Mechanism Of Cold Resistance On Kiwifruit Based On Genome Re-sequencing And BSR-Seq

The freezing injury,which is one of the important abiotic stresses in horticultural crops,can influence the growth and development of kiwifruit（Actinidia Lindl.）,which results in a dramatical reduction or destroying the garden,breeding fine cultivar with strong cold tolerance is one of the important targets in kiwifruit breeding.At present,our understanding of cold resistance of kiwifruit is scarce,there were fewer studies on molecular mechanisms.In this study,four genetic populations were constructed,through the analysis of nuclear genome and chloroplast genome,we aimed to study the cold resistant trait in Actinidia arguta,learning the heredity laws of cold resistance in F1populations,and screening the key genes of cold resistance by BSR-Seq method,one candidate gene was chosen to identify the function through transgene in Arabidopsis,which may provide a scientific basic for kiwifruit cold resistance breeding.We mainly did five aspects which are as follow:1. Study on the genetic rule of cold resistance in F1 populations in A.arguta.Four biparental populations were constructed.The analysis of leaf color in autumn implied that the time when leaf became yellow in autumn（deciduous time）was related to male parent.Cold resistance identification was performed in all the 492 F1 populations in‘Ruby-3’בKuilv male’.REL of F1 populations showed normal distribution when treated with-30℃,and super parent trait was appeared in populations.The highest LT50 was-7.5℃,and the lowest LT50 was-36.9℃in populations.F1 populations provide basic materials for cold resistance study.2. Study on change rule of physical index of cold resistance in A.chinensis,A.deliciosa and A.arguta at overwintering stage.The results showed,as winter temperature dropped,the content of soluble sugar showed increasing trend,the content of soluble protein,proline and SOD raised first and fell later.Comprehensive analysis of cold resistance by subordinative function value showed that the cold resistance order was:‘Kuilv male’>‘Yongfengyihao’>‘Boshanbiyu’>‘Hongbei’>‘Hongyang’>‘Xuxiang’,which was consistent with LT50.In winter,‘Kuilv male’accumulated the most physical index.Correlation analysis with temperature showed soluble sugar（SS）and mean highest temperature had significant negative relationship except in‘Boshanbiyu’,SS might response for low temperature firstly.Physical index represent the cold resistance to a certain degree,this study provide physical support for cold resistance gene mining.3. Development and identification of mutant genes related to cold based on nuclear genome and chloroplast genome re-sequencing.Re-sequencing was performed in cross parents（‘Ruby-3’and‘Kuilv male’）and two other males（‘Yongfeng male’and‘Hongbei male’）.In total,4,710,650,4,646,026,4,787,750 and 4,590,616 SNPs and 1,481,002,1,471,304,1,534,198 and 1,425,393 In Dels were detected in the‘Ruby-3’,‘Kuilv male’,‘Yongfeng male’and‘Hongbei male’genomes,respectively,compared with the reference genome of cv‘Hongyang’.Genes related to non-synonymous SNPs and In Dels in coding domain sequences were screened for functional analysis.GO and KEGG analysis showed that genes involved in cellular responses to water deprivation,sucrose transport,decreased oxygen levels and plant hormone signal transduction were significantly enriched in A.arguta.A subset of 120 In Dels were selected for re-sequencing validation,81 primer pairs showed amplification,64 primer pairs were polymorphism.16 variable genes contained CBF transcription factor were selected for q RT-PCR verification random,and except 4 genes had not been detected,other genes were response to low temperature.Chloroplast（cp）genome of female parent（‘Ruby-3’）was sequencing using Illumina and Pac Bio RS II sequencing technologies.The cp genome from A.arguta was 157,611bp in length and composed of a pair of 24,232 bp inverted repeats（IRs）separated by a20,463 bp small single copy region（SSC）and an 88,684 bp large single copy region（LSC）.Overall,the cp genome contained 113 unique genes,containing 79 coding genes,4 r RNA genes,30 t RNA genes.The cp genomes from A.arguta and three other Actinidia species from Gen Bank were subjected to a comparative analysis.Indel mutation events and high frequencies of base substitution were identified,Forty-seven simple sequence repeats（SSRs）and 155 repetitive structures were identified.rbc L and rpo A genes were different expression in low temperature and were used as candidate genes of cold resistance.The analysis of cp genome provide polymorphism information to develop cold resistant genes in chloroplast.4. Development of cold resistant genes.F1 genetic populations with 20 cold sensitive populations and 20 cold tolerant populations were sequenced by BSR-Seq method.A total of 28,496 genes were obtained,126 genes were identified by gene expression analysis,59 genes were up-regulated and 67 genes were down-regulated.Gene Ontology（GO）analysis showed there were 21 GO terms in up-regulated genes;KEGG enrichment analysis showed genes involved in starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism and plant hormone signal transduction.There were main 10 key enzyme encoding genes and two regulatory genes.The encoding genes were ADP glucose pyrophosphorylase（AGP）,granule-bound starch synthase,sucrose synthase（SUS）,1,4-alpha-glucan-branch enzyme,alpha-1,4 glucan phosphorylase,beta-amylase（BAM）,glucan water dikinase（GWD）,neutral alpha-glucosidase,disproportionating enzyme 2（DPE2）and Proline rich protein（PRP）,the regulatory genes were EIN-binding F box（EBF）and 14-3-3,these two genes could also regulate the expression of CBF genes.BSR-Seq results were consistent with q RT-PCR results.5. Function identification of cold resistance of CBF gene.CBF gene was cloned from A.arguta and A.chinensis,CBF was responded to low temperature,the expression of CBF was increased at first and then decreased as air temperature dropped in winter.When overexpression Aa CBF in Arabidopsis,transgenic plant showed the dwarf phenotype.After-2℃treatment,overexpression plant of Aa CBF showed the lower REL and accumulated the higher O^2-,the content of soluble sugar,proline,the activity of SOD and beta-amylase were higher in transgenic plants than in WT.We speculate Aa CBF gene may activate the COR genes and then improve cold resistance.