Construction Of Molecular Linkage Map And Genetic Analysis Of Hybrids And Polyploidy Of Citrus Based On EST-SSR,Genomic-SSR And SCoT Markers

Citrus is one of economically the most important fruit trees in the world. The genetic improvement programs of citrus has been subjected to the attention of global researchers, many new cultivars have been released in the world via bud sport selection, cross, polyploid and other breeding channels.The promotion of these cultivars has played an important role in promoting local economic development and the improvement of living standards of the people. The molecular biotechnology provide new technical means for improvement programs of citrus, have greatly promoted the citrus breeding. Identification and location of important qualitative genes and quantitative trait loci on a chromosome has also become a hot spot of citrus genetics and breeding programs with the rise of genetic and genomic studies,and genetic linkage mapping is one of the important foundation. The progress of the above aspects are a profound impact on research and applications for citrus. Therefore, we constructed the genetic linkage map of citrus with different molecular markers, and which were used for comparison with other citrus genetic linkage maps. Then, we obtained some hybrid progenies throgh different cross combinations, and identification and analysis by cell and molecular biology methods.The results are as follows:1. An optimized SSR analysis system on citrus were obtained, through building a new method to isolate DNA from microamount of citrus leaves rapidly and testing three PAGE concentrations and three silver staining methods. The results showed that the method had the characteristics of little material and shorter time, and its primary application showed that the optimization SSR system could provided a technical support for citrus molecular assisted breeding and constructing of molecular linkage map.2. Orthogonal design was applied to optimize SCoT-PCR amplification system of citrus in five factors such as Mg2+, dNTPs, primer, Taq DNA polymerase and template DNA. An optimal reaction system of Citrus was established, PCR reaction mixtures contained Mg2+1.5mmol· L-1, dNTPs0.35mmol· L-1, primers0.625μmol· L-1, Taq polymerase0.25U, template DNA10ng. The most suitable annealing temperature of primers was49℃. Amplifications and genetic analysis were carried out on progenies from Wan2[tangor, Citrus unshiu (Mark.) Marc. x Citrus sinensis (L.) Osb.] x Li2[sweet orange, Citrus sinensis (L.) Osb.]. The result showed that the SCoT system established in this report is useful for genetic diversity analysis and constructing linkage map of Citrus, which possesses considerable potential for citrus breeding.3. The genetic diversity of92F1individuals derived between Wan2(tangor, Citrus unshiu (Mark.) Marc, x Citrus sinensis (L.) Osb.) and Li2(sweet orange, Citrus sinensis (L.) Osb.) was analysized. A total of294polymorphism bands were amplified by112EST-SSR and56SSR merkers,with an average of1.7bands.and20SCoT primers produced32polymorphism bands,with an average of1.6bands. UPGMA cluster analysis showed that92F1individuals could be grouped into eight distinct families, and the resuits from two different marker were similar,which all showed that the F1population has high genetic diversity and greater genetic variation, can be speculated that the separation of population is diversity.4. Expressed sequence tag-simple sequence repeat (EST-SSR), simple sequence repeats (SSR),and start codon targeted (SCoT) polymorphism markers were used to establish genetic linkage map of citrus using a population of92F1individuals derived between Wan2(tangor, Citrus unshiu (Mark.) Marc.×Citrus sinensis (L.) Osb.) and Li2(sweet orange, Citrus sinensis (L.) Osb.). We initially tested518primers or primer pairs, and219(42.28%) gave polymorphic markers,which included67SSR(37.02%of187SSR),127EST-SSR (47.57%of267EST-SSR) and25SCoT primers(35.57%of267EST-SSR). Among all,206primers, including65SSR,121EST-SSR and20SCoT primers were used in linkage analysis.EST-SSR, SSR, and SCoT analyses produced213(1.8per primer),114(1.7per primer) and32(1.6per primer) polymorphic bands, respectively. Total number of polymorphic bands were359by206primers, with an average of1.7bands. Among all,232markers, including69SSR,131EST-SSR, and32SCoT markers were found segregating in the progeny, and113(48.7%) showed distorted segregation.A genetic map was constructed using JoinmaP4.0setting CP type data based on232markers, a total of226markers,including67SSR markers,129EST-SSR markers and30SCoT markers were assigned to9linkage groups. The groups ranged in size from5to122.3cM, with an average length of77.1cM, The total map length was693.7 cM, with an average distance of3.1cM between adjacent markers. the total number of markers per linkage group ranged from4to104, with an average number of25.1markers, ESS-WL1has the most number markers,and the least number of markers was detected in the ESS-WL7.The effects of SCoT markers and distorted markers on genetic linkage maps were analyzed. Distribution of SCoT markers was evenly on chromosome, which were beneficial in the construction of linkage maps, and were valuable for genetic mapping. The distorted markers also will not have a great effect in linkage maps. Which shows that the genetic map constructed in this paper is accurate and reliable, can be used for determining locations of genes on a chromosome or QTL mapping of citrus.5. Through comparison of shared markers in the map constructed by us and6maps constructed by other researchers, we found that some markers in this study could show better identity with other maps. However, some markers were placed on difference postion in different linkage groups.Which show that construction of a reference map of citrus is very necessary.6. The hybrid nature and the genetic relationship of offsprings from'Shatianyou'×'Hongjiangcheng'was analyzed with optimized SSR system.The results showed that all the offsprings from'Shatianyou'pummelo×'Hongjiangcheng'were proved to be hybrids with5pairs SSR primers, one homozygous maker AAT12was obtained, and some markers were absent in55F1seedlings in primer GA18and AGC9, which may be related to chromosome abnormality exchange. The cluster analysis of SSR data showed that65.45%progenies could be grouped with'Shatianyou'together, and34.55%grouped with'Hongjiangcheng' together, which indicated that the most of offsprings were partial maternal inheritance and the genetic variation of hybrids from the cross combinations was obviously.7. A total of5fruits and32seeds were obtained from the cross between tetraploid lemon and'Chandler'pummelo,and1fruit and5seeds were obtained from tetraploid lemon xtrifoliate orange. The hybrid fruits and seeds are significantly smaller than normal tetraploid fruits and seeds. A total of29progenies (27from lemon×'Chandler' pummelo,2from lemon×trifoliate orange) were obtained.Cytological analysis indicated that all the29hybrids were triploid (2n=3x=27). Two progenies from tetraploid lemon×trifoliate orange were considered to be hybrids by morphology identification and number of chromosomes.25triploid from tetraploid lemon× 'Chandler'pummelo were hybrids by EST-SSR verification. Genetic analysis were carried out on25triploid from tetraploid lemon×'Chandler'pummelo and the parents by13pairs EST-SSR primers. The13primers generated a total of87fragments,and82(94.03%) were polymorphic, some progenies had non-parent markers and some markers were absent in progenies, UPGMA cluster analysis showed that27plants could be grouped intotwo distinct families:25triploid hybrids were grouped with female parent together;'Chandler'pummelo was grouped alone.Which showed that the hybrids included different genetic information from female and male parent. EST-SSR markers can reflect plant genetic material contained in the amount of the hybrid, which can Provide a basis for ploidy confirmed.8. Amplifications and genetic analysis were carried out on four tetraploids of Shatianyou pomelo [Citrus grandis(L.)Osb.'Shatianyou'].84bands were amplified from Shatianyou pomelo with11primers, of which46(54.8%) were polymorphic. UPGMA cluster analysis showed that four tetraploids could be grouped into three distinct families based on similarity of0.825, and three homologous tetraploids were clustered in different locations, which showed that different degree of genome variation was detected in tetraploids compared with the diploid. And the tetraploids can provid important genetic resources for breeding programs of citrus.