Heterosis Analysis And Mechanism Research Of Cold-resistant Heterosis In Fraxinus Mandshurica Hybrids

Fraxinus mandshurica Rupr.(Fraxinus L.,M),is one of the most important large-diameter timber tree species in Northeast China,which has a wide range of application scenarios.In response to the lack of excellent germplasm in the production of F.mandshurica and the vulnerability of young seedlings to cold and pests,the interspecific(MA,MV,MS)and intraspecific(MM)hybrid breedings between the F.mandshurica(as female parents,FPs)and the tree species in Fraxinus(The F.americana Linn.(A),F.velutina Torr.(V),F.sogdiana Bunge(S)and F.mandshurica,as male parents,MPs)were conducted.In this research,basing on the seeds and seedlings obtained by interspecific hybridizations of F.mandshurica,the intraspecific hybridization(100 hybrid combinations consist of 15 MPs and 17 FPs)was further carried out.After the systematic analysis of setting effects in hybridizations and heterosis in multiple traits(seed trait and cold adaptability,growth and pest resistant traits of seedlings)of hybrid F1 progenies(F1)and the excellent selection of multiple traits of hybrid germplasm,the explanation of formation mechanism of the cold-resistant heterosis(affect wood formation)among many heterosis of F1 was conducted.Main results were as follows:1.Analysis of setting effects in hybridizations:Through seed collection and seedling breeding,24,165 hybrid seeds and 55 hybrid combinations of intraspecific hybrid F1 were obtained.The setting of interspecific hybridization under the high-voltage electrostatic field(HVEF,20 KV,10 cm,30 min)had been significantly improved(The number of seeds and seedlings in 100 baged inflorescences increased 22.4?517.5%and 190.3?927.8%),so,the technology of HVEF promoting the setting in interspecific hybridizations has been proposed.2.Heterosis analysis of F1 in multiple traits:The hybrid F1 in F.mandshurica showed heterosis in seed trait(the female parental heterosis(HFPs)in TGW index of interspecific hybrids were 10.5?14.2%).Results of the progeny tests in many years indicated that,the interspecific hybrid F1 of mandshurica can live through winter normally in Northeast China.their survival rates and growth trait showed significant heterosis(the highest HFPs in the 12-yr survival rate and volume indexes were 35.6%and 29.6%,respectively),and the HFP of 12-yr pest-resistant trait in MS hybrid was 9.7%,which indicated that the breeding strategy of interspecific hybridization could be significantly effective in genetic improvement of F.mandshurica.Meanwhile,through regression analysis of 14 curve equations and their adaptability tests,tree height growth models of the hybrid F1 and heterosis estimate models of interspecific hybrid F1 were established.3.Selection and breeding of excellent hybrids in multiple traits:The number of seeds and thousand-grain weight(representative breeding traits in the popularization of good varieties),the adaptability,growth,pest-resistant and cold-resistant of the hybrid seedlings were selected and the breeding effects were further evaluated.1)Breeding for seed setting and 1000-grain weight:The selected parents were 45.6?98.8%and 11.6?26.3%higher than the average in seed setting and 1000-grain weight,and the 34 hybrid combinations were 43.1?167.8%and 11.6?26.6%higher than the average.2)Breeding for adaptability,growth and pest-resistant:Parents and hybrid combinations with excellent adaptability were selected.The highest survival rate of the parents exceeding the average of 27.0%(MV)and the highest HFPs of 25 hybrid combinations were 85.2%(MV).Parents,hybrid combinations and single plants with excellent growth and pest-resistant were selected.The volume genetic gain of the parents was 16.8%(MV)and the pest-resistant genetic gain was 2.1%(MS).The highest volume HFPs of 25 hybrid combinations was 164.7%(MV),and the pest-resistant HFPs of 10 hybrid combinations were 43.3%(MS)and 15.9%(MM).The highest volume of 27 MM plants with excellent straightness was 167.5%higher than the over-average value and the pest-resistant of 27 MM and 8 MS plants with excellent pest-resistant were 181.7%and 67.1%to their averages.3)Breeding for cold-resistant hybrid combinations:4 hybrid combinations were selected based on comprehensive values of survival rates,growth and physiological indicators.The heterosis of the representative high cold-resistant hybrid combination(2×8)was evaluated from many aspects.Among them,the 3-yr survival rate HFPs reached 62.6%,and the 3-yr typical single plants had no obvious branches in comparison to the controls,who had more branches after going through the cold winter in Maoershan.The new branches and leaves of grafted clones in F1 also showed significant cold-resistant heterosis.4.Analysis of the formation mechanism of cold-resistant heterosis in hybrid F1 based on DNA sequence differences(nuclear genetic basis):Mechanism analysis of the cold-resistant heterosis from the differences between 2×8 and 2ck at multiple levels:1)Adaptability and growth differences:2×8 seedlings have significant heterosis in their adaptability and growth traits(HFPs of the 6-yr survival rate,volume and 5-yr straightness were 63.1%,66.5%and 7.7%,respectively).2)Cold-resistant coefficient differences of physiological and biochemical indexes:2×8 in osmotic system(HFP of soluble sugar was 292.0%),membrane system(HFP of relative conductivity was 13.7%),ROS(Reactive oxygen species)system(HFP of POD activity was 155.6%)and ABA(abscisic acid)content(HFP was 67.9%)showed significant heterosis.3)Gene expression differences:After cold treatment,genes expression of 2×8 in CBF(C-repeat binding factor)dependent pathway(HFPs of FmLHY(LATE ELONGATED HYPOCOTYL),FmCBF1 and FmCOR413(Cold-regulated 413)were 160%,109%and 495%),ABA pathway(HFP of FmPYR1(Pyracbactin Resistant 1)was 109%),ROS-related genes(HFP of FmPOD was 155%)and 6 cold-resistant related WRKYs(HFPs of Fm WRKY21 and FmWRKY40 were 289%and 240%)all showed significant heterosis.4)Differences analysis in DNA sequences of cold-resistant transcription factors:2×8 had a SNP(Single nucleotide polymorphism,T to A)site,a small 18 bp fragment loss and an extra 58 bp intron at 355 bp,395 bp and 80 bp downstream from the ATG in the FmCBFl,FmWRKY40 and FmWRKY7 gene CDS regions,respectively.In the promoter of FmWRKY7 gene(148 bp upstream of ATG),2X8 had a SNP(G to A)site.According to these results of differences,the specific mechanism of the cold-resistant heterosis in F.mandshurica intraspecific hybrids was that:The intraspecific hybridization recombined the genomes of different hybrid parents.During the DNA recombination process,compared to the female parental control,some important cold-resistant transcription factors(such as FmCBF1.FmWRKY40 and FmWRKY7)mutations in the F.mandshurica intraspecific hybrids with cold-resistant heterosis,like loss,insertions,or mutations in the genes or upstream promoter DNA sequences had been completed.Then,these changes directly affected the expression of key transcription factors such as FmCBF1 and FmWRKY40 genes in hybrid F1 seedlings.Cooperating with rhythm genes,high expression of cold-related genes(CBF pathway genes(FmCIHK and FmICE1),stress response genes CORs(FmCOR413)),ROS and ABA related genes)caused a series of physiological indexes(like osmotic system,membrane system and ROS system)and endogenous hormones(ABA)to respond to cold stress,thereby forming the cold-resistant heterosis in F.mandshurica intraspecific hybrid F1.In this study,the technology of high-voltage electrostatic field treatment to pollen promoting the high-efficiency setting of interspecific hybridization,based on the setting effects analysis,has been proposed.The heterosis analysis of F1 in multiple traits confirm that the interspecific hybrid breeding strategy by introducing pollen can overcome the introduction bottleneck(It is difficult to survive in the Northeast because of the cold envirment)of the three Fraxinus tree species,and feasibly introduce their excellent traits for genetic improvement of F.mandshurica.The excellent selection and breeding of hybrid germplasm of F.mandshurica with multiple traits provide excellent materials and breeding strategies for the construction of large-diameter timber forests in our country and Heilongjiang province.The cold-resistant heterosis mechanism of hybrid F1 through the DNA sequence variation of key cold-resistant genes,gene expression and physiological and biochemical analysis were revealed,and breakthroughs of DNA sequence differences in the key transcription factors between parent and F1 progeny has been made,which lays the foundation in the genetic improvement and further revealing of the heterosis mechanism of the cold-resistant trait in F.mandshurica.