New opportunities for using WUS/BBM and GRF-GIF genes to enhance genetic transformation of ornamental plants

Broad application of plant transformation remains challenging because the efficiency of plant regeneration and regeneration-based transformation in many plant species is extremely low.Many species and genotypes are not responsive to traditional hormone-based regeneration systems.This regeneration recalcitrance hampers the application of many technologies such as micropropagation,transgenic breeding,and gene editing in various plant species,including ornamental flowers,shrubs,and trees.Various developmental genes have long been studied for their ability to improve plant meristematic induction and regeneration.Lately,it was demonstrated that the combined and refined expression of morphogenic regulator genes WUSCHEL and BABY BOOM could alleviate their pleiotropic effects and permit transformation in recalcitrant monocots.Moreover,ectopic expression of plant growth-regulating factors(GRFs)alone or in combination with GRF-interacting factors(GIFs)improved the regeneration and transformation of dicot and monocot species.Fine-tuning the expression of these genes provides new opportunities to improve transformation efficiencies and facilitate the application of new breeding technologies in ornamental plants.

Genome-wide association studies for inflorescence type and remontancy in Hydrangea macrophylla

Inflorescence type and remontancy are two valuable traits in bigleaf hydrangea(Hydrangea macrophylla L.)and both are recessively inherited.Molecular marker-assisted selection(MAS)can greatly reduce the time necessary to breed cultivars with desired traits.In this study,a genome-wide association study(GWAS)using 5803 single-nucleotide polymorphisms(SNPs)was performed using a panel of 82 bigleaf hydrangea cultivars.One SNP locus(Hy_CAPS_Inflo)associated with inflorescence type was identified with general linear model(GLM)and mixed linear model(MLM)methods that explained 65.5%and 36.1%of the phenotypic variations,respectively.Twenty-three SNPs associated with remontancy were detected in GLM whereas no SNP was detected in MLM.The SNP locus(Hy_CAPS_Inflo)was converted to a cleaved amplified polymorphic sequence(CAPS)marker that showed absolute identification accuracy(100%)of inflorescence type in a validation panel consisting of eighteen H.macrophylla cultivars.The SNP was investigated in 341 F1 progenies using genotyping by sequencing(GBS)and co-segregated with inflorescence type(χ^(2)=0.12;P=0.73).The SNP was subsequently used for breeding selection using kompetitive allele specific PCR(KASP)technology.Future directions for the use of genomics and MAS in hydrangea breeding improvement are discussed.The results presented in this study provide insights for further research on understanding genetic mechanisms behind inflorescence type and remontancy in H.macrophylla.The CAPS and KASP markers developed here will be immediately useful for applying MAS to accelerate breeding improvement in hydrangea.

Identification of Tsuga Germplasm by Morphological Characters and RAPD Markers

Germplasm collection is important to preserve and maximize genetic diversity for germplasm conservation.Tsuga dumosa(D.Don)Eichler in Engler & Prantl.and T.chinensis var.forrestii(Downie)Silba germplasm was collected from three localities in China:Mt.Yulong,Wenfeng Temple and Mt.Dishiergu,Yunnan Province.Accessions were identified based on morphological characters and RAPD markers.The shapes of the apices and margins of needles were examined,and the length and width of needles,cones and seeds from accessions of mature plants were used to compare the morphological differences and to identify the germplasm.Molecular markers generated by randomly amplified polymorphic DNA(RAPD)were also used to characterize the taxa.Although the clustering based on RAPD markers was inconsistent with the morphological characters of the needles,based on the overall morphological characters and on RAPD markers,the accessions from Mt.Yulong and Wenfeng Temple were identified as T.chinensis var.forrestii,and those from Mt.Dishiergu identified as T.dumosa.Taxonomic identification of the accessions was made based on morphology and by RAPD markers concurred.The results indicate that the shapes of the apices and margins of needles particularly from young plants could not be used as a possible key to identify T.dumosa and T.chinensis var.forrestii.Fig 6,Tab 3,Ref

A hybrid swarm population of Pinus densiflora × P. sylvestris inferred from sequence analysis of chloroplast DNA and morphological characters

To confirm a hybrid swarm population of Pinus densiflora × P. sylvestris in Jilin, China, we used needles and seeds from P. densiflora, P. sylvestris, and P. densiflora × P. sylvestris collected from natural stands or experimental stations to study whether shoot apex morphology of 4-year old seedlings can be correlated with the sequence of a chloroplast DNA simple sequence repeat marker （cpDNA SSRs）. Total genomic DNA was extracted and subjected to sequence analysis of the pine cpDNA SSR marker Pt15169. Results show that morphological characters from 4-year old seedlings did not correlate with sequence variants of this marker. Marker haplotypes from all P. sylvestris trees had a CTAT element that was absent from all sampled P. densiflora trees. However, both haplotype classes involving this insertion/deletion element were found in a P. densiflora × P. sylvestris population and its seedling progeny. It was concluded that the P. densiflora × P. sylvestris accessions sampled from Jilin, China resulted from bi-directional crosses, as evidenced by both species’ cpDNA haplotypes within the hybrid swarm population.

Engineered Cleistogamy in Camelina sativa for bioconfinement

Camelina sativa is a self-pollinating and facultative outcrossing oilseed crop.Genetic engineering has been used to improve camelina yield potential for altered fatty acid composition,modified protein profiles,improved seed and oil yield,and enhanced drought resistance.The deployment of transgenic camelina in the field posits high risks related to the introgression of transgenes into nontransgenic camelina and wild relatives.Thus,effective bioconfinement strategies need to be developed to prevent pollen-mediated gene f low(PMGF)from transgenic camelina.In the present study,we overexpressed the cleistogamy(i.e.f loral petal non-openness)-inducing PpJAZ1 gene from peach in transgenic camelina.Transgenic camelina overexpressing PpJAZ1 showed three levels of cleistogamy,affected pollen germination rates after anthesis but not during anthesis,and caused a minor silicle abortion only on the main branches.We also conducted field trials to examine the effects of the overexpressed PpJAZ1 on PMGF in the field,and found that the overexpressed PpJAZ1 dramatically inhibited PMGF from transgenic camelina to non-transgenic camelina under the field conditions.Thus,the engineered cleistogamy using the overexpressed PpJAZ1 is a highly effective bioconfinement strategy to limit PMGF from transgenic camelina,and could be used for bioconfinement in other dicot species.

Integrative genomics reveals paths to sex dimorphism in Salix purpurea L

Sex dimorphism and gene expression were studied in developing catkins in 159 F 2 individuals from the bioenergy crop Salix purpurea,and potential mechanisms and pathways for regulating sex development were explored.Differential expression,eQTL,bisulfite sequencing,and network analysis were used to characterize sex dimorphism,detect candidate master regulator genes,and identify pathways through which the sex determination region(SDR)may mediate sex dimorphism.Eleven genes are presented as candidates for master regulators of sex,supported by gene expression and network analyses.These include genes putatively involved in hormone signaling,epigenetic modification,and regulation of transcription.eQTL analysis revealed a suite of transcription factors and genes involved in secondary metabolism and floral development that were predicted to be under direct control of the sex determination region.Furthermore,data from bisulfite sequencing and small RNA sequencing revealed strong differences in expression between males and females that would implicate both of these processes in sex dimorphism pathways.These data indicate that the mechanism of sex determination in Salix purpurea is likely different from that observed in the related genus Populus.This further demonstrates the dynamic nature of SDRs in plants,which involves a multitude of mechanisms of sex determination and a high rate of turnover.