1052 Comparative genomic analyses in Asparagus Joseph C. Kuhl, Michael J. Havey, William J. Martin, Foo Cheung, Qiaoping Yuan, Lena Landherr, Yi Hu, James Leebens-Mack, Christopher D. Town, and Kenneth C. Sink Abstract: Garden asparagus (Asparagus officinalis L.) belongs to the monocot family Asparagaceae in the order Asparagales. Onion (Allium cepa L.) and Asparagus officinalis are 2 of the most economically important plants of the core Asparagales, a well supported monophyletic group within the Asparagales. Coding regions in onion have lower GC contents than the grasses. We compared the GC content of 3374 unique expressed sequence tags (ESTs) from A. officinalis with Lycoris longituba and onion (both members of the core Asparagales), Acorus americanus (sister to all other monocots), the grasses, and Arabidopsis. Although ESTs in A. officinalis and Acorus had a higher average GC content than Arabidopsis, Lycoris, and onion, all were clearly lower than the grasses. The Asparagaceae have the smallest nuclear genomes among all plants in the core Asparagales, which typically have huge genomes. Within the Asparagaceae, European Asparagus species have approximately twice the nuclear DNA of that of southern African As- paragus species. We cloned and sequenced 20 genomic amplicons from European A. officinalis and the southern Afri- can species Asparagus plumosus and observed no clear evidence for a recent genome doubling in A. officinalis relative to A. plumosus. These results indicate that members of the genus Asparagus with smaller genomes may be useful genomic models for plants in the core Asparagales. Key words: asparagus, GC content, duplication. Résumé : L’asperge comestible (Asparagus officinalis L.) appartient à la famille monocotylédone des Asparagacées au sein de l’ordre des Asparagales. L’oignon (Allium cepa L.)etl’A. officinalis sont 2 des plantes les plus importantes sur le plan économique au sein des Asparagales nucléaires, 1 groupe monophylétique bien supporté au sein des Asparaga- les. Les régions codantes chez l’oignon ont un contenu en GC plus faible que chez les graminées. Les auteurs ont comparé le contenu en GC de 3374 EST (étiquettes de séquences exprimées) uniques de l’A. officinalis avec ceux ob- servés chez le Lycoris longituba et l’oignon (2 autres membres des Asparagales nucléaires), l’Acorus americanus (ap- parentée à toutes les autres monocotylédones), les graminées et l’Arabidopsis. Bien que les EST chez l’A. officinalis et l’Acorus présentaient un contenu en GC plus élevé que chez l’Arabidopsis,leLycoris et l’oignon, le contenu en GC chez tous ceux-ci étaient nettement plus faible que chez les graminées. Les Asparagacées ont les plus petits génomes nucléaires au sein des plantes appartenant aux Asparagales nucléaires, lesquelles ont typiquement d’énormes génomes. Au sein des Asparagacées, les espèces européennes de l’Asparagus ont environ 2 fois plus d’ADN nucléaire que les es- pèces d’Afrique méridionale. Les auteurs ont cloné et séquencé 20 amplicons génomiques chez des A. officinalis euro- péennes et chez l’espèce africaine Asparagus plumosus et les auteurs n’ont observé aucune évidence d’un doublement génomique chez l’A. officinalis par rapport à l’A. plumosus. Ces résultats montrent que les membres du genre Aspara- gus qui ont un génome plus petit pourraient constituer des modèles génomiques utiles pour l’études des Asparagales nucléaires. Mots clés : Asparagus, contenu en GC, duplication. [Traduit par la Rédaction] Kuhl et al. 1060 Introduction Conran 1987), of which garden asparagus (Asparagus officinalis L.) is the most economically important. The ge- The genus Asparagus contains a diversity of species nus Asparagus belongs to the family Asparagaceae of the spread throughout Europe, Asia, and Africa (Clifford and monocot order Asparagales. The Asparagaceae, Agavaceae Received 6 November 2004. Accepted 22 June 2005. Published on the NRC Research Press Web site at http://genome.nrc.ca on 8 December 2005. Corresponding Editor: T. Schwarzacher. J.C. Kuhl and K.C. Sink.1 Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA. M.J. Havey and W.J. Martin. Agricultural Research Service, US Department of Agriculture, Vegetable Crops Unit, Department of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706, USA. F. Cheung, Q. Yuan, and C.D. Town. The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850, USA. L. Landherr, Y. Hu, and J. Leebens-Mack. Department of Biology and Life Sciences Consortium, 612 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA. 1Corresponding author (e-mail: [email protected]). Genome 48: 1052–1060 (2005) doi: 10.1139/G05-073 © 2005 NRC Canada Kuhl et al. 1053 (agave and yucca), Amaryllidaceae (amaryllis and narcis- on September 8, 2004, from GenBank (www.ncbi.nlm.nih. sus), and Alliaceae (garlic, leek, and onion) are all members gov). The 5′ ATG codons were aligned as described in Kuhl of the core Asparagales, a well supported clade within the et al. (2004) and were used to generate 2 novel independent Asparagales (Chase et al. 1995, 1996, 2000, 2005). The or- datasets, one with 60 sequences for A. officinalis, onion, and der Asparagales is sister to the commelinid clade (Chase et rice, and the other with 76 sequences for A. officinalis,on- al. 2000; Fay et al. 2000), a monophyletic group that in- ion, and Arabidopsis. cludes the orders Poales (grasses and relatives), Zinger- berales (includes banana), Commelinales, and Arecales Cloning and sequence analyses of Asparagus genomic (palms). Kuhl et al. (2004) observed important genomic dif- regions ferences between these 2 groups of monocots; for example, Following the procedures of Kuhl et al. (2004), 51 A. of- the GC (guanine plus cytosine) content of coding regions in ficinalis ESTs that were highly similar (>75% identity over onion (Allium cepa L.) were much lower than that in the >70% of the length of the asparagus TC–singleton) to sin- grasses and more similar to that in the eudicots than in the gle-copy sequences randomly distributed in the rice genome grasses. were identified. An additional 14 A. officinalis ESTs that Plants in the order Asparagales have some of the largest were strongly similar (BLASTX scores <1.0E-20) to a single nuclear genomes among all plants (Leitch et al. 2005). For orthologous pair of rice and Arabidopsis genes defined in example, agave, garlic, and onion are diploid plants with nu- cluster analysis of the 2 proteomes (http://fgp.bio.psu.edu/ clear genomes approximately 2.4, 5.9, and 6.1 times larger PlantTribes) were also identified. These 65 A. officinalis than maize, respectively (Bennett and Leitch 2003). Aspara- ESTs were compared with TIGR onion gene index (release gus species have the smallest nuclear genomes known among 1.0) using BLASTN to identify highly similar (<1.0E-10) all members of the core Asparagales. Diploid (2n =2x = 20) onion sequences. Sixty-five nested primer sets were de- European Asparagus species possess approximately twice signed according to Kuhl et al. (2004) from these sequences, the nuclear DNA of diploid southern African species 51 and 14 based on onion and asparagus sequences, respec- (Stajner et al. 2002); for example, Asparagus officinalis L. tively. has 1308 Mbp (Arumuganathan and Earle 1991) and aspara- DNA was purified from single plants of A. officinalis gus fern (Asparagus plumosus Baker) has 695 Mbp of DNA MSU-A19 and A. plumosus, as described by Kuhl et al. per 1 C (Stajner et al. 2002). One explanation for an in- (2001). DNA of the A. officinalis doubled haploid line G769 creased genome size with the same chromosome number is was a gift from Dr. David Wolyn (University of Guelph, polyploidization followed by genomic rearrangement and Guelph, Ont.). G769 was selected from microspore regener- diploidization, such as that proposed for the maize genome ated plants of G133, a single male plant selected from the since divergence from a common ancestor with sorghum cultivar ‘Jersey Giant’. The 65 primer sets were screened us- (Gaut et al. 2000). Another possibility is genome expansion ing Taq polymerase (Promega, Madison, Wis.) and DNA of through the proliferation of transposable and repetitive ele- A19 and G769, from which 30 and 31 genomic amplicons ments, resulting in greater nuclear-DNA content. In this study, were cloned, respectively. For 29 of these amplicons, at least we undertook sequence analyses of expressed regions in the 4 TA clones from A19 and G769 were sequenced, revealing, A. officinalis nuclear genome, compared GC content with respectively, 25 and 24 that were highly similar to the origi- other important plants in the eudicots and monocots, and as- nal asparagus or onion EST. The 14 amplicons with the best sessed whether a recent genome doubling occurred in A. of- alignments (no relation to the above-mentioned 14 primers) ficinalis relative to the southern African A. plumosus. were then amplified from the G769 asparagus line and the single A. plumosus plant (Table 1). An additional 6 regions Materials and methods (ACADQ29, ACADZ08, TC2026, TC2545, TC3239, TC3464) were added using primers described by Kuhl et al. (2004), Expressed sequence tags (ESTs) of A. officinalis yielding a total of 20 genomic regions. PCR conditions were Premeiotic male flower buds were collected from 1 plant optimized for the 20 primer sets to produce single amplicons (MSU-A19) of A. officinalis. Messenger RNA was isolated from G769 and A. plumosus using Platinum Taq DNA Poly- and directionally cloned using the Stratagene (La Jolla, Ca- merase High Fidelity (Invitrogen, Carlsbad, Calif.), which lif.) Zap cDNA synthesis kit (http://fgp.bio.psu.edu/). Bacte- were then excised from agarose gels, purified (QIAEX II rial colonies carrying recombinant plasmids were randomly Extraction Kit; Qiagen, Valencia, Calif.), TA cloned using picked and subjected to single-pass sequencing from the 5′ the pGEM-T Easy vector (Promega, Madison, Wis.), and se- end (Kuhl et al.