Development Of SSR Molecular Markers For Citrus And Genetic Identification And Agronomic Trait Analysis Of ’Yanxiwanlu Pokan’ Early Maturing Bud Variation

Citrus is the number one fruit in the world.China is one of the countries with the most abundant citrus germplasm resources,and with the development of citrus industry,more and more new high quality varieties are selected and bred.The national standard document for the registration of new citrus varieties has been issued,and it is necessary to evaluate the biological and agronomic traits of the citrus varieties to be registered;previously,the development of citrus SSR molecular markers was mostly carried out for a certain variety and its close relatives.Therefore,the development of new citrus SSR molecular markers and the discovery of citrus germplasm resources for genetic identification and agronomic trait analysis are of great significance in citrus breeding.In this study,57 citrus varieties in China were collected and divided into seven groups,and DNA was extracted from each sample separately,and genomic DNA from each group was mixed to form seven mixed genomic DNA libraries,and SSR loci enrichment libraries were constructed for each group separately for sequencing,so as to obtain sequence information of SSR loci for the seven groups of materials,and to develop and design specific primers,hoping to develop a set of We also developed and designed specific primers,hoping to develop a set of efficient SSR molecular markers for genetic identification among citrus varieties.Secondly,this study was conducted to genetically identify the early maturing bud variant strains of ’Yanxi Late Rus’(which matured about50-60 d earlier than ’Yanxi Late Rus’),and to observe the biological traits and agronomic traits for the later stage of ’Yanxi Late Rus’.The genetic identification was carried out,and the biological and agronomic traits were also observed to provide data support for the selection of new early maturing bud-variant varieties of ’Yanxi late reu’ Ponkan.The main results were as follows:1.SSR locus enrichment sequencing and sequencing data analysis of seven groups of citrus materialsIn this study,57 citrus varieties were collected and divided into seven groups according to the characteristics of each variety,and seven sequencing libraries were constructed.In total,9.76 GB of raw data were obtained,and 69.63 Mb of raw reads were processed and analyzed,and 66.43 Mb of high quality fragments were obtained,accounting for 95.40%of high quality fragments;1,561,367 SSR loci were searched for all reads;the types of SSR loci searched were analyzed The number of sequences with one SSR site was335,428,and the number of sequences with compound form(containing two or more SSR sites)was 372,698;in the analysis,it was also found that the most common two-nucleotide repeat model of SSR was AG/CT,which had 265,545 motifs,accounting for 55.58%of the two-nucleotide repeat model.In addition,among the trinucleotide repeat models,the most abundant was AAG/CTT with 212,043 motifs,accounting for 50.84%of the trinucleotide repeat models.2.Development of SSR molecular markers for citrus and genetic identification of Yanxiwanlu ponkanSSR locus analysis and primer openings were performed based on the SSR locus enrichment sequencing results of 57 citrus varieties.A total of 280 pairs of SSR-specific primers were designed and synthesized,and 150 pairs of SSR primers were randomly selected and validated for analysis:(1)A total of 38 pairs of highly polymorphic primers were obtained,of which the number of specific bands was 2-5,of which two primers,cs-9and cg-33,had the highest polymorphism,and the number of polymorphic bands of the highest primer was 5;(2)All polymorphic primers,which could amplify(2)all polymorphic primers could amplify clear bands,and the percentage of designed and synthesized high polymorphic primers was 25.33%,and their amplified fragment sizes were in the range of 100 bp-400 bp;(3)SSR primer combinations were used to achieve identification of a single species,and primer combinations were developed to identify sugary orange,cocktail grapefruit,and Dianjiang white grapefruit in the broad-skinned orange and sweet orange groups,respectively;(4)primers found in the literature and newly developed primers were used to(4)The early maturing bud variant of ’Yanxi late reed’ and’Yanxi late reed’ were identified,and no differences were identified.3.Comparative analysis of agronomic traits of Yanxiwanlu ponkan and its early maturing bud variantThe biological characteristics,phenological period and agronomic traits of ’Yanxiwanlu Ponkan’(WT)and’Yanxiwanlu Ponkan’early maturing bud variant(MT)were observed,and it was found that WT and MT were almost the same in the periods of tree shape,tree potential,tufts,flowering and fruit set,etc.,but The fruit color change period of MT was 25-30 d earlier than that of WT,and its ripening period was 50-60 d earlier than that of WT;moreover,the fruit of MT was slightly larger than that of WT during the whole fruit development stage.Compared with WT fruits at maturity,MT fruits were flattened,and the MT fruit shape index 0.71 was significantly different from WT fruit shape index 0.82,and the flesh capsule was more uniform;MT fruit weight per fruit was also larger than WT after full maturity,and the average fruit weight per fruit was about 20 g higher than WT;MT soluble solids content was 12.4%,5.6%higher than WT,and titratable acid content was 0.51%,significantly lower than WT.The titratable acid content was 0.51%,which was significantly lower than that of WT.In a comprehensive comparison,MT matured 50-60 d earlier than WT,and the overall quality was better than WT.