| During the process of plant evolution, polyploidization has long been recognized as an important force, and is an important way of plant evolution and the formation of new species.70% of angiosperm experienced one or more times polyploidization process. There are many natural polyploid plants in nature, such as autopolyploid alfalfa, potatoes, mulberry, sugar cane and grapes; allopolyploid cotton, Brassica napus, wheat, chrysanthemums, tulips, coffee and so on. Genome duplication makes polyploidy increase genetic material available so that it can accommodate a wider range of the natural environment. Duplicate genes may lead to functional differentiation, get stronger drought tolerance and pest resistance, and make the flowering and organ size change, so as to make polyploid species have a greater advantage in natural selection. People often use the newly synthesized polyploid to simulate the process of polyploidy generation. Comparing the various aspects of cellular, molecular biology, common genetics and molecular genetics of polyploidy to the parent species found that significant changes occurred in many aspects in polyploidy. The genetic variation caused by polyploidization is very complex, including heritable variation, such as chromosomal rearrangement and elimination sequences, and epigenetic changes, such as methylation, gene silencing, and gene activation, and changes of the small RNA. small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), trans-acting siRNAs (tasiRNAs), small nucleolar RNAs (snoRNA) and small nuclear ribonucleic acids (snRNA), regulate gene expression by epigenetics.However, what changes various types of small RNA take place and what roles they play in polyploidization remains unclear.B. napus is also an important allopolyploid oil crops. The average acreage of canola is 6.7 million hectares in China and is China’s largest oil crop acreage, accounting for 45% of edible oil consumption. B. napus only has 400 years’cultivation history and exhibits narrower genetic base. China has a long history of cultivation of B. rapa and B. oleracea and rich genetic resources. Introgression of the genetic resources of diploidy parental species of B. rapa and B. oleracea into B. napus is meaningful and important to broaden the genetic base of B. napus. Hybridization between B. oleracea and B. rapa and subsequent chromosome doubling is an important way of synthetic B. napus. However, in this process, there will be some non-parental genetic phenotypes, such as long siliques, large blades. What causes these variations? In synthetic B. napus, what roles various types of small RNA play in the process of polyploidization of B. napus did not answer. The specific questions are as follows:(1) In different generations, what are laws of miRNA changes and regulation laws of regulating small RNA target sites? (2) In the process of polyploidization, what are laws of various types of non-coding RNA (miRNA, siRNA, snRNA, snoRNA) variation, and whether similar laws exist? (3) What is the difference of miRNAs and target sites between the nature polyploidy and current parental polyploid species? What are roles of these differences played in polyploidy generation? (4) Whether are there significant differences between newly synthesized polyploid and naturally polyploidy? What are roles of these differences played in polyploid evolution?There are two strategies to study small RNA variation law in the process of B. napus formation. The first method is that the total six samples, including four samples from synthetic B. napus S1-S4 generations and the two DH parent materials B. oleracea and B. rapa, are used to conduct the high throughput sequencing to obtain a variety of different types of small RNA. Then, these miRNAs were classified, located, and also analyzed toward length of the small RNA, precursor sequence, base composition, and differences of structure, expression pattern and target gene function and other aspects, comparing parental to different generations, so as to elaborate on the change laws of small RNA in B. napus formation.Second, we collected published B. oleracea, B. rapa and B. napus sequence miRNA, names and other information and established the pool of three species of collected miRNA, combining the miRNA data above. Then, the miRNA pool is used to perform blastn analysis against the genomes of three species, respectively. Two neighboring sequences of each hit are extracted to predict their secondary structure prediction. The analysis of miRNA target sites is also conducted. We also seek some laws of miRNA sequence itself of B. oleracea, B. rapa and B. napus, including base composition, the length distribution and discrepancy analysis of miRNA type, and analyzed the regularities of one miRNA regulating multiple target genes and more than one miRNA regulating target genes. All these analysis is intended to exploit the laws of changes of miRNA position on the genome, and their nucleotide composition, structure and function and types of repetitive sequences generating miRNAs.1 The variation analysis of sRNA and miRNA of parental species and different generations of resynthesized B. napus(1) S population of resynthesized B. napus derived from B. oleracea and B. rapa has been constructed, and has four generations of S1-S4, containing 29 lines. S1 is maintained by tissue culture and the others are transmited by single seed descent.(2) By high-throughput sequencing,11412769,25393355,47982866,12566574, 10029041 and 16421620 reads with the removal of low-quality sequences, were obtained from B. oleracea, B. rapa and future generations S1-S4. From the transfer process of different generations of P-S1, S1-S2, S2-S3, and S3-S4, in the synthetic B. napus, the common sequence average ratio of unique sRNAs were 15.91%,15.87%, 18.29% and 21.64%, respectively, showing a gradual upward trend, and also reflecting the gradual slowdown trend of sRNA sequence variation in the process of selfing. As a whole, the proportion of unique sRNAs in the total sequence were very low, all below 25%, illustrating the tremendous changes of sRNAs occur in the process of formation of polyploid species.(3) From the transfer process of different generations of P-S1, S1-S2, S2-S3, and S3-S4, in the synthetic B. napus, the proportion of common sequence of sRNAs was averagely 71.22%,61.88%,70.77% and 70.97%, respectively, showing a trend of decrease and then increase of variation, respectively, in early generations and later generations.(4) The number of known and new miRNA identified in B. oleracea, B. rapa and synthetic SrS4 of B. napus were 2947,3005,3270,3114,3176 and 3226, respectively. The number of miRNAs expressing in the offspring of synthetic B. napus was higher than that parents by 4.64-9.88%, suggesting that polyploidzaiton of synthetic B. napus promoted the increase of miRNAs types. In moreover, the number of 24nt miRNAs was the most, followed by 23nt,22nt and 21nt miRNA.(5) Through the analysis of bases of different positions of miRNAs in synthetic B. napus and their parents, the different bits of the base were significantly found. For example, the first base showed the preference of U; G base frequently occurred in the third base position; the C base easily happened in the No.19 base position. The first base of miRNA also obviously showed a base preference.(6) The number of transposon generating miRNA of S1-S4 was significantly higher than the parental species B. oleracea and B. rapa, which indicated that the process of polyploidization can induce activation of transposons producing miRNA. The activation of transposons was obvious in S1 generation of interspecific hybridization and then lowered in the offspring.(7) Expression patterns of different types of small RNAs were significantly different. miRNA mainly showed additive effect, followed by dominant effect and super-dominant effect, and the numbers of Si-specific and parent-specific expression patterns were low. The expression patterns of the rest of the snRNA, snoRNA and small RNA fragments derived from exons and introns exhibited parental specificity and Si specificity, but additive, dominance and super dominance were relatively small.(8) The percentage that miRNAs expressed in offspring but not in parents (B. oleracea and B. rapa) reached 33.34%. The percentage that miRNAs expressed in parents but not in offspring accounted for 6.39%, while the percentage that miRNAs expressed both in parents and in offspring was 60.27%. These indicated that the new miRNAs played an important role in generation of synthetic B. napus and silent miRNAs less functioned, besides the main expressed pattern that miRNAs expressed both in parents and in offspring.(9) The number of target genes controlled by each miRNA in B. oleracea and B. rapa were averagely 1.2 and 1.6, respectively, while the corresponding number of S1-S4 generations were over 3, which was twice higher than that of parents. These indicated that the number of target genes regulated by each miRNA significantly increased after polyloidization, and enhanced the complex of gene expression regulation of polyploid species.2 Analysis of miRNA variation of parental species, and traditional B. napus miRNA(1) A total of 5104 miRNA from 2050 family are collected from 22 articles published, including 555 miRNAs of B. oleracea,2582 of B. rapa,1967 of B. napus. A total of 6138 miRNAs were collected, combing miRNAs above. The number of miRNA only detected in the parent species were 387 (6.3%), while the number in B. napus was 80, accounting for 1.3%. The number of miRNA both in the parent species and S1-S4 offspring the parental species and polyploid offspring were 5671 (92.4%). This shows that, in the offspring of interspecific hybridization of B. oleracea and B. rapa, the species-specific miRNA only occupied a small proportion of the total, but the miRNAs present in both parental species and offspring accounted for most of the miRNA.(2) Analyzing of base compositions of Brassica miRNA paves the way for the identification and prediction of miRNA. The analysis of base compositions of A, U, G, C in each of the miRNAs of three species reveals the presence of significant differences. Overall, the content of the base U is significantly higher (33.66%) than others, the content of G is significantly lower (19.97%), and the rest of the A and C were basically consistent with the theoretical value. AU content is higher than GC content, and in particular the AU content of 1 and 23 loci is higher than 66%, which is perhaps related to their function. There is no significant difference through the base comparison of miRNAs of the three species, indicating that related species showed the similar base composition of miRNA. Further, the AU content (>60%) of the first base of all miRNAs was much higher than that of GC content, and the content of C was significantly lower than that of other types.(3) In B. oleracea, B. rapa and B. napus, more than 85% of miRNA genes were located in intergenic sequences, followed by exons (3-7%) where more were distributed in the antisense strand than in the sense strand.2-6% of miRNA genes were distributed in intron regions, and there were not different between on the sense strands and on antisense strands. The proportion of miRNAs located in the repeat region was 1% or less. By the comparison of miRNAs distribution of three species, the origin of miRNA of B. napus were more diverse and wide.(4) In order to analyze the new features of repeats, transposon sequences were detected in the genomic loci of miRNA. A total of 425,247 and 506 transposon sites containing miRNA were detected in B. oleracea, B. rapa and B. napus, repestively. The number of transposon sites generating miRNAs in B. napus was higher than that of B. rapa by 72%, indicating that in the generation process of B. napus, the number of transposon generating miRNA sharply increased, and becomes one possible reason of an increase of miRNAs after polyploidization.(5) The number of target genes controlled by miRNAs in B. napus significantly increased. The percentage of lower regulatory network (e.g. a miRNA regulating 7 or less target genes) decreased, while the number of regulatory networks that one miRNA controlled 25 or more target genes has increased significantly. This shows that after polyploidization, the number of miRNA target genes increases, enhancing the complexity of miRNA regulation of polyploid.In summary, in the process of polyploidization of B. napus generation, a large variation of various types of small RNA occurred, showing a tendency to become stable in each generation. Most sites exhibited the performance of additive expression, followed by dominant effect and super-dominant effect. Newly-expressed miRNA plays an important role in the process of synthetic B. napus. In contrast to parental species, the number of miRNA of polyploid, the number of transposons generating miRNAs, and the number of target genes controlled by miRNAs significantly increased, which increased the regulation complex and diversity of B. napus and enhanced ability to adapt to the external environment. |
