Analysis Torreya Grandis ‘Xifei’ High Oil Content Molecular Mechanism And SSR Markers Development Based On RNA-seq

Torreya grandis ‘Merrillii’ is known as an excellent variety of Torreya grandis ‘Xifei’ is one of the good varieties in Torreya grandis Fort.ex Lind.It is discovered that the Torreya grandis ‘Xifei’ ripe seeds had about 53% fatty acid,and the content of unsaturated fatty acid was 78%,it is crisp and delicious with great commercial value.In this paper,q RT-PCR was adopted to study the expression of key genes in fatty acid synthesis of Torreya grandis ‘Xifei’,three c DNA sequences KAS1,FAD2-2 and GPAT6 were obtained by cloning,with low oil content Torreya grandis ‘Dielsii’ for comparison.In addition,16 pairs of SSR primers were developed based on the transcription data of Torreya grandis.All of these results helped to establish the molecular mechanism of high oil content and molecular marker assisted breeding.The main results are as followed:1.In order to study the molecular mechanism of Torreya grandis ‘Xifei’ fatty acid synthesis,with Torreya grandis ‘Xifei’ and Torreya grandis ‘Dielsii’ as materials,the expression of 17 genes related to fatty acid synthesis were detected by qRT-PCR.The results showed that RNA-Seq selected 10 genes had similar expression pattern to those of qRT-PCR.Same genes had similar dynamic change rules in Torreya grandis ‘Xifei’ and Torreya grandis ‘Dielsii’,but they had differences in relative transcript level.In Torreya grandis ‘Xifei’ seeds,the continuous up-regulation of KAS1 accelerate the C16 to C18 fatty acids conversion,meanwhile the up-regulation of FAD2-2 promotes the synthesis of unsaturated fatty acids.Combined with previous studies,it is speculated that the unsaturated fatty acids accumulation in Torreya grandis ‘Xifei’ seeds due to the co-work of FAD2-2 and KAS1.In addition,according to the data of Torreya grandis transcriptome and qRT-PCR experiments,it is inferred that KAS1,FAD2-2 and GPAT6 are key genes in fatty acids synthesis.2.The cDNA sequences of KAS1,FAD2-2 and GPAT6 were cloned based on the transcriptome data of Torreya grandis.The sequence of KAS1 cDNA was 1574 bp,encoding 492 aa.The FAD2-2 cDNA sequence was 1131 bp and encoded 376 aa.The GPAT6 cDNA sequence was 1476 bp and encoded 491 aa.The proteins encoded by KAS1 and GPAT6 in Torreya grandis ‘Xifei’ were highly homologous to the proteins which encoded by KAS and GPAT genes in Picea sitchensis,while the FAD2-2 gene encoded protein was highly homologous to the FAD2 in Ginkgo biloba.3.Statistical analysis of Torreya transcriptome data,all high quality reads were assembled into142,213 unigene.A total of 5458 SSRs containing inserts with various microsatellite motifs(di-to penta nucleotides)were identified from 22,976 unigenes(> 1 kb).From these,16 polymorphic SSRs were characterized.The number of alleles of each locus ranged from 2-6,which was equally 3.4 per locus.The average value of polymorphism information content(PIC),observed heterozygosity(Ho)and expected heterozygosity(He)was 0.46,0.28,0.50 respectively.Hence,development of EST-SSRs markers from transcriptome data should be more efficient,and these EST-SSRs markers will be powerful for evaluation on genetic diversity,construction of fingerprint and molecular marker-assisted breeding in Torreya.