The Genetic Analysis Of The Helical Growth Mutant Helical(hel) And Gene Cloning In Tomato Plants

Tomato is an important economic crop.the helical growth of the plant often has an important effect on the growth and development.The molecular mechanisms underlying helical growth in Arabidopsis have been well characterized.However,the regulatory pathway controlling helical growth remains largely unknown in tomato.The study of the helical growth of tomato not only has important theoretical significance,but also set a good foundation for us to further understand the growth habits in Solanaceous species.The hel mutant obviously showed right-handed helical growth with the cotyledons,true leaves,stem and flower compared with its background material.The hel mutants can also provide valuable experimental materials for further research on the function of tomato microtubules and the research of plant type.In this research,we studied a helical(hel)mutant of tomato.To explore the molecular mechanism of mutant phenotype,the phenotypic identification,genetic analysis and construct generation F2 segregation population with LA1589,development of molecular marker for gene cloning.explore the mutation mechanism of mutant,set a good foundation for us to research on the function of tomato microtubules and the research of plant type.The results of the study are summarized as follows:1.The phenotypic of hel mutant was stable.which obviously showed right-handed helical growth with the cotyledons and petiole of the seedlings compared with its background material.We observed the whole plantlet and found that the leaves and stems showed right-handed organ torsions.The mutated phenotype continued throughout the whole growth period in tomato.2.Measureing the two Cotyledons helical angle between the main leaf veins and petioles of hel mutant found it is about 39.8 ° and 40.1 °respectively.indicating that the mutant of cotyledon is symmetrical spiral growth.3.Paraffin section observation found that spiral growth part of petiole epidermis cells arranged unevenly,and appeared arc arrangement.4.F2 genetic segregation population was constructed by crossing LA1589 with hel mutant.Genetic analysis indicated that the mutant phenotype of hel mutant was controlled by recessive nuclear gene.5.Using the BSR-Seq method for sequencing analysis,the data analysis showed that there was an obvious SNP site on chromosome 4,indicating that the target gene was on chromosome 4.6.We then developed three types of molecular markers to localize the target gene,including insertion-deletion length polymorphysim(InDel),single nucleotide polymorphism(SNP)and cleaved amplified polymorphic sequences(CAPS);By using 8 pairs of InDel markers with polymorphism and 108 progenies showing the helical growth phenotype separated from the F2 mapping population,We delimited the hel gene in an approximately 2.4 cM fragment between the makers CH4-25 and CH4-35;Then we further expanded the F2 mapping population of the cross LA1589 × hel.Exploiting newly SNP and CAPS markers.Fine mapping of hel was then carried out using 1136 individuals showing the helical growth phenotype separated from the F2 mapping population.We finally delimited hel to an approximately 389-kb fragment flanked by makers SNP4-3 and SNP4-6.7.Twenty putative candidates(ORF1–ORF20)were predicted in this fragment between SNP4-3 and SNP4-6 based on the tomato genome annotation.The expression analysis of the partial open reading frame(ORFs)showed that ORF2 was significantly less expressive in the mutants hel than in the isogenic line CR291.We designed specific primers for full-length coding sequences of these candidates,then amplified and sequenced the genomic sequences of these candidates from hel mutant and its background CR291.We aligned the genomic sequences and determined that there were 18 nucleotide changes in the predicted coding sequence of ORF2.Subsequently,we analyzed the amino acid sequences encoded,which showed that the amino acid sequence of the hel mutants was missing from the large segment Compared with CR291.8.Determine the of auxin content in hel mutant as well as the background material CR291 by liquid chromatography.The results show that the auxin content in the hel mutant is only about a third of the controlled CR291,namely,the auxin content decreased significantly in helical growth mutant hel compared to its background materials CR291.9.Expression analysis of some key genes in auxin biosynthesis pathway and signaling pathway of in hel and CR291 by BSR-Seq data.The result showed that in auxin biosynthesis pathway,the expression of P450 and ISS1 in hel was significantly higher than that in CR291,the expression of AMI1 in hel was less than that in CR291.The expression of BSP1 and AXR3 in the hel was significantly higher than that of the control materials CR291 in signaling pathway while the ARF11 have lower expression in hel than that in CR291.