| Lilium spp.is a famous bulbous flower in the world.It has a long history of medicinal,edible and ornamental cultivation.China is the natural distribution center of Lilium in the world,and its wild species distributed are the important parents of Lilium breeding.The investigation and systematic evaluation of lily germplasm resources are the basis of germplasm innovation;molecular assisted breeding is one of the important development directions of modern lily breeding,but the related research is less and the foundation is weak.Therefore,based on the systematic investigation and collection of lily resources,phenotypic diversity of wild species and cultivar populations was evaluated;EST-SSR markers were developed from three transcriptome databases of lily;genetic diversity and population genetic structure were analyzed.Hybridization was widely carried out,and phenotype and genetic diversity of hybrid and inbred populations were analyzed which would provide scientific basis for molecular marker assisted selective breeding.The main results and conclusion in this study as follows:1.The phenotypic characters of 233 resources were investigated and analyzed.The results showed that there were great disparities among Lilium hybrids on the leaf width and flower characters(flower color,number of spots,length of style,width of inner tepal,length of filament and length of pistil,etc.).The phenotypic diversity varied greatly among different hybrids,and population of the wild species was the highest,followed by Asiatic hybrids(A)> Oriental trumpet hybrids(OT)> Oriental hybrids(O)>Longiflorum asiatic hybrids(LA)> Trumpet hybrids(T)> Formolongi Hybrids,Longiflorum hybrids,Longiflorum oriental hybrids(F / L / Lo)> Martagon Hybrids(M).The correlation analysis showed that there was significant or extremely significant positive correlation among plant height,stem diameter,leaf and flower characters.The principal component analysis showed that the main characters of different hybrids were pistil length,filament length,inner tepal length and width,diameter of stigma top.By cluster analysis and principal component analysis,the germplasm resources of Lilium can be divided into six types: O and OT hybrids(phenotype biased O);L,T and OT hybrids(phenotype biased T);LA and A hybrids(spotted);LA and A hybrids(spotless);wild species with small flowers and M hybrids;double-flowered varieties and new flower types.2.Based on the rich variations in flower colors,pattern chart of lily's flower color was first proposed,taking the flower color,spot or spotless and spots raised or unraised as three key classification characters.Then,different types of flower color are divided into 13 types according to the standard,which would provide valuable reference for lily variety identification,application and new variety selection.The flower color value of each variety was measured by instrumental color measurement.The suitable position for the measurement of flower color was discussed from three positions: inner and outer valve,front and back,upper and lower parts.Through comparison of different methods of color classification(Q cluster analysis and ISCC-NBS color name chart),the quantitative classification of flower color phenotypes of lilies was studied for the first time.The flower color of lily was divided into 8 groups,which filled the gaps of research on flower color phenotypes of lily.From the perspective of the color distribution of different lily hybrids,A,LA and OT are the three hybrids with the widest color distribution,and the A and OT hybrids contain the most multicolor varieties.3.EST-SSR markers were developed by using transcriptome database.32611 SSR loci were identified from 188606 Uni Gene transcripts of three transcriptome databases,with an average distribution frequency of 17.29% and an average of 4.48 KB per SSR locus.The most abundant SSR motifs were trinucleotide and dinucleotide.21862 pairs of primers were designed by software.151 pairs of primers were screened by multi sequence comparison and 43 pairs of primers were selected for subsequent tests after preliminary screening and rescreening.In addition,6 SSR primers were developed from the transcriptome database of potted asiatic variety,total 49 pairs of SSR primers with high amplification efficiency,good polymorphism and stability were used as follow-up experiments.The results show that the primer screening method based on multiple sequence comparison can predict some invalid or failed primers in advance,and effectively improve the efficiency of screening.4.The genetic diversity and population structure of 240 lily varieties and wild species were analyzed by 49 pairs of core primers.The genetic relationship between the hybrids and wild species was revealed at the molecular level.The population of wild species possed the highest genetic diversity,and then the LA,OT and A hybrids,higher than that of other hybrids.Cluster analysis showed that,L,F and LA clustered into one group,O,T and OT clustered into one group,M independent into one group;the wild species that participated in the modern lily breeding were concentrated in each hybrid group,while the wild species that rarely participated in the modern lily breeding were concentrated into another group.These data partly reflect the phylogenetic relationship among wild species in China,and show the original parent composition of each hybrids of Lilium.Based on the analysis of genetic composition and phenotype,it can be concluded that T biased varieties in OT hybrids have more breeding advantages than O biased varieties.5.Nine pairs of SSR molecular markers with high polymorphism information content were screened.Combined with flow cytometry(FCM)detection and root tip squash test,we tried to use microsatellite markers to predict the ploidy of polyploids in the population.Among 116 varieties,109 were correctly predicted as diploid or polyploid,and the accuracy was 93.97%.6.After 997 cross combinations of Lilium,322 were successfully obtained with embryonic offsprings,including the triploid LA and OT hybrids as the female parent.It was found that 45 species or varieties were self-fertility after self cross test of 85.The highest fruit setting rate and embryo rate were wild species,T and A hybrids.The ploidy of parents and the distance of genetic relationship have great influence on cross compatibility.234 F1 progenies from 7 cross combinations of A hybrids were authenticated as true hybrids by using identification of 21 pairs of SSR primers.Through the analysis of the phenotypic character F1 progenies from 7 cross combinations of A hybrids,it was showed that A50 × A1 and AT8 × AT11 had more advantages in the cultivation of large flower varieties.The flower color of most F1 progenies from different cross combinations were between parents,and the cross combinations with the largest variation of flower color were A49 × A4,A48 × A4 and A41 × AT11.The number of spots in each cross combination showed continuous separation,and the number of spots in offspring was correlated with that in parents,while the number of spots in inner tepal was positively correlated with that in outer tepal.In the same flower,the number of spots in outer tepal was always lower than that in inner tepal.Pollen free progeny appeared in 4 combinations,and there were five types of stamens phenotype in pollen free plants.The flower phenotypic characters of sterile plants were significantly smaller than those of fertile plants.A41,A49 and A50 are all good breeding female parents for pollen free breeding.No pollen character can be inherited stably in two years.The phenotypic analysis of trumpet inbred population showed that the variation of flower number,leaf width and other characters was rich,and the flower color of the progeny was separated,and there were three types: light yellow green(near milky white),yellow,orange,and yellow were the most.24 SSR primers were used for genetic diversity analysis.The genetic diversity of the A hybrids was significantly higher than that of the trumpet self cross population.The SSR genetic diversity of A29 ×AT3,A50 × A1 and AT14 × AT11 was higher than that of other combinations.The clustering results based on molecular markers mightbe correlated with some phenotypic characters of flowers.Cluster analysis showed that: Lilium lancifolium(W1),Lilium leichtlinii.var.maximowiczii(W2),Lilium davidii var.davidii(W4),Lilium davidii(W5)and Lilium amabile(W26)might be the original parents of these A hybrids varieties;Lilium leucanthum(W16),Lilium regale(W17)and Lilium sulphureum(W37)might be the original parents of the tested T hybrids varieties. |
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