Seed Dormancy Process And Dormancy Release In Ginkgo Biloba

Ginkgo biloba is one of the oldest extant gymnosperm.Although Ginkgo seeds have important economic values,the seed dormancy release mechanism is still unclear.This paper used the newly harvested seeds（September）,dormant seeds（November）and dormancy released seeds（February）of G.biloba as the materials,investigating the different development characteristics of the embryo,and trying different stratification to break seed dormancy.Meanwhile,we tested the endogenous hormones of embryo and endosperm respectively.Combining with high throughput sequencing and bioinformatics,we further analyzed miRNAs associated with the process of seed dormancy release.The main results are as follows.（1）Morphological observation and statistical anaslysis showed that the length and weight of the embryo were both significantly increased from September to February.During early stag in dormancy from September（mature seed harvest）to October（the embryo developed slowly in appearance）,the embryo was about 0.78 cm,and its different parts were hard to distinguish.However,the embryo grew rapidly from November to December and the average length is about 1.2 cm.The morphology of embryos changed significantly,and the tissue structure differentiated into the radicle,the hypocotyl,the germ and the cotyledon.The morphological development of embryo was largely completed in February over winter,except the continuing growth in cotyledons.（2）By different stratification to release seed dormancy,we found that the rate of seed germination increased gradually with the time of outdoor stratification.The rate of seed germination was similar in the outdoor stratification between 90 d and 100 d,and the 100 d reached 42.9%,which was 19.93%higher than the control group（35.77）.After 4 weeks of constant 4℃ stratification,seed germination rate of 4℃stratification in 50 d and 60 d were 36%and 35.5%respectively.The seed germination rate of 40 d in 4℃stratification（about 26.5%）was not significantly different from that of the control group.It indicates that the germination rate increased by 4℃ stratification over 50 d.Compared with outdoor stratification,4℃ stratification is an effective method to shorten seed dormancy duration.（3）We used tandem quadrupole mass spectrometer to test endogenous hormones in the dormancy process of seeds.The results showed that the content of ABA was high during three dormancy stages,especially in the Novemeber embryo,the content of ABA was the highest（about 570 ng.g-1）.And the content significantly decreased in February（about 365.6 ng.g-1）.The content of GA was low in three dormancy stages,and the lowest stage was in September embryo（about 5.8 ng.g-1）.The relative content of GA was up to 10.94 ng.g-1 in February embryo.These results indicate that ABA played a major role in maintaining dormancy during the dormancy process of G.biloba seeds.（4）We obtained 13292093,13404160 and 14141212 raw sRNA reads in the Sep,Nov and Feb embryos respectively by high-throughput sequencing.After removing low-quality reads,adapter sequences,poly A sequences,sequences of<18nt,and other artifacts,12749995,13026297 and 13664058 high-quality clean reads were obtained between 18 nt and 30 nt in length respectively.The length of small RNAs was concentrated in 21 and 24 nt,which conforms to a feature of plant miRNA in length distribution.（5）We mapped all the sRNA sequence of the embryos with miRBase database in known mature and precursor miRNA sequences,and obtained 256 conservative sequences,belonging to 49 miRNA families.Among the miRNA results,miR156 and miRl 59 were the two large families with members.We also discovered 198 novel miRNAs,which contain a hairpin structure.In addition,those miRNAs with a threshold qvalue<0.01&|log2（foldchange）|>1 were considered to be significantly up-regulated or down-regulated in expression.A total of 415 differentially expressed miRNAs were screened.Comparison of 3 databases in September,November and February showed that 90 differentially expressed miRNAs in GEN vs GES,174 in GEF vs GES and 151 in GEF vs GEN.（6）To identify putative targets for known and novel miRNAs,we applied the web-based psRNATarget to hunt for potential miRNAs target genes.The results showed that total 1710 and 1168 genes were obtained for predicting targets of known and novel miRNAs respectively.The analysis of GO enrichment showed that predicted targets of differently expressed miRNAs were annotated as biological process,cell components and molecular function.The KEGG pathway analysis showed that differently expressed miRNA targets in GEF vs GES were mainly concentrated in biosynthesis and plant hormone signal transduction pathways.GEN vs GES were enriched in metabolic and plant hormone signal transduction pathways,and GEF vs GEN were in biosynthesis,metabolism and other ways.（7）Further combined with the data of G.biloba genome,transcriptome of embryo and target gene functional annotation,the expression patterns of miRNAs in the process of seed dormancy were analyzed.Among them,miR156 family members were related to embryo development and had high expression levels.miR156a decreased and then increased,while miR156b generally increased,with their highest expression levels in February.The predicted target of miR156 was Gb₂7198,which displayed a decreased（functional annotation SPL gene）tendency in all three dormancy stages,and was contrary to the expression pattern of miR156b.MiRNAs associated with hormonal signaling,such as miR159 family,did not change significantly in the first two stages,and decreased significantly in the third stage.The predicted target of miR 159 is Gb₀2586（functional annotation is MYB gene）which increased in all three dormancy stages,and was contrary to the expression pattern of miR159 in late two stages.In addition,we used the Cytoscape software to construct the interaction networks of key miRNAs and their target genes.The results showed that multiple genes could be targeted by the same miRNA,and some miRNAs targeted to one gene or common genes.（8）We used qRT-PCR experiments to validate the expression patterns of 6 known miRNAs（miR156a,miR56b,miR159a,miR166c and miR172a）,2 novel miRNAs（novel₈9 and novel₁41）and part of their targets in three dormancy stages.The results were mainly consistent with the sRNA-Seq data,suggesting that the high throughput sequencing results were reliable.The results also indicate that these miRNAs play an important role in the dormancy process of G.biloba embryos.In summary,based on the morphological changes,physiological indexes,different stratification and comprehensive analysis of small RNAs,our results provide a comprehensive reference resource for seed dormancy studies in G.biloba seeds.