%,respectively;the polymorphism rates of heat-stressed and untreated wild-type Moso bamboo half-sib seedlings were 98.48% and 96.23%,respectively.Statistical analysis showed significant differences between the two populations(P<0.01),indicating that the half-sib Moso bamboo seedlings’ polymorphism increased after cold and heat stressed treatment.The reason for the increase in polymorphisms was possibly because cold and heat stresses activated the LTR retrotransposons in Moso bamboo.In conclusion,five stable and efficient SSAP molecular markers were developed in this study and were applied to identify the genetic polymorphism of forms of Moso bamboo,half-sib Moso bamboo seedlings,heterozygous Moso bamboo seedlings,flowering Moso bamboo,non-flowering Moso bamboo,Moso bamboo seedlings under cold and heat stress treatments.The results showed that SSAP molecular markers were sensitively distinguished the genetic diversity of Moso bamboo forms than other DNA-based molecular markers.The statistical results of SSAP molecular marker diversity also showed that heterozygous Moso bamboo seedlings had higher genetic diversity than half-sib Moso bamboo seedlings.However,there was no significant difference in genetic diversity between flowering and non-flowering Moso bamboos.Transposition of LTR-retrotransposon in Moso bamboo can increase the genomic polymorphism;hence,SSAP technology could also be used to identify the transposition activities of LTR retrotransposons.