| Rice is one of the most important crops in the world, and it is also a model plant in the study of functional genomics of monocotyledon cereals. The seed setting rate is one of the important factors markedly affecting rice yield and has been focused in rice genetic breeding and production. After transducing T-DNA(MIM164c, OE164 c,MIM168a, OE168a) into kasalath, a large of transgenic mutants were achieved. Through the successive selection and identification of biological characteristics of these transgenic lines from T1 to T4 generation, a transgenic line(3) 19-9-4 was screened out, whose seed setting rate is significantly lower than wild type. In this study, we attempt to provide the theoretical bases to explore the molecular mechanism associated with seed setting rate of rice, the fertility and viability of pollens of(3) 19-9-4 was measured and the alignment of whole genome resequencing(WGR) of this mutant line was compared with the wild type.The main results are as follows:1. From T1 to T4 generation lines created by transforming exogenous T-DNA(MIM164c, OE164 c, MIM168 a, OE168a) into conventional rice kasalath, many phenotypes of mutation were acquired,including plant height, tiller number, leaf shape, heading date, grain shape,effective panicle number, 1000 grain weight, seed storage tolerance etc,all of these exhibit significantly different from the wild type. Among them, the MIM168 a transgenic line(3)19-9-4(hereinafter referred as(3)19-9-4) has a remarkable reduce in seed setting rate, which attracted the most attention.2. Compared with the wild type,(3)19-9-4 exhibits additional growth period, shorter plant height, thin stem and easily lodging, more tillerings,smaller grain size. The anther of this mutant line is not plump, the fertility of its pollens is 70% lower than that of the wild type, the seed setting rate is 42% lower than the wild type.3. Using Nipponbare genome as reference, the whole genome resequencing(WGR) of(3)19-9-4 and the wild type was compared. The results indicated that the number of total reads of(3)19-9-4 and the wild type was 91,410,984 and 88,022,138, with the average depth 22.7X and23.2X, and the quality score 93.99% and 92.61% respectively; the coverage reached 89.88% and 89.77% of the Nipponbare genome 430 Mb respectively, the total mapped ratio was 67.35 % and 71.71 %, unique mapped ratio was 46.3% and 49.69%,no-mismatch mapped ratio was21.06% and 22.19% respectively. Mutant unique SNPs and In Dels was1,618,456, and the most varianty frequency was located in Chr.10, which possessed 520 varianties per 100kb; the minimum varianty frequency was on Chr.2, which was 377 varianties per 100 kb. Total base Transitions(TS)including T/C and A/G of(3)19-9-4 was 2259837, 105060 more than that of the wild type. Among them, T / C transition was 53603 more than the wild type; Total transversions(TV) including T/G、T/A、C/G、C/A of(3)19-9-4 were 928351, 47532 more than the wild type;(3) 19-9-4 had the maximum transversion of T/A, 14310 more than that of the wild type.(3)19-9-4 SNPs and indels resulted in 864 stop gain(gene terminator) and363 stop loss(gene terminator lost) of genes in total which may change the function of genes, and were 44 and 16 more than that of the wild type respectively. The SNPs of(3) 19-9-4 lead to 51727 non-synonymous SNVs, which were 1665 more than that of the wild type. The In Dels of(3) 19-9-4 lead to 873 nonframeshift insertions and 939 nonframeshift deletions respectively, which were 113 and 97 more than the wild type.The SNPs of(3)19-9-4 result in 82647 nonsynonymous amino acid, 2039 more than that of the wild type, in which nonsynonymous alanine(A)was the maximum(7990), tryptophan( W) was the minimum(917);Among all types of non-synonymous amino acids, 206 serines(S) was the largest difference quantity between(3) 19-9-4 and the wild type, the second difference quantity was 203 arginine(R), the least was 6 tyrosines(Y).(3)19-9-4 possessed 405274 structural varianties(SVs), 35934 more than that of the wild type, with the exception of chromosome translocation(INT), the quantity of other types of SVs including long fragment insertion(LI), short fragment insertion(SI), deletion(DEL) and inversion(INV) were all more than that of the wild type, and the unique SVs of(3) 19-9-4 were 99037, 35934 more than that of the wild type,including 42993 deletions(DEL) which was the type with the maximum quantity. Furthermore, blasting to the genome of the wild-type,(3)19-9-4possessed 106 copy number variations(CNVs), Both of Chr. 4 and Chr.12 had 16 CNVs, Chr. 3 had only 2 CNVs.4. On the basis of WGR analysis and PCR verification, twocandidate insertion sites of the exogenous T-DNA(GCS-IPS1-MIM168a)in kasalath chromosomes were identified, one of which was behand of2609601 bp on Chromosome 3; the other was between 604415 bp and604502bp of Chromosome 5. Bioinformation analysis showed that the first insertion site may be involved in two genes, one of which is LOC-Os03g5334, a potential gene of encoding protein, but the relative biological function has not been reported; the other one is OS03G0146800(non coding RNA gene). Whether these two genes are related to the regulation of seed setting rate needs further study. The second insertion site belongs to intergenic region, which may not affect gene function.In general, the results of whole genome resequencing(WGR) of(3)19-9-4 to the wild type showed that the insertion of exogenous T-DNA lead to significant varianties of wild-type genome structure including SNPs, In Dels, SVs and CNVs, which could result in the changes of the related gene expression and amino compositions. These results have provided important clues for the in-depth exploration to the molecular mechanism acting on the trait of seed setting rate. |
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