based on random amplified polymorphic DNAs and recombinant inbred lines
A. Yezerski,* L. Stevens† and J. Ametrano*
*
King’s College, Biology Department, 133 North River Street, Wilkes-Barre, PA, USA; and
†
University of Vermont, Biology Department, Marsh Life Science Building, Burlington, VT, USA
Abstract
Tribolium
beetles provide an excellent and easilymanipulated model system for the study of genetics.However, despite significant increases in the availabil-ity of molecular markers for the study of genetics inrecent years, a significant genetic linkage map forthese beetles remains undeveloped. We present thefirst molecular genetic linkage map for
Triboliumconfusum
using random amplified polymorphic DNAmarkers. The linkage map contains 137 loci mapped onto eight linkage groups totaling 968.5 cM.
Genetic linkage maps are very useful in describing thearrangements of genetic markers based on patterns of theirinheritance. Creating such a map is a vital step in under-standing an organism’s genome. Genetic linkage mapsare the basis of studies such as quantitative trait loci (QTL)studies that strive to explain the genetic architecture ofimportant phenotypes. By creating genetic linkage mapsfor easily manipulated model systems, complex behavi-oural and physiological traits can be better defined in thehope that such knowledge can clarify complex processes inhumans.
Tribolium
beetles were originally the subject of researchto discover a method of controlling this stored grain productpest, but more recently these beetles have become more
useful as a model system (see Sokoloff, 1972, 1977 forsummary). Despite their many uses as genetic models,only recently has significant research been done to elucidateour knowledge of the arrangement of loci on the genome ofthis insect genus (see Beeman & Brown, 1999). Previously,genetic linkage mapping for
Tribolium confusum
and
Tribolium castaneum
has been limited to morphologicalmarkers, especially those of homeotic mutants (Sokoloff
et al
.,1967; Dawson & Jost, 1983; Beeman
et al
., 1996; Stuart
et al
., 1998). If
Tribolium
species are to continue to expandin their usefulness as genetic models, an intensive geneticlinkage map must be created. Because morphologicalpolymorphisms are limited in
Tribolium
compared with
Drosophila
(Kafatos
et al
., 1991; Hartl
et al
., 1992), a geneticlinkage map as intensive as that available for the fruit fly isnot as easily created. Therefore, molecular markers mustbe used to expand upon the limited knowledge that themorphological markers have given us about the
Tribolium
genome.Because comparatively little is known about
Tribolium
genomes, the most efficient way to incorporate molecularmarkers on to a genetic linkage map is to use a techniquethat does not require any a priori information about thegenome itself. One technique that fits this criteria is that ofrandom amplified polymorphic DNAs (RAPDs). As RAPD-PCR reactions utilize a single short primer (
≈
10-mer) thatpotentially anneals in many places throughout the genome,amplifying several unique regions at one time, the genomecan be screened for polymorphic markers without havingany previous sequence information. Genetic linkage mapsusing RAPD primers are beginning to be made for manyorganisms. Example studies include others on insects(Hunt & Page, 1995; Promboon
et al
., 1995; Antolin
et al
.,1996; Dimopoulos
et al
., 1996; Laurent
et al
., 1998). How-ever, many more are found in plants because of the easeof producing recombinant inbred (RI) lines by selfing(Reiter
et al
., 1992; Tulsieram
et al
., 1992; Echt
et al
., 1993;Giese
et al
., 1994; Grattapaglia & Sederoff, 1994; Nelson
et al
., 1994; Kurata
et al
., 1994; Bryne
et al
., 1995; Plomion
et al
., 1995; Kesseli
et al
., 1994; Stockinger
et al
., 1996).RAPDs are especially useful for small insects because theamount of DNA required for an RAPD reaction is about
Received 13 June 2002; accepted after revision 27 June 2003. Correspond-ence: Dr Ann Yezerski, King’s College, Biology Department, 133 North RiverSt., Wilkes-Barre, PA 18711, USA. Tel.: +1 570 208 5900 x. 5602; fax: +1 570208 6024; e-mail: [email protected]
100th the amount necessary for other molecular markertechniques such as microsatellites (Williams
et al
., 1990).The small DNA template requirement allows the techniqueto be repeated for many primers on one individual. In thecase of
Tribolium
beetles, up to 2000 mapping reactionscan be completed per individual.
One of the major criticisms of using RAPD-basedmarkers is that they are considered solely dominant(Williams
et al
., 1990, 1993). Therefore, heterozygotes goundetected unless RAPD primers are coupled with atechnique for separating heterozygotes from homozygousdominants, such as single stranded conformation poly-morphism (SSCP) (Antolin
et al
., 1996). This difficulty canbe especially problematic in linkage mapping.
One way of solving the problem of having dominantrather than codominant markers is to only measurehomozygous individuals. The lack of detection of heterozy-gotes can be alleviated by using RI lines. Bailey (1971) firstdescribed the creation of RI lines for mice. RI lines are con-structed by crossing two highly inbred parental strains of anorganism and then subsequently inbreeding the F
2
progenythat result. Although inbreeding can be relatively easilydone in plants that self-fertilize, other organisms are mostefficiently inbred to homozygosity via full sibling mating.This requires many more generations of mating beforesufficient homozygosity is reached (Haldane & Waddington,1931; Falconer & Mackay, 1996). Therefore, the numberand types of animals for which RI lines are available andwell-documented is limited largely to laboratory rodents(Oliverio, 1979; Taylor, 1989). These RI lines are onlybeginning to be used in a genetic linkage mapping capacityas their advantages become more apparent (Siracusa
et al
., 1989; Routman & Cheverud, 1995; Markel
et al
.,1996; Pravenec
et al
., 1996).Having RI lines has two main advantages for genetic link-
age mapping. Because RI lines have high homozygosity,the main problem of using the dominant RAPD-basedmarkers is curtailed. Therefore, the need to distinguishheterozygotes from homozygotes is diminished. A secondadvantage of using RI lines is that all representatives of aparticular RI line are nearly genetically identical, allowingfor replication in measuring either a genotype or a pheno-type even months or years after the line was measured pre-viously simply by using a new representative from the line.This is especially useful in using a map created with theselines to detect QTL, because, once the genotypes of anRI line are known, different individuals can be used tomeasure important phenotypes (Neumann, 1990, 1991; Burr& Burr, 1991; Plomin
et al
., 1991; Dixon, 1993).Despite the obvious advantages of coupling RAPD-
based markers with RI lines, this combination has beenused only rarely for animals (Cheah
et al
., 1994). Here weuse 182 RI lines of
Tribolium confusum
to map 138 RAPD-based markers into eight linkage groups. This map provides
new opportunities in using
Tribolium
beetles as a geneticmodel and will be the basis for not only more intensivemapping in the future but also future QTL studies that canelucidate the genetic basis of unique behavioural andphysiological traits present in these beetles.
Results
Identification of useful markers
Of the 240 primers screened, 114 primers (about 48%)had at least one polymorphic band between the b-+ andb-Pak strains. One hundred and fifty-eight total polymorphicmarkers were identified for an average of 1.4 markers perprimer. Primers that showed polymorphic bands had betweenone and six markers each. Not all 182 RI lines were scoredfor each of the 158 potential markers, but 90 markers werescored in almost all individuals of both crosses.
Segregation analyses
A number of loci had segregation ratios that were skewedsignificantly from the 1 : 1 ratio as determined by the
G
-test.Twenty-eight loci of the 158 loci scored (17.7%) did not fitthe criteria of equal segregation. Twenty-four of these lociwere included on the completed map (Fig. 1). Because rep-resentatives of reciprocal crosses were used and alleleswere scored based on their origin in one or the other ulti-mate parental line, the skews are categorized into twocategories: skewed towards the b-+ allele (shown by anasterisk on Fig. 1) or skewed towards the b-Pak allele(shown as a cross on Fig. 1). Many more loci (twenty of thetwenty-four) are skewed towards the b-+ parental source. Infact, a large region of Group V is highly skewed towards theb-+ form of the allele. Skewed loci could represent direc-tional selection in the lines against the one allele, althoughnot to the level of a lethal allele as there was still represen-tation of some of the under-represented allele.
Linkage analysis and loci ordering
The 137 loci placed on this map are ordered into eight link-age groups shown from largest size in centiMorgans (cM)to smallest size in centiMorgans (Fig. 1).
Tribolium confu-sum
has a karyotype distinctive to the genus with eightautosomes plus one neo-XY chromosome that is believedto have arisen via a translocation of the sex chromosomeson to an autosome (Smith, 1952a; Samollow
et al
., 1983;Juan & Petitpierre, 1990a,b). With eight linkage groups, thelinkage map almost mirrors the known karyotype but eitherindicates a higher level of interaction between the loci thanwould be expected by physical linkage alone or one chro-mosome has no significant representation by any set oflinked markers. The data in this study did not include aknown sex-linked marker or other methods to locate the sexchromosome and, therefore, there was no attempt to iden-tify the neo-XY chromosome. However, data collected in a
Figure 1. Linkage groups derived from linkage analysis of 137 RAPD markers on 182 recombinant inbred lines of Tribolium confusum beetles. Linkage was determined by a Chi-square test using an α level of 0.05. Genetic distance (in cM) values are shown cumulatively for each group along the left-hand side of the linkage group. Marker names run alongside the right-hand part of the linkage group. Results of a G-test for segregation ratios are shown as follows: skewed to b-+ originated alleles at P values of 0.05 (*), 0.01(**), 0.001(***) and 0.0001 (****), and skewed to b-Pak originated alleles at P values of 0.05 (†) and 0.01 (††). Pairs of markers outlined with a black box fit the criteria to be considered for additional experimentation to be considered truly codominant markers (see text).
QTL study using some of the same lines suggests LinkageGroup II as a candidate (A. Yezerski, unpubl. data).
Not all 158 loci that were scored were able to be placedon this map. The remaining twenty-one loci did not havehigh enough likelihood of data (LOD) scores to be placedwith any of the extant linkage groups. This is most likely theresult of the large number of missing data for these loci. Ofthese twenty-one errant loci, eight could not be sufficiently
linked with any other loci in the data set (OPAY17-580,OPC06-780, OPG08-1840, OPG10-1190, OPI12-680,OPJ01-695, OPJ04-600 and OPY16-650). Eight otherswere paired with only one other locus and not able to besufficiently placed on the map [OPAY14-1500 with OPI06-1030 (LOD 1.1), OPG10-450 with OPG18-480 (LOD 1.7),OPJ17-1170 with OPJ17-1180 (LOD 1.7) and OPY15-450with OPY15-800 (LOD 1.2)]. The last five of these twenty-one questionable loci had low, but significant, linkage withinan extant linkage group, but adding them to the groupdestroyed the linkage that had already been established.These include OPK20-1000, which links with OPG04-600on Group II (LOD 1.5), OPAY07-1860, which links withOPH11-1240 on Group VI (LOD 1.0), OPJ17-906, whichlinks with I07-975 on Group II (LOD 1.3), OPG18-1170,which links with OPH06-690 on Group I (LOD 2.6), andOPG02-1190, which links with OPH11-1060 on Group VIII(LOD 1.3).
The total map size is shown at 968.5 cM giving an aver-age of just over 7 cM between markers. However, thesemarkers are not spaced evenly throughout the linkagegroups. Linkage Group V, although not the largest, has thehighest apparent density at just 4.68 cM average spacingbetween markers and Linkage Groups VI and VII tie forthe least dense with just over 10.5 cM between markers.However, these averages are slightly misleading because,in several cases, two or more loci mapped to identicalpositions. This could occur because the markers are legiti-mately linked so as to have not segregated for ten or moregenerations of inbreeding. However, two other reasonsfor such a tight linkage are also possible. Because ofthe nature of RAPD markers, primers with very differentsequences originating from unrelated kits may span thesame exact region of the genome and, thus, find the samepolymorphism. This may be the case for the cluster ofmarkers in the middle of Group V stemming from primersfrom Kits AY, G, J and K. Of course this region might alsosimply be highly polymorphic and coincide with a hetero-chromatin region. Another possibility is discussed below iftwo markers actually represent one codominant marker.
Possible codominant loci
Although RAPD-based primers are considered dominant,there is a possibility of codominance. The method by whichthis could arise is shown in Fig. 2 and is explained furtherin the discussion. The linkage map figure indicates poten-tially codominant loci as locus names surrounded by ablack box. In order to be considered as codominant, twobands must be created by amplification with the sameprimer, their fragment sizes must differ by less than 2% insize, and one band must be consistently found in oneparental strain while being consistently absent in the other.In this case, the presence of both bands would indicate aheterozygous individual. In order to avoid the presumption
Figure 2. Schematic drawing representing the method by which random amplified polymorphic DNAs can be detected. (A) This schematic represents the amplification of a single strand of DNA from two individuals, each representing genetically differentiated strains. In Strain I, the RAPD primer finds two primer annealing sites and amplifies the region of DNA between them. In Strain II, a point mutation indicated by the black box prevents the primer from annealing on the complementary strand of DNA and amplification does not occur. (B) The agarose gel resulting from such an event would show a band (indicated by the arrow) for Strain I and the absence of that band (lane 7) for Strain II. A heterozygote in this situation would appear the same as Strain I because it has one copy of the amplified region. (C) This schematic represents the codominant situation where Strain I is amplified as noted before in A, but Strain II has an insertion of additional DNA between the primer annealing sites causing the amplified band to be larger that the band in Strain I. (D) The resulting gel in the codominant situation would have a high molecular weight band for Strain II (lane 1) and a low molecular weight band for Strain I (lane 3). A heterozygote can be detected in this situation by the presence of both bands as seen in lane 2. Further experimentation would be necessary to determine definitively this situation to be codominance (see text).
of codominance, each band was scored individually andtheir juxtaposition to each other later noted. The abovecriteria suggested that fourteen locus pairs could possiblybe codominant markers. However, in order for the markersto be truly codominant, they would have to map next to eachother on the map.
Of the fourteen potential codominant loci pairs, ninewere juxtaposed to each other on the map and are shownboxed in Fig. 2. Of these, only four were given the exact sameposition on the map. Of the remaining five of these, all werequite close in position except for the OPJ17 pair, whichwere placed 10.8 cM apart making true codominanceextremely doubtful. It is possible, however, that any dis-tance between connected pairs represents residual heter-ozygosity in the RI lines as homozygosity is a functionresulting in a parabolic approach to 100% homozygosityand, thus, heterozygosity can always still be present(Falconer & Mackay, 1996). Five other potential pairs thatmight have been cases of codominance by the initial criteriadid not map to the same site, and sometimes not even tothe same linkage group (OPAY11-1020 and OPAY-1030,OPG19-600 and OPG19-620, OPI12-675 and OPI12-680,OPG08-1820 and OPG08-1840, and OPK06-680 andOPK06-690).
Discussion
We mapped 137 RAPD-based markers on to eight linkagegroups with an average spacing of 7 cM between loci. Theresults of mapping these loci appear to suggest that the
T. confusum
genome is around 968 cM. We believe thatthe use of RI lines might cause this measurement to be anoverestimate. Although the use of RI lines is allowed usingthe
MAPMANAGER
program, it has been shown that usingRI lines often overestimates genetic linkage distancesbecause of the increased opportunities for recombinationas inbreeding occurs through several generations in creat-ing the lines (Silver & Buckler, 1986; Neumann, 1990;Pravenec
et al
., 1996). Although the
MAPMANAGER
programallows for choosing ‘full-sib’ inbreeding as an option, it doesnot allow for a selection of the number of generations thatthe lines were inbred. Each generation of inbreeding is anadditional opportunity for crossing-over events. Whereasthe self-fertilization process used in plants leads to 99%probability of homozygosity in just ten generations, fullsibling inbred lines are only at an inbreeding coefficientof 0.886 in the same number of generations (Falconer &Mackay, 1996). Instead it would take nearly twice thenumber of generations of full sibling mating to reach thehomozygosity level of self-fertilized lines. Therefore,although the program does modify the recombination frac-tion using specific RI line functions, it does not allow for theever increasing number of recombinants generated witheach subsequent generation of inbreeding.
MAPMANAGER
does not allow an input of the number of generations thelines were inbred, but it does offer calculations that aresuited for best use with RI lines because it considers inter-ference negligible (Manly, 1993).
The possible overestimation in map size is apparent whencompared with the estimated size of the sister species
T. castaneum
at 570 cM (Beeman & Brown, 1999). Althoughthe total map size estimate is not directly comparablebetween these two species, especially considering theirvarying chromosome numbers (
T. castaneum
has ten), thiswould be the closest comparison between publishedgenetic linkage maps for this genus. One of the causes ofthe disparity between map sizes could actually be based inthe methods chosen for the mapping process. Beeman &Brown used slightly fewer backcrosses as data points vs. theRI lines utilized in this study. The current computer modelsfor mapping deal with intercrosses and backcrosses ina much more accurate manner and, thus, Beeman & Brown’sestimate of total map size map may more accurately reflectthe recombinant of a typical
Tribolium
genome. However,rates of recombination have not been reported in thesespecies and may vary considerably. One advantage tohaving several RI lines available vs. using intercrosses orbackcrosses is that it allows for the more efficient additionof loci to the
T. confusum
map in the future.Total map size can also be reduced by some somewhat
arbitrary changes to the map. Mapping with RI lines usuallyresults in more linkage groups than results when usingbackcrosses or intercrosses (Pravenec
et al
., 1996). It isthen somewhat arbitrary whether to set a goal of creatingfew large linkage groups that might encompass a largergenetic distance or instead opt for many smaller linkagegroups that would give a smaller map size but have lesssuggestion of linkage with low LOD scores. The choice toleave the map at eight linkage groups with reasonablyspaced markers is based on the fact that it almost mirrorsthe number of chromosomes for this species, and the linkedmarkers had LOD scores similar to other maps utilizing RIlines. As will be discussed later,
Tribolium
beetles tend tohave less DNA in their chromosomes than equivalentcategories of beetles (Juan & Petitpierre, 1990a,b). Otherresearch has suggested that smaller chromosomes actu-ally have greater numbers of recombination events per unitof physical size in insects (Hunt & Page, 1995). Therefore,a small genome such as that of
Tribolium
, at approximately250 Mb (Alvarez-Fuster
et al
., 1991), might consistentlyhave overestimated total map sizes because it may beundergoing more recombination events per generation,allowing for relatively tightly linked loci to dissociate. Addi-tionally, this suggests that a single algorithm in a computerprogram such as
MAPMANAGER
cannot sufficiently accountfor different recombination fractions. The program does notprovide an opportunity to input a hypothesized recombina-tion fraction as does its competitor
The order of the loci within the linkage groups canalso be difficult to determine when using RI lines (Silver &Buckler, 1986). The accepted LOD scores for the order ofthe loci also tend to be lower with RI lines than when usingbackcross or intercross data because of increased recom-bination events between linked markers (Lander & Botstein,1989). Therefore, although the loci were considered linkedby our statistical criteria, several loci had LOD scores below2.0 when considering their order. Most studies not utilizingRI lines set a preliminary minimum LOD score at 3.0 ormore (e.g. Nelson
et al
., 1994; Postlethwait
et al
., 1994;Antolin
et al
., 1996; Davis & Yu, 1997). For instance, Link-age Group I with twenty-two loci has three instances ofLOD scores below 2.0. The three loci attached to thislinkage group by these low scores, OPK14-1000, OPAY10-1230 and OPG19-600, were all situated on the end of thelinkage group. Most loci linked with low LOD scores arelocated on the end of the linkage groups. The ends of thelinkage groups tend to have an inflation of centiMorgandistances for this reason (Dixon, 1993). However, the vastmajority of loci were ordered with LOD scores easilyexceeding even the 3.0 level. However, there has not beena threshold of LOD scores given for determining linkage forRI lines.
Despite the difficulties in ordering with RI lines and theinability to input numbers of generations into
MAPMANAGER
,there are numerous advantages to using RI lines with thisprogram and for future research that outweigh the potentialproblems with exact map distances. Unlike other forms ofbreeding crosses set up for mapping, RI lines continue tosupply individuals for future testing. A single beetle can pro-vide enough DNA for up to 2000 RAPD-PCR reactions (farmore than with other techniques), and if additional reac-tions need to be done, an additional beetle from the sameline can provide virtually identical DNA for future RAPD-PCR reactions or even other techniques because all indi-viduals within the same line can be considered virtuallyidentical. This is especially useful when using the mappinginformation for QTL studies. Because these studies striveto correlate information about the alleles present in an indi-vidual to the measurement of a phenotype, the replicationability of RI lines can be crucial. Information from lines usedin mapping studies from prior years can still be correlatedwith the measurements on other representatives of thelines for various phenotypes along with additional genotyp-ing information. This ability to use repeatedly an additionalmember of a certain RI line to represent the whole is anadvantage that certainly outweighs the possible difficultieswith overestimated map size.
As stated previously, RI lines are also very well suited touse with RAPD-PCR-based markers. The high homozy-gosity in the lines significantly reduces the chances ofencountering problems with the tendency of RAPD markersto be considered solely dominant. Although RAPD-based
markers must be considered dominant and bands scoredsimply as present or absent, it is possible that codominantmarkers exist in this data set. There are two establishedmethods by which RAPD markers produce polymorphism(Welsh & McClelland, 1990; Williams
et al
., 1990) (Fig. 2).However, with no a priori information about an organism’sgenome, actual codominance could not be defined herewithout additional experimentation to prove this situation.This would be done in two steps. A cross betweenhomozygous individuals for each of the fragment polymor-phisms would have to result in 100% of the progeny beingheterozygous demonstrated by having both bands present.Additionally, a cross between F
1
individuals would haveto produce a ratio that is not significantly different from1 : 2 : 1 in the F
2
generation. An additional check ofsequencing the region to determine sequence similaritywould confirm that it was a codominant marker. This wouldbe most easily done using direct amplification of lengthpolymorphism (DALP) (Desmarais
et al
., 1998).Sequencing with arbitrary primers such as RAPDs
can be difficult and require additional knowledge of thegenome. Therefore, our suggestion of codominance in ourmap is not based on these aforementioned experimentalcriteria, but suggested codominant loci could later betested in this manner for assurance. Our codominancecriteria include any loci that were produced by the sameprimer, differed by 2% or less in fragment size and eachlength was consistently scored in the opposite parentalstrains. The nine loci pair that fit the a priori criteria and thenwere found to be tightly linked could be considered forbreeding and sequencing studies in the future. If found to betruly codominant, these loci would be excellent candidatesfor determining the true level of heterozygosity in therecombinant inbred lines. Additionally, one potentiallycodominant pair, OPJ17-605 and OPJ17-715, may indicateyet another genetic phenomenon if this pair proved to betruly codominant. The apparent large distance betweenthese two markers may actually be an indicator of hetero-zygosity present in the lines beyond what is expected to beresidual at this level of inbreeding. This pair, if truly codom-inant, could indicate a locus experiencing heterosis inwhich a heterozygous condition is selected for throughoutthe inbreeding process. This is still possible in these linesbecause so many lines are lost to inbreeding depressionduring their development that it is likely that more linessurvived that have a nonlethal mix of these alleles at this,and possibly other, loci.
The RAPD markers also are beneficial in that they pro-vide many markers per reaction. Our results averaged 1.4markers per primer. This value is generally lower than otherinsect mapping projects using RAPD markers (7.7 markers/primer, Hunt & Robert, 1995; 1.45 markers/primer,Laurent
et al
., 1998; 6.6 markers/primer with SSCP, Antolin
et al
., 1996). It is a value, however, that matches well with
that found by Beeman & Brown (1999). However, the levelof polymorphism is comparable with the genetic variationbetween categories of organisms being studied and thislevel can vary considerably depending on strain or specieschoice for the particular experiment. Additional primerkits are available from Operon Technologies and othercompanies that might be able to increase that average.
Future studies should not only strive to add loci to thismap by additional RAPD markers and other techniquessuch as amplified fragment-length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs) and micro-satellites, but also compare total map sizes to the resultsreported here. Although additional RAPD polymorphismswould be reasonably easy to find because the techniquesare already available and with at least 960 other primersavailable from Operon Technologies alone, any problemsinherent in using such markers, such as lack of definablecodominance if the lines are not completely homozygous,would continue to plague the map unless other unrelatedtechniques could verify the level of recombination.
As additional markers derived from various techniquesare added to the map, identifying the chromosomes onwhich they reside will become more important. Morpholog-ical markers are available and have been mapped to spe-cific chromosomes in both
T. confusum
and
T. castaneum
(Sokoloff
et al
., 1967; Dawson & Jost, 1983; Stuart
et al
.,1991, 1998, 1993; Beeman
et al
., 1996; see also
Tribolium
website: http://bru.usgmrl.ksu.edu/beeman/Tribolium/maps/map.html for the current morphological marker-basedmap for
T. castaneum
). Backcrossing of the RI lines to themutant lines of
T. confusum
beetles could allow these largenumbers of molecular markers to be positioned on specificchromosomes. Those markers found in T. castaneum couldbe used to find probes for the actual loci in order to look forpolymorphisms. Two isoenzymatic loci, for malic enzymeand hexokinase-1, are sex linked and could further defineother loci that are carried on one of the sex chromosomes(Dawson & Jost, 1983). Unfortunately, most of the knownmutations mapped in T. confusum are homeotic mutantsthat tend to reduce survivorship, making the large numbersof scorable progeny that are necessary difficult to acquire.Ultimately, defining which chromosomes contain whichlinkage groups will be necessary for further detailed geneticcharacterizations of T. confusum.
Associating certain linkage groups to chromosomescould be a first step in physically mapping the Triboliumgenome. Although a daunting task for larger genomes suchas human and mouse, the Tribolium genome is smaller atan estimated 250 Mb (Alvarez-Fuster et al., 1991). If theestimated genetic linkage map size of 968.5 cM is accu-rate, then there should be approximately 2.1 × 105 bp/cM.With approximately half of this genome being noncodingsatellite DNA (Plohl et al., 1993), physical mapping wouldbe very possible for these beetles.
One use of a genetic linkage map is to begin definingthe genetic elements underlying phenotypes. QTL studieshave become very powerful in defining the major and minorgenes responsible for variation in a phenotype (Lander &Botstein, 1989; Jansen & Stam, 1994; Zeng, 1994; Zhenget al., 1996). RI lines have been shown to be very effectivein these studies, especially for the ability of using the samelines redundantly for separate traits (Plomin et al., 1991;Dixon, 1993). Using each RI line as a separate experi-mental unit that compares the quantified phenotype to thescored genotypes of the line allows for the use of a simpleregression to begin detecting a genetic basis for the trait(Haley & Knott, 1992). The RI lines that have been mappedfor this research differ in many interesting traits, includingemigration behaviour, fecundity and survivorship (A. Yezerski,unpubl. data), benzoquinone production (Yezerski et al.,2000), and parasite susceptibility (Yan & Norman, 1995;A. Yezerski, unpubl. data). We are currently in the processof quantifying the extant RI lines for the benzoquinoneproduction, parasite susceptibility, emigration behaviourand fecundity. In addition, using RI lines for QTL studieseliminates the criterion that the parental generation mustdiffer significantly in the trait in order to be detected usingQTL methodology because the process of inbreedingincreases the number of rare genotypes (Klein, 1978;Plomin et al., 1991). Therefore, any trait that can be sufficientlyquantified in the beetles from morphology, through physiol-ogy and even behaviour, could ultimately be mapped usingthe large number of lines and markers that will becomeavailable. Thus, a complex trait such as parasite suscepti-bility can not only be defined as a whole but also by meas-uring subset phenotypes such as feeding behaviour anddigestive physiology, which also contribute to the ultimatemeasurement of the trait.
This first genetic linkage map for T. confusum is the foun-dation on which a physical map can be developed and onwhich future studies of pleiotropy and epistasis within andbetween traits can be based. The 400 or so lines developedin anticipation of this project along with an additional 400 orso created from a cross between the b-I and b-IV strains(see Experimental procedures for the origins and nomen-clature of these lines) are also available for additionalresearch. Future work should increase the amount of avail-able markers, include detailed QTL studies and increasethis Tribolium species’ usefulness as a genetic model.
Experimental procedures
Derivation of recombinant inbred lines
The parental strains for these recombinant inbred lines are derivedfrom crosses between the laboratory b-+ and b-Pakistan (b-Pak)strains of T. confusum (following the convention of Thomas Park,strains of the species T. confusum have the prefix b; see Park et al.,1961; 1964 for origins of b-+, b-I and b-IV; b-Pak was obtained fromDr Ralph Howard, Kansas State University). These strains are from
different geographical locations and have been maintained asseparate laboratory stocks for over 10 years. Laboratory stocks aregenerally kept at relatively high population levels to keep within-strainvariance high. Therefore, the parental strains used in this experi-ment were first inbred by full sibling mating for six generations toensure a high homozygosity within the strains before intercrossing.Fifteen single pair, full-sibling crosses between a b-+ female andb-Pak male and fifteen reciprocal crosses between a b-Pak femaleand b-+ male were used to create heterozygous progeny. From eachof these fifteen crosses, up to thirty single pair crosses betweenthe F1 progeny were initiated. Successful crosses from thesematings between the F1s continued to be inbred by way of full-siblingmating for 10–12 generations, creating lines that are virtuallygenetically identical within lines and genetically distinct betweenlines. By the end of the twelve or so generations, 320 RI linesfor each of the two cross types for a total of 640 RI lines had beencreated. Of the 640 RI lines initially created, only 200 of the b-+ byb-Pak cross (female stated first) and 210 of the b-Pak by b-+ crosshave survived to this date. It is not uncommon to lose lines toinbreeding depression (Dixon, 1993). Of these, 106 of the b-+ byb-Pak crosses and seventy-six of the b-Pak by b-+ crosses wereincluded in this mapping study for a total of 182 RI lines utilized.
DNA extraction
Total nucleic acids were isolated from adult beetles by homogeni-zation in 100 µl lysis buffer (1% CTAB, 50 mM Tris (pH 8), 10 mM
EDTA, 0.75 M NaCl) followed by an addition of 100 mg/ml of Pro-teinase K and a 2-h incubation period at 60 °C. An additional35 µl of high-salt lysis buffer (1% CTAB, 50 mM Tris (pH 8), 10 mM
EDTA, 1.5 M NaCl) was added approximately half-way through theincubation period. The homogenized and incubated samples werethen subjected to the standard phenol/chloroform extraction pro-cedure (Sambrook et al., 1989). Isolated DNA was precipitatedwith sodium acetate (pH 5.2) and ice-cold ethanol. After air-dryingthe samples, they were resuspended in 20 µl TE buffer at 65 °C.Several representative samples were quantified using UV spectro-photometry. These samples determined that the extraction proce-dure resulted in consistent DNA template concentrations that weresubsequently diluted 1 : 100 to approximately 4 ng/µl before inclu-sion in the reaction.
RAPD-PCR
The reaction mix for each of the polymerase chain reactions con-sisted of the following: 6.98 µl of distilled and sterile water, 1.2 µlof 10× Reaction Buffer (Gibco), 0.5 µl of 50 mM MgCl2 (Gibco),1.2 µl of 10 mM dNTP (Gibco), 1 µl of 10 mM RAPD primer, 0.12 µlof Taq polymerase, and 1 µl of DNA template at 4 ng/µl. The 12-µlreaction mixes went through the following temperature cycle on anMJ Research thermocycler: four cycles of 1 min at 94 °C, 1 min at35 °C, a ramped increase of temperature of 0.3 °C/s until a tem-perature of 72 °C was reached, and 2 min at 72 °C; a thirty-onelooped cycle of 10 s at 94 °C, 30 s at 35 °C and 1 min at 72 °C; andfinally a 10 min hold at 72 °C.
Visualization and scoring of amplification products
PCR products were run on 2% or 3% agarose gels (depending onthe level of separation needed) at 120 V for 3.5 h and stained withethidium bromide for 15–30 min and then destained for 1 h in dis-tilled water in order to increase contrast.
Polymorphic bands (determined as described below) wereverified by the presence of parental PCR products. RI line repres-entative progeny were then scored as presence or absence of thissame product. Some amplifications were repeated in order toassess repeatability of the technique or when necessary to clarifyamplifications that were not easily scored in the first run. Size ofthe fragments was verified using an extended 100 bp ladder (FMCBioProducts).
RAPD primer screening and designation
Primers were screened for polymorphism between the parentalstrains by amplifying DNA from 4–6 representatives each from theb-+ and the b-Pak strain and comparing the results on an agarosegel for consistent presence or absence of a certain amplified frag-ment. Two-hundred and forty Operon Technologies primers fromKits A–K, Kit AY and Kit Y were screened in this manner. Thosethat showed polymorphism between strains while being homoge-neous within strains were used on representative individuals fromthe 120 RI lines. When screening the RI line representatives, 4–6representatives from the original parental lines were included inthe reaction set to verify that the scored bands were indeed poly-morphic. Polymorphic marker designations all begin with ‘OP’ forOperon Technologies. This designation is followed by the OperonKit letter(s) and then the specific primer number within the kit in atwo-digit format. The approximate fragment size in base pairsdetermined by comparison with an extended 100 bp ladder followsthe alphanumeric designation preceded by a hyphen.
Linkage analyses
Genotypes were determined for all appropriate lines and enteredinto MAPMANAGER QT (version b28 for the Power PC) as full sib-mated RI lines (Manly & Elliot, 1991; Manly, 1993; Manly & Olson,1999). All identified loci were first tested for 1 : 1 segregation ofthe parental alleles using a G-test at a 95% confidence interval(Manly, 1998).
The MAPMANAGER program uses a Chi-squared distributionbased on the number of recombinants and the number of inform-ative progeny to determine significant linkage between loci whenthere are more than 100 progeny scored (Manly, 1993). Once agroup is determined, the order of the loci within this group is deter-mined by maximum likelihood methods assuming no interference(Manly, 1993). This assumption is valid in this case because RIlines created by full-sibling mating have negligible interference(Manly, 1993). Initially, loci with very high likelihood of linkage weregrouped together. Orders within the linkage groups were deter-mined by using the ‘rearrange’ command, which uses the Metrop-olis algorithm (Press et al., 1990) in order to minimize interlocusintervals. Each linkage group was subjected to three repetitions ofthis algorithm and the order minimizing the linkage group lengthwas accepted. Some additional loci were added to linkage groupsafter linkage to two or more loci within an established group wasdetermined. After adding additional loci, the group was again ‘rear-ranged’ to search for the arrangement with the highest LOD score.
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