| Tendrils are climbing organs in cucumber(Cucumis Sativus L.).Allowing tendrils freely growing results in serious shading between plant and support rope,which influences ventilation and transmitting and makes field management hard to carry out.What's more,frizzy tendrils can bind to branches and fruits.Most important of all,inserting in the same node order,tendrils strongly compete nutrients against female flowers,and therefore,cucumber production will dramatically increase by removing tendrils as early as possible.For reducing consuming of nutrients and avoiding difficulties in management,tendrils are often wiped off artificially.However,as a result,such practice will certainly increase the amount of labor and cost of production..Thus,tendril-less character is theoretically and practically important to high production and substrate culture.Since all cucumber germplasm have tendrils in axil and the tendril-less mutant “B007”had no tendrils during whole growing process,which was important material for researching formation mechanism of tendrils in cucumber.Here,two F2 genetic populations were constructed by crossing tendril-less mutant “B007”(P1)with North America phenotype“Gy14”(P2)and North China phenotype “9930”(P3)respectively to analysis inheritance and map the td(Tendril-less)gene.At meantime,BSA-seq was introduced to screen the mutant site and the candidate genein theinterval of Cstd.In addition,above-ground morphogenesis was determined by shoot apical meristem(SAM),hence,knowledge on occurrence and formation of cucumber tendrils could not be gained without anatomy study of developing SAM.Our research improved paraffin sections protocol and by which impact on cucumber SAM was discussed to lay a foundation for understand tendrils formation.Main research results were followed:1.Inheritance and preliminary mapping of Cstd geneF1 progeny of B007 with Gy14 and 9930 were selfed to acquire 1673 F2 progeny plants in P1/P2 and 834 F2 progeny plants in P1/P3,which was used for statistics of tendrils segregation ratio.Results demonstrated that P1/P2 population was composed of 347tendril-less plants and 1326 normal plants while P1/P3 population was made up of 196tendril-less plants and 638 normal plants.The segregation ratio was almost 1:3 by chi-square test(P=0.05),which indicated cucumber tendril-less gene Cstd was a qualitative trait controlled by a single recessive gene.Screening 2489 pairs of primers resulted in 193 pairs of polymorphic primers in P1/P2 and 129 pairs of polymorphic primers in P1/P3 and their polymorphism ratio was 7.8% and 5.2% respectively.Selecting 5 individuals from each population to construct tendril and tendril-less gene mixing pool to analyze the polymorphic primers above by taking bulked segregant analysis.New markers were developed according to the linkage markers to gain more linkage ones.Finally,16 linkage markers and 5 linkage markers were used to map Cstd into 197 kb interval on the terminal of chromosome 6 in cucumber.2.BSA-seq for screening the mutant site and candidate genes102 dominant plants and 102 recessive plants were taken from F2 population in P1/P2 for equal DNA mixing to be high-through re-sequenced.Comparing with genome of 9930 and wildtype ones,impact of mutant sites on genes was comprehensively analyzed according to SNP index.A nonsynonymous mutation was detected in mapping interval of 197 kb,which was(G28893996A)on chromosome 6 in cucumber based on genome information of 9930.3.Inheritance linkage and diversity analysis of candidate geneThe mutant site filtered by gene mapping and BSA-seq was utilized to design a pair of dCAPS marker to be tested in 500 individuals in P1/P2 and P1/P3.Results showed that the mutant site was con-segregated with Cstd.Moreover,unicity of the site was verified in about400 different cucumber germplasms and which indicated that the very site existed only in B007,which proved that the candidate gene was the target gene controlling tendril-less phenotype of cucumber.4.Cloning of the candidate genePredicted in Cucurbitaceae database,the gene where Cstd site laid was used to designed amplification primers.The candidate gene was amplified from 9930 and B007 respectively and was sub-cloned by TA cloning.Results revealed that length of the candidate gene in 9930 and B007 was both 1686 bp,and the corresponding protein was predicted to be made up of562 amino acid residues.The 82 th AA was changed from D to N in wildtype5.Improvement on methods of cucumber SAM paraffin sectionsExperiment was conducted with apical meristem of cucumber as material for paraffin sectioning to improve transparenting,waxing and staining.The results showed that combining entire staining with section staining can improve the efficiency of paraffin section and quality of staining.Using stearic alid as clearing agent can keep colour fresh and make no significantdifferent with general xylene clearing agent in paraffin section expect that stearic alid may cause some dropping of sections.Before being dewaxed with xylene for 50 min,heating slides as well as dye vat to 40° so that dewaxing can be thorough.Section staining was performed with safranine and fast green double dyeng,which was the best method of staining.The quality of staining was good and homogeneous,and the process was simplified. |
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