| Radish (Raphanus sativus L.), an annual or biennial herb of the Brassicaceae family, is an important root vegetable crop with high nutritional and medicinal value in the world. Radish is a typical cross-pollination vegetable crop, with high genetic variation and obvious heterosis. Compared with haploid breeding, it needs longer time and maybe appears economic traits degradation by conventional breeding methods, due to multi-generational selfing. Moreover, haploid, with sigle set of genomes, can be doubled into diploid (or double haploid, DH), which combining with conventional breeding, molecular biology, and genetic engineering would promote breeding process greatly. Isolated microspore culture is an important means of haploid plant breeding. Presently, there are still some problems, such as low inductivity of somatic embyo, narrow embryogenesis genotype, which limits the application of this technology in breed improvement in radish; moreover, the limited number of SSR markers developed based on coding sequences make it difficult to construct high-density genetic maps and locate the key traits. Thus, this study would be conducted to isolate a number of embryogenesis related genes, miRNAs as well as their targets at the genome-wide level, and develop a large number of EST-/genic-SSR markers based on ESTs in NCBI database and transcriptome sequences in radish. The main achievements obtained were as follows.1) To isolate the differentially-regulated genes involved in radish embryogenesis networks and improve the embryo induction rate in radish, the 0_DAP, 7_DAP and 15_DAP RNA libraries constructed from radish olvue were sequenced and analyzed using RNA-Seq approach. In all,7,191,808 (97.31%),7,235,080 (98.25%), and 6,963,130 (97.1%) clean reads were obtained. Moreover, after BLAST against reference genome and analysis of gene coverage, a total of 3,314 (7_DAPvsO_DAP),4,268 (15_DAPvsO_DAP) and 1,213 (15_DAPvs7_DAP) significantly differentially-regulated genes were successfully identified, respectively. GO analysis showed that these differentially-regulated genes could take part into a number of cell development, unique organ development, metabolism and stimulant response. And these genes were shown to be involved in 127 pathway, such as Starch and sucrose metabolism (ko00500), Plant hormone signal transduction (ko04075), viarous amino acid (ko00400, ko00260, ko00250) and organics (ko00908, ko00790, ko00940) biosynthesis, notably, a number of genes, such as AUX1 and ARF were shown to play a key role during radish embryogenesis. These results would provide theoretical basis for identification of radish embryogenesis related genes.2) Using a radish advanced inbred line ’NAU-DY13’, the RsSERK1, RsAGL15, RsCDC2 and RsLEA3 genes were obtained by homology-based cloning technology. The full ORF sequence of RsSERKl, RsAGL15, RsCDC2 and RsLEA3 genes were 1905 bp,818 bp,882 bp and 285 bp with encoding 635,279,293 and 94 amino acids, respectively. Analysis of the two proteins on the basic physical and chemical properties, hydrophilic and hydrophobic, transmembrane domain structure, coiled-coil, the secondary and tertiary structures were predicted by multiple bioinformatics online softwares. Genome walking method was used to clone 5’UTR region of RsBBM and RsCDC2 gene, and the flanking region upstreams from ATG were obtained containing a number of putative cis-acting elements, such as TATA-box and CAAT-box, as well as some auxin response and seed specific elements, which could play critical roles in growth and development of radish. Furthermore, qRT-PCR analysis revealed that RsSERK1, RsAGL15, RsBBM, RsCDC2 and RsLEA3 genes exhibited different expression and regulation during radish embryogenesis and embryo development. RsSERK1 had higher expression profiles in floral organs in florescence.3) In this study, two small RNA libraries were constructed from radish ovules before and after fertilization. Both libraries were sequenced by next generation sequencing (NGS) technology. This analysis identified 144 conserved and 34 non-conserved miRNAs (representing 43 known miRNA families) and 38 novel miRNAs (representing 28 miRNA families). Comparative analysis revealed that 29 known and 10 novel miRNA families were differentially expressed during embryogenesis. QRT-PCR analysis confirmed miRNA expression patterns and revealed tissue-specific and/or developmental stagedependent expression for some miRNAs. Moreover, potential target predictions indicated that most of these targets were transcription factors involved in regulating plant growth, development and metabolism. Notably, target transcripts such as squamosa promoter-binding protein, auxin response factor and agamous-like MADS-box protein participated in radish embryogenesis.4) In this study,51,625 unique expressed sequence tag (ESTs) from a total of 289,621 radish ESTs in the National Center for Biotechnology Information (NCBI) database were used to search for simple sequence repeat (SSRs) by SSRLocate, and 2,917 SSRs in 2,891 ESTs were identified. The SSR marker motifs contained di-, tri-, tetra-, penta-, and hexa-nucleotide repeats, and the number was 945 (32.40%),1,300 (44.57%),179 (6.14%),262 (8.98%) and 231 (7.92%), respectively. The motifs AG/CT (16.15%) and GA/TC (13.58%) were the most abundant type. Among 20 amino acids encoded by trinucleotide, Ser (16.17%) was the most common transcript, followed by Leu (13.00%) and Glu (9.90%). A total of 1,082 EST-SSR primers in Raphanus sativus were designed and synthesized, and 864 (79.85%) EST-SSRs were successfully amplified. Polymorphism of these loci was evaluated in a panel of 48 genotypes in Raphanus. The polymorphism information content (PIC) values of those primers varied from 0.33 to 0,84, with a mean value of 0.58. Moreover, it was found that 25 out of 53 EST-SSR primers could be successfully amplified in Brassica species. For its polymorphism, reproducibility and functionality, these novel EST-SSR markers could be used as a powerful tool for marker-assisted selection (MAS) and genetic mapping in radish.5) In the present study, from a total of 73,084 unigenes and 150,455 contigs which were assembled from 71.95 million Illumina sequence reads of a radish taproot library, a collection of 11,928 genic-SSR loci were successfully identified in 11,311 unigene sequences. Trinucleotide repeats were the most abundant repeat units, as in many other plants, with a frequency of 52%. Furthermore, a total of 5,503 genicSSR primers were developed, from which 1,052 SSR primers were synthesized, and a subset of 823 (78.23%) primers could generate stable bands. Moreover,67 selected informative genic-SSR markers were used to determine the genetic diversity of 32 radish genotypes, in which the polymorphism information content values ranged from 0.49 to 0.89. For effective cultivar identification, a novel strategy called manual cultivar identification diagram was employed. Thirty-two radish accessions were clearly separated by six genic-SSR markers. Additionally, the cross-species/genera transferability of these SSRs was further validated in nine relatives in Brassicaceae. These results suggested that the novel genic-SSR markers, as a basis for future genetic linkage and gene tagging analysis, could be very valuable in facilitating genetic mapping, marker-assisted selection and comparative genome analysis. |
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