LIBRO-CONGRESO-CITRUS

S03
of large genomic regions. Sequence analyses have also shown that natural and induced mutations that
produce similar changes in the genome also generate similar phenotypes. From the commercial standpoint
this information may help to authenticate varieties, to support marker-assisted breeding and to identify
genes of major agronomic interest.
This work was conducted with funding of projects: PSE-060000-2009-8 and IPT-010000-2010-43
S03O03
Whole genome sequencing and mapping analysis for identifying polymorphism among 11 citrus
varieties
Shimizu T. 1 , Yoshioka T. 1 , Nagasaki H. 2 , Kaminuma E. 2 , Toyoda A. 2 , Fujiyama A. 2 , and Nakamura Y. 2
1 NARO Institute of Fruit Tree Science (NIFTS), Citrus Research, Japan; and 2 National Institute of Genetics (NIG), Genome Informatics
Laboratory, Japan. tshimizu@affrc.go.jp
NIFTS has been conducting cross-breeding of citrus to develop new promising scions with unique and
attractive features for consumers. Developing new scions, however, requires 14 to 20 years due to long
juvenile period of citrus. Marker assisted selection of important traits at the seedling stage is anticipated
to reduce the total cost of the breeding, and it requires sufficient numbers of genome-wide DNA markers
to achieve efficient selection. In this study, we sequenced 11 citrus varieties that were widely used in our
breeding program to obtain sufficient amounts of polymorphic loci among them. Nine of them include
satsuma, ‘Ponkan’, sweet orange and clementine. Two hybrids were also selected to confirm inheritance
of called polymorphisms. Next generation sequencing of the citrus varieties was conducted by pairedend
analysis with HiSeq 2000 DNA sequencer (101 bp for both ends). A total of 100 x genome sequence
coverage were obtained for each variety, and they were de novo assembled individually. Mapping analysis
with the clementine haploid sequence as a reference detected more than 100,000 SNPs. Number of SNPs
and ratios of homozygous SNPs to heterozygous SNPs were different among varieties. Obtained sequences
for individual varieties with called SNPs would be useful to develop high-throughput genotyping platforms
and to promote cross-breeding with less cost.
S03O04
Sweet orange genome: sequencing, annotation and beyond
Xu Q. 1 , Ruan X. 2 , Chen L.L. 2 , Chen D.J. 2 , Zhu A.D. 1 , Chen C.L. 1 , Ruan Y. 2 , and Deng X.X. 1
1 Huazhong Agricultural University (HAU), Horticulture Department, China; and 2 Huazhong Agricultural University (HAU), Colleage of
Life Science and Technology, China. xuqiang@mail.hzau.edu.cn
Sweet orange (Citrus sinensis) is one of the most important fruit crops in the world. Orange is responsible
for approximate 70% of total citrus production, and is used both as fresh fruit and processed juice. Most
oranges are diploids, with the haploid chromosome number x=9 and estimated genome size of 370 Mb.
On a double-haploid line of ‘Valencia’ sweet orange, we employed the Illumina/Solexa platform to produce
785 million high quality paired-end-tag sequencing reads (2×100 bp) from various DNA fragment sizes (~300
bp, 2 Kb, 10 Kb, and 20 Kb). The sequence reads were assembled by SOAPdenovo and Opera, resulting in
16,890 assembled sequence contigs and 4,811 scaffolds (N50=1.7 Mb). The total contig sequence length
covers nearly 90% of the estimated sweet orange genome. We also sequenced more than 5000 BAC-ends, the
alignments between these BAC sequences and the citrus genome assembly showed an overall high identity.
To annotate the citrus genome for protein coding genes, a comprehensive strategy that combined ab initio
gene predictions, homology searches, and experimental supports (EST, RNA-seq and RNA-PET) was employed.
We generated 965 million RNA-seq reads and 100 million RNA-PET reads from four citrus tissues (callus, leaf,
flower and fruit) to characterize the orange transcriptomes. In total, 29,655 protein-coding loci (gene models)
with 44,645 transcripts were identified, with 99% of predicted coding sequences (CDS) supported by RNAseq
data, demonstrating the high accuracy of gene annotation in the orange genome. The sweet orange
genome, as a complementary source to the clementine genome released by the International Citrus Genome
Consortium, represents a valuable resource for genetic understanding of orange biology and manipulation of
many important traits for citrus breeding.
62 - VALENCIA CONFERENCE CENTER, 18th-23rd NOVEMBER 2012