| Chrysanthemum(Chrysanthemum morifolium)is one of the four important cut flowers and Chinese traditional ornamental flowers,and occupies a crucial position in the flower industry.Tubular flower in the middle inflorescence will shed pollen outside-in during flowering period,and huge quantities of pollen grains fell on ray flowers,reducing the ornamental effects,and many pollen grains will float through the air result in pollen pollution and affect viewers' health even suffer from hay fever.Therefore,chrysanthemum without pollen contamination has a broad application value.For the moment,cross-breeding is the most universal and effective breeding means in selection.Thus,it is necessary to study on inheritance patterns of pollen dispersal for chrysanthemum cultivars,which will provide guidance to crossbreeding,establiah the theoretical foundation for molecular marker and quantitative trait locus(QTL)mapping,meanwhile provide theoretical basis for choosing and breeding non-pollen contamination germplasm.This cross-breeding chose non-pollen contamination varieties(no pollen grains in anther or anther indehiscence)as female parent,such as 'Kingfisher','Nan Nong Yu Hu',Q1-62,QX-107,'Nan Nong Bing Ji Ling' and QX-008.And different kinds of pollen quantity varieties as male parent,such as 'Nan Nong Fei Zi','Nan Nong Hong Cheng','Nan Nong Yin Shan','Nan Nong Hong Yan' and 'Nuo Ya'.Cross incompatibilities,the flower traits and characteristics which are related to the pollen dispersal and the levels of pollen contamination were investigated,and on the basis we screened non pollen contamination strains.Then,a cross combination with larger discrepancies in parents were selected,genetic analysis was performed for two pollen quantity traits based on phenotypic data collected in 129 plants of a F1 population producted from the crossing between Q1-26(female parent)and 'Nan Nong Hong Yan'(male parent)with single generation segregation analysis method of major gene plus polygene mixed genetic model.Moreover,we performed transcriptome analysis between the critical periods of 'Kingfisher' pollen abortion by high throughput sequencing.The main results and conclusions are listed as follows:(1)Among 20 cross combinations,the number of pollen germination on stigma of QX-107x'Nan Nong Hong Cheng',QX-107×'Nan Nong Yin Shan' and Q1-62x'Nan Nong Hong Yan' were maximum with 19.93,17.53 and 15.63,respectively;the number of pollen pollen germination on stigma of QX-008x'Nan Nong Hong Cheng'?'Nan Nong Bing Ji Ling'×'Nan Nong Hong Cheng' and QX-107×'Nan Nong Fei Zi' were middle with 6.87,9.87 and 10.87,respectively;and the number of pollen germination on stigma of QX-008×'Nuo Ya'?'Nan Nong Yu Hu'×'Nan Nong Hong Yan' and Q1-62×'Nuo Ya' were low with 0,0.02 and 0.12.The seeding rate of QX-107×'Nan Nong Hong Cheng' and QX-107x'Nan Nong Yin Shan'were high with 53.02%and 51.73%,respectively;while the seeding rate of Q1-62×'Nan Nong Fei Zi' and QX-008×'Nuo Ya' were low with 0.63%and 0%.The seeding rate/ovary full rate after 8 DAP of cross 2,cross 3 and cross 4 were 97.43%,94.40%and 104.0%,respectively;while the seeding rate/ovary full rate after 8 DAP of QX-107×'Nan Nong Hong Cheng'?QX-107×'Nan Nong Yin Shan' and 'Nan Nong Bing Ji Ling'×'Nan Nong Fei Zi' were low with 7.23%,2.00%and 7.73%,respectively.In addition,we obtained 57 strains with non-pollen contamination,23 strains showed non pollen grains in anther,and the others contains pollen grains but anther indehiscence.The result indicated that low seeding rate may relate to poor pollen-stigma compatibility,immature embryo sac,pollen tube elongation blocked and embryo abortion.The number of pollen germination on stigma and the seeding rate were higher in the cross combination which female parent with non-pollen grains in anther.Conversely,it was lower in the cross combination with varieties contain pollen grains but anther indehiscence as female parent.(2)Pollen quantity per anther and pollen quantity per inflorescence were widely segregated in F1 population with high coefficient of variation(CV),80.63%and 88.64%,respectively,and the ratio of heterosis value of mid-parents(RHm)for the two traits were 29.33%and-4.11%.The mixed genetic analysis suggested that pollen quantity per anther fitted to B-2 model,that is 'two pairs major gene,additive-dominant genetic model',the heritability of major gene was 85.56%;pollen quantity per inflorescence was accorded with B-1 model,'two pairs of major genes,additivity-dominance-epistasis genetic model,and the heritability of major genes was 80.15%.The results indicted that:pollen quantity per anther and pollen quantity per inflorescence were widely segregated in F1,controled by two pairs major gene,and the heritability of major gene were high with little environment affection.(3)Illumina HiSeqTM 2000 platform was utilized to perform high-throughput sequencing between pollen aborting stage and pollen aborted stage of 'Kingfisher'.25.39 Gb bases in total were obtained,after the low-quality,adaptor-polluted and high content of unknown base(N)reads being removed,we obtained 98,459 unigenes,57,226 unigenes be annotated with at least one functional database,and a total of 19,671 unigenes were classified into 25 COG functional classes,8,214 unigenes were annotated into 45 functional categories in GO,including 19 biological processes,12 cellular components and 14 molecular functions,and a total of 40,068 unigenes were assigned to the 135 KEGG pathways.With Nr annotation,we statisticed the distribution of annotated species,11.58%,8.2%,6.25%and 5.16%unigenes closely matches with Vitis vinifera,Coffea canephora,Nicotiana sylvestris and Nicotiana tomentosiformis.The number of DEGs was 18,663,among which,there were 8,448 and 10,215 up-and down-regulate.Several genes associated with PCD,protein degradation,reactive oxygen species scavenger and pollen development are activated or suppressed between pollen aborting stage and pollen aborted stage.The results suggested that the cysteine protease,aspartic protease,metacaspasc,26S/proteasome,ethylene,AMS,ZAT1 and CalS5 in the regulation of pollen abortion of chrysanthemum is likely to play an important role in the development of metabolic network,which provide a basis for study of molecular mechanism of chrysanthemum pollen fertility and pollen sterility gene cloning. |
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