Dissertation
Dissertation > Agricultural Sciences > Crop > Cereal crops > Rice

The Screening of T-DNA Insertional Mutants Pool in Rice

Author TangZuo
Tutor PanXueBiao;ChenZongXiang
School Yangzhou University
Course Crop Genetics and Breeding
Keywords T-DNA insertional mutagenesis phenotype identifying genetic analysis flaking sequence rice
CLC S511
Type Master's thesis
Year 2006
Downloads 204
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In this study, we got 4416 rice T-DNA tagged lines from two transformed parents, zhonghua11 and zhonghua15. By phenotype identifying and mutagenesis analyzing in T1 and successive generations, we could distinguish the heritable mutation from non-heritable variation of the morphological characteristics. The heritable mutants can be divided into two types: the variation among different lines or among different individual plants of a line. Based on the proportion of mutants and detection of co-separation between the result of special PCR based on T-DNA segment and the mutant phenotype in a line,we could screen functional-losing mutations that caused by T-DNA insert. Then we carried on the further segregation of sequences flanking the insertions and the co-segregation detection between the flanking sequence and the mutant phenotype. And then we forecasted the function of some genes that were destroyed by a single T-DNA insert, which lay the foundation to the segregation and clone of function genes. The main results are as follow:(1) We screened nearly 520 lines that appeared obvious morphological mutation from 4416 rice T1 tagged lines with a mutation ratio of 11.8%. Many of the mutations were heritable, which could be divided into two types, the like-homozygous mutation of all plants in a line and the separated mutation of some plants in a line.(2) There were abundant mutation phenotypes, related to 14 kinds of traits, such as heading date, shape of plant and leaf (plant height, tiller number, leaf shape, leaf color), shape of panicle (panicle number, panicle shape, grain shape, male sterility, awn), disease-like mutation, and so on.(3) In total, there was no obvious difference of the mutation frequencies between the two varieties, zhonghua11 and zhonghua15, the frequencies were 9.52% and 9.58%, respectively. There was a comparatively high mutation frequency (over 1%) for plant height, heading date, leaf color and male sterility. No significant change of the mutation frequency in plant height and leaf color existed in different years or transformed parents. But, the mutation frequency in heading date and male sterile varied heavily, which suggested that the environment had a great effect on their phenotypes. We should be especially careful in the judgment of mutants in the latter characteristics.(4) Generally, there was only one morphological character mutated in some individual plants of a line. But in individual cases, there were two or more mutational phenotypes in the same lines. In most of these lines, different mutational characters performed independently, i.e. different mutational phenotypes mainly appeared in different mutant plants, most mutant plant were just exhibited one mutational character. But in a few lines, all mutant plants in a line had two or more mutational phenotypes similarly.(5) By continuously co-segregating analyses in T1 and T2 generations, we found that most of the heritable mutations did not belong to T-DNA insertional mutation.(6) As for T-DNA insertional mutation, we found different situations, i.e. T-DNA inserted into receptor parent with not only single copy or series-wound multi-copies but also independent multi-copies. We could clone the functional genes based on the flaking sequence analysis of the inserted DNA segment with single copy or independent multi-copies. Besides, for some interested somaclonal variations, we could clone based on the strategy of map-based cloning, which could be help to raise the using efficiency of rice mutant pools.(7) Based on the detections of co-segregation among the inserted segment, its flanking sequences and the mutation phenotypes, we primarily found 5 T-DNA insertion mutants. We will further clone the relative genes.

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