| Citrus is one of the most important fruit crops in the world.The traditional breeding in citrus is hindered by the interference of nucellar embryo,which makes it difficult to obtain true hybrid.Citrus nucellar embryony is a unique apomictic phenomenon.Nucellar seedlings has same genetic composition as the mother plant,which are widely used for producing identical offspring in citrus industry.The mechanisms involved in citrus nucellar embryony have been widely studied.Previous studies have genetically mapped nucellar embryoy of Citrus and Poncirus using different populations.However,so far,the candidate genes controlling nucellar embryony have not been identified.In this study,short juvenile materials of Hongkong kumquat(Fortunella hindsii)were used to construct the hybrid segregating populations.These segregating populations in together with the Citrus and Poncirus hybrid segregating populations were applied for mapping candidate chromosome region controlling citrus nucellar embryony to Gene expression of the ovules from monoembryonic and polyembryonic Hongkong kumquatswas analyzed by RNA-seq analysis.The expression of the genes in candidate chromosome regions was analyzed.A candidate gene CitRWP was found to control citrus nucellar embryony.Moreover,an effective molecular marker was developed to distinguish monoembryonic and polyembryonic varieties in Citrus.The main findings are as follows:1 Using several genetic populations to locate candidate region controlling citrus nucellar embryonyIn this study,we used peculiar short juvenile materials of Hongkong kumquat to construct F1 segregating population,with five monoembryonic varities from Guangdong and Fujian province as the female parent,and a polyembryonic varity from Jiangxi province as the male parent.A total of 1321 hybrids were obtained and confirmed by molecular markers with the hybridization rate of 95.24%.The segregation ratios of monoembryonic and polyembryonic progeny in five segregating populations were all chose to 1: 1.The BSA-seq method was used to analyze the two DNA pools from 30 monoembryonic and 30 polyembryonic progenies,respectively.The main locus of the nucellar embryony of kumquat was locked to the specific locus on chromosome 4 refering to the sweet orange reference genome.A total of 36 recombinant plants were obatined from the population to narrow the locus to the region 3.51Mb-3.75 Mb on the chromosome 4.The locus contains 35 genes,of which 17 genes are linked to co-segregating marker.The phenotypic analysis of the F1 populations from a cross of HB pummelo(Citrus grandis)and Fairchild(FC)tangelo(C.reticulata×Citrus grandis)was carried out for 5 consecutive years.There were 66 monoembryonic hybrids and 58 polyembryonic hybrids.The segregation ratio of monoembryonic and polyembryonic progeny was approximately 1: 1.The BSA-seq method was used to analyze two DNA pools of 20 monoembryonic and 20 polyembryonic progenies,respectively.The gene of nucellar embryony was located in the 2.5Mb to 3.9Mb interval of chromosome 4.The locus was validated in the HB × FC population using molecular markers.Molecular markers were used to narrow the region to a span of 480 Kb.It is confirmed that the nucellar embryony was controlled by the same locus in Citrus and Fortunella,which is linked to the locus from previous reports.The phenotypic analysis of the F1 populations from a cross of two polyembryonic parents red tangerine(C.reticulata)× trifoliate orange(Poncirus trifoliata)was carried out for three consecutive years.There were 28 monoembryonic hybrids and 39 polyembryonic hybrids.The segregation ratio of monoembryonic and polyembryonic progeny was approximately 2:1 or 1:1.The candidate locus from previous analyses was also validated in the offspring.It was found that the phenotype of 59 progenies is linked to the locus,but 8 polyembryonic trees were not linked to this region.The markers linked to the traits were all from the parent red tangerine.The heterozygous markers in the trifoliate orange were not linked in the region,indicating that there is another control locus in Poncirus.2 RNA high-throughput sequencing to dicover the dicovery the genes related to the nucellar embryony development in Hongkong kumquatCytological analysis of sexual and apomictic ovule revealed that two key stages: stage A,a few NEICs(nucellar embryo initial cells)appeared histologically in apomictic ovule at uni-nucleate embryo sac stage of mega-gametogenesis prior to anthesis;stage B,an increasing number of NEICs occurred at bi-nucleate embryo sac stage.The ovules of these two stages were used for RNA high-throughput sequencing.Through de novo assembly,a total of 66,699 unigenes and 156040 transcripts were found with an average length of 1647 bp and 900 bp,respectively.All unigenes were searched for homologous sequences in the Nr,Nt and Swiss-Prot databases.As a result,23,665(35.48%),15,959(23.92%),18,267(27.39%)unigenes were aligned against these three databases,respectively.At stage A,316 and 557 unigenes were respectively up-and down-regulated in nucellar genotypes.At stage B,532 and 541 unigenes were up-and down-regulated,respectively.Moreover,KEGG-related pathway analysis showed that differential expressed genes enriched in the pathways of hormone signaling,where 25 differentially expressed genes were detected in relation to auxin and cytokinin transduction pathways.The expression analysis of genes within the candidate region showed that the CitRWP gene might be responsible for citrus nucellar embryony.Twenty differential genes were randomly selected for q PCR validation.The results indicated that RNA-seq data were reliable.3 Functional analysis of the gene CitRWP and molecular marker developingThe CitRWP genes,from HB pomelo,Fairchild tangelo,clementine mandarin,and Hongkong kumquat were cloned and only few SNPs were found between these cultivars.RT-q PCR analysis showed that the expression of CitRWP gradually decreased with the development time of ovule.CitRWP had a very low expression in ovules of monoembryonic varieties,such as Wanbai pumello,citron and clementine mandarin but ahigh expression in polyembryonic varieties,such as flame grapefruit,lemon,ponkan and Valencia sweet orange.Meanwhile The expression levels of Cit RKD1 and Cit RKD2 in the RWP-RK family were not significantly different from those in the monocytic progeny in the multi-embryonic progeny of the HB × FC hybrid population.Subcellular localization indicated that CitRWP was localized mainly in the nucleus.Evolutionary analysis showed that CitRWP did not belong to the RKD family reported in Arabidopsis thaliana,and its phylogenetic position indicated that it was similar to Pp RWP3 and Pp RWP4 in Physcomitrella patens and Mp RWP1 in Marchantia polymorpha,with an independent origin.A polyembryony specific MITE transposon was found in the CitRWP promoter region.We developed molecular markers through this transposon and validated them in 786 citrus germplasm resources.The marker mite_p1 was found to be completely linked to polyembryony traits.Overexpression vectors of the CitRWP were constructed to verify the gene function by genetic transformation in Arabidopsis thaliana. |
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