Genetic Diversity Identification Of Hunan Sour Orange Genotypes And Genomics Analysesof Thornless Sour Orange Mutant

Citrus aurautium L.is an important material for genetic improvement and germplasm innovation of citrus in China.It is widely distributed and rich in genetic diversity which has long history of cultivation.With developed roots,cold resistance,drought resistance and strong disease resistance,Citrus aurautium L.played an important role in the classification and origin evolution of citrus.It is not only one of the parents of many citrus such as lemon,but also one of the important rootstocks resources in the world citrus industry.Hunan residents always use it due to its moisturizing,antitussive and medicinal effects.Sour orange and its cultivated varieties have been widely concerned and applied as Chinese herbal medicines in production.Besides,variation and hybrid materials of sour orange appeared in the long-term artificial cultivation process.However,with the gradual expansion of human activities,many rare resources are facing the danger of extinction due to the damage of environment.Citrus always has thorns,especially Poncirus trifoliata（L.）Raf,C.aurautium L.,C.jambhiri Lush.,C.ichangensis Swingle,and other species.Industry practice shows that pericarp can easily punctured by thick thorns which result in significantly decrease of fruit commerciality.It will prick the hands by thorns during fruit picking which directly reduced the productivity.During grafting of seedlings,the cutting of thorns on rootstocks is a waste of labor force and make against of mechanization.It has a great value for citrus seedling and production to understand the growth and development characteristics and formation mechanism of citrus thorn traits.In this study,a number of domestic and foreign varieties of sour orange were collected and preserved,and the genetic diversity and classification relationship were also analyzed by morphology,molecular markers and genomics.Then,the genetic relationship and genetic background of some local citrus hybrids‘Huarongdao Zhoupigan’and‘Local Juhong’were identified,and their parental types and possible evolutionary relationships were primarily clarified.Finally,analysis of genetic variation in Thornless sour orange provides basis for the identification and utilization of thorn trait by using morphological,genetic and genomic techniques.The main results are as follows:1.A total of 112 samples were collected.Among them,88 samples were from48 counties and districts in 14 cities and prefectures of Hunan Province,8 samples were from the Citrus Research Institute of Southwest University,which belong to the United States,Brazil,Morocco,Italy,Japan,Australia,Mexico,16 samples were from Hubei,Shaanxi,Chongqing,Guizhou,Guangdong,Jiangxi,Fujian,Jiangsu,Zhejiang,Shanghai provinces.2.The morphological diversity of fruits and leaves of sour oranges and its hybrids and varieties was analyzed.The results showed that the fruit polar diameter was 40.21-98.95 mm,equatorial diameter was 47.28-107.03 mm,fruit shape index was 0.70-1.08,pericarp thickness was 3.10-10.64 mm,single fruit weight was 46.9-475.6 g,and single fruit seed number was 8.2-58.8.Leaf length was 24.56-144.36mm,leaf width was 14.74-65.32 mm,leaf shape index was 1.67-2.52,leaf thickness was 0.163-0.354 mm,length of petiole wing was 5.63-35.10 mm,width of petiole win was 1.64-25.11 mm.3.The cluster analysis of morphology of leaves,fruits and molecular marker were carried out.According to the leaf phenotype,the leaves were divided into small and circular,middle and large petiole wing,middle and small petiole wing,small and long leafe,large and large petiole wing,and small-leaf sour orange types.According to the fruit phenotype,they were divided into medium fruit thick pericarp,medium fruit thin pericarp,medium fruit mediumthick pericarp,small fruit,large fruit thick pericarp,and small-leaf sour orange types.According to the results of molecular marker,they were divided into intra-provincial sour orange resources I（Yuanjiang River Basin and Dongting Lake Region）,intra-provincial sour orange resources II（other regions in the province）,intra-provincial sour orange resources III（Changde Region）,inter-provincial and foreign sour oranges and hybrid resources,and large petiole wing sour orange hybrid resources.Finally,the taxonomic results of molecular marker,leaf and fruit phenotypes were compared and discussed4.Sixty-one representative sour orange resources were used for genome-wide resequencing and phylogenetic analysis,which was selected by the cluster analysis results of SSR molecular marker,combined with the geographical distribution,leaf and fruit characters and cluster analyses,and elimination of obvious hybrid and approximate repeat materials.The results showed that 61 representative sour oranges were divided into three groups:Group I was 38 local sour orange resources in Hunan Province,Group II was composed of 12 sour orange resources outside Hunan Province,2 local resources in Hunan Province and 2 foreign,Group III was composed of 4 foreign resources and 3 resources outside Hunan Province.The correlation between genetic distance and geographical distribution is more obvious.5.On the basis of phenotypic observation,n SSR and cp SSR markers were used to determine the parental types,and the parental hybridization and evolutionary relationship were speculated according to the proportion of genome similarity.The results showed that the pollen parent of‘Huarongdao Zhoupigan’may be Nanju,and the seed parent may be orange.The genomic sequence similarity rate of‘Huarongdao Zhoupigan’with mandarin was 36.09%,with mandarin heterozygosity was 46.5%.It was speculated that‘Huarongdao Zhoupigan’may be a natural hybrid with sour orange（a sexual hybrid of pummelo and mandarin）as the seed parent and mandarin as the pollen parent.Similarly,the seed parent of‘Local Juhong’was closely related to orange and pomelo,its pollen parent may be mandarin.Its main gene component was mandarin-pummelo hybrid,with a similarity rate of 70.88%.So,it is speculated that‘Local Juhong’may be the F₁generation with pummelo as the seed parent and mandarin as the pollen parent,and it is sister relationship with‘Huangpi’sour orange.6.Genome-wide resequence was performed between Thornless and ordinary sour orange,then analyzed their SNP,In Del and SV.The results showed that most of the variations of the two materials were distributed in the intergenic region,followed by introns and exons,compared with the reference genome of sweet orange.The number of In Del in coding region was significantly lower than that in non-coding region,and the number of hybrid In Del was as high as 6800,while that of homozygous In Del was only 400-500.the number of structural variation sites was obviously less than that of SNP and In Del,the Deletion（DEL）were maximum which both more than 2000,however,the Inversion（INV）were only 20 and 26.The SV became much more intuitionistic when the variation point located to reference chromosomes.Hormone related gene IAA,transcription factor AP2/ERF and MADS-Box related genes were screened by functional annotation analyses with In Del variation genes in coding region.Finally,the genes Cs3g01570,Cs9g13620,Cs6g21640,Cs5g29870,Cs4g02210,Cs3g20200,Cs7g11800,Cs5g17670 were speculated may be involved in regulation of the formation of thorns incorporation with related literature.