Distribution and characterization of over 1000 T-DNA tags in rice genome

We generated T-DNA insertions throughout the rice genome for saturation mutagenesis. More than 1,000 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T-DNA tags were not randomly spread on rice chromosomes and were preferentially inserted in gene-rich regions. Few insertions (2.4%) were found in repetitive regions. T-DNA insertions in genic (58.1%) and intergenic regions (41.9%) showed a good correlation with the predicted size distribution of these sequences in the rice genome. Whereas, obvious biases were found for the insertions in the 5'- and 3'-regulatory regions outside the coding regions both at 500-bp size and in introns rather than in exons. Such distribution patterns and biases for T-DNA integration in rice are similar to that of the previous report in Arabidopsis, which may result from T-DNA integration mechanism itself. Rice will require approximately the same number of T-DNA insertions for saturation mutagenesis as will Arabidopsis. A database of the T-DNA insertion sites in rice is publicly available at our web site (http://www.genomics.zju.edu.cn/ricetdna).     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

Features of the expressed sequences revealed by a large-scale analysis of ESTs from a normalized cDNA library of the elite indica rice cultivar Minghui 63

The indica subspecies of cultivated rice occupies the largest area of rice production in the world. However, a systematic analysis of cDNA sequences from the indica subspecies has not been performed. The aim of the present study was to collect and analyze the expressed sequence tags (ESTs) of indica rice on a large scale. A total of 39 208 raw sequences were generated from a normalized cDNA library prepared by use of 15 different tissues of the indica cultivar Minghui 63. After trimming, processing and analysis, 17 835 unique sequences were obtained, each of which presumably represents a unique gene. Of these sequences, 2663 were novel, and at least 70 were indica specific. Comparison of the Minghui 63 sequences with the ESTs/full-length cDNAs in GenBank revealed a large number of deletion/insertion/substitution (DIS) at both the inter- and intra-subspecific levels. The overall number of polymorphisms in the expressed sequences was higher in the inter-subspecific comparisons than in the intra-subspecific comparisons. However, the extent of DIS-based polymorphism was highly variable among different rice varieties. In total, 15 726 unique sequences, including 697 novel sequences, were assigned to regions where large numbers of quantitative trait loci (QTLs) for agronomic traits had been detected previously. These results may be useful for developing new molecular markers for genetic mapping, detecting allelic polymorphisms associated with phenotypic variations between rice varieties, and facilitating QTL cloning by providing the starting points for candidate-gene identification.