| Polyploidazation plays an important role in driving the plant evolution process, nearly 70% of angiosperms plants experienced once or more polyploidy events. Polyploidization can remarkably sharp the genome of plants and result in genetic changes, like morphological modification, chromosomal structure variation, genome restructuring, etc. Polyploidy also has epigenetic effects such as DNA methylation, nucleolar dominance, transposon activation, gene expression variation, etc. Allopolyploid is the main target for gene expression ressearch and transcriptome analysis. It is revealed that it is the genome merger rather than genome doubling per se that responsible for considerable transcriptomic alterations in natural and synthetic allopolyploids. While the gene expressions in autopolyploid turns to be limited. Distant hybridization is an effective method of crop genetic improvement for cultivated species. Isatis indigotica Fort. (Chinese woad,2n=14), a member of Isatideae tribe of Brassicaceae family, is a biennial herbaceous plant which has been used as an important and popular medicinal and indigo-producing plant with a long history in China. The medicine derived from its dried roots (Radix Isatidis) can be used for antibacterial, antiviral, and immune regulatory effects in the treatment of cold, fever, and influenza. This plant is also utilized as valuable germplasm for resistance breeding of Brassica crops because of its resistance to tobacco mosaic virus (TMV) and stem rot (Sclerotinia sclerotiorum), which is the most destructive disease of rapeseed in China. In this research, we studied the phenotypic, cytological and physiological changes by the comparison of diploid and synthetic autopolyploid. A comprehensive survey of global gene expression in ploidy level was performed using Illumina RNA-Seq among its diploid, autotriploid and autotetraploid. Brassica rapa-woad allohexaploid (AAIIII) which was synthesized by predecessors via somatic cell fusion was used to cross with Brassica napus to generate F2. Then F2 was backcrossed by B. rapa. The cytological analysis was carried out on the cross and backcross generations, and the results are as follows:1. The cytology and transcriptome analysis of the woad autopolyploid. In this study, its autopolyploids (3x,4x) were produced and characterized together with the 2x donor with their phenotype, cytology and transcriptome analysis. With the increase of genome size, it showed obviousgiantism in cells and organs and higher photosynthetic rate. During the meiotic division of pollen mother cells (PMCs) of 4x plants, the chromosomes were paired with variable numbers of bivalents and quadrivalents and segregated equally, which resulted in high pollen fertility. The ratio of bivalents inmultivalents is significant higher than the theoretical value that should accelerate the cytological stabilization of the autotetraploids. From transcriptomic data of 2x,3x,4x plants, differentially expressed genes (DEGs) including both up- and down-regulated genes were detected with their pair-wise comparisons. These DEGs were mainly involved in cell growth, cell wall organization, secondary metabolite biosynthesis, response to stress and photosynthetic pathways.2. The cytology analysis of the progenies of B. rapa-woad allohexaploid and B.napus, B. rapa. B. rapa-woad allohexaploid (AAIIII) was crossed with B.napus to generate F2. From the point of phenotype, the F2 was more similar to B. rapa except the tall plant type. GISH analysis of the F2 plant showed that the F2 plant had 9 C chromosomes and 14 I chromosomes. Meiotic analysis of the F2 PMC revealed that the meiosis of the F2 was irregular. Then F2 was backcrossed by B. rapa for the first time, the BC1 plants only kept 1-3 woad chromosomes while 9 C chromosomes were reserved. We speculated that the I chromosomes might be eliminated when crossing F2 with B. rapa, as B. rapa is much more closely related to B.napus than I. indigotica.48 B. rapa-woad or B. rapa-C chromosome addition lines were selected after several times of backcross. GISH and FISH analysis revealed that all of the 48 plants were C chromosome additions. Nearly all of the additions had a phenotype of B.napus, some of the plants had degenerated anther. The additions with normal anther were fertile or partially fertile. Some plants with 20 chromosomes were obtained from the backcross progenies. Genomic changes and differences ahout absent DNA bands and novel bands was found when compared the induced B. rapa with the B. rapa and I. indigotica. These genome changes may be helpful to broaden the gnentic basis of the B. rapa and to breed disease-resistant B. rapa. |
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