Nuclear DNA amounts in angiosperms: targets - Annals of Botany

124 Pages·2011·1.15 MB·English

Annalsof Botany107:467–590, 2011
doi:10.1093/aob/mcq258,availableonlineat www.aob.oxfordjournals.org
Nuclear DNA amounts in angiosperms: targets, trends and tomorrow
M. D.Bennett* and I. J.Leitch
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
*For correspondence. E-mail: [email protected]
Received:25August2010 Returnedforrevision:18October2010 Accepted:24November2010 Publishedelectronically:21January2011
CONTENTS Do
w
INTRODUCTION 468 nlo
Extendingthe range of genome sizes encounteredin angiosperms 469 ad
e
The needforreference lists 470 d
TARGETSIN GENOMESIZE RESEARCH 470 fro
Meetingtargetsforspeciesrepresentation 470 m
h
Progresstowardstargetsforfamilialrepresentation 471 ttp
Improved systematicrepresentation forgenera 472 s
Improved representation ofother groups 472 ://a
c
TRENDSIN TECHNIQUESUSED TO ESTIMATE GENOMESIZE 473 ad
The risein flowcytometryasthe techniqueof choiceforgenome size estimations 473 em
Developmentofdifferentisolation buffersforflow cytometry 473 ic
.o
The applicationof flowcytometry to plantsystematics 474 u
p
Recent developmentsin the application of flow cytometryto genome size studies 474 .c
o
(i)The useof seeds 474 m
(ii)Ease of accessto methodologicaldata 474 /a
o
(iii)Newequipment 474 b/a
Are there anynew techniques forestimating genome size onthe horizon? 475 rtic
(i)Canreal timePCR beusedforestimating plantgenome sizes? 475 le
(ii)Will ‘complete’ genome sequencinggive useable genome size estimates? 475 -a
b
TOMORROW 479 stra
Uncoveringandcollatinggenome size datafromdiversepublishedsources 480 c
Screeningex situ andinsitu collectionsas sources of target taxa 481 t/1
0
DEDICATION 481 7/3
LITERATURECITED 482 /4
6
APPENDIX 485 7
Notesto the Appendix 485 /14
6
Original referencesforDNAvalues 587 1
4
6
b
†BackgroundandAimsTheamountofDNAinanunreplicatedgameticchromosomecomplementisknownasthe y
g
C-valueandisakeybiodiversitycharacteroffundamentalsignificancewithmanypracticalandpredictiveuses. u
e
Since1976,BennettandcolleagueshaveassembledeightcompilationsofangiospermC-valuesforreferencepur- s
posesandsubsequentlythesehavebeenpooledintotheAngiospermDNAC-valuesDatabase(http://data.kew.org/ t on
cvalues/).Sincethelastcompilationwaspublishedin2005,alargeamountofdataonangiospermgenomesizehas 2
8
beenpublished.ItisthereforetimelytobringthesedatatogetherintoaninthcompilationofDNAamounts. M
†ScopeThepresentworklistsDNAC-valuesfor2221speciesfrom151originalsources(includingfirstvalues arc
for 1860 species not listed in previous compilations). Combining these data with those published previously h 2
showsthat C-values are nowavailable for6287 angiospermspecies. 0
1
†KeyFindingsAnalysisofthedataset,whichisbyfarthelargestoftheninecompilationspublishedsince1976, 9
showsthatangiospermC-valuesarenowbeinggeneratedatthehighestratesincethefirstgenomesizeswereesti-
matedinthe1950s.Thecompilationincludesnewrecordholdersforthesmallest(1C¼0.0648pginGenliseamar-
garetae) and largest (1C¼152.23 pg in Paris japonica) genome sizes so far reported, extending the range
encountered in angiosperms to nearly 2400-fold. A review of progress in meeting targets set at the Plant
GenomeSizemeetingsshowsthatalthoughrepresentationforgenera,geographicalregionsandsomeplantlife
forms (e.g. island floras and parasitic plants) has improved, progress to increase familial representation is still
slow.Intermsoftechniqueitisnowclearthatflowcytometryissoonlikelytobecometheonlymethodavailable
forplantgenomesizeestimations.Fortunately,thishasbeenaccompaniedbynumerouscarefulstudiestoimprove
thequalityofdatageneratedusingthistechnique(e.g.designofnewbuffers,increasedawarenessandunderstanding
ofproblemscausedbycytosolicinhibitors).ItisalsoclearthatalthoughthespeedofDNAsequencingcontinuesto
rise dramatically with the advent of next-generation and third-generation sequencing technologies, ‘complete
genomesequencing’projectsarestillunabletogenerateaccurateplantgenomesizeestimates.
Keywords: DNAC-value,nucleargenome size, PlantDNAC-values Database, flow cytometry.
#The Author2011.Publishedby OxfordUniversity Presson behalfofthe AnnalsofBotanyCompany.Allrights reserved.
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468 Bennett & Leitch – Nuclear DNA amounts in angiosperms
INTRODUCTION amount in the unreplicated gametic nuclear chromosome
complement [defined by Swift (1950) as the 1C-value) of
It has been possible to estimate the amount of DNA in plant
different taxa. [Recently this was defined as the holoploid
and animal nuclei using various methods for over 60 years.
genome size by Greilhuber et al. (2005).]
Following the discovery of the key role of DNA in biology
Easy access to data for these key characters is clearly an
in1953(WatsonandCrick,1953)suchresearchhasincreased
important facility. Yet in practice it has often been difficult
ineachsuccessivedecade,andthistrendhasfurtherintensified
to know whether a C-value exists for a particular taxon,
in the 21st century. These decades saw the loss of many who
and if so, where to find it, especially as estimates are
played key roles in founding and developing this interest
widely scattered in the literature or even unpublished.
[including Hewson Swift (1920–2004), John McLeish
Although small lists of nuclear DNA amounts were published
(1929–1971), Arnold Sparrow (1914–1976) and Hugh Rees
in reviews and research papers as genome size data started to
(1923–2009)]. Yet until now the remarkable birth and accumulate in the 1960s and 1970s, the first large compi- D
explosion of genome size data in ‘the DNA age’ occurred in ow
lation of DNA amounts for angiosperms, compiled primarily n
the scientific lifetime of some individuals who witnessed or as a reference source, was published in 1976 with data for loa
experienced the entire development of this field. However, d
753 species from 54 sources (Bennett and Smith, 1976). e
the death of Jim Price (1944–2005 – Arnold Sparrow’s post- d
doctoralstudent)in2004,andtheretirementofthefirstauthor This noted an intention to publish supplementary compi- fro
lations for reference purposes at intervals and seven followed m
(Mike Bennett – Hugh Rees’s postdoctoral student) in 2006 h
and Johann Greilhuber in 2010 shows that an historic change (see Table 1). ttp
hstaasrtboefgutnheasDtNheAbaagtoen, wpahsossees ktonogwenleedrgateioonfs euanrblyorngeantomthee amIonucnretsaswinagsamccaedsesibpiolistsyibalnedthearoseugohf apcocoelsinsgtoonfudcaletaarinDNthAe s://aca
published reference compilations into one electronic database d
size research reflects written accounts alone, with no further e
available on the internet. The Angiosperm DNA C-values m
recourse to supplementary first-hand sources. Happily, recent ic
Database (release 1), which collated all the data from the .o
trends noted in this review show that tomorrow’s studies of u
first five published compilations (Table 1), went live in April p
nuclearDNAamountsinangiospermsrestwithanenthusiastic 1997. After several updates, a major new release (3.1) of .co
new cohort of practitioners focused on carefully improving m
angiosperm data was combined with databases for gymnos- /a
techniques and data representation to better understand the o
perms, pteridophytes and bryophytes, into the Plant DNA b
oriAgisn,neoxtetedntbyanBdeenfnfeectttsaonfdvLareiiattcihon(2in00p5laan),t g‘NenuocmleearsiDzeN.A fCu-rvthaleuresexDpaatnadbeadsein(re2le0a0s4e (1r.e0l)eaisne230.001). tTohiisncdoartpaobraastee wnaost /article
amount and genome size (C-value) are important biodiversity only first values for 628 species not previously listed in the -ab
characters, whose study provides a strong unifying element in s
btaixoolongoymwicitghropurpacstisctualdiaendd, BpreendniectttivaendusLese’i.tcOhf(2a0ll0t5hae)mnoatjeodr Atimngei,osDpNerAm aDmNoAunCts-vfaolrue2s5D3ataalbgaasle,spbeuctieasl.soA,nfoardtdhietiofinraslt tract/1
update of the Plant DNA C-values Database (release 4.0), 0
that angiosperms were probably the most intensively studied. adding data for over 300 angiosperm species (from 7/3
With published prime C-values for over 6280 species now /4
Zonneveld et al., 2005), went live in 2005. Release 5.0 of 6
available (Table 1), they remain so today. Indeed, work on the Plant DNA C-values Database (December 2010) 7/1
plants has played a leading part in the research on the DNA 46
1
4
6
b
y
g
TABLE 1. Total numbers of first estimates for angiosperm species and of original references from which they come in nine ue
compilationsofnucleargenomesizeestimatescollatedforreferencepurposessince1976 st o
n
2
No.offirstestimatesfor No.ofprimeestimatesforspecies No.oforiginal No.offirstestimatesper 8
‘species’inabstractof inthePlantDNAC-valuesDatabase referencesin originalreference(column M
a
Originalcompilation compilation (release5.0) compilation 2/4) rc
h
2
1.BennettandSmith(1976) 753 596† 54 13.9 01
2.Bennettetal.(1982) 240 195† 53 4.5 9
3.BennettandSmith(1991) 588 552† 56 10.5
4.BennettandLeitch(1995) 899 868† 106 8.5
5.BennettandLeitch(1997) 471 481† 37 12.7
6.Bennettetal.(2000) 691 686† 71 9.7
7.BennettandLeitch(2005a) 628 636† 88 7.7
8.Zonneveldetal.(2005) 308 308 1 308.0
9.Presentwork 1860 1974† 151 13.1
Total 6438* 6287* 617 –
*ThetotalnumberoftaxainthePlantDNAC-valuesDatabase(i.e.6287)islowerby151thanthetotalforpapercompilations(i.e.total¼6438)because
severaltypesofentryincludedinthelatterareomittedfromtheformer.Theseinclude:87DNApercellvalues,35genussp.valueswhereothervaluesfor
namedspeciesinthesamegenusareincluded,and29valuesforsomevarietiesofspecieswherevaluesforothervarietiesofthesamespeciesareincluded.
†ThenumberofprimeestimatesforspeciesinthePlantDNAC-valuesDatabase(release5.0)(BennettandLeitch,2010)maydifferfromthenumberof
firstestimatesforspecieslistedintheabstractofpapercompilations(column2)owingtothereallocationofprimestatusfromthefirstestimatepublishedfor
ataxontoanestimatepublishedlater.
Bennett & Leitch – Nuclear DNA amounts in angiosperms 469
incorporates all the data presented in the Appendix here, and relationship between genome size and pollen size (Knight
brings the total number of angiosperms with C-value data et al., 2010).
available in the database to 6287 (http://data.kew.org/cvalues/ Given their ongoing uses as reference sources, the publi-
, Bennett and Leitch, 2010). The database is in a format cation of a further supplementary compilation of angiosperm
which can be queried through a variety of search options, C-values is needed, and indeed, it is overdue. Whereas the
thus enabling users to analyse, sort and filter the data, while first three compilations were published in journals of the
at the end of each query, the number of records returned Royal Society (London), the five later ones, from 1995
together with their summary statistics (i.e. minimum, onwards, have all been published in Annals of Botany, a
maximum,meanandstandarddeviation)aregiven.Inaddition decision based, in part, on its wider availability to intended
taxonomic(i.e.family,highergroup,voucherstatus),cytologi- users, especially those in institutions in the USA. Annals of
cal (i.e. ploidy level, chromosome number), technical (i.e. Botany (AoB) has played a major role in plant genome size
methodusedtoestimategenomesize)andbibliographicinfor- research through its sponsorship of discussion meetings and D
o
mation can be displayed where such data are available. workshops on plant genomes [at the Royal Botanic Gardens, w
n
Compiling the reference lists and, more recently, the elec- Kew (RBG, Kew) in 1997, 2003 and 2007, and at the XVII lo
a
tronic databases has clearly been of value, given the scope International Botanical Congress held in Vienna, Austria, in de
d
and range of uses made of them by the science community. 2005]. These led to the publication of three special issues fro
An indication of their value can be gleaned from noting that ‘Genome size in Plants’ (December 1998), ‘Plant Genome m
the published compendia listing angiosperm C-values have Size’ (January 2005) and ‘Plant Genome Horizons – Vistas h
been cited over 2100 times and over 85 citations for just the and Visions’ (April 2008). Partly as a consequence of these ttps
seventh compilation (Bennett and Leitch, 2005a). In addition, developments, AoB has played a growing role as a vehicle ://a
c
theelectronicdatabaseshavebeencitedover230timesandthe for research papers on plant genome size and has rapidly a
d
e
total number of hits on the Plant DNA C-values Database is becomethejournalofchoiceforfirstpublicationofsignificant m
now well over 250000 (most of which are enquiries for work. Indeed, Bennett and Leitch (2005a) noted that the ic.o
angiosperms). highest proportion of papers containing C-values were pub- up
Particular uses of DNA C-values for individual taxa have lished in AoB – a role maintained until now. Thus, 17% .co
m
been reviewed previously (Bennett et al., 2000; Bennett and (15 of the 88) of the original sources cited in Bennett and /a
Leitch, 2005a; Leitch and Bennett, 2007). Together, these Leitch (2005a) were originally published in AoB, which is ob
rdeastoauercsesesnhtiaavlefoclreamrlaynycodnitvineurseedctoompproavraitdievelasrtguediseasm. pRleecseonft sthimeiplarersteont1c6o%mp(e2n4dioufm1.5I1t)isofaltshoewoorirgthinnalotsionugrctheastciintebdotihn /article
examples of large surveys include studies of the relationship cases the proportion of first estimates for species (26.7 and -a
b
between genome size and B-chromosomes (Levin et al., 34.7%, respectively) published in AoB was much higher stra
2005), duration of cell cycle (Francis et al., 2008), seed size than the proportion of original sources. c
and mass (Beaulieu et al., 2007b), photosynthetic rate Over theyears, the citation impact factor for AoB has risen t/10
(Beaulieu et al., 2007a), plant growth form and distribution from 1.127 in 1995 to 3.501 in 2010 and plant genome size 7/3
(Ohri, 2005), leaf cell size and stomatal density (Beaulieu papers have contributed strongly to this increase. For /46
7
et al., 2008; Hodgson et al., 2010), and patterns of genome example, compilations published in 1995–2000 together /1
4
size evolution (Leitch et al., 2005, 2009, 2010; Beaulieu havereceivedover600citations,whiletwopapers,oneonter- 6
1
et al., 2010). minology (Greilhuber, 2005) and the other on technique 46
Variation in genome size has interesting relationships with (Dolezˇel and Bartos, 2005), each with over 100 citations, by
characters at many levels ranging from the nucleus and cell were bothamong the five most cited papers in AoB published gu
e
to tissues and whole organisms (Bennett, 1987; Bennett and in 2005. Clearly AoB is an important platform to showcase s
Leitch, 2005c), and recent work continues to confirm this. plant genome size research and reference data, and prac- t on
For example, relationships between genome size and a titioners would be wise to maintain a critical mass of such 28
range of nuclear characters have been detected in surveys work to benefit their field into the future. M
a
using the Plant DNA C-values Database since 2003. rc
h
Houben et al. (2003) showed that the extent of methylation 2
Extendingtherangeofgenomesizesencounteredinangiosperms 0
of histone H3 in euchromatin of plant chromosomes 19
depends on basic nuclear DNA content. Prokopowich et al. The minimum and maximum DNAC-values listed in 1972
(2003) found a positive correlation between rDNA copy forArabidopsisthalianaandFritillariaassyriaca,respectively
number and genome size in eukaryotes (Gregory, 2005), (Bennett, 1972), remained unchanged for over 30 years,
whilst Ross-Ibarra (2007) found that genome size is signifi- although the number of species listed increased more than
cantly correlated with recombination rate, with changes in 15-fold from 273 (Bennett, 1972) to more than 4100 (Plant
genome size explaining a meaningful proportion (approx. DNA C-values Database, release 4.0 October 2005). Lower
20%) of variation in this character. In contrast, Barow and values were claimed for several taxa (including A. thaliana),
Meister (2003) reported that genome size had only a but most of these were discounted or withdrawn later,
minor, albeit significant, effect on endopolyploidization in mainly on technical grounds (Bennett and Leitch, 2005a).
seed plants (Barow and Jovtchev, 2007), with other factors For example, the very low value for Cardamine amara of
such as phylogeny and life strategy having a stronger 1C¼0.051 pg reported by S. R. Band in 1984 as a personal
impact. Similarly, a large-scale analysis of a phylogenetically communication to the first author has subsequently been
diverse sample of species failed to find a strong significant shown to be an underestimate. Three independent
470 Bennett & Leitch – Nuclear DNA amounts in angiosperms
measurementsusingflowcytometryobtainedavalueof1C¼ 24.3%. [Actually, Dolezˇel et al. (1998) reported a mean
0.24 pg (Bennett and Leitch, 2005b). 2C-valueof1.22and1.26pgforRaphanussativusdepending
Nevertheless, data listed in the present work have recently on which species was used as the primary standard.] Had
extended the range of values for angiosperms at both ends of Schmuthsetal.(2004)usedthevalueof1.11pgtheirestimate
the scale. At the lower end, Greilhuber et al. (2006) reported for A. thaliana ‘Columbia’ would have been 0.166 pg, and
convincing C-values much lower than for A. thaliana for within 2% of the prime C-value estimate for A. thaliana
several Lentibulariaceae species, including 1C¼0.09 pg for ‘Columbia’ of 0.163 pg (Bennett et al., 2003). The electronic
Utricularia gibba, 0.065 pg for Genlisea aurea and a new databaselackssuchdetailedcommentsbutitcitesthecompen-
record minimum angiosperm C-value estimate of 0.0648 pg diumreviewtowhichreferencecanbemadetocheckifuseful
for Genlisea margaretae. This drop in the minimum known extra information is available and analysis provided.
C-value was not small, but constituted a remarkable 60% Theexampleaboveconcernsonetaxon.However,compen-
reduction to well under half the value for A. thaliana (0.16 dia reviews also provide opportunities to publish results of D
o
pg). Indeed, Bennett and Leitch (2005a) previously discussed usefulwidercomparisonstoidentifytrends,ortestperceptions w
n
what was the lowest C-value fora free-living angiosperm and (and correct misconceptions). For example, the compendium lo
a
suggested that the theoretical minimum monoploid genome papers give overviews of trends in the latest sample of esti- de
size (1Cx value) may approach 0.05 pg for a true diploid. mates and papers, and report on progress towards reaching d fro
The results for Genlisea (Greilhuber et al., 2006) confirm milestones andtargets in gapfilling and inthe take upof gui- m
this prediction, and should provide a practical rationale and dance regarding best practice issued by the GEnome Size h
spur for new surveys to see if other taxa have yet lower Initiative (GESI – Bennett and Leitch, 2005b) by the plant ttps
C-values and approach the suggested theoretical minimum genome size community (http://data.kew.org/cvalues/pgsm/ ://a
c
even more closely. #9%20Key%20recommendations). In the past, none of these a
d
e
At the other end of the scale, the maximum angiosperm was routinely included with updates or new releases of the m
C-value has also increased recently. Zonneveld (2010) electronic database, but instead may be mentioned there, ic.o
reported a 1C-value of 132.5 pg for a hexaploid hybrid citing a compendium as the reference for full details. Unlike up
Trillium×hagae (2n¼6x¼30; Melanthiaceae), just less the Plant DNA C-values Database, compendia also often .co
m
than the reported maximum genome size for an animal (133 draw attention to some of the novel and key uses to which /a
pg in the marbled lungfish Protopterus aethiopicus, DNAC-value metadata have been put, and review the results ob
PgeenduerssePna,ri1s97(1al)s.oYMeteleavnetnhiatcheiase)haisnbweehnichexace1edCe-dvaliunethoef adnatdactoongceltuhseironwsithofssuucchhasntauldyiseess. aHnadvirnegvitehwesciosllaecvteadluanbelwe /article
152.23 pg for the octoploid Paris japonica (2n¼8x¼40) resource providing overviews inthe broadestpossiblecontext. -a
b
was recently estimated by Pellicer et al. (2010). This value However, given the retirement of the first author and the stra
is 15% larger than all previous plant genome size estimates need to update the Plant DNA C-values Database on a more c
and extends the range encountered in angiosperms to nearly regular basis than in the past, future releases of new data t/10
7
2400-fold. will be uploaded with or without publication of any compen- /3
Suchstudiesillustrateagainthatwedonotyetknowthefull dium paper such as this. Thus, the database will continue to /46
7
ranges of basic genome parameters (cf. Bennett, 1998) and be updated and made available on the internet through the /1
4
confirm the continued need for new surveys of plant DNA website of the RBG, Kew, who are committed to maintaining 6
1
C-values, chromosome numbers and ploidy levelsto establish and managing this important biodiversity resource for the 46
their full ranges experimentally. genome size community. by
g
u
e
s
The need for reference lists TARGETS IN GENOME SIZE RESEARCH t o
n
Asthedatagatheredinthiscompendiumhavebeen entered The present work marks several major milestones in plant 28
into the Plant DNA C-values Database, it might be asked if genome size research which concern both quantitative and M
a
publishing them in this form has added value to their elec- qualitativeaspectsoftheDNAC-valuedata.Thecompendium rc
h
tronic listing. Indeed it does, and at various levels ranging lists DNA C-value estimates for 2221 angiosperm species 2
0
from individual values to overviews and reviews of the meta- taken from 151 original references (including first values for 19
data.Forexample,givenonlythenumberspublisheditispuz- 1860 species from 132 sources). Thus, it is by far the largest
zling why Schmuths et al. (2004) obtained a 1Cx value of number of new and novel species’ values, and the largest
0.206 pg for A. thaliana ‘Columbia’, which was 26% higher number of new sources, published together, among the nine
than the value of 0.163 pg for the same species measured compendia published since 1976 (see Table 1). So how do
using the completely sequenced genome of Caenorhabditis the new data contribute to the targets set for plant genome
elegans as the calibration standard (Bennett et al., 2003). size research?
However,thisseemscleargiventheextrainformationinfoot-
note‘ac’(fororiginal reference number 494).This showsthat
Meeting targets for species representation
Schmuths etal.(2004)choseRaphanussativusastheinternal
calibration standard using a 2C-value of 1.38 pg, which they Previous compilations have tracked the annual output of
reported had been taken from Dolezˇel et al. (1998), rather DNA C-value estimates and shown a strong rising trend in
than the 2C-value estimate for Raphanus sativus ‘Saxa’ of the number of total and first estimates since 1960 (Bennett
1.11 pg given by Dolezˇel et al. (1992) – a difference of+ and Leitch, 2005a). Updating this analysis to include values
Bennett & Leitch – Nuclear DNA amounts in angiosperms 471
publishedhere(comprisingdataincludedincompilations1–8 analysis shows better progress as the addition of C-values
and the present Appendix – see Table 1) shows that these for a further 2155 species compiled over this time frame
trends were strongly maintained. Thus, the mean total shows that 86% of the target was met.
number of C-values for angiosperm species per year rose 69 However, recently the total number of angiosperm species
% from 311 in 2000–2004 to 527 in 2005–2008, whilst the has been revised upward to a widely accepted 352000
mean total number of first values for angiosperm species (Paton et al., 2008). Using this higher value, the percentage
rose 72% from 219 in 2000–2004 to 378 in 2005–2008 of species with C-values still stands at only 1.8%.
(Fig. 1). Clearly, encouraging progress towards increasing Nevertheless, if present rates of data acquisition are main-
the representation of angiosperm species with genome size tained, the sample of species for which C-values are listed
data continues to be made. Indeed the slope of the graph in should reach 2% (i.e. 7040) and 3% (i.e. 10560) of the
Fig. 1 shows that the last 3-year period (2005–2008) had the higher number within about 3 and 12 years, respectively.
highestrateofgenomesizedatagenerationforanyperiodana- Clearly the setting of targets has been invaluable for focus- D
o
lysed since the first plant genome sizewas estimated in 1950. ingandimprovingtherepresentationofspecieswithC-values. w
n
Itisthereforetimelytoreviewandassesshowfarprogresshas It therefore seems reasonable to maintain a target of lo
a
been made towards meeting targets set for increasing species first C-values for angiosperm species at 2500 for the next de
d
representation for angiosperms. 5 years. If rates of data acquisition continue at the same rate fro
Bennett and Smith (1976) listed C-values for 753 angios- then such a goal seems reasonable, but attaining 10% m
perm species, and it was not until 1995 that data for 2500 species representation (i.e. approx. 35200, the ultimate goal h
species were available. It was then conservatively estimated which seemed sensible to Bennett and Leitch, 2005a) would ttps
that the total number of angiosperm species was still be estimated to take 60–80 years. ://a
c
about 250000, thus C-value data were already available for a
d
e
1% of species. At the 1997 Angiosperm Genome Size m
Workshop, with C-values for 2802 species compiled into the Progress towardstargets for familial representation ic.o
Angiosperm DNA C-values Database, a target was set to In1997,C-valueswerestillunavailablefor68%ofangios- up
measure a further 1% of species (i.e. approx. 2500 species) perm families recognized by the first Angiosperm Phylogeny .co
m
by 2003. Analysis shows that this target was aspirational, as Group (APG) classification published in 1998 (APG, 1998). /a
a review at the Plant Genome Size meeting held in 2003 Thus, at the first Angiosperm Genome Size workshop (1997) ob
sbheoewnedcoltlhaattedC,-vcaolrureesspofonrdionnglytoa jfuusrtthe4r1%103o2fsptheecietsargheatd, aThgiosaplrowvaesdsoevtetro-ocpotimmpisletitce. Pfarmogilrieaslsrweparsesineinttiaatliloynvebryy2s0lo0w2., /article
although knowledge of further data as yet unpublished and/ as only 12 (1.7%) of the 691 species’ values in the fifth -a
b
or collated suggested that approx. 66% of the target may DNA compilation (Bennett et al., 2000) were also first stra
have been met (Bennett and Leitch, 2005a). Nevertheless, values for families. Nevertheless, new work focused to c
the 2003 meeting reset the target to measure a further 2500 addressthe shortfall produced first C-values forover 80 unre- t/10
7
species in the next quinquennium (i.e. 2003–2008) and presentedfamiliesby2003(e.g.Hansonetal.,2001a,b,2003; /3
Leitch and Hanson, 2002). This increased familial represen- /46
tation to 47.5% (Bennett and Leitch, 2005a), based on 7/1
4
C-value data for 217 families out of the 457 families recog- 6
500 Total 1
First nized in the revised angiosperm phylogeny (APG II, 2003) 46
(Fig. 2). As this still fell well short of the original target, it by
ates 400 was revised down at the Plant Genome Size Meeting held in gue
m 2003 to a new goal of 75% familial representation. Based s
of esti 300 ownouthlderneuqmuibreerthoef fmameaisluiersemreecnotgonfizaendabdydiAtioPnGalII11(240f0a3m),iltiheiss. t on 28
er Analysis of the 1860 first C-values for species listed in the M
b a
m current Appendix shows that only 24 (0.13%) are also first rc
n nu 200 estimates for families, representing just 21% of the target of h 20
ea 114 families. Combining these with data in the Plant DNA 19
M
C-values Database shows that in total there are currently 240
100
angiosperm families with genome size data. Given that a
further revision by the APG (APG III, 2009) now recognizes
0 only 415 families (10% down on the number recognized
54 59 64 69 74 79 84 89 94 99 04 08 in APG II), the current familial representation now stands at
9 9 9 9 9 9 9 9 9 9 0 0
1 1 1 1 1 1 1 1 1 1 2 2 58%, so progress towards achieving even the reduced target
– – – – – – – – – – – –
50 55 60 65 70 75 80 85 90 95 00 05 set of 75% familial representation in the last quinquennium
9 9 9 9 9 9 9 9 9 9 0 0
1 1 1 1 1 1 1 1 1 1 2 2 (i.e. 2003–2008) has remained poor. Major factors limiting
Period progress have been discussed previously (Hanson et al.,
2003)ashastheexpectationthatsuchdifficultieswillintensify
FIG. 1. Mean number per year of total and ‘first’ DNAC-value estimates as familial representation approaches 100%. Nevertheless,
communicatedin11successive 5-yearperiodsandthe4-yearperiod2005–
2008, between 1950 and 2008. Data taken from the Plant DNA C-values progress since 2003 has been disappointing in relation to the
Database(release5.0,December2010). size and distribution of the shortfall and has again depended
472 Bennett & Leitch – Nuclear DNA amounts in angiosperms
60 representation would require a first C-value for an additional
approx. 400 genera and so this target was set. Subsequently,
the total number of genera was revised down to 12962
50
s (Mabberley, 2008), which would make achieving 10% rep-
e
mili resentation easier, requiring a first C-value for only approx.
G fa 40 2ta5i0nsgCen-evraal.uAesnafolyrs5is5o4fgtehneedraataofinwthhiechAp3p2e9n(d5ix9.s4h%ow)sarietcfiorns-t
P
A C-values for a genus. Overall the 6287 species with genome
% of 30 size data include 1444 genera, an increase of 402 genera
e since 2003. Thus, work in the last 5 years has made consider-
v
ati 20 able progress towards meeting the generic target, increasing
ul representation to 11.2%. Indeed, progress towards this target D
m o
u was outstanding, greatly exceeding that at both specific and w
C 10 familial levels noted above. Given the current focus on nlo
a
genera it seems reasonable to keep a similar target of first de
d
0 C-values for a further 450–500 genera over the next 5 years, fro
1950 1960 1970 1980 1990 2000 to enable representation to approach approx. 15%. m
Year h
ttp
s
AFInGg.io2sp.erCmumPhuylaltoigveenpyeGrcreonutpag(AePoGf)a(nAgPioGspIeIIr,m20f0a9m)iwlieitshraecfiorsgtnCiz-evdalubeyrethpe- Improved representation of other groups ://ac
resentedinthepresentPlantDNAC-valuesDatabase(release5.0,December Although the Plant Genome Size Workshops have noted ad
2010). other gaps in general discussions, they have not set any em
targets to fill them, choosing to focus first on improved sys- ic.o
mainlyonworkbyjustoneinstitution – RBG,Kew[i.e.RBG, tematic representation in angiosperms, and on widening rep- up
Kew, estimated 65 of the 74 (87%) first values for families resentation of other plant groups, such as gymnosperms, .co
m
added in 1997–2002, and 18 of the 25 (75%) first values pteridophytes (i.e. monilophytes and lycophytes), bryophytes /a
for families added in the present Appendix]. Bennett and and algae. Nevertheless, previous papers in this series have ob
oLreaitticohn(t2o00lo5caa)teprmevatieoruisallys nanodtedestthimatat‘e..g.einntoemrneatsiiozneaslfcoorllsatibl-l ipdeernmtifiCed-vpaalurteiscul(aer.gm. aBjoerngnaeptts ianndourLekintcohw,le1d9g9e5o,f2a0n0g5ioas)-, /article
unsampled families will be essential if the long term goals which merit research (but without recommending targets and -ab
of 75% and then complete familial representation are to be time frames). Clearly this was influential as many of these stra
athcihsietvaregde’t.,Scuocllhabpororagtrieosnstwoisllharerequtihree lcohaadmmpioornesbtoroafodclyusanodn gexaapms phlaev,eBesunbnseettquaenndtlyLerietccehiv(e1d99s5ig)nnifiocteadntthaattten‘teisotnim. aFteosr ct/10
7
funds for collecting exceptional and esoteric material of the wererareformanytypesofangiospermssuchas:palms,para- /3
approx.70or175familiesstillneededfor75or100%familial sitic plants, noxious weeds and succulents’. Studies soon /46
7
representation, respectively. addressed some, but not all, of these groups listing first /1
4
C-values for 74 palms (Ro¨ser et al., 1997) and 39 weeds 6
1
(Bennett et al., 1998). 46
Althoughsomeprogresshadbeenmade,BennettandLeitch by
Improved systematic representation for genera (2005a) noted several ongoing gaps in geographical and plant gu
e
Within the keyrecommendations forangiosperms set at the life form, including ‘island floras rich in endemics’ and ‘taxa s
Plant Genome Size meeting in 2003 (http://data.kew.org/ from bog, fen, tundra, alpine and desert environments, and t on
cvalues/pgsm/#9%20Key%20recommendations) was a target halophytic, insectivorous, parasitic, saprophytic and epiphytic 28
of achieving 10% generic representation. Although targets species andtheirassociated taxa’. Examining the 151original M
a
for species and familial representation have been set and references contributing to the Appendix shows they include rc
h
tracked since 1997, none was set for genera prior to 2003, so significant contributions to filling some of the above gaps. 2
0
2003–2008 is the first quinquennium for which this target Several provide data for island floras. Murray et al. (2003, 19
has been monitored. The reason for adding this new level 2005) give genome sizes for 158 endemic and indigenous
was to reflect developments in the field of angiosperm phylo- grasses of New Zealand, whilst Suda et al. (2003, 2005) list
genetics based on molecular studies. By 2003 the APG had values for species representing 40% of the Macaronesian
transformed the view of higher order angiosperm phylogeny, angiosperms. Data for 13 Oncidiinae orchids (Chase et al.,
bringing new certainty and stability at the familial level and 2005) and 18 Cuscuta vines (McNeal et al., 2007) have use-
replacing several different conflicting non-molecular systems. fully increased representation for epiphytes. The latter,
Consequently, the focus of new molecular-based work was together with estimates for approx. 40 taxa of holoparasitic
shifted to the generic level. Orobanche (Weiss-Schneeweiss et al., 2005) and for several
Analysis of the 4119 species listed by 2003 showed DNA taxa in two genera of Cistaceae (Halimium and Xolantha;
C-values for 1042 genera, out of the then approx. 14000 Boscaiu et al., 2008) have also significantly improved rep-
genera recognized (M. Chase, Rotal Botanic Garden, Kew, resentation for parasitic plants with first values for approx.
London, pers. comm.), equivalent to approx. 7.4% represen- 70 species. Finally, estimates for four Adriatic seagrasses
tation overall. Thus, it was calculated that achieving 10% (Dolenc Koce et al., 2003) and 13 marine halophytes
Bennett & Leitch – Nuclear DNA amounts in angiosperms 473
(DolencKoceetal.,2008)havemadeapositivestartindirect 300
A
response to address a gap identified in Bennett and Leitch Fe (Fe and CIA)
(2005a).
ar 250 FC (all fluorochromes)
e
y
s/
e
TRENDS IN TECHNIQUES USED TO ESTIMATE mat 200
GENOME SIZE sti
e
Therise in flowcytometryasthe technique ofchoiceforgenome st
size estimations of fir 150
A continued interest in genome size research has focused on ber
m 100
the techniques used to obtain such data. Previous analyses u D
n o
haveshownatrendtowardstheincreasinguseofflowcytome- n w
trysince 1983 (Galbraith et al., 1983) to reach 58.4% of first ea 50 nlo
M a
estimates listed in the seventh compilation (i.e. Bennett and de
d
Leitch, 2005a). Updating this analysis shows a further strong 0 fro
rise in the use of flow cytometry in recent years, with m
4 4 4 4 4 4
Feulgen microscopy methods continuing to fall (Fig. 3A). 195 196 197 198 199 200 http
Indeed, of the 1860 first estimates listed in the present – – – – – – s
1A5p.p4e%ndixu,se8d4.5F%eulwgeenre mmiacdreosucsoipnyg flmoewthcoydtso.mDetirvyidbiuntgonthlye 1950 1960 1970 Yea1980r 1990 2000 ://acad
e
latter into traditional Feulgen microdensitometry and m
computer-basedimageanalysis(CIA)ofFeulgen-stainedprep- 100 ic.o
arorasteionbset(wFeige.n3B2)00sh0owansdtha2t0a0lt6h,oumgohrethereucseenotlfyCIiAtsguesneerahlalys B Traditional Fe up.co
CIA m
declined (contributing just 31 C-value estimates in 2007 and 80 /a
2008, Fig. 3B) along with that of the more traditional s ob
FpsCaiceetIccouAetmlesgdseektntibeot,rumslteaiaarncednrfloidanedggcterhtnelseaesbtidfhtoaoerimbalfiueteornstertariyletpsolrmoafdsoceserttivhocteroefaldoitnsrCpa.inIdTaAgith‘iaiosstntneadcatnrhltedennpacirdhqhdouniteosionlmoneaxgoincyptdreoutnrdneasaeenivsnxsye--- of first estimate 60 /article-abstrac
wtfoeor’ufl,lodcwobnecdyiettisoosmnensettiwraylhiffcoohrr CBesIetAnimnteaotttibnaegncdoCm-Lveeailttuhceehs.r(e2Ia0nli0ds5etieacd),aslCutegIrAgneasthtievades Number 40 t/107/3/46
20 7
advantages for some materials in providing cytological sights /1
4
and insights into the material being studied (such as chromo- 6
1
some number, aneuploidy, B-chromosomes and nuclear stab- 46
ility) unavailable to users of flow cytometry without also 0 by
making separate cytological preparations. Clearly, the use of 2000 2001 2002 2003 2Y0e0a4r 2005 2006 2007 2008 gue
Feulgen microscopy with CIA has not been favoured by most s
practitioners in the plant genome size community. FIG. 3. (A) Mean number per yearof ‘first’ DNAC-value estimates made t on
Analysis of data in the Animal Genome Size Database usingFeulgenmicrodensitometry(Fe)andflowcytometry(FC)communicated 28
(Gregory, 2006) shows a different picture. Here, 1151 esti- in 11 successive 5-year periods and the 4-year period 2005–2008, between M
1950 and 2008. (B) Number of first estimates made using traditional a
mates were made using CIA for the period 1991–2009 (com- Feulgen methods (Traditional Fe) and Feulgen staining measured using rch
pared with 190 in plants). Nevertheless, of the 29 original computer-aidedimageanalysis(CIA)since2000.DatatakenfromthePlant 2
0
sources giving such data, 13 (which list data for 549 taxa) DNAC-valuesDatabase(release5.0,December2010). 19
were published by T. R. Gregory, so (as with many other
aspects of genome size research) this trend has been strongly
of some equipment made for a different prime use in a
influenced by the output of just one productive practitioner
larger (e.g. medical) market.
and his colleagues. In plants, all recent estimates made using
CIA were co-authored by Vilhar in Slovenia or come from
the University of Vienna where Greilhuber and colleagues
Development of different isolation buffers for flow cytometry
use the CIRES system, which is now unavailable to purchase.
Inviewoftheabove,theuseofCIAtoestimateplantgenome Since 1983,flowcytometry hasbeen increasinglyusedasa
sizesisunlikelytoincreaseagainunlessmanufacturerssupply method to estimate genome size (Galbraith et al., 1983), and
and support complete and inexpensive CIA packages which the composition and number of different types of isolation
compete with the options provided by flow cytometry buffers has grown considerably, reflecting the complex diver-
makers. This now seems unlikely given the small size of the sity of biochemistry in plant cells. Indeed, in the FLOWer
genome size market, but could still occur as a marginal use database for plant flow cytometry (Loureiro et al., 2007b,
474 Bennett & Leitch – Nuclear DNA amounts in angiosperms
2008) there are 28 different buffers which have been used Recent developments in the application of flow cytometry to
based on 826 research articles included in the database. genome size studies
Recently, the performances of four of the most commonly
(i)Theuseofseeds. Recent reports suggest that dryseeds may
used flow cytometry buffers (Galbraith’s, LB01, Otto’s and
be suitable material for genome size estimations made using
Tris.MgCl ) on seven plant species were compared by
2 flow cytometry (Sliwinska et al., 2005, 2009; Jedrzejczyk
Loureiro et al. (2006a). By analysing various parameters of
and Sliwinska, 2010). This approach, if shown to be widely
the flow histogram including forward scatter, side scatter and
applicable, has several advantages: (1) it enables the direct
relative fluorescence intensity of propidium iodide-stained
analysis of seed stored in seed banks (provided the owners
nuclei, the performance of each buffer was assessed. They
of the genetic resources approve), (2) it overcomes the pro-
concluded that no single buffer worked well for all species.
blems associated with analysing fresh material collected in
Instead, the optimal buffer for a particular species depended
the field within a reasonable time frame before material has
on a number of factors, although Otto’s and LB01 buffers D
degraded, and (3) it provides an alternative source of tissue o
wereconsideredtobethebestforthemajorityofspeciesana- w
for analysis if the accumulation of staining inhibitors in n
lysed. Subsequently, Loureiro and colleagues have developed lo
other tissues has been shown to be problematic. Indeed, dry a
twonewbuffers,ageneral-purposebufferandonespecifically seed may contain a lower level of cytosolic inhibitors than ded
developed for use with woody material (Loureiro et al., other parts of the plant and hence may be easier to analyse fro
2007a). m
Tworecentstudieshaveextendedourunderstandingonhow (Greilhuber et al., 2007). However, there is the potential that h
cytosolic compounds, which are released into the isolation the seeds are intra- or inter-specific hybrids rather than ttps
bufferduring nucleiisolation,can interferewith fluorochrome representing the genomic make-up of the parent. In addition, ://a
using whole seeds for genome size studies means that all the c
stainingandleadtoerroneousresultsandpseudo-genomesize a
material is destroyed, thereby preventing further genomic de
plasticity (Loureiro et al., 2006b; Bennett et al., 2008). m
Bennett et al. investigated the effects of anthocyanin, while aisnatolysgiserm(cihnraotmeotshoemceolcleocutnetds seetecd.).aAndn aanltaelrynsaetivtheeagprporwoaincgh ic.o
pLoouunredisr,o wethiaclh. eaxraemiwneiddestpanrenaidc aaccirdo.ssBoathnggiorospueprsmosf, cwoemre- pSluadnat metataelr.ia(l2.0I0n5a) lsaurcghe saunrvaepyproofacthhewMaascrtoakneensi.anSefleodrsafboyr up.com
shown tohavepotenteffects onDNA stainingleadingto stoi- over 100 taxa were collected from the Canary Islands during /a
ciinnhthieoirbmniateoltrryisctacneodrmraorprdsoi.uznaStduisocnhasastnurdedcioethsmememiemnpdpheoadrstiazbneycethDeooflneetzˇeeesdltin(to1g9u9fos1er) figaneeralmdlywisnoisar.tkioanndantdhesnubbsreoquugehnttbflaocwk ctoytothmeeCtrzicecahndRecpyutoblloicgicfoarl ob/article
and as more recently discussed in detail by Greilhuber et al. -a
b
(2007). (ii) Ease of access to methodological data. The launch of the stra
FLOWer database (Loureiro et al., 2007b, 2008) in 2007 has ct/1
providedavaluableresourceenablingusersofflowcytometry 0
The application of flow cytometry to plant systematics 7
toaccessandassessdetailsoftheprotocolsusedbyotherprac- /3
Withtheabilityofflowcytometrytoanalyselargenumbers titionerstoassistthem inchoosing themostlikelymethod for /46
7
of individuals within and between populations, genome size analysing the plant species of interest. /1
4
data are now increasingly being used to help resolve taxo- 6
1
nomic issues. These have included examples where genome (iii) New equipment. Another development which may assist 46
size has been used to provide new insights into species in extending the ease of genome size analysis includes by
relationships (e.g. Agapanthus, Zonneveld and Duncan, the launch of a new type of flow cytometer, the Accuri C6, gu
e
2003; Galanthus, Zonneveld et al., 2003; Nerine, Zonneveld by Accuri Cytometers in December 2006 (www. s
and Duncan, 2006; Curcuma, Leong-Sˇkornickova` et al., AccuriCytometers.com). The company promised a revolution t on
2007; Hieracium, Suda et al., 2007; Narcissus, Zonneveld, in flow cytometry with ‘reliable, high performance fluidics, 28
2008) as well as for recognizing new taxonomic entities (e.g. inexpensive, robust optics, and sophisticated and simplified M
a
Cochlearia borzaeana, Kochjarova´ et al., 2006; Pandanus electronics’ – allforapprox.£30000.Oneofthemaindiffer- rc
h
fascicularis, Panda et al., 2009), supporting existing ones ences between such a machine and conventional flow cyt- 2
0
(e.g. Lilium bosniacum, Muratovic et al., 2005) or rejecting ometers such as those of Partec, Beckman Coulter or 19
them (e.g. Lamium endtmannii, Rosenbaumova´ et al., 2004). Beckton Dickinson is the greater dynamic range of operation
Genome size has also been used as a reliable guide in the (as it uses a 24-bit analog-to-digital converter for signal pro-
separation of closely related species and their hybrid such as cessing), enabling species with widely different genome
shown in studies of Elytrigia repens, E. intermedia and their sizes to be analysed together in a single sample. Indeed,
hybrid (Mahelka et al., 2005) (original reference 593 in the tests on the machine carried out by Galbraith (2009) have
Appendix). Mahelka et al. (2005) analysed 238 individuals demonstrated that species with 1C-values ranging from 0.16
from 55 different populations and found that there was so to40pgcanbevisualizedandanalysedonthesameflowhis-
little intraspecific variation in DNA amount for each of the togram. Galbraith demonstrated that the machine had the
two species and the hybrid that genome size could be used potential to measure genome sizes in species with C-values
as a reliable taxonomic indicator. Indeed, genome size was ranging from 0.032 to 185 pg and hence was unlikely to be
showntobemorerobustandlesssubjectivethanmorphologi- limited by this character. Galbraith concluded that ‘for
cal characters that varied more or less continuously between routine analysis of plant nuclear DNA contents, ploidy or
the species and hybrid. investigations of other issues requiring C-value
Bennett & Leitch – Nuclear DNA amounts in angiosperms 475
determinations, the Accuri C6 flow cytometer provides an interest and (2) an accurate method to estimate concentration
excellent platform’. He also suggested that given the reprodu- of genomic DNA. qPCR was then performed and genome
cibility and simplicity of use it might now be feasible to rea- size calculated by dividing the amount of DNA present in
listically consider estimating genome sizes in the remaining the sample by the number of copies of the single copy gene
98% of angiosperm species (Galbraith, 2009). Nevertheless, detected. The method has since been used to estimate
althoughGalbraithconsideredthatitmightbepossibletoesti- genome sizes in several eukaryotic species with genomes up
mate genome sizes in 12 species per hour, in reality he also to 2900 Mb. However, comparisons with estimates made
recognized that potential problems arising from the need to using other methods have not always produced similar
optimize buffers and test for secondary products would inevi- results (Table 2) and reasons for the discrepancies remain
tablyslowthingsdown.Inaddition,theneedtomakechromo- unclear. Recent modifications to the technique may overcome
some counts will further impede output – a factor often some of the initial problems identified (e.g. Jeyaprakash and
omitted by flow cytometer practitioners when extolling the Hoy, 2009), but given that (1) considerable time is required D
o
greater speed of flow cytometry compared with Feulgen to optimize conditions for qPCR and (2) prior molecular w
n
microscopy. Nevertheless, the availability of such a compact, knowledge is needed to identify suitable, specific primers for lo
a
inexpensive, yet easy to use machine is likely to make an amplifying a unique sequence, whether the method is widely de
d
impactinthefieldofgenomesizeresearchasolder,moretra- applicable remains unknown even for species with small fro
ditional flow cytometers are replaced and updated. genomes. Moreover, there are as yet no data to demonstrate m
howaccuratethemethodisforgenomeslargerthan3000Mb. h
Indeed, the general suitability of the method for angios- ttps
Are there any new techniques forestimating genome size on the perms remains unclear as there are no reports of plant ://a
c
horizon? genome size estimations using qPCR. Nevertheless, given ad
e
(1) the propensityof plant genomesto undergo both localized m
bySaintceletahset 1e9ig5h0ts,dniufcfelereanrtDNteAchanmiqouuens,tsihnacvluedbineegn: ecshtiemmaitceadl andwholegenomeduplicationsand(2)theneedtoempirically ic.o
extraction, Feulgen microdensitometry, flow cytometry, CIA, sperliemcetrsafsouriteaabcleh ssipnegclieescoinpvyesgtiegnaeteadn,ditdseeveemlospuanplipkreolpyritahtee up.co
reassociation kinetics, pulse field gel electrophoresis and m
‘complete’ genome sequencing. From an analysis of all the method will be widely used. It is too time consuming and /a
gasennoomteedsiazbeodvaet,ajuinstthtweoPla(FnetuDlgNenA mC-ivcraoludeesnsDitoatmabeatrsye aanndd eoxfpgeennsoivmeeesvieznesifeintccoaunnbteeresdhoiwnnpltaonwtso.rkacrossthespectrum ob/artic
flow cytometry) have contributed over 96% of first estimates le
-a
forangiosperm species and it seems clear that flow cytometry (ii)Will‘complete’genomesequencinggiveuseablegenomesize b
s
will remain the dominant method of choice for estimating estimates? It is now 10 years since the first complete genome tra
c
DNA C-values in plants in the near future. Indeed, it may sequence for an angiosperm was published (Arabidopsis t/1
become almost the sole method unless CIA or some new Genome Initiative, 2000), and to date similar data for a 07
method takes off. So are there any realistic alternatives on further eight angiosperm species has followed (Table 3). It /3/4
the horizon? istherefore timely to assessthe actual contribution that com- 67
pletegenomesequencinghasmadetoourknowledgeofplant /14
6
(i)Canreal-timePCRbeusedforestimatingplantgenomesizes? DNA C-values, and to ask if this molecular approach has 1
4
In 2003, a molecular-based method to estimate genome size yielded more accurate genome size measurements than were 6 b
was published which used the quantitative real-time polymer- previously available, and thereby provided the new ‘gold y g
asechainreaction(qPCR)(Wilhelmetal.,2003).Themethod standard’ calibration standards against which other species ue
s
required (1) a cloned single copy gene from the species of can be compared. Bennett and Leitch (2005a) discussed t o
n
2
8
M
a
TABLE 2. Comparison of genome sizes estimated by quantitative real-time PCR (qRT-PCR) with other methods for various rc
h
eukaryotic species (PFGE, pulse field gel electrophoresis; FC, flow cytometry; Fe, Feulgen microdensitometry; RK, reassociation 2
0
kinetics;Ch,chemicaldetermination). 1
9
Species Group 1CvalueestimatedusingqRTPCR Reference Rangeof1Cvaluesestimatedbyothermethods*
Saccharomycescerevisiae(yeast) Fungus 12.1Mb Wilhelmetal.(2003) 12.06–12.2Mb(completegenomesequencing)1
Piriformosporaindica Fungus 15.6–24Mb Zuccaroetal.(2009) 15.7Mb(PFGE)2
(dependingongeneused)
Thalassiosirapseudonana Diatom 35.9Mb vonDassowetal. 31.3Mb(completegenomesequencing)3
(2008)
Muscadomestica(housefly) Insect 295Mb GaoandScott(2006) 950Mb(spectrophotometry)4,900Mb(FC)5
Drosophilamelanogaster(fruitfly) Insect 184Mb GaoandScott(2006) 157–206Mb(FC)4
Xiphophorusmaculatus(platyfish) Fish 550Mb Wilhelmetal.(2003) 410–550Mb(Fe,RK)6,760–990Mb(FC)4
Homosapiens(human) Mammal 2900Mb Wilhelmetal.(2003) 2940Mb(Ch)7to3355Mb(Ch)8
*1FungalGenomeSizeDatabase(Kullmanetal.,2005);2Zuccaroetal.(2009);3Armbrustetal(2004);4AnimalGenomeSizeDatabase(Gregory,2006);
5J.S.Johnston(TexasA&MUniversity,pers.comm.,2008);6Wilhelmetal.(2003);7VendrelyandVendrely(1948);8Mandeletal.(1950).
TABLE 3. ComparisonbetweentheamountofDNAsequencedandtotalgenomesizein‘complete’plantgenomesequencingprojects 47
6
Actualamountof
Totalgenomesize assembledsequence
reportedbythe generatedbythe Percentageofgenome
‘complete’genome ‘complete’genome estimatedtohavebeen
sequencingproject sequencingproject sequenced(column3/ 1C-valueestimatedbyother
Species‡ (1C-valueinMbp) (Mbp) column2) methods Comments D
o
w
Arabidopsisthaliana 125† 115.4 92%assuminga 157MbusingFC:PIand A1C-valueof125MbgivenbytheArabidopsisGenome nlo
‘Columbia’1 1C-valueof125Mbbut Caenorhabditiselegansas Initiativewasbasedonadding115.4Mbofassembled ad
(Arabidopsis) actuallyonly73.5%if standard(1C¼100Mb) sequencetoanestimateof10Mbfortheunsequenced ed
FamilyBrassicaceae thehigher1C-valueof (Bennettetal.,2003) centromeresandribosomalDNAregions(Arabidopsis fro
157Mbisused GenomeInitiative,2000). m
Oryzasativasubsp. 466† 362 77.6% 510MbusingFeandVigna Yuetal.(2002)estimatedthattherepetitivesequenceswhich Be http
i(nRdicicea)‘93-11’2 r(Badeinantaett(1aCnd¼S0m.5it3h,p1g9)91) wadedreeddtioffitchuelttototaallaigmnoaucnctooufnatelidgnfoerd1s0e4quMenbceofdDatNaAth.isWghaevne nnett s://ac
FamilyPoaceae anestimatedgenomesizeof466Mbasstatedincolumn2. & ad
e
Oryzasativasubsp. 420* 389.8 n/a 510MbusingFeandVigna Noattemptwasmadetoestimateatotalgenomesize.Instead, L m
j‘aNpiopnpiocnabare’3 r(Badeinantaett(1aCnd¼S0m.5it3h,p1g9)91) aSy1nCg-evnatlauegroofup4)20alMthobupgwhatshelisstoeudrcbeyoGfothffisetvaallu.e(,2a0n0d2h–ence eitch ic.oup
(Rice) howitwasdetermined,wasnotstated. – .c
o
Oryzasativasubsp. 389† 370 95% 510MbusingFeandVigna ThisworkbuiltonthatofSyngenta(seeabove,Goffetal., Nu m/a
japonica radiata(1C¼0.53pg) 2002)toproduceahigherqualitysequenceestimatedto c o
‘Nipponbare’4 (BennettandSmith,1991) comprise95%ofthewholegenome,includingallthe lea b/a
(Rice) eainudccdlhiunrdogemdthanetiicneserteicmgeinaottnersod.mlTeehnreegst1haCno-dfva61l27uegteaolpofsm3(8e¼9reMs18tbo.1pthMweabts)o,btawalsheliedcnhognth rDNA rticle-ab
ofalignedsequence(¼370Mb). am stra
Populustrichocarpa 485†+10 410 85%oftotalgenomebut 686MbusingFC:DAPIand Theauthorsconsideredthatthe410Mbofassembled ou ct/1
genotype‘Nisqually approx.90%of chickenredbloodcellsas sequencedatarepresentedtheeuchromaticportionofthe n 0
(1b’5lackcottonwood) euchromaticregion s(Btarnaddasrhdaw(1Can¼dS1t.e2t1tleprg,) g7e5nMombeo.fTgheenotomtailcgseenqoumenecesiztheawtcaosubladsendotobneaadsdsienmgbtlheidstionto tsin 7/3/4
FamilySalicaceae 1993) anyofthemajorscaffolds.Theauthorssuggestedthiswas a 67
cthoensgiesnteonmtewwithaschyetotegreoncehtricomevaitdice.nTcehisshwowasinbgasaepdproonx.D3A0P%I of ngios /1461
stainingofsevenprophaseandmetaphasecellsfollowedby pe 46
physicalmeasurementsofheterochromaticbands(NB: rm by
adjustmentsweremadeforthedifferentialcontractionof s g
heterochromatinversuseuchromatin). ue
s
Vitisvinifera 475* 487 Seecomments 415MbusingFC:PIand Noattemptwasmadetoestimateatotalgenomesize.Instead, t o
n
genotypePN400246 chickenredbloodcellsas the1C-valueof475MbgivenbytheFrench-ItalianPublic 2
8
(grapevine) standard(LodhiandReisch, ConsortiumforGrapevineGenomeCharacterization(2007) M
FamilyVitaceae 1995) wastakenfromLodhiandReisch(1995).Actually,thisfigure a
comesfromtheabstractofLodhiandReischwhostatedthat rc
h
‘theVitisgenomesizeis475Mb’basedonmeasurementsfor 2
0
19speciesincludingV.vinifera.ThemeasurementsofLodhi 1
9
andReischweremadeusingflowcytometryandchickenred
bloodcellsasstandard(assuming2C¼2.33pg)andshowed
therewasintraspecificvariationbetweendifferentvarietiesof
Vitisviniferarangingfrom1C¼415to511Mb.Thecultivar
‘PinotNoir’,whichisclosesttothevarietyusedforgenome
sequencing,wasestimatedtohaveagenomesizeof415Mb.