High-Resolution Crossover Maps for Each Bivalent of Zea mays Using Recombination Nodules

18 Pages·2003·0.41 MB·English

Copyright2003bytheGeneticsSocietyofAmerica
High-Resolution Crossover Maps for Each Bivalent of Zea mays Using
Recombination Nodules
LorindaK.Anderson,*,1GregoryG.Doyle,†BrianBrigham,*JennaCarter,*KristinaD.Hooker,*
Ann Lai,* Mindy Rice* and Stephen M. Stack*
*DepartmentofBiology,ColoradoStateUniversity,FortCollins,Colorado80523and
†DepartmentofAgronomy,UniversityofMissouri,Columbia,Missouri65211
ManuscriptreceivedApril11,2003
AcceptedforpublicationJune17,2003
ABSTRACT
Recombinationnodules(RNs)arecloselycorrelatedwithcrossingover,and,becausetheyareobserved
by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs
providethehighest-resolutioncytologicalmarkercurrentlyavailablefordefiningthefrequencyanddistri-
butionofcrossoversalongthelengthofchromosomes.UsingthemaizeinbredlineKYS,wepreparedan
SCkaryotypeinwhicheachSCwasidentifiedbyrelativelengthandarmratioandrelatedtotheproper
linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce
high-resolution maps of RN frequency and distribution on each bivalent. RN frequencies are closely
correlatedwithbothchiasmafrequenciesandSClength.ThetotallengthoftheRNrecombinationmap
isabouttwofoldshorterthanthatofmostmaizelinkagemaps,butthereisgoodcorrespondencebetween
the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a
unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe
reductionofRNsatandnearkinetochores.ThefrequencyofRNsatknobsiseithersimilartoorhigher
thantheaveragefrequencyofRNsalongtheSCs.TheseRNmapsrepresentanindependentmeasureof
crossingoveralongmaizebivalents.
MAIZE(ZeamaysL.)isoneofthefirstmodelorgan- ern1967;delaTorreetal.1986;Jones1987;Sherman
ismsinwhichthepowerofgeneticswasproduc- and Stack 1995).
tively merged with cytology to create the new field of One way to relate linkage maps to the structure of
cytogenetics (reviewed by Rhoades 1984). The ability chromosomes isto observethe locationand frequency
to recognize all 10 maize chromosomes in pachytene of crossing over along chromosomes by means other
squash preparations was key for relating each chromo- thanlinkageanalysis.Toadegreethishasbeenaccom-
some to a specific linkage group (McClintock 1931, plishedinsquashpreparationsbyobservingthenumber
1933;RhoadesandMcClintock1935).Subsequently, andpositions ofchiasmataon latediplotene-diakinesis
maizechromosomecytologyhasremainedafertilefield bivalents(Jones1987).Unfortunately,formostspecies,
for investigation (e.g., Dawe et al. 1994; Freeling and includingmaize,thisisarelativelyinaccurate,low-reso-
Walbot1994;Bassetal.2000;Muehlbaueretal.2000; lution method because (1) twists can be mistaken for
Sadder and Weber 2002) that has been accompanied chiasmata, (2) nearby chiasmata may not be resolved,
bythedevelopmentofsophisticatedmaizelinkagemaps (3) chromosomes are comparatively short when chias-
(e.g.,Davisetal.1999;Sharopovaetal.2002).However, mata are visible, and (4) it is difficult to relate most
integrationoflinkagemapswithchromosomestructure bivalentstospecificpairsofchromosomes(Stacketal.
hasbeendifficultinmaize(aswellasinotherorganisms) 1989;ShermanandStack1995;Stevensonetal.1998).
because crossing over is not evenly distributed along Anothermethodforcytologicallyassessingcrossingover
thelengthofchromosomes.Forexample,crossingover hasbeendevelopedrecentlyusingfluorescentantibod-
istypicallyuncommoninheterochromatinandcentro- ies to label MLH1p, a mismatch repair protein that is
meres compared to euchromatin (e.g., Mather 1939; present at crossover sites during pachytene (Baker et
forreviewsseeComings1972;Resnick1987),andeven al. 1996; Hunter and Borts 1997; Anderson et al.
ineuchromatin,wherethebulkofcrossingoveroccurs, 1999;Moensetal.2002).BecauseMLH1signalsappear
the frequency of crossing over can vary considerably asdiscretefluorescentfoci onpachytenebivalentsthat
fromonepartofachromosometoanother(e.g.,South-
are5–10timeslongerthanbivalentsatdiakinesis,MLH1
focicanbemappedathigherresolutionthanchiasmata
can.However,theapplicationofthistechniquetomap-
1Correspondingauthor:DepartmentofBiology,ColoradoStateUni-
pingcrossovershasbeenlimitedtobirdsandmammals
versity,FortCollins,CO80523.
E-mail:[e-mail] so far (e.g., Pigozzi 2001; Froenicke et al. 2002), per-
Genetics165:849–865(October2003)
850 L.K.Andersonetal.
hapsbecausetheantibodieswereraisedtomammalian grown in a temperature-controlled greenhouse with supple-
mentallighting.AllinversionheterozygoteswereinKYSback-
MLH1 proteins, and these antibodies do not bind to
ground.
theirplantcounterparts(ourobservations).Whilerep-
Diakinesischromosomesquashes:Antherscontainingdiaki-
resenting a significant improvement over chiasmata to
nesis-stage cellswere fixed for 1–24hr in 1:3acetic ethanol.
mapcrossoverevents,analysisofMLH1fluorescentfoci Afterclearingtheanthersfor1–5minin45%aceticacid,the
isstilllimitedbytheresolutionofthelightmicroscope. meioticcellsweresqueezedoutoftheanthersinafreshdrop
The highest-resolution method available to map the of45%aceticacidandsquashedlightlyunderasiliconizedcover
glass.Thecoverglasswasremovedusingdryice,theslidewas
frequencyandlocationofcrossovereventscytologically
allowed to air dry, and chromosomes were stained with 2%
remains analysis of late recombination nodules (RNs;
aceto-orcein under a cover glass with brief heating over an
sometimes abbreviated as LNs) on synaptonemal com- alcohol lamp. After staining, cover glasses were removed by
plexes(SCs;e.g.,Carpenter1975;reviewedbyZickler invertingtheslideover95%ethanol.Beforethepreparations
andKleckner1999;AndersonandStack2002).RNs dried, new cover glasses were mounted with Euparal. Com-
are proteinaceousellipsoids, (cid:1)100 nm intheir longest pletesetsinwhichallchromosomeswereseparateandinter-
pretablewerephotographedusinga(cid:1)100PlanApoobjective
dimension, which lie on SCs (that is, pachytene biva-
and a digital camera attached to an Olympus Provis light
lents). RNs are closely correlated with crossovers and
microscope.
lieatsiteswherechiasmatawillformlater(Carpenter PachyteneSCspreads:SCspreadswerepreparedonplastic-
1975, 1979; von Wettstein et al. 1984; Marcon and coated slides as described by Stack and Anderson (2002).
Moens 2003). The proposed role of RNs as molecular SC spreads were stained with 2% uranyl acetate followed by
Reynold’sleadcitrate(UP)orwith33%silvernitrate(Sher-
factories for crossing over has been corroborated re-
man et al. 1992). The slides were scanned using phase light
cently by work showing that the MLH1 protein is a
microscopy, and good SC spreadson plastic were picked up
component of late RNs (Moens et al. 2002). The high
onto50-or75-meshgrids.ThegridswereexaminedinanAEI
resolutionofRNanalysisisduenotonlytotheobserva- 801electronmicroscope,andSCspreadswithoutdetectable
tionofRNsonrelativelylongpachytenechromosomes, stretchingandwithkinetochoreswerephotographedatamag-
butalsotothesmallsizeofRNscomparedtochiasmata nification of (cid:1)1600 or (cid:1)2500. In total, 2080 individual SCs
(0.1(cid:2)mvs.1(cid:2)m,respectively)andtotheuseofelectron from290setswereidentifiedandmappedwithregardtoRNs.
BothtotalRNnumberandtotalSCsetlength(thecombined
microscopy to resolve RNs.
lengthofallSCsinacell)couldbeassessedfor206ofthese
Themostusefulcytologicalmapsofcrossingoverare SCsets.Intheremainderofthesets,certainoftheSCscould
thoseinwhicheverybivalentcanbeidentifiedunequivo- not be identified, usually due to unclear or missing kineto-
cally and related to a specific linkage group. This has chores.SomeSCshadasmallamountofasynapsis,particularly
not been possible in many organisms, and, in lieu of neartheends, andmayhavebeeninthe veryearlieststages
ofdiplotene.
this,somestudieshavepooledcrossoverdatafromchro-
Measurements:Electronmicroscopenegativeswerescanned
mosomes of similar size and arm ratios (Southern
intoacomputerusingaHewlett-PackardScanJet4candAdobe
1967; Anderson et al. 1999). In other studies, it was Photoshop (version 5.0) software. Montages of SC spreads
possible to identify some of the bivalents using either were assembled using Adobe Photoshop. Proper tracing of
a combination of relative lengths and arm ratios (e.g., eachSCandthepositionofkinetochoresandRNsweredeter-
mineddirectlyfromthenegativesusinga(cid:1)8magnifyingloupe
LaurieandJones1981;Pigozzi2001)orfluorescence
and recorded onto prints of the montages. One lateral ele-
insituhybridizationofchromosome-specificsequences
ment from each SC was measured in micrometers using the
(Lynnetal.2002;Teaseetal.2002).Thusfar,onlytwo
computer program MicroMeasure (Reeves 2001). Total SC
studieshavemappedcrossingoveroneverybivalentin length varied from set to set, but the relative length of SCs
aset,oneusingRNsontomatoSCsthatwereidentified andSCarms,i.e.,armratios,withineachsetremainedconsis-
by relative lengths, arm ratios, and patterns of hetero- tent. SCs were identified by relative lengths (percentages of
totalSClengthfortheset)andarmratios(lengthofthelong
chromatin (Sherman and Stack 1995) and the other
armdividedbylengthoftheshortarm).RNswererecognized
usingMLH1focionmouseautosomalSCsafterchromo-
usingcriteriaofsize,shape,stainingintensity,andassociation
some-specific painting (Froenicke et al. 2002). Here with SC as described by Stack and Anderson (2002). RN
we report such an analysis in maize. For this, we first positions were measured and expressed as a percentage of
preparedanSCkaryotypeonthebasisofrelativelengths the arm length from thecentromere. Using average lengths
andarmratiosforthemaizeinbredlineKansasYellow for eachof the10 SCs andtheir averagearm ratios,each of
theSCswasdividedinto0.2-(cid:2)msegments,andeachobserved
Saline (KYS). Each SC was identified and related to a
RNwasplacedintooneofthesesegmentsonthebasisofits
specific maize chromosome and linkage group. Most SC
originalrelativepositionfromthecentromere.ForthoseSCs
identificationswereverifiedusinginversionheterozygotes. inwhichanarmwasnotdivisiblebytwo,themostproximal
We then determined the frequency and distribution of intervalwasmade0.3(cid:2)mratherthan0.2(cid:2)m.Aftercompiling
RNs (crossing over) on each of the 10 maize SCs. theRNdata,thegeneticmaplengthofeachSCwascalculated
bydeterminingtheaveragenumberofRNsperSCandthen
multiplyingby50(oneRN(cid:3)onecrossover(cid:3)50cM).
MATERIALS AND METHODS Statistics: The program Minitab (version 13) was used for
statisticalanalysesandforpreparinghistograms.Thesmooth-
Plants:MaizeinbredKYSandheterozygotesforinversions ing (Lowess) lines were based on the histograms (Minitab
1d, 2i, 3c, 4c, 5d, 6b, 7a, 8c, 9b, and 10a (Doyle 1994) were degreeofsmoothing(cid:3)0.1;steps(cid:3)0).
RNCrossoverMapsinMaize 851
Figure 1.—Complete set
of silver-stained spread SCs
from maize KYS. Each SC
has been identified by its
relative length and arm ra-
tio and numbered accord-
ingly. Arrows indicate the
location of RNs. The two
RNsontheshortarmofSC
6aresoclosetogetherthat
theywouldlikelyresultina
structure that would be in-
terpreted as a single chi-
asmaduringdiakinesis.Lat-
eral element thickenings
can be observed on several
SCs (e.g., 1S, 2S, 7S, 10L).
Such thickenings are more
commonlyobservedwithsil-
ver staining than with ura-
nylacetate-leadcitratestain-
ing.K,kinetochore;S,short
arm;L,longarm.Bar,5(cid:2)m.
RESULTS in relative length between 2 (or sometimes 3) SCs was
minimal, but the arm ratios were noticeably different.
SCkaryotype:SCspreadswerepreparedbyexposing
The average relative length and average arm ratio for
protoplasts to a hypotonic solution containing a small
eachrankedSCfromthe30setsarepresentedinTable
amount of nonionic detergent (Stack and Anderson
1alongwiththesameinformationforfiveotherkaryo-
2002). During this procedure, the cytoplasm as well as
types produced from squashes, three-dimensional re-
the chromatin surrounding each SC decondenses to
become almost invisible, while SCs and RNs are rela- constructions,andSCspreads.Exceptforabsolutetotal
tivelyunaffected(Figure1).Dispersionofthechroma- lengths of complete sets and the arm ratio of SC 6
tinmeansthatsuchfeaturesaschromomeresandknobs thatcarriestheNOR,thesimilarityofthekaryotypesis
are not visible to help identify SCs. In addition, the striking.Overall,theSCsdecreasegraduallyinaverage
nucleolus is usually dispersedin these preparations, so relativelengthfromSC1(14.8%)toSC10(6.8%),but
itisnotpossibletodetecttheassociationofthenucleo- thelengthpositionsofSC4andSC5havebeenreversed
lus with the nucleolus organizer (NOR) on the short to reflect the standard pachytene chromosome karyo-
arm of SC 6 (McClintock 1934). This means that SC type.Withregardtoarmratios,eachrankedSCgroup
identificationmustrelyonrelativelengthandarmratio. is statisticallydifferent fromthe SCgroup immediately
Armratioscanbedeterminedonlywhenkinetochores precedingorsucceedingit(P(cid:4)0.002,two-samplet-test).
are visible in SC spreads that are probably in mid- to Thus, each SC in a set can be identified accurately on
late pachytene. the basis of its relative length and arm ratio.
To prepare the karyotype, SC spreads were selected To verify that our SC identifications were consistent
usingthefollowingcriteria:(1)eachofthe10SCscould withthegeneticlinkagegroups,weanalyzedspreadsof
befollowedalongitsentirelength,(2)thekinetochore SCs from plants that were heterozygous for one of the
was visible on each SC, and (3) SCs were not visibly following inversions: 1d, 2i, 3c, 4c, 5d, 6b, 7a, 8c, 9b, or
stretched.ThirtysetsofSCsthatmetthesecriteriawere 10a (Figure 2; Doyle 1994). Unfortunately, obtaining
measuredforlengthsandarmratios.ThenSCsineach spreadsthatcontainedinversionloopsalongwithdistin-
set were ordered according to their relative lengths. If guishable kinetochores on each SC proved to be diffi-
necessary, the order of an SC was changed so that the cult. For example, no SC spreads from the inversion
arm ratios for each SC were consistent with pachytene heterozygotesIn8candIn10afulfilledthesetwocriteria,
maps(Table1).Outofthe(10(cid:1)30(cid:3))300SClength andonly27SCsets(In1d(cid:3)2;In2i(cid:3)6;In3c(cid:3)3;In4c(cid:3)4;
positions, 55 (18%) were changed on the basis of the In5d (cid:3) 2; In6b (cid:3) 1; In7a (cid:3) 5; In9b (cid:3) 4) for the other
armratiodata.Formostofthesechanges,thedifference inversion heterozygotes could be used to verify the SC
852 L.K.Andersonetal.
TABLE1
Comparisonofselectedkaryotypesformaize
Chromosomesquashes
3Dfluorescence:
SCspreads
Chromosomeor McClintock Longley(1939); Daweetal. 3Dsections:
SCrank etal.(1981) Rhoades(1950) (1992,1994) Gillies(1973) Gilles(1981) Thiswork
1 14.9(1.27) 14.8(1.30) 15.8(1.30) 14.2(1.26) 14.9(1.24) 14.8(1.27)
2 12.0(1.20) 12.1(1.26) 12.3(1.68) 12.4(1.20) 11.9(1.15) 11.9(1.08)
3 11.2(2.03) 11.7(2.01) 10.8(2.06) 11.1(2.03) 11.3(2.06) 11.2(2.02)
4 10.6(1.57) 10.6(1.59) 10.0(1.39) 10.8(1.68) 10.5(1.62) 10.7(1.56)
5 10.7(1.16) 10.7(1.20) 10.1(1.08) 10.9(1.21) 11.0(1.13) 10.8(1.10)
6 8.9(3.14) 8.4(3.22) 9.5(3.41) 9.2(3.01) 9.1(2.75) 8.9(2.63)
7 8.4(2.58) 8.3(2.72) 9.0(2.38) 8.5(2.38) 8.8(2.67) 8.7(2.76)
8 8.8(3.29) 8.3(3.26) 8.2(2.83) 8.8(2.92) 8.2(2.99) 8.5(3.04)
9 7.7(1.82) 8.1(1.85) 7.4(1.69) 8.0(1.89) 7.7(2.09) 7.7(1.92)
10 6.7(2.83) 6.9(2.80) 6.7(2.31) 6.2(2.43) 6.5(2.54) 6.8(2.43)
Averagetotal — 556 — 353 421 331
lengthofsets((cid:2)m)
No.ofobservations — 28 2 5 14 30
The karyotypeswere prepared usinga variety oftechniques including lightmicroscopy of pachytenechromosome squashes,
deconvolution-based, three-dimensional fluorescence light microscopy of intact pachytene nuclei, three-dimensional electron
microscopic reconstructions of pachytene nuclei from serial thin sections, or electron microscopy of SC spreads. In all cases,
primarymicrosporocytesfrommaizeinbredKYSwereused.Foreachkaryotype,theaveragerelativelengthofeachSCispresented
firstasapercentageofthetotallengthoftheSCset,followedbytheaveragearmratioinparentheses.
identifications (Figure 2). Because an inversion loop evenifnotalloftheSCscouldbeindividuallyidentified
with associated asynapsis on its borders can alter the (usually because of the absence of discernible kineto-
expectedrelativelengthandarmratiofortheinversion chores).ThesecompletesetsofSCsaveraged20.7RNs
loop SC, the nine normal chromosomes in a set were per SC set, a difference of (cid:4)1% when compared to
identified using relative lengths and arm ratios, with usingRNaveragesforindividualSCs.Thus,usingRNs,
the “missing” SC being identified as the SC with the weestimatethatthetotalmaplengthformaizeinbred
inversionloop.Foreachoftheeightinversionheterozy- KYS is between (20.5 RNs (cid:1) 50 cM/RN (cid:3)) 1025 cM
gotes in which this test was possible, the identification and (20.7 RNs (cid:1) 50 cM/RN (cid:3)) 1035 cM.
oftheinversionloopSCcorrespondedwiththeappro- Chiasma frequency per cell: To compare rates of
priatechromosome.ThisresultconfirmsthatourSCiden- crossing over in KYS maize determined from RNs to
tificationsare consistentwithestablished linkagegroups. those determined from chiasmata, we analyzed at least
Weexamined290setsofSCswithRNsandwereable 50squashesofchromosomesetsatdiakinesisfromeach
toidentify2080((cid:1)72%)individualSCs(Table2,Figure of 5 plants (Figure 4) and at least 10 SC spreads from
3). Some SCs were easier to identify than others, so eachof10plants(Table3).Nosingleplantwasanalyzed
the number of each SC analyzed for RNs varies. For for both chiasmata and RNs. Each plant, regardless of
example, SC 2, a long SC with an arm ratio near 1.0, the method of analysis, demonstrated large cell-to-cell
andSC10,theshortestSC,wererelativelyeasytoiden- variability (up to twofold) in the number of crossovers
tify,andas aresult,wemade247 observationsofeach. observed. While there were no significant differences
In contrast, the number of observations for SC 6 and amongplantsinvarianceforchiasmataorforRNs(Bart-
SC7waslower(n(cid:3)176and178,respectively)because lett’stestandLevene’stest,P(cid:5)0.2),thereweresignifi-
they are similar in both relative length and arm ratio cant differences among plants in the mean number
and more difficult to distinguish from one another. of crossovers per cell on the basis of both chiasmata
Nevertheless, thesedata represent thehighest number (ANOVA; P (cid:4) 0.001) and RNs (ANOVA; P (cid:4) 0.001).
of observations of RNson individual SCs made for any Becausetheplantswereallfromthesameinbredstrain
organism except tomato (Sherman and Stack 1995). and presumably had the same genetic makeup, we ex-
RN frequency per cell: On the basis of the average plored the possibility that environmental conditions
frequency of RNs per SC, there is an average of 20.5 wereresponsible forthedifferencesin meancrossover
RNs per cell (Table 2). To verify that this number is frequency. The plants analyzed for chiasmata were all
representative, we examined 239 complete sets of SCs exposedtothesamegreenhouseconditionsduringthe
inwhicheach SCcouldbeanalyzed forRNfrequency, summer of 2002. The RN data were accumulated over
RNCrossoverMapsinMaize 853
used.Bivalentswithtwocrossoverswereobservedmore
often for chiasmata than for RNs, while bivalents with
threeormorecrossoverswereobservedmoreoftenfor
RNs than for chiasmata. The difference in resolution
ofthetwotechniquesmaycontributetotheseobserved
discrepancies.
Relationship between RN frequency and SC length:
For 206 SC sets, we were able to determine both total
RN number and total SC set length (although not all
SCscouldbeidentifiedineachspread;Figure5A).The
slope of the regression is significantly different from
zero[totalRNs(cid:3)(0.026(cid:1)totalSClength)(cid:6)11.8,P(cid:4)
0.001,r2(cid:3)16.3%].Thus,SCsetlengthandRNnumber
persetarepositivelyrelated,with(cid:1)16%ofthevariation
in RN number explained by SC set length.
When the 10 maize bivalents are considered sepa-
rately(n(cid:3)2080SCs,Tables1and2),thereisastrong
positiverelationshipbetweenaverageRNfrequencyand
averageSClength,with96%ofthevariabilityinaverage
RNfrequencyrelatedtoaverageSClength[Figure5B;
y(cid:3)(0.042(cid:1)SClength)(cid:6)0.66,r2(cid:3)96.2%].Asimilar
relationship is observed when average SC arm lengths
are compared to the average number of RNs per arm
Figure2.—CompleteSCspreadstainedwithuranylacetate- [Figure5C,y(cid:3)(0.044(cid:1)SClength)(cid:6)0.31,r2(cid:3)96.5%].
lead citrate from a maize plant heterozygous for In7a. Each
For both regressions, the slope and y-intercept are sig-
SC has been labeled at the kinetochore (K) with the appro-
nificantlydifferentfromzero(P(cid:4)0.001).Thus,average
priateSCnumberonthebasisofitsrelativelengthandarm
ratio.TheinversionloopinthelongarmofSC7canbeseen RN frequency is closely correlated with individual SC
in the lower left of the spread. Kinetochores from different length, whether one considers arm lengths separately
SCs are often fused (6K and 8K, 1K and 10K, 4K and 5K). or bivalent length as a whole.
There are no obvious lateral element thickenings on any of
theSCs.Bar,5(cid:2)m. Distribution of RNs along SCs: Histograms showing
thedistributionofRNsalongeachSCarepresentedin
Figure 6. Each SC is represented by the x-axis with the
fouryearsfrom1998to2002,againusingplantsgrown shortarmto theleftandthe kinetochore(kc)marked
in the same greenhouse. SC spreads from 8 of the 10 withaverticallinebeneaththeaxis.EachSCisdivided
plantswerepreparedinthesummer(April–September) into0.2-(cid:2)msegmentswiththenumberofRNsobserved
and SC spreads from 2 of the plants were prepared in ineachsegmentrepresentedbyaverticalbar.Twolines
thefall(October–March).TheaveragenumberofRNs are superimposed over each distribution. One is a
percellwas19.8and21.5forthe2winter-grownplants smoothing(Lowess)linederivedfromthedata(Cleve-
and17.0–23.1forthe8summer-grownplants.Although land1979),whichshowsthegeneraltrendofcrossing
the data are limited, this pattern of RN numbers does overalongeachSC.Thesmoothinglineminimizesvaria-
not support the hypothesis that environmental factors tion regardless of whether the variation is caused by
are responsible for the differences in RN numbers ob- samplingerrorsorbylocalizeddifferencesincrossover
served. frequency.Thesecondsuperimposedlineishorizontal
Because the ranges of values for chiasmata and for and represents the average number of RNs observed
RNsweresimilaramongplants(eventhoughtherewere per 0.2-(cid:2)m interval for each SC. Intervals that differ
significant differences in mean crossover frequency considerably from the horizontal lines represent hot
among plants), the data for chiasmata and RNs were (above average) or cold (below average) regions for
pooledseparatelyandcompared.Theaveragenumber crossing over.
ofRNspercellwas(cid:1)10%higher(20.6)thantheaverage All of the SCs share the general characteristics of a
number of chiasmata (18.9; Table 3). In addition to high frequency of RNs in distal regions (including the
determiningtheaveragenumberofcrossoverspercell, veryendsofSCs)andalowfrequencyofRNsinproximal
we also examined the frequencies of bivalents with 0, regions, i.e., around kinetochores (Figures 6 and 7).
1,2,and 3ormoreRNsand chiasmata(Table3).The The distributions of RNs on SCs 2, 4, and 9 indicate
frequenciesofbivalentswithzeroandonecrossoverper that a few RNs occur within kinetochores. This is an
cellwerethesamewhetherRNsorchiasmatawereused. artifactduetocompilingdatafromSCsthatvarysome-
However, the frequency of bivalents with two or more whatinarmratiosandplottingthedistributionsonan
crossoversdiffereddependingonthemethodofanalysis “average” SC. While RNs sometimes occur quite close
854 L.K.Andersonetal.
TABLE2
PredictedmaplengthbasedontheaveragenumberofRNsperSCcomparedto
classicalgeneticandmolecular(IBM)linkagemaps
SC Geneticmapa,b IBMmapa,c
No.ofSCs Average Equivalent Equivalent Equivalent
SCno. observed no.ofRN cMd cM RNe cMf RNe
1 216 2.69 134.3 258 5.2 325.3 6.5
2 247 2.37 118.4 224 4.5 204.2 4.1
3 189 2.22 110.9 216 4.3 228.4 4.6
4 196 2.14 107.1 172 3.4 228.3 4.6
5 203 2.21 110.6 185 3.7 202.6 4.1
6 176 1.81 90.6 144 2.9 164.2 3.3
7 178 1.86 93.0 128 2.6 188.7 3.8
8 194 1.80 90.2 177 3.5 189.2 3.8
9 234 1.85 92.7 178 3.6 193.9 3.9
10 247 1.54 76.9 174 3.5 157.4 3.1
Total 2080 20.5 1024.7 1856 37.1 2082.2 41.8
aFromhttp://www.agron.missouri.edu/cMapDB/cMap.html(March2003).
bGeneticmapistheclassicalgeneticmap,compiledbyEdCoefromcooperators.
cIBMisanintermatedB73/Mo17recombinantinbredhigh-resolutionmolecularmap.
dTheaveragenumberofRNs(cid:1)50cM/RN.
eCentimorgansdividedby50.
fThismolecularmapincludesrecombinationeventsfromthreemeioses(K.Cone,personalcommunication),
so the centimorgans presented on the web site have been divided by 3 here for the purpose of comparison
usingthestandarddefinitionofcentimorgans(Tamarin2002).
to the kinetochore, we did not observe any RNs that segmentscontain48%(2061/4271)ofallRNsobserved
wereclearlywithinkinetochores(Figure7).Inaddition while the most proximal segments contain 4% (156/
tothegeneraltrendsinthedistributionofcrossingover 4271) of all RNs observed. Thus, over the same SC
that all SCs share, each SC also has a distinct pattern length,distalregionshave12timesmorecrossingover
of RNs along its length. than proximal regions do.
ToquantifythedifferenceinRNfrequencybetween MaizeKYShasfiveknobs,twoonshortarms(1S,9S)
distal andproximal segments, we dividedeach SC arm and three on long arms (5L, 6L, 7L; Dawe et al. 1992;
into five equal (20%) segments, pooled the total num- Chenetal.2000).Thelocationofeachknobisindicated
berofRNsobservedforallofthemostdistalsegments, byahorizontallinebeneaththeappropriateSCinFig-
and pooled the total number of RNs for all proximal ure6.Overall,thefrequencyofRNsinknobregionsis
segments. The combined distal segments for all SCs either about the same as the average for the SC as a
represent (0.20(cid:1) 331(cid:2)m total SClength (cid:3)) 66.2(cid:2)m whole(5L,6L,7L)orhigher(1S,9S).Thisisparticularly
in length as do the combined proximal segments. If true for the knob on the tip of the short arm of SC 9,
RNsweredistributedevenlyalongthelengthofallSCs, which has a frequency of RNs that is twice as high as
theneach20%segmentwouldhave(cid:1)20%ofthetotal theaverageforSC9(7vs.3.4RNsper0.2-(cid:2)minterval).
number of RNs observed. Instead, the most-distal SC In contrast, the NOR region on the short arm of SC 6
Figure3.—Close-upofSC9fromasetofSCs
stained with uranyl acetate-lead citrate. A small
portion of the distal end of the long arm is asy-
napsed, indicating that the SC has begun desy-
napsisandisintheearlieststageofdiplotene.SC
9 has one RN (inset a) in the short arm. The
dispersedchromatinaroundSC9isvisibleasan
amorphous coating. A portion of SC 2 with an
RN(insetb)isalsoshown.Double-headedarrows
show the same RNs at higher magnification in
insets.K,kinetochore.Bar,2(cid:2)m(1(cid:2)mininsets
aandb).
RNCrossoverMapsinMaize 855
different maize lines, aside from the observation that
thenumberandlocationofheterochromaticknobsmay
vary(Longley1939;McClintocketal.1981;Dempsey
1994).However,whilethepresenceofknobscanaffect
thelengthandarmratiosofmitoticchromosomes,the
presence or absence of knobs has little effect on the
relative length and arm ratios of SCs because hetero-
chromatin is underrepresented in SC length (Stack
1984; Jones andde Azkue 1993). Thus, itis likely that
the KYS SC karyotype applies in large measure to all
maize lines.
Within animal and plant species (including maize)
and even within individuals, there may be as much as
Figure4.—DiakinesischromosomesquashfrommaizeKYS. atwofoldvariationintheabsolutelengthsofsetsofpachy-
Thecounted numberofchiasmata isindicatednext toeach tenechromosomesorSCs.However,therelativelength
bivalent.Withtheexceptionofchromosome6thatisassoci- andarmratioforeachchromosomeorSCinasetremains
ated with the nucleolus (N), none of the chromosomes can
beidentified.Bar,10(cid:2)m. constant (Moses et al. 1977; Gillies 1981; Sherman
andStack 1992;ourobservations).This indicatesthat
each chromosome/SC in a set responds in a propor-
has a slightly reduced level of RNs compared to the tional way to changes in the length of the entire set.
average for SC 6 (1.6 vs. 2.2 RNs per 0.2-(cid:2)m interval), Chromosomenumberinginmaizeisbasedprimarily
butthislowerlevelcouldbeduetoitsproximitytothe on relative length with the longest chromosome num-
kinetochore (Figure 6). bered 1, ranging down to the shortest numbered 10.
However, in each karyotype reported for maize, chro-
mosome/SC 5 is slightly longer than chromosome/SC
DISCUSSION
4(Table 1).Thisdiscontinuityin numberingarosebe-
SC identification: We have prepared a karyotype for cause maize chromosomes were numbered initially us-
SCsfromKYSmaizeonthebasisofrelativelengthsand ing mitotic chromosomes that differ slightly in relative
armratios(Figure1;Table1).ThisSCkaryotypeisvery length from pachytene chromosomes (McClintock
similar to other maize pachytene karyotypes that have 1929;Rhoades1955;Carlson1988).Inaddition,chro-
beenpreparedusinganumberofdifferenttechniques mosome/SC 8 is longer than chromosome/SC 7 in
(aceto-carmine-stainedpachytenechromosomesquashes, some karyotypes but not in others (Table 1). These
4(cid:7),6-diamidino-2-phenyindole-stained intact pachytene differences in relative length between different maize
nuclei,three-dimensionalreconstructionsofpachytene karyotypesaresmallandmayrepresenteithermeasure-
nuclei from serial thin sections, and SC spreads). The ment error or natural variation within populations. In
inbred KYS line of maize generally has been used in either case, these chromosomes are still readily distin-
thesestudiesbecauseKYSpachytenechromosomessep- guished from each other on the basis of differences in
aratewellduringsquashingandspreading,makingthem theirarmratios.Inaddition,wewereabletoverifythe
easier to analyze than pachytene chromosomes from identity of SCs 1, 2, 3, 4, 5, 6, 7, and 9 using inversion
many other maize lines (Dempsey 1994; our unpub- heterozygotes. Although not verified by inversion het-
lishedobservations).Inanycase,thereislittledifference erozygotes, our identification of SCs 8 and 10 is also
inthebasicpachytenechromosomekaryotypebetween firmbecausebothSC8andSC10canbereadilydistin-
TABLE3
ComparisonofcrossoverfrequencyformaizeKYSusingchiasmataandRNs
No.ofcells Meanno.ofRNs No.ofbivalentswith0–6crossoversa
(bivalents) orchiasmata(SD)
observed andrange 0 1 2 3 4–6
RNs 151 20.6(3.3) 14 363 743 312 78
10plants (1510) 13–33 (0.9) (24.0) (49.2) (20.7) (5.2)
Chiasmata 278 18.9(2.5) 31 667 1673 402 7
5plants (2780) 12–26 (1.1) (24.0) (60.2) (14.5) (0.3)
Pvaluesb (cid:4)0.001 0.56 0.97 (cid:4)0.001 (cid:4)0.001 (cid:4)0.001
aNumbersinparenthesesrepresentpercentages.
bStudent’st-testusingmeanfrequencyofbivalenttypepercell.
856 L.K.Andersonetal.
location of the NOR in spreads. However, we did not
observe any unusual fragmentation or any detectable
stretching of the SC in the short arm of maize SC 6.
Whatever the cause of the differences in arm ratio for
chromosome/SC6,weareconfidentthatwecaniden-
tify SC 6 because the arm ratio for SC 6 is consistent
betweensetsof SCspreads,SC6can beseparatedreli-
ably fromthe otherSCs, andSC 6has been verifiedto
correspondtochromosome6byanalysisofSCspreads
from inversion heterozygotes for In6b.
Estimatingcrossoverfrequencyusingdifferentmeth-
ods:Somecontroversysurroundsestimatesofcrossover
frequencythathavebeendeterminedusingchiasmata,
RNs,andlinkagemaps(e.g.,Nilssonetal.1993;Sher-
manandStack1995;Sybenga1996;Kingetal.2002a).
Typically,saturatedornearlysaturatedclassicallinkage
maps indicate higher crossover rates than chiasma
counts,andmolecularlinkagemapsoftenindicateeven
highercrossoverrates(e.g.,Nilssonetal.1993;Moran
etal.2001;Kingetal.2002a;Table3).Thisalsoappears
tobethepatterninmaizewherechiasmacountsrange
fromanaverageof17–21percellwhendiakinesis-meta-
phase I chromosomes are used (Beadle 1933; Ayo-
noaduandRees1968;Pagliarinietal.1986;Table3)
although higher averages (27–37 per cell) are possible
whenlongerdiplotenechromosomesareused(inwhich
chiasmata are more difficult to distinguish from twists;
Beadle 1933; Darlington 1934). In comparison, ge-
netic and molecular linkage maps indicate 37 and 42
crossoverspercell,respectively(Table2).Thisproblem
Figure 5.—Scatter plots and regressions for (A) total SC hasbeenaddressedrecentlybyKingetal.(2002a),who
set length and total RN number (y (cid:3) 0.026x (cid:6) 11.82, r2 (cid:3) introgressed a single chromosome of Festuca pratensis
0.16),(B)averageSClengthandaverageRNfrequency(y(cid:3)
into Lolium perenne to create a monosomic substitution
0.042x (cid:6) 0.66, r2 (cid:3) 0.96), and (C) average SC arm length
andaverageRNfrequency(y(cid:3)0.044x(cid:6)0.31,r2(cid:3)0.97). line (hereafter referred toas the Festuca/Lolium biva-
lent).TheF.pratensischromosomesynapsesandrecom-
bines with the homeologous L. perenne chromosome,
guished from the other SCs and from each other on although the two chromosomes can be distinguished
the basis of their relative lengths and arm ratios. from one another using genomic in situ hybridization.
Themoststrikingdifferenceamongmaizekaryotypes King et al. (2002a) compared the recombination fre-
involvesthearmratioforchromosome/SC6.Theaver- quencyforthishomeologousbivalentusingbothampli-
age arm ratio is 3.0 or higher for squashed pachytene fiedfragmentlengthpolymorphismmarkersandchias-
chromosomes and sectioned SCs, but only 2.6–2.7 for mata.Theyfounda1:1relationshipbetweencrossovers
SC spreads (Table 1). Since the short arm of chromo- detectedbythetwomethods.Theysuggestthatchiasma
some/SC6carriestheNOR,itislikelythatdifferences counts tend to somewhat underestimate the amount of
betweenthekaryotypessomehowinvolvethenucleolus. crossing over due to difficulty in resolving nearby chias-
Typically, a single large nucleolus is visible in primary mata,whilemolecularmapstendtoinflatetheamount
microsporocytesthatarefixedbeforesquashingorsec- of crossing over due to errors in typing and the use of
tioning(Rhoades1950;Daweetal.1994).Incontrast, differentcomputerprogramswithdifferentalgorithms
SCspreadsarepreparedbyexposinglivecellstoahypo- to generate the maps. King et al. (2002a) also pointed
tonicsolutioninwhichnucleoliandchromatinaredis- out that differences in map lengths are not surprising,
persedbeforefixation. Thelargerarmratioof SC6in given that various investigators employed a variety of
squashescomparedtoSCspreadscouldbeduetorela- mappingtechniquesondifferentmappingpopulations.
tiveshorteningoftheNORshortarminsquashes(per- Recently,KnoxandEllis(2002)showedinpea(Pisum
haps because the nucleolus obscures part of the arm) sativum)thatexcessheterozygosityinthemappingpop-
or lengthening of the short arm in SC spreads. In this ulationcanalsoleadtoinflationofthemolecularmap
regard, Sherman and Stack (1992) observed that the comparedtochiasmacounts.Theyconcludethatanum-
short arm of SC 2 in tomato was often broken at the ber of factors contribute to map inflation when using
RNCrossoverMapsinMaize 857
Figure 6.—Histograms showing the distribution of RNs along the length of each maize SC. Each SC is represented on the
x-axis with the short arm to the left and the position of the kinetochore (kc) marked with a vertical line. The positions of the
NOR(6S)andknobs(1S,5L,6L,7L,9S)areindicatedasshorthorizontalbarsbeneaththex-axis.Thehistogrambarsrepresent
thetotalobservednumberofRNsineach0.2-(cid:2)mSClengthinterval.Superimposedovereachdistributionisathicksmoothing
linethatshowsthegeneraltrendoftheRNdistributionaswellasathinnerhorizontallinethatrepresentstheaveragenumber
ofRNsineach0.2-(cid:2)minterval.
molecular markers and that chiasmata and RNs yield zero or one RN (Table 3). When higher categories of
good estimates of crossover frequency. crossing over are compared, the number of bivalents
Here,weshowthatinKYSmaizethefrequencyofchias- with two chiasmata is higher than the number of biva-
matacompareswellwiththefrequencyofRNs,particularly lents with two RNs, while the number of bivalents with
when the frequency of homologous pairs with zero or three or more chiasmata is lower than the number of
onechiasmaiscomparedwiththefrequencyofSCswith bivalentswiththreeormoreRNs.Giventhedifference
858 L.K.Andersonetal.
Figure6.—Continued.
inresolution betweenRNs andchiasmata (Stacket al. Inbred KYS and the rate of crossing over in maize:
1989;ShermanandStack1995),itislikelythatmulti- IstherateofcrossingoverininbredKYSrepresentative
plecrossoversaremoredifficulttoidentifyusingchias- of the rate of crossing over for maize in general? This
matathanusingRNs,andsomeofthebivalentsclassified question is of importance because KYS has long been
withtwochiasmataprobablyhadthreeormore.Overall, used as the favorite inbred line for the study of maize
weestimatethatourcountsofchiasmatainmaizeunder- chromosomes, but genetic maps and molecular maps
estimate the amount of crossing over by (cid:1)10% com- typicallyusedatafromdifferentlinesandfromcrosses
pared to our estimates of crossing over based on RNs. that do not include KYS (http://www.agron.missouri.