| Citrus is one of the most important fruit trees with cultivation for long centuries, which had been widely used for fresh fruit and juice. However, there are many challenges for citrus traditional breeding, such as most important agronomical traits are quantitative trait loci(QTLs), long cycle of generation, complicated inheritance origin, apomixes and gametophyte sterility. Markers that associated with important agronomical trait can be obtained by construction of high-density genetic map and QTL analyses using molecular markers. Marker-assisted selection(MAS) breeding can greatly reduce the time of breeding and improve the breeding efficiency. In this study, we first developed a large number of SSR markers based on whole genome sequences of Clementine mandarin; second, we developed many Indel markers based on whole genome resequencing data of trifoliate orange; third, the highest genetic map of citrus until now has been constructed based on SSR and SNP markers; finally, after combined the leaf abscission trait and the constructed genetic map, we identified the QTLs linked with citrus deciduous trait for the first time. Following are the main results achieved in this study.1. Whole genome sequences of Clementine mandarin were downloaded from citrus database. A total of 80,708 SSR loci were obtained using MISA software, and the average density of SSR loci was 268/Mb, dinucleotide and trinucleotide types are the most abundant. A total of 8,989 SSR loci were obtained from 6,834 transcripts(a total of 33,929 transcripts). Furthermore, these transcripts that contained SSR loci were functional annotated using Blast2 GO, and the percentage of different SSR types were analyzed.2. A total of 105 pair primers(27 located in genomic region and 78 located in CDs) were designed based on SSR loci. Finally, 95 polymorphic and reliable SSR markers(90.5% markers were successfully identified) were obtained after validation against 18 citrus different cultivars. The phylogenetic tree showed that the 18 citrus species divided into three genera, and the relationship among these species is consistent with previous studies.3. Deep resequencing was performed toward the hybrid derived from clementine mandarin and trifoliate orange(CP) and trifoliate orange(PT),(depth of resequencing was 22.86 X and 25.98 X, respectively). By alignment with reference genome, a total of 2,591,103 SNPs were obtained from CP and 3,105,640 SNPs were obtained from PT, the common SNPs were 503,132 between the two species. Moreover, a total of 722,298 Indels and 895,643 Indels were obtained from the CP and PT, respectively, and the common Indels were 503,132 between the two species.4. A total 82 Indel loci were random selected and designed primers. Characterization was performed against 32 citrus species using these markers. Eventually, a total of 74 reliable and polymorphic Indel markers were obtained(null amplification and non-polymorphic markers were deleted). The phylogenetic relationship among 32 species is consistent with previous studies. The results demonstrated that the efficiency and reliablility of developed Indel markers based on whole-genome resequencing data is high.5. The two parents’ high-density genetic maps were constructed by using SSR and SNP markers(SNP markers were developed using SLAF-seq method), and integrated genetic map of the two parents was also constructed. The maternal genetic map contained 1491 markers with a total of genetic distance of 1773.53 c M, and the average distance is 1.19 c M; The parental genetic map contained 2,980 markers with a total of genetic distance of 1530.88 c M, and the average distance is 0.51 c M; The integrated genetic map contained 4179 markers with a total of genetic distance of 1770.37 c M, and the average distance is 0.42 c M. Furthermore, the number of segregation distortion markers was identified, and the collinearity analysis demonstrated that the constructed genetic maps are in good quality.6. The leaf abscission trait of the F1 population was calculated with two years data(the phenotype data showed normal distribution, suggesting that it belongs to QTLs). The QTL mapping was performed using the phenotypic data and genetic map. Finally, two leaf abscission associated regions were obtained, which are located on the chromosome 1 and 8, respectively; one region on chromosome 1 contained 1.37 Mb with 36 genes, and the other region on chromosome 8 contained 0.67 Mb with 107 genes. Importantly, the region on chromosome 8 is much more significant, and with a higher density of genes.7. Functional annotation was conducted for the 143 identified genes that located in the two regions. Several markers were developed based on the Indel loci in the region on chromosome 8, and four markers with good amplified results showed that they are likely linked to leaf abscission trait. Leaf abscission trait is closely linked with low temperature or freezing, hence, we performed a treatment to trifoliate orange with cold stress condition. Subsequently, according to functional annotation and transcriptome data published before, 7 and 23 candidate genes were selected on chromosome 1 and 8, respectively. The expression patterns were performed of these genes using q RT-PCR. The results of m RNA expression level demonstrated that 12 genes were up-regulated significantly, 2 genes were down-regulated greatly, and the other 16 genes showed no significant change. In a word, these results laid a solid foundation for further characterization of leaf abscission-associated genes. |
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