Studies On Seedless Mechanism And Molecular Markers Linked To Seedless Trait In Citrus

Seedlessness is an important horticultural trait and so it is selected and reserved in producing practice for a long time.The peoples prefer choosing the seedless fruit for its easy-eating and better quality,and the seedless trait has important significance for fresh eating and processing of citrus.Since 1960s fresh-eating citrus cultivars have become seedless step by step overseas and all famous-brand species are seedless or low-seedy in international market of citrus.In china citrus resource are very rich along with many regional excellent cultivars but most of them are seedless,even with more seeds,so which reduces commercial values and competitive in international market.Therefore,breeding seedless or low-seedy cultivars is an important goal.To date researchs on citrus seedless mechanism are put focus on morphology and anatomy et al.,however few works are made in the aspect of gene level using the molecular biology means as well as get little knowledge about molecular basis of citrus seedlessness.In present research 'Lipeng No.2',a novel seedless Ponkan cultivar,served as material and common seedy cultivar was used as control,investigating fertility of male,female gametophyte and compatibility systemly.Simultaneously in order to search the linkage markers related to seedless trait we performed the AFLP analysis using the 8 Ponkan cultivars and 4 Bendizao mandarin accessions as materials respectively.And sequences of the obtained AFLPs were compared homology with known functional genes.Primary results were as follows:1.Seedless reasons of 'Lipeng No.2' Ponkan(1).Pollen ferlity descending.Pollen grains per anther,pollen germinability,pollen stainability of 'Lipeng No.2' were low obviously compared with seedy control.Pollen grains per anther of 'Lipeng No.2' and CK was 25300,41500 respectively,which giving distinct difference;pollen germinability of 'Lipeng No.2' and CK were 1.18%,15.05%and 0.82%, 14.35%at screened optimal concentration medium in two years;and pollen stainability were 9.27%,50.23%and 9.18%,45.07%.(2).Microstructure of two cultivars had remarkable difference by using EMC.Pollen of seedy Ponkan was long-global or near long-global shape,plumb,and net-hollow on outer wall are big with 4-5 germinable grooves.Nevertheless pollen of 'Lipeng No.2' showed concave or jujube-nuclus shape,strip sunken in many areas,small net hollow;and number of germinable groove was 4-5,narrowband shape,morever germianable hollow sinked insidely.(3).Pollen mother cells of 'Lipeng No.2' were abnomal at meiosis phase.PMCs divided abnormally including chromosome chain,chromosome loop,lagging chromosome and chromosome unequal distribution.Choromosome chain rate was as high as up to 37%and chromosome chain was composed of 3-9 chromatids;chromosome loop rate was approximate 8%.These abnomal chromosomes resulted in monstrous pollen and then bring on pollen fertility descending.(4).'Lipeng No.2' embryo sac sterility.Paraffin section result incidated that zygote of embryo sac had developed to become globular stage embryo at the 7th day after fertilization. However in the process of embryo sac development of 'Lipeng No.2',two-nucleus stage was observed at the 2th day after blooming as well as four-nucleus stage in ovule at 4-5th day after blooming,but 8-nucleus stage embryo sac was not observed.Embryo sac sterility begined from 4-nucleus stage and subsequent with development,embryo sac of 'Lipeng No.2' appeared middle-hollow and the shape became round from long.(5).Cross test.Not only self-pollination but also pollinated by xuegan orange had high fruit-setting rate,which showed self-incompatibility was not the reason for seedless trait.In cross test 'Lipeng No.2' produced only one seed and the fruits were almost seedless,which proved that seedless trait was resulted from the female sterility.2.Studies on molecular markers linked to seedless trait of citrus(1).Combining the AFLP with DNA pool 8 respresent Ponkan cultivars were analysised,19 polymorphic bands were obtained by 13 primer combinations screened from 72 primer pairs and then these specific bands were identified in 8 individuals.Five markers,named as AFLP-1,AFLP-2,AFLP-3,AFLP-4,AFLP-5,were linked to seedless gene probably.Band size ranged from 128bp to 267bp and all sequences were submitted to Genebank database, BLAST result showed that marker AFLP-1 had 73%identify with Arabidopsis seed development defect mutant coding gene ttn8 and chromosome structural maintance protein cds;AFLP-2 had 80%identify with GTPase-activating protein cds;AFLP-3 had 71%and 69%identify with ABC transporter protein family PGP18 cds and ABC transporter protein; the identify of AFLP-4 with Pyrus communis putative ATP synthase partial cds and partial sequence of ferredoxin-dependent glutamate synthase-like gene;AFLP-5 had the same identify(73%)with Arabidopsis thaliana glycosyl hydrolase family 17 protein cds and beta-1,3 glucanase-like protein cds.These results showed that 5 isolated fragments had partial homology with functional genes aboved.(2).Four bendizao mandarin accessions were conducted AFLP analysis using the same primer combinations with Ponkan.Twelve good polymorphic primer pairs screened from 72 primer combinations were performed final PCR amplification,obtaining 9 clear and stable bands named as SL₁-SL₉.These fragments were cloned and sequenced and then Blast result showed that SL₁ had 71%homology with putative auxin efflux carrier protein 9(PIN9)gene, SL₂ had 86%homology with retrotransposon Rtsp-1 DNA sequence and SL₄ had 94% homology with chloroplast genomic DNA sequence.These valuable information helped to realize the mechanism of seedless variation and locate the seedless gene of citrus.