Development Of Alien Addition Lines From Cucumis Hystrix In C.sativus And Observation Of Chromosomal Behavior In Meiosis

The genus Cucumis contains more than 50 species distributing mainly in Asia,Africa and Australia,of which the cultivated species,Cucumis sativus（CC,2n=2x=14）,originating in India,have a narrow genetic base.The cultivated cucumber is sensitive to biotic and abiotic stress.And important resistance genes were absent in the species.C.hystrix（HH,2n=2x=24）,as a wild species of Cucumis,possesses many excellent resistant genes,such as resistant to powdery mildew,downy mildew and gummy stem blight.Therefore,introducing these valuable resistant genes of wild speices into cultivated species through interspecific hybridization would be of significance for crop breeding.At the present,transferring excellent genes by AALs（alien addition lines）has become an important way of crop breeding.One or several alien chromosomes in AALs are more likely to pair with homoeologous chromosomes in background of recipient genome,resulting in the possibility of recombination and exchange of chromosomes and realizing the introgression of alien genes.Therefore,the development of Cucumis sativus-hystrix AALs can make full use of excellent germplasm resources and mining valuable genes of wild species.Meanwhile,AALs provide excellent materials for interspecific genetic improvement of cucumber.In this research,using the synthetic allotetraploid C.hytivus,which derived from the cross of C.sativus and C.hystrix,two backcrossed with diploid cultivar were carried out to create Cucumis sativus-hystrix alien addition lines.Secondly,we screened AALs and determined the number of C.hystrix chromosomes using GISH（genomic in situ hybridization）technique.And high-resolution chromosome painting technique and a set of chromosome-specific sequences markers distributed on C.hystrix chromosome were applied to determine the identities of alien chromosomes.AALs were self-pollinated and screened for DAALs（disomic alien addition lines）by chromosome-specific sequence markers and GISH technique.Further,we investigated the meiotic behavior of alien chromosomes in partial AALs and analyzed the chromosome pairing between alien chromosomes of C.hystrix and C.sativus chromosomes.Finally,we observed the phenotypic characteristics of different AALs.The results are as follows:1.The creation of Cucumis sativus-hystrix alien addition lines.Using allotetraploid,C.hytivus,two backcrossed with cultivated cucumber were carried out to obtain candidate alien addition lines.After GISH analysis,77 out of 110 BC2 progenies were identified as AALs,including 45 monosomic alien addition lines（MAALs）and 32 multiple addition lines（MALs）.We randomly selected 28 AALs plants to determine the composition of alien chromosome by oligonucleotide-fluorescence in situ hybridization（oligo-FISH）technique.Finally,we obtained 10 different types of AALs with one or several alien chromosomes,including five Cucumis sativus-hystrix MAALs（CC-H01,CC-H06,CC-H08,CC-H10,CC-H12）and five MALs（CC-H06+H09,CC-H06+H10,CC-H06+H12,CC-H08+H10,CC-H01+H06+H10）.The chromosome-specific sequence markers of C.hystrix were used as an auxiliary way to further verify the chromosome composition of these AALs,and the results were consistent with the cytological identification results.2.The creation of disomic alien addition linesThe selfed progenies of AALs were screened for AALs by specific sequence markers of C.hystrix chromosome,and then the number of alien chromosomes in AALs was determined by GISH analysis.Finally,we screened and obtained disomic alien addition lines（DAALs）from the selfed progenies of CC-H10 plant,carrying a pair of C.hystrix chromosome H10（CC-2H10）.3.The analysis on meiotic behavior of alien chromosome in AALsThe C.hystrix chromosome pairing behaviors of three AALs,including CC-H01,CC-H06+H10 and CC-H01+H06+H10 at meiosis process were analysed.A total of 134 pollen mother cells（PMCs）were detected at the meiotic metaphase I stage in this research.The alien chromosomes were unpaired as univalent in 122 PMCs,and in 9 PMCs,C.hystrix chromosome paired with a pair of C.sativus chromosome to form a trivalent.Interestingly,the alien chromosomes paired with only one C.sativus chromosome to form a bivalent in 3 PMCs,while the other C.sativus chromosome existed as an univalent.4.Phenotypic traits of alien addition linesWe conducted preliminary phenotypic observations on Cucumis sativus-hystrix alien addition lines,and found that the phenotypes of most AALs were biased toward cultivated cucumber,such as leaf shape,growth potential,branching.However,some traits of partial AALs are different from cultivated cucumber.The leaves edge of AALs with chromosome H06 of C.hystrix was wavy,and the leaves edge of AALs with chromosome H12 was serrated.