Mutant Construction And Function Analysis Of Potato Haploid Induction Gene StDMP

Potato（Solanum tuberosum）is an annual dicotyledonous plant in Solanaceae,originating in the Andes mountains of South America.It has been cultivated in China for more than 400 years.Because of its strong tolerance,high yield,wide adaptability and complete nutrition,potato has become the fourth largest food crop after wheat,rice and corn.China is the largest potato producer in the world.Developing potato industry is of great significance for guaranteeing food security and increasing farmers’ income.Tetraploid potato genome is highly heterozygotic and genetic analysis is complex,resulting in long breeding cycle and slow variety update.Diploid potato genome is relatively simple with rich genetic variation.Diploid potato hybrid breeding will greatly help the development of potato industry.However,most diploid potatoes are selfincompatibility,and after a long time of asexual reproduction,a large number of harmful mutations have accumulated in the genome,and inbreeding recession often occurs in the breeding process,which brings challenges to the creation of high-purity diploid inbred lines.Compared with the traditional method of multi-generation selfcrossing,the haploid induction technique can obtain highly homozygous potato material in one generation,which has obvious advantages such as low workload and short cycle.At present,a number of studies have shown that functional loss of Domain of unknown function membrane protein（DMP）can induce haploid formation in a variety of plants,but its application in potato has not been reported yet.In this study,St DMP gene was identified in potato by constructing phylogenetic tree,and then mutants with St DMP gene dysfunction were constructed by combining CRISPR/Cas9 system with RFP screening markers specifically expressed in embryos.On this basis,nearly 100% homozygous diploid potato material was obtained by hybridization,molecular marker identification,flow cytometry determination and colchicine doubling,and a method system for the construction of high purity diploid potato inbred line using double haploid technology was preliminarily established.The main research results are as follows:1.A total of 6 DMP genes were identified in potato genome.In this study,the phylogenetic analysis of DMP genes in tomato,maize,Arabidopsis and potato was conducted.The results showed that Soltu.dm.05G005100 was compared with Zm DMP（Zm00001d044822）,At DMP8（AT1G09157.1）,At DMP9（AT5G3965.1）and Sl DMP（Soly C05G007920.2.1）.It was named St DMP because of its closest relative.St DMP contains one DUF679 domain and four transmembrane domains.Transient expression of St DMP in tobacco showed that St DMP was located on the cell membrane,and tissuespecific expression pattern analysis showed that St DMP was highly expressed in pollen,which was consistent with previous reports in maize,Arabidopsis and tomato.It indicates that the loss of St DMP can also induce haploid formation in potatoes.2.In order to study the function of St DMP gene,this study constructed a doubleallelic mutant stdmp with function loss on the background of S.tuberosum Group Phureja S15-65（S15-65 for short）in diploid potato.Compared with wild-type plant S15-65,there was no significant difference in vegetative growth of stdmp mutant.However,pollen viability of stdmp mutant decreased significantly,but pollen tube germination and fertilization were still possible.3.In this study,tens of thousands of potato seeds were obtained by crossing stdmp mutant with diploid potato PG6359 and Up1（a hybrid）.Some haploid plants without fluorescence in embryo and radicle but with weak fluorescence in endosperm were identified by fluorescence labeling screening.Subsequently,further molecular markers and flow cytometry were used to identify 7 haploid materials from two cross combinations.Compared with diploid parents,haploid plants were weaker,showing yellow leaves,shortened internodes,and sparse roots.4.In order to study the genetic background of haploid plants,this study carried out resequencing analysis on 4 haploid plants.The analysis results showed that the haploid genomes were all from the mother and there was no fragment of the male induction line,indicating that stdmp mutant is an ideal induction line for maternal parthenogenesis.In addition,the distribution of heterozygous SNPS in the haploid genome was also analyzed in this study,and it was found that the haploid genome contained almost no heterozygous fragments compared with the high-generation inbred line,and the genome homozygous degree was significantly higher than that of the high-generation inbred line.5.In order to obtain homozygous diploid inbred lines,haploid plants were treated with colchicine at different concentrations in the hope of determining the effective colchicine concentration that can promote the doubling of haploid.Results showed that Haploid was Doubled to diploid by continuous treatment at 0.5% colchicine concentration,and 3 of the 12 treated haploids were successfully converted to double Haploid（DH）.The growth of the double haploid is similar to that of the parent.Compared with the haploid,the leaves of the double haploid are obviously larger,internode elongation,root system is strong,and it has the potential to be further cultivated into an excellent inbred line.To sum up,this study built using double haploid technology to obtain high purity of diploid potato material method,compared with the traditional method of selfing,don’t have to overcome the types of potato structural obstacles,and greatly shorten the breeding cycle,about it can be finished within 8 months,this will bring the diploid potato cross breeding to provide strong technical support.