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A Hexaploid Wheat BAC Sequencing and Analysis of Microcolinearity between Wheat and Rice at the Rht-D1 Locus

Jiajie Wu, Xiuying Kong, Yue Liu, Jianhui Xiao, Cuiyun Jin, Chunqing Zhang and Jizeng Jia

Key laboratory of Crop Germplasm and Biotechnology, Ministry of Agriculture, Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences, Beijing 100081,,


About 200kb BAC from hexaploid wheat Aibai/10*CS BAC library was sequenced. Random-sheared subclones of BAC 1J9 containing Rht-D1c were sequenced at both ends to give a total of 2877 reads, 1.5x107 base pairs. The final assembled sequence covered BAC 1J9 with 7.5 x coverage. Sequence analysis showed that repetitive sequences accounted for about 73% of the DNA sequenced. The LTR retrotransposons made up to 83% of the repetitive sequences. Five genes were identified and distributed in 100kb at one side of the BAC sequence. Three of the five genes were clustered in a 16kb gene-enriched island, while the other two were not in the cluster and separated by repetitive DNAs. Comparison with the orthologous rice BAC AC087797 revealed that three genes shared the same gene order and orientation, but small rearrangements were also detected in the compared orthologous region.

Media summary

Hexaploid BAC sequencing revealed the conservation and rearrangement between wheat and rice at Rht-D1 locus.

Key Words

Wheat, BAC, sequencing, microcolinearity


Wheat and rice are staple food in the world. Comparative genetic maps have revealed a good conservation of the marker order (colinearity) among grass species (Moore et al. 1995, Gale et al 1998), while the information of their microcolinearity is limited. Compared to other grasses, rice has a small genome. In 2002, the draft sequences were released (Yu et al. 2002, Goff et al. 2002) and International Rice Genome Sequencing Project rapidly develops more complete draft sequence. The rice genomic sequence data provided a powerful new resource for studies in microcolinearity. However, wheat is a hexaploid crop with a huge genome size. The whole genome sequencing is more difficult. With the rapid development of the techniques of sequencing and BAC library construction, some BACs were sequenced in Triticum monococcum L., Aegilop tauschii Coss and Triticum turgidum L.. Comparison of Waxy gene locus suggested intron number variation but exon content conservation in the several cereal species (Bennetzen and Ma, 2003, Ma et al. 2004). The intergenic regions of wheat and barley orthologous regions have shown no similarity (Ramakrishna et al., 2002; Gu et al. 2003). However, no BAC sequencing of hexaploid wheat is published. We present here an about 200kb BAC sequencing and analysis of microcolinearity between wheat and rice at the Rht-D1 locus.

Material and Methods

BAC 1J9 was selected from hexaploid wheat Aibai/10*CS BAC library by screening with PCR marker cosegregating with Rht-D1c. The insert size of the BAC clone was about 200kb based on PmeI digestion. BAC sequencing and sequence analysis were as in Kong et al (2004).

Results and Discussion

Sequence organization of BAC 1J9

Annotation of the 200 kb region of BAC1J9 is presented in Figure 1.

Figure 1. Annotation of the BAC 1J9 sequence. Structural features identified are coded by the symbols presented in the box.

Gene density and gene order

FGENESH and GENESCAN soft wares predicted five genes. The gene density of BAC 1J9 sequence was about one gene per 41.4kb. The previous reports varied from 1 gene per 5-45kb. CDS1 and CDS2 had homology with wheat ESTs and unknown genes. They were active in wheat. CDS5 had a very high similarity to rht-D1a and OsGAI, while CDS3 and CDS4 haven’t matched any EST or gene. CDS3, CDS4 and CDS5 formed a 16kb gene-enriched island and separated from the other two genes.

Repetitive DNAs

Seven intact LTR retrotranspsons, nine partial LTR retrotransposons, one non-LTR retrotranspson LINE, two CACTA transpsons and three MITEs were identified in the sequenced region. These transpsons were abundant and mainly distributed in the first 100kb and intergenic region.

Orthologous region between wheat and rice

Comparative genetic maps revealed that gene controlling gibberellin insensitivity and plant height gene were conserved in wheat and rice (Figure 2).

Figure 2. Comparison of gene content and order between wheat and rice at the target region

At the gene level, our data showed three genes–CDS1, CDS2 and CDS5 (Rht-D1c) had conserved gene order and transcriptional orientation with rice BAC AC087797. The similarity was 90.7%, 75.7% and 83.3% at amino acid level, respectively. However, small numerous rearrangements occurred during evolution. CDS3 and CDS4 had no homologous sequences found in orthologous rice region. One rice active

gene, 1 kb downstream from the homologous CDS2, was lost in wheat. In addition, seven predicted genes in rice BAC showed no similarity to orthologous wheat region. As Rht-D1c was almost at the end of BAC 1J9, the BAC adjacent to the gene-enriched island of BAC 1J9 needs to be identified and sequenced. Details about this orthologous region in rice and wheat will be revealed.


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