IRGSP Sequencing Meeting 2002

Highlights:

• The IRGSP is on track to complete the genome at the phase 2 level by the end of 2002.
• Three chromosomes (1, 4, 10) are essentially complete. There are interesting lessons from the preliminary chromosomal analyses as reported by Takashi Matsumoto (RGP) and Bin Han (Shanghai).
• Significant advances is the rice physical map, the size of the genome, and the structure and sizes of the centromere were reported by Minsheng Chen, Jianzhong Wu, and Jiming Jiang and their colleagues.
• The IRGSP is committed to go on to complete the genome with finished quality sequence. Cogent reasons are presented for completing the genome with high quality sequence. Furthermore, Dick McCombie presented strategies that would make the endeavor, at less than $.05 / base, relatively inexpensive.

Sequencing Round Table February 5, 7 pm Epochal Center, Tsukuba

As at TIGR, this evening meeting was held for P.I.s and essential colleagues; funding agency representatives and other auditors were also welcome. The point of the round table sessions is to cover those issues that can be best resolved by discussion among the P.I.s.

1) Sequencing progress and anticipated output in the next four months:
 

Chrom.	Total1	Phase 3	Remain3	Expected.	Deficit5	Expected
	(Mb)	or 22	(Mb)	by May4	(Mb)	Completion6
1	47a	42	5	2	0	yes
2	40b	19.4	20.6	5.8	14.8	yes
3	41b	21.4	19.6	6	13.6	probably
4	36a	35.7	0.3	0.3	0	yes
5	33b	10	23	8	15	yes
6	32b	22.6	9.4	5.8	3.6	yes
7	35b	21.9	13.1	5.8	7.3	yes
8	28b	23	5	5	0	yes
9	22b	3.8	18.2	6.4	11.8	yes
10	23a	23	0	0	0	yes
11	30b	2	28	7.5	20.5	probably
12	31b	1.2	29.8	15	14.8	yes
Totals	398	226	172	67.8	104.4

¹ Current size of chromosomes estimated by sequencing groups^a or by Chen et al^b.
² Estimated non-overlapping amount submitted to public databases.
³ Amount remaining to be sequenced.
⁴ Additional sequence expected to be submitted by May, 2002.
⁵ Deficit remaining after May.
⁶ Expected completion of phase 2 sequence by December, 2002.
 

• Based on progress over that last three months, the group appears more likely to be on target to complete the phase 2 draft by the end of 2002.
• Antonio Costa de Oliveira has received startup funding and will collaborate with the RGP on chromosome 9 to assess his group's capacity.

2) Revision of annotation standards.

• Katsumi Sakata made the following presentation of differences in annotation outcomes at TIGR and the RGP. Based on these observations he made recommendations for amending the Annotation Standards.
• The group recommended the following amendments to section 2:

All groups agree on a standard nomenclature for predicted proteins:

Sequences with 100% identity at the amino acid level to known proteins will receive the same, original gene name.

Sequences with less than 100% identity but with significant homology to known proteins will be called "putative" proteins of the same name. The name of the nearest hit will be included as a note. Sequences that are clearly related to a gene family can be called "XXX-like" or "similar to XXX" protein.

Protein matches with BLASTP bit scores of >100, e-values of < e-20, or equivalent criteria, will be regarded as significant homologies.

Sequences with homology to unknown ESTs will be called "unknown." The EST hit will be included in a note. The homology standard is at least 95% identity at the nucleic acid over ~90% of the length of the entire EST, and should cover two adjacent exons.

Sequences predicted by multiple gene prediction programs with no homology to an EST will be called "hypothetical protein." The gene prediction programs will be included in a note.

Homology to proteins with higher, but still significant, e values or bit scores should be examined to estimate the function of as many predicted genes as possible.

3) Plans for Clemson workshop to make a minimum tiling path. Takuji Sasaki and Jianzhong Wu explained how other groups using RGP clones could integrate them into the Clemson physical map. There seemed to be diminished interest in holding such a workshop.

4) BAC Registry. The group decided not to act on Dick McCombie's earlier proposal to participate in the NIH registry of BACs in progress.

5) Commitment to completing the genome with finished quality sequence.

Reasons rice and cereal grass communities support obtaining finished quality sequence:

• Because of syntenic relationships with the other cereal grasses, and the potential for the rice sequence to help determine gene order in other species, money spent on the rice sequence will leverage otherwise more costly endeavors in the other grasses. Examples are mouse-human and Brassica-Arabidopsis.
• Rice as model plant is the most likely species where homologous genes will be tested for functionality. Their identification, plus their well established location in the rice genome, will facilitate functional assignments in other species.
• Not all genes can be identified in phase 2 sequence because gaps remain. Jo Messing gave the example from completing 2.8 Mb of high quality phase 2 sequence. In the finished sequence, which filled in 6,000 N's, the number of conservatively scored genes rose from 338 to 360.
• Some scientists are interested in repetitive sequences and insertions of organellar DNA. These are the regions and sequences that are ignored or under-represented in draft sequences. One class of genes which we know very little about is non-coding RNAs. They are not recognized by conventional annotation software and may be missing from phase 2 sequence.
• Mapping genes associated with traits found by plant breeders and plant geneticists demands correct identification of genes and the ability to detect polymorphisms. The basis for the latter comes from having a high quality reference sequence from a single cultivar.

Representatives of the participating countries were polled about their commitment to continue to obtain finished quality sequence past the end of 2002. Representatives of Japan, Korea, India, Brazil, and France said that they were committed to complete the finished sequence and believed that they had support to do so. Representative of Taiwan and the US said that while they were committed to finishing the genome with high quality sequence, they were uncertain of continued support.

6) Publications of completed chromosomes.

Takuji Sasaki said that Japan would be submitting a paper on chromosome 1 within a month. Dick McCombie said that the US was about ready to start writing and was assigning tasks in preparation. They would submit by mid May at the earliest. Bin Han said that annotation would be completed soon and they were prepared for writing. They could submit within two months.

There was strong support for submitting the papers at the same time. This would have a greater impact and, as no paper submitted now would be published in time for the expected Bejing and Syngenta publications, it was most important for the papers to have a 2002 publication date.

Dick McCombie further made the point that it was most important for these papers to be intellectually interesting. Possible comparisons with draft sequence and between japonica and indica were examples.

7) Problem of exploitation of genome wide annotation work.

Francis Quetier pointed out that while genome sequencers want and are happy to have their sequence used with appropriate attribution, they rightfully regard the use of their sequences for whole genome analysis as theft of unpublished material. This is a difficult area wherein responsible journal editors generally keep unscrupulous authors in check. The only solution proposed was to follow the example of sequencing groups who post use policies on sequencing group web sites stating that the material is unpublished and should be treated as such by groups who want to use it to prepare publications. Dick McCombie suggests that the Data Release Policy, especially the wording for microbial organisms, posted by the Sanger Center (http://www.sanger.ac.uk/Projects/release-policy.shtml) is an appropriate model.

8) There will be two round table format meetings in 2002. The first will take place at Genoscope May 23-24. The second will take place at Cold Spring Harbor in October.

9) The draft of a letter from the U.S. Advisory Committee to support the completion of a high quality public rice sequence was read and suggested amendments were made and transmitted.
 

Public Sequencing Workshop, February 6, 9 am to 5 pm Epochal Center, Tsukuba

Sequencing Reports:

Rice Genome Research Program, NIAS/STAFF-Institute, Tsukuba, Japan, K. Yamamoto, H. Kanamori, S. Hosokawa, M. Hamada, H. Yamagata, S. Hijishita, M. Nakamura, K. Kamiya, K. Machita, M. Nigishi, A. Kikuta, Y. Nakama, N. Ono, T. Mizubayashi, K. Tsuji, H. S. Zhong, S. Ito, N. Namiki, Y. Katayose, K. Sakata, T. Matsumoto, T. Sasaki.

Progress report of RGP genomic sequencing

The rice genome sequencing project in Japan has started in April 1998 with the aim of sequencing the entire genome of rice. The project is also a part of the International Rice Genome Sequencing Project (IRGSP), in which Japan is in charge of sequencing chromosomes 1, 2, 6, 7, 8 and 9.

In an effort to facilitate the immediate and efficient completion of genome sequencing, we have introduced a semi- automated laboratory system and fully automated data analysis system. The system includes an automated liquid handling system (Biomek FX) for preparation of sequence sample with 384-well plate; sequencing with 384-well plate by ABI3700 capillary sequencers; automatic transfer of sequence data files; automatic assembly by Phred/Phrap; and phase classification.

As a result, we have already released ca. 136 Mb sequence data to the public domain corresponding to 1003 PAC/BAC clones. As for chromosome 1, about 90% (47Mb) of phase 2 sequence has been published as of January 2002 and 67% (34.8Mb) is completed as phase 3.
 

National Centre for Gene Research, Shanghai, Bin Han

Rice chromosome 4 sequencing progress

To completely sequence the chromosome 4 of Oryza sativa ssp. japonica cultivar Nipponbare, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Nipponbare chromosome 4 through an integration of 114-sequenced Oryza sativa ssp. indica Guangluai 4 BAC clones and 182 RFLP and 407 EST markers with the fingerprinted data of the Nipponbare genome from the Clemson University Genomics Institute. The map consists of nine contigs accounting for 35.2 Mb covering 96% of the estimated chromosome size (36.6 Mb). BAC clones corresponding to telomeres as well as the centromere position were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH) giving rise to an estimated length ratio of the long arm vs. the short arm of 5.13 bigger than 2.9 based on the physical map, indicating that the short arm is a highly condensed arm. Combining the FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin occupied about 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence.

We have completely sequenced 282 BAC clones. 235 of the sequenced BACs have been released to the public databases. The total length of the sequenced BACs is 34.8 Mb excluding the overlaps (15.1% overlap of the sequenced BACs). We therefore believe that 95% of the chromosome 4 has been sequenced completely. The total length of remaining 8-physical gaps should be less than 1.4 Mb. Most of the physical gaps are located in the high heterochromatic regions, indicating that these gaps will be difficult to be filled. The annotation and findings of chromosome 4 were presented in the meeting.
 

Academia Sinica Plant Genome Center, Taipei, Teh-Yuan Chow

Progress report of rice chromosome 5 sequencing project in Taiwan

The Academia Sinica Plant Genome Center (ASPGC) working on rice chromosome 5 DNA sequencing has submitted about 90 BAC/PACs, with more than 10 Mega base pairs at the phase 2 level (http://genome.sinica.edu.tw/). Although most of the submitted BACs DNA were isolated from the Monsanto"s BAC library and the sequences assembled using Monsanto's provided raw sequence data, some of the BACs DNA sequences were finished with our own sequence data. Another 22 BACs were at the shotgun library and sequencing stages. BAC contigs cover about 30 % of the chromosome 5. Physical mapping of the BAC clones in the gaps of BAC contigs are supported and cooperated with RGP, STAFF.
 

Our data is updated regularly and can be viewed at http://www.genome.clemson.edu/projects/rice/ccw/.
Funded by: ¹ USDA-CREES/NSF/DOE Rice Genome Sequencing Program, ² Rockefeller Foundation, ³ Novartis
 

The Institute for Genomic Research, Robin Buell

Progress of chromosomes 3, 10 & 11 sequencing

TIGR is participating in the International Rice Genome Sequencing Project and has been assigned portions of chromosomes 3, 10, and 11. We currently have ~30 Mb of rice genomic DNA in our high throughput sequencing pipeline (http://www.tigr.org/tdb/e2k1/osa1/). All sequence is released to Genbank/DDBJ/EMBL to either the High Throughput Sequence (HTGS) or the PLANT division. A total of ~25 Mb has been deposited in Genbank. We have in production all clones for our allocation on chromosome 10 (10S and 10L) with the exception of one small (30 kbp) gap. All completed BACs are manually annotated for genes and this information can be accessed through Genbank and the TIGR web site (http://www.tigr.org/tdb/e2k1/osa1/). We provide automated annotation of all rice BACs (>1200), for which this annotation is available on the TIGR web site. We have extended our bioinformatic analyses of rice and have identified putative orthologues of rice using the TIGR Orthologous Gene Alignments (http://www.tigr.org/tdb/tgi/ego/). We also have performed global alignments of rice genome sequences with all available plant transcripts to further identify candidate orthologous genes. As with all other TIGR Rice information, these data can be accessed on the TIGR Rice web site at http://www.tigr.org/tdb/e2k1/osa1/.

At the TIGR Rice Genome Project Site (http://www.tigr.org/tdb/e2k1/osa1/) can be found the newly updated Rice Gene Index and Rice Repeat Database. Also the The TIGR Orthologous Gene Alignment (TOGA) database has recently been updated.

Robin described automated annotation tools at TIGR and said that while five gene prediction programs are used, they have found that FGENESH is the most robust. A Whole Automated Genome Annotation Database is available constructed from processing all the rice BAC/PAC sequences (phase 2 and phase 3) in GenBank.

Other tools that are available are a whole rice genome BLAST search, and databases of in silico mapping of rice genetic markers to rice STCs and to rice BAC/PAC sequences.
 

Plant Genome Initiative at Rutgers, Joachim Messing

Progress of chromosome 11 sequencing

Chromosome 11 has been divided between India, France, and the United States. The region north of the centromere has been divided between France, TIGR, Wisconsin University and our group PGIR. The region South of the centromere has been assigned to India and TIGR. Within the southern region is an area of collaboration between India and PGIR around position 97.3 cM. Because France is sequencing chromosome 12, which contains a segmental duplication from 0.3 - 8.5 cM of rice 11, US will coordinates its efforts with France. The regions assigned to PGIR include the sequence between marker C104B at position 4.1 and R2916 at position 10.3 and the sequence between marker S20163S at position 27.8 and G320 at position 34.8. From each region 6 clones have been sequenced. An additional 3 clones from the region around 97.3 cM have been sequenced as well, yielding a total of 15 new clones in about 6 months. The region between 4.1 and 10.3 is contained within the FingerPrintedContig (FPC) #203. A Minimum Tiling Path (MTP) has been selected that spans 4 cM from position 4.1 to 8.1. To complete this sequencing contig, one more clone north and 4 clones south will be necessary. The second region contains two FPCs, #205 and #206, which have been merged by the sequence of a single BAC clone. Here we will need only one or two more clones south to complete this sequence contig.
 

Korea Rice Genome Research Program, Ho-il Kim

Progress of chromosome 9 sequencing

The National Inst. of Agricultural Science and Technology (NIAST) /RDA has participated in IRGSP to sequence whole rice genome since 1998. Now we focused on the 68.2-77.7cM and 93.2-94.4 cM region of chromosome 9. We have screened some seed BACs by RFLP markers on our maps and combined the CUGI BAC fingerprinting contigs and RGP map data. We finally selected 16 sequence-ready BAC clones with about 1.7Mb on the 68.2-77.7cM and 3 BACs with 0.4Mb on 93.2-94.4 cM region. Shotgun libraries of those BACs were constructed by sonication, then sequenced and assembled by PhredPhrap. Currently 2.1 Mb(11 BACs from chr.1 and 3 BACs from chr. 9) were registered in GenBank and 12 sequences from chr. 9 were ready to submit as phase 2. Our rice genome sequence information has been released in NIAST web site (http:// biogen.niast.go.kr).
 

Indian Initiative for Rice Genome Sequencing, Akhilesh K. Tyagi

Progress of Indian Initiative for Rice Genome Sequencing (IIRGS)

The Indian Initiative for Rice Genome Sequencing was started in June 2000 with the financial support from the Department of Biotechnology, Government of India, with the main purpose of sequencing a region spanning 56.9 to 109.3 cM on the long arm of chromosome 11 as part of IRGSP. The work is being carried out at the University of Delhi South Campus and the Indian Agricultural Research Institute, New Delhi. The infrastructure for physical mapping, sequencing and genome informatics has been established and two ABI3700, two ABI377 and one MegaBace1000 DNA sequencers have already been made functional.

By screening one PAC library at RGP and two BAC libraries at CUGI, along with analysis of fingerprints and in silico information, it has become possible to develop a physical map covering ~80% of the region assigned to India in the form of 13 major contigs encompassing ~80 genetic markers and ~200 ESTs. Attempts have also been made to overlap and integrate PAC and BAC physical maps. About 100,000 sequencing reactions have been performed to generate data of ~50 million bases at phase 0 level from 2K/5K shotgun clones of 10 PACs and 14 BACs. Several of them have been processed to phase 1 and phase 2 level with a potential to achieve >3 Mb sequence coverage at >10X level. The sequencing of six more BACs is in progress. Assembled and ordered sequences of 5 BAC/PAC clones have already been submitted to GenBank. In these assembled BAC/PAC sequences, the overall ge! ne frequency is estimated to be about 1 gene/6 kb based on annotation using RiceGAAS software, notwithstanding individual BAC/PAC variations.

IIRGS appreciates RGP, Japan, as well as CUGI, TIGR, PGIR and CSH, USA, for hosting short duration visits of various Indian scientists.
 

GENOSCOPE and UMR , Nathalie Choisne, Gisela Orjeda, Nadia Deivange, Eric Pellitier, Marcel SalanoubatT, Jean Weissenbach and Francis Quetier

Progress report on the sequencing of rice chromosome 12

The chromosome 12 of the rice genome is estimated at 30 Mb. Physical mapping contructed on FPC basis by CUGI (USA) is currently checked and completed by PCR and hybridizations. The anchoring of seed BACs is achieved by using genetical markers complemented with the set of EST markers recently published by the Japanese group.

34 BACs from the Clemson library are under sequencing. In addition, 75 BACs sequenced by MONSANTO with a moderate coverage are receiving a further 2-3 x coverage to be finished.

Segmental duplication between chromosome 12 and chromosome 11, which is estimated at 2.5 Mb, is worked out in collaboration with the Rutger University group (USA) and TIGR (USA).
 

Universidade Federal de Pelotas, Brazil, Antonio Costa de Oliveira, Fernando Iraja Felix de Carvalho, Paulo Dejalma Zimmer, Luiz Anderson Teixeira de Mattos.

The Brazilian Rice Genome Initiative - BRIGI

Rice is an important crop in Brazil as it is worldwide and the demand is currently above internal supply. Advances in the discovery of the rice sequence can lead to increases on productivity and yield. Also, rice can be seen as a model crop for the grasses, leading to further gains on other important cereal crops. Officially a member of IRGSP since September 2000, the Brazilian Rice Genome Initiative (BRIGI) has obtained support from the Ministry of Education to start sequencing part of the rice genome on January 2002. Reorganized in 1995, the Setor de Fitomelhoramento at the Faculdade de Agronomia Eliseu Maciel (FAEM) from the Universidade Federal de Pelotas (UFPel), is now entering a new era. Two main labs, one called Genomics and Plant Breeding (Laboratorio de Genomica e Fitomelhoramento - LGF) and the other called Hydroponics and Tissue Culture (Laboratorio de Hidroponia e Cultura de Tecidos - LHCT), give support to studies focusing inheritance, morphological and molecular characterization, genetic and physical mapping, mutation induction and now sequencing of genes related to root development and agronomically important traits. Comparative genetic and genomic studies of rice-oats and rice- maize involving those traits are becoming the major focus of our research group.

We propose to IRGSP, to sequence a region of ~1.4 Mb spanning Contig number 263 from chromosome 9 obtained from CUGI's library. Sixteen BAC clones aligned on a minimum path will be shotgun sequenced and released to public databases as a contribution to the goal of having the complete high quality draft of the rice genome by December 2002.

Laboratorio de Genomica e Fitomelhoramento - LGF, Departamento de Fitotecnia, FAEM, UFPel. Campus Universitario, P.O. Box 354, Capão do Leão - RS, 96001-970, Brazil.
 

Special Topics:

Why we must finish the rice genome and how we can do it, W. Richard McCombie

The draft of a complex genome such as rice is a valuable resource for the plant biology and agronomic communities. However, it is an inadequate resource for many purposes, particularly studies of the structure of the genome (as opposed to the genes) and especially for comparative genomics. The lack of completion in the draft as well as the lack of order provided to the islands of sequence it contains make overall genome structure studies and comparative studies difficult to carry out. Since rice is to serve as a reference sequence for monocot genomics, it is crucial that the sequence be brought to finished levels of quality and contiguity so that it will provide a structural and genetic anchor for studies on other cereals.

To do this we have been developing software and lab protocols designed to rapidly and cheaply finish draft level sequence, even if subclones from the draft are not available. We have been optimizing a transposon based finishing strategy and directed primer walking of BAC templates. The software we have developed generates a worklist for this finishing process. It also monitors the progress of clones through finishing to allow efficient management of the process. In combination, these tools will rapidly accelerate finishing. Dick's presentation may be viewed here.
 

Current status of sequence-ready physical maps constructed for rice chr. 1, 2, 6, 7, 8 and 9, Jianzhong Wu, Satoshi Katagiri, Yoshino Chiden, Mika Hayashi, Masako Okamoto, Yukiyo Ito, Shoko Saji, Hiroshi Mizuno, Shoji Yoshiki, Wataru Karasawa, Yoko Ichikawa, Maiko Ikeno, Rie Yoshihara, Masao Hamada, Marina Nakashima, Tomoya Baba, Takashi Matsumoto and Takuji Sasaki

RGP is now constructing sequence-ready physical maps of rice chromosomes 1, 2, 6, 7, 8 and 9 as part of an international collaboration to sequence the rice genome. BAC clones with 5 x sequences provided by the Monsanto company were used after BLASTing the BAC sequences against about 8,000 RGP genetic and EST marker sequences to determine the position of the clones on these six chromosomes. The resulting physical maps showed a coverage of 35~50% varying in six chromosomes. In regions where no Monsanto BAC clones were confirmed, we used our STS / EST markers to screen the RGP PAC library by PCR and then fingerprinted the selected PAC clones. As a result, the coverage increased to 70~75% for each chromosome. At present, we are filling the gaps by STC strategy using the CUGI (Clemson Univ. Genomics Institute) BAC clones. BLASTing our finished BAC/PAC sequences against the STC database of CUGI could obtain bridge clones thereby closing the gaps between contigs or elongating the contigs. The remaining gap regions correspond mainly to regions close to centromere and we are now making efforts to cover these regions by end-walking method. These combined strategies should enable us to construct a minimum number of contigs covering the entire region of each chromosome to complete the rice genome sequence. This work is supported by the MAFF Rice Genome Project grant GS-1101.
 

An integrated physical and genetic map of the rice genome, Mingsheng Chen, CUGI

Rice was chosen as a model organism for genome sequencing due to its economic importance, its small genome size, and its syntenic relationship with other cereal species. We have constructed a BAC based fingerprint physical map of the rice genome (Oryza sativa ssp. japonica cv. Nipponbare) to facilitate whole genome sequencing of rice. Over 91% of the physical contigs are genetically anchored by overgo hybridization, Southern hybridization, and in silico anchoring. Genome sequencing data were also integrated into the rice physical map. Correlation of the genetic and physical maps reveals that recombination is severely suppressed in centromeric regions as well as the short arms of chromosome 4 and 10. This integrated physical and genetic map of the rice genome will greatly facilitate whole genome sequencing by identifying a minimally redundant tiling path of clones to sequence and organizing the efforts of the many sites participating in this effort. Furthermore, the high-resolution physical map will aid map-based cloning of agronomically important genes, and will provide an important tool for comparative genomic analysis of grass genomes.
 

Chromosome	Genetic Markers	Probes	Contig Number	Previously Estimated	Predicted Chromosome	Size of Anchored	Coverage (%)
				Size (Mb)	Size (Mb)	Contigs (Mb)
1	231	413	32	51.5	44	42.7	97
2	184	316	26	43.4	39.8	35.8	90
3	224	364	26	47.5	40.8	35.7	87.5
4	119	273	24	36.8	39	34.5	88.5
5	139	239	27	33.6	33.2	30.9	93.1
6	129	229	22	35.1	31.8	28.2	88.7
7	158	292	25	33.1	35	30.3	86.6
8	88	181	18	33.6	27.6	25.8	93.5
9	80	139	16	27	21.6	20.3	94
10	136	337	20	23.7	26.8	24.6	91.8
11	118	245	28	33.7	30.3	28.6	94.4
12	98	171	20	30.9	30.6	25.5	83.3
Total	1704	3199	284	430	400.5	362.9	90.6

 Landscape of rice chromosome 1, Takashi Matsumoto, Baltazar A. Antonio, Masatoshi Masukawa, Atsuko Idonuma, Manami Negishi, Michie Shibata, Yuichi Ito, Kimiko Yamamoto, Katsumi Sakata and Takuji Sasaki, RGP

The Rice Genome Research Program (RGP) has been participating in the worldwide sequencing collaboration centered at the International Rice Genome Sequencing Project (IRGSP). Our initial target for constructing a physical map and for sequencing analysis was chromosome 1 which is the longest (51 Mb) of the 12 rice chromosomes. Since sequencing of 80% of chromosome 1 except the centromere, telomeres and some gap regions in the physical map was finished at either phase 2 or 3 HTG grade in March 2001, we started a chromosome-level annotation. Sequences from individual PAC or BAC clones were connected considering the overlapped regions, and the resulting long sequence contig was divided into 1-3 Mb sections. Utilizing the newly equipped high performance auto-annotation program, RiceGAAS we could predict the location of genes coding for proteins, tRNA, ESTs (including genetic markers) and some repeats on the chromosome. Statistical analysis of the gene structure has elucidated that the rice genome has more GC-rich exon and longer intron as compared with Arabidopsis. About 7000 genes were predicted by multiple gene prediction programs including RiceHMM which is tuned for the gene prediction from rice genome, but only 30% of them were functionally categorized. Many transposons and MITES were found throughout the chromosome, indicating that rice genome is abundant in repeat structures. Many genes are redundant and arrayed tandemly. Forty predicted genes showed strong homology to Arabidopsis proteome, while many has no shared sequences.
 

Two relevant reports from the Chromatin Dynamics Workshop (February 7) and the Rice Genome Forum (February 8):

Characterizing the genomic regions surrounding rice centromeres, Jianzhong Wu, Kimiko Yamamoto, Takashi Matsumoto and Takuji Sasaki, RGP

Centromeres play a key role in the process of chromosomal segregation and transmission in cell divisions, in the karyotypic stability, and in generating artificial chromosomes as cloning and expression vectors. As the central site for mitotic and meiotic spindle fiber attachment, molecular organization of centromeres has been studied extensively in some eukaryotes, the majority of these species have plenty of highly repetitive DNA (~ several Mb). In the plant kindom, for instance, the 180 bp repeat forms large tandem arrays and occupy the central domain of all five chromosomes in Arabidopsis centromeres (Fransz et al. 1998, 2000. Heslop-Harrison et al. 1999). In rice, the RCS2 family is a tandem repeat of a 168 bp (about 6000 times in the genome) and is organized into long uninterrupted arrays with the centromeric regions of Oryza species (Dong et al. 1998). We have screened and mapped a number of PAC or BAC clones onto the regions close to the centromeres of chromosomes 1, 2, 6, 7 and 8. Some of these clones have been sequenced. Repetitive sequences including the RCS2 family have been identified. Details on the possible structure and sequence organization within these rice centromere regions will be reported. This work is supported by the MAFF Rice Genome Project grant GS-1101.

References
Dong et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 8135-8140.
Fransz et al. (1998) Plant J., 13, 867-876.
Fransz et al. (2000) Cell, 100, 367-376
Heslop-Harrison et al. (1999) Plant Cell, 11, 31-42
 

Rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon, JIMING JIANG, University of Wisconin

The centromere is the most characteristic landmark of chromosomes in high eukaryotic species. Centromeres are responsible for sister chromatid cohesion and is the site for kinetochore assembly and spindle fiber attachment, allowing for faithful pairing and segregation of sister chromatids during cell division. In the majority of eukaryotic species, centromeres are embedded within megabases of highly repetitive DNA. Thus determining the precise DNA boundaries of centromeres has proven to be a difficult task. Several centromeric repetitive DNA elements have been reported in rice (Oryza sativa) (Dong et al., 1998; Nonomura and Kurata, 1999). Sequence analysis revealed that most of these centromeric DNA elements are derived from a Ty3/gypsy class retrotransposon family that is specific to the centromeric regions of grass chromosomes. However, a 155-bp satellite repeat RCS2 is unique to rice and is exclusively located in the rice centromeres. We have mapped the RCS2 satellite within the chromosomal regions to which the spindle fibers attach. RCS2 is organized as long tandem arrays and is interrupted irregularly by centromere-specific retrotransposon elements. Each rice centromere contains from 100 kb to 2 Mb of the RCS repeat. Quantification of the RCS2 satellite in normal and telocentric rice chromosomes revealed that the breakpoints for several centromere misdivision events are located in the middle of the RCS2 arrays. Our results demonstrated that the centromeric DNA of rice chromosomes is organized similarly to several other model eukaryotic species, including humans and Arabidopsis thaliana, and that the RCS2 satellite is a key DNA element of rice centromeres.

rgpwebmaster@ml.affrc.go.jp Copyright (C) The International Rice Genome Sequencing Project (IRGSP). 2005 All rights reserved.