Silkworm genomics progress and prospects

Transcription

1 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY Silkworm genomis progress n prospets J. Ngrju, n M. R. Golsmith** Lbortory of Moleulr Genetis, entre for NA Fingerprinting n ignostis, EIL Ro, Nhrm, Hyerb , Ini **eprtment of iologil Sienes, University of Rhoe Isln, Kingston, RI, , USA The biology n genetis of silkworm, ombyx mori, is the most vne of ny lepioptern speies. Its rih repertoire of geneti resoures n potentil pplitions in seriulture n s moel for other Lepiopter le to the initition of genomis reserh. uring the pst ee muh effort hs been me in the res of mrker evelopment, n moleulr mps hve been onstrute in stnr strins with the use of RFLPs, RAPs, ISSRs, STSs, n mirostellites. The potentil pplitions of moleulr mrkers n linkge mps inlue stok ientifition, Mrker Assiste Seletion (MAS), ientifition of Quntittive Trit Loi (QTL), n, ultimtely, positionl loning of visible muttions n QTL. To these ens, A librries hve been onstrute n re being use to mke lrge-sle physil mps, with mrkers bse on ESTs s frmework nhors. Altogether this work provies fountion for ientifition of gene funtion, gene n hromosome evolution, n omprtive genomis. THE silkworm, ombyx mori, omestite for silk proution for bout 5000 yers is the most well-stuie lepioptern moel system beuse of its rih repertoire of well hrterize muttions ffeting virtully every spet of the orgnism s morphology, evelopment, n behviour n its onsierble eonomi importne. y virtue of long history of silkworm rering for ommeril purpose, silkworm genetis hs been the subjet of onsierble reserh interest resulting in reful olletion, tloguing n mintenne of vrious silkworm geneti stoks of onsierble sientifi n eonomi interest. Toy, the opportunities for geneti mnipultion n stuy of the silkworm,. mori inlue more thn 400 visible muttions out of whih 200 hve been ssigne to onventionl linkge groups overing M (ref. 1). These muttions ffet mny funmentl spets of the inset s life yle, inluing egg n egg shell formtion, erly embryoni evelopment n pttern formtion, lrvl feeing behviour, molting, embryoni ipuse, et. In ition, vst rry of istint geogrphil res n inbre lines tht represent vrition for number of qulittive n quntittive trits of bsi biologil n eonomi interest inluing For orresponene. (e-mil: jngrju@ boy size, silk qulity, feunity, pthogen resistne, n het tolerne re vilble. In ft, the silkworm. mori is genetilly the best known inset next only to the fruitfly, rosophil melnogster. The hploi genome size of. mori is estimte to be 530 Mb (ref. 2), 3.8 times tht of. melnogster n one-sixth the size of the mmmlin genome. Supporte by the infrstruture of t n geneti resoures vilble for reserh, the silkworm is key moel orgnism in Lepiopter whih inlues more thn 160,000 speies, of whih ombyoi moths inlue silkmoths of eonomi importne n Notui moths, the lrgest group in the orer n inlues some of the most evstting pests of griulture, prtiulrly Heliothis (H. viresens, H. rmiger, H. ze). The genomi informtion of the moel speies. mori shoul be pplible to the most importnt speies in Lepiopter. omprtive stuies on genome sequenes between Lepiopter n other publishe genome sequenes oul unover lepiopter-speifi genes. Prouts of suh novel genes oul serve s trgets for lepiopternspeifi insetiies. ombyoi moths serete iverse vrieties of silk fibres. These speies inlue. mori of fmily ombyie n wil silkmoths tht belong to Sturniie, Anthere mylitt (Inin tropil tsr silkmoth), A. pernyi (hinese ok tsr silkmoth), A. ssm (Inin golen silkmoth), A. ymmi (Jpnese ok silkmoth) n Philosmi ynthi riini (Inin stor silkmoth). Silk proution bse on these moths, speilly. mori, A. pernyi, A. mylitt n A. ssm plys importnt role in rurl eonomies of mny populous eveloping ntions. Six million people in Ini lone re involve in seriulture, whih involves intensive lbour n provies the key to improving lol qulity of life. In orer to mke seriulture eonomilly vible, genes ffeting growth rte, yiel, fibre qulity, virus resistne n be tgge with moleulr mrkers for rpi onstrution of genetilly improve strins. onsiering the unique experimentl vntges of this orgnism n its eonomi importne, n Interntionl onsortium on Lepioptern Genomis ws reently forme to promote interntionl oopertion to sequene the genome of. mori n to unertke omprtive genomis of other eonomilly importnt Lepiopter 3. Suh interntionl oopertion is expete to foster knowlege both in the bsi n pp- URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST

2 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY lie spets of inset siene. The urrent efforts of severl lbortories ontributing to the silkworm genome projet hve, s their immeite objetive, the genertion of high ensity moleulr linkge mp, evelopment of tools n tehniques to fingerprint silkworm vrieties, ientifition of lepiopter-speifi genes, n genertion of lone-bse physil mp tht will llow virtully immeite ess to ny efine region of the euhromtin. The long-term objetive is to ientify the position, NA sequene n funtion of ll the genes in the orgnism. These re hievble gols onsiering the revolutionry evelopments in nlytil tools n tehnil fesibility of lrge-sle NA sequening s emonstrte reently in omplex genomes suh s rosophil n humn 4 6. In this review, we summrize the results rue in vrious genome nlysis projets now unerwy in severl lbortories in the re of moleulr genetis n genomis of the silkworm. Geneti mrkers In the pst ee, severl key vnes in moleulr biology hve gretly vne the impt of moleulr genetis in biology. Most importnt hve been: (i) evelopment of the polymerse hin retion (PR), whih mplifies speifi strethes of NA to usble onentrtions, (ii) the pplition of evolutionrily onserve sequenes s PR primers, (iii) the vent of hypervrible mirostellite loi s geneti mrkers n (iv) the vent of routine NA sequening in biology lbortories. These innovtions, ouple with the reent explosion of powerful nlysis n reltively user-frienly omputer progrms, hve elerte the pe t whih moleulr geneti t n be use for vrious progrmmes. These evelopments le to the etetion, nlysis n exploittion of nturlly ourring NA sequene polymorphisms in eukryotes n li fountion for ontemporry genomis of severl orgnisms. Polymorphi geneti mrkers hve foun potentil pplitions in niml n plnt improvement progrmmes s mens for vrietl n prentge ientifition, onstrution of moleulr linkge mps n evlution of polymorphi geneti loi ffeting quntittive eonomi trits. A wie rry of NA mrker tehniques is vilble for geneti stuies. All NA mrkers reflet ifferenes in NA sequenes. Typilly, in iploi orgnism, eh iniviul n hve one or two ifferent sttes (lleles) per hrter (lous). The hoie of prtiulr geneti mrker often epens upon the purpose of the stuy n is usully tre-off between prtility n preision of geneti mrkers. One mnifesttion of this is the ihotomy between multilous NA tehniques whih inlue Rnom Amplifie Polymorphi NAs (RAPs), Amplifie Frgment Length Polymorphisms 416 (AFLPs) n Inter-Simple Sequene Repet PR mrkers (ISSR-PR) n single lous tehniques whih inlue single opy nuler NA regions n mirostellites. Multilous methos result in simultneous visuliztion of mny ominnt mrkers n re tehnilly onvenient, but hve some mrke weknesses n limittions, inluing ominnt inheritne the NA frgment n be sore only s present or bsent, n in mny ses substntil proportion of vrition they etet n be non-heritble or not even erive from the trget orgnism. y ontrst, single lous mrkers re quite informtive beuse they provie lleles whose zygosity sttus n be ssye esily. evelopment of moleulr mrkers is importnt in the silkworm for onstrution of linkge mp, fingerprinting of strins for breeing, n mrker-ssiste seletion. In the erlier stuies, fingerprinting nlyses of silkworm strins hve been rrie out using RAPs 7, heterologous ministellite probes, km(2)8 (refs 8, 9), M13 (ref. 10), n ISSR-PR (ref. 11). Geneti linkge mps of silkworm hve been onstrute using RAPs 12 14, RFLPs 15, n AFLPs 16. Most of these mrkers, exept RFLPs, re ominnt n strin-speifi. onsiering the geneti informtiveness offere by mirostellite loi, whih re o-ominnt, mirostellite isovery progrmme in the silkworm genome ws initite 21. Mirostellites, lso known s simple sequene repets (SSRs), re short strethes of nuleoties in whih motif of one to six bses is tnemly repete, hve emerge s iel mrkers in eukryoti genetis. They re ubiquitous in prokryoti n eukryoti genomes n re rnomly istribute, both in protein oing n non-oing regions. The sequenes in mirostellite motifs n iffer in repet number mong iniviuls, proviing rey soure of polymorphism. With the vent of the PR, this property of mirostellite NA ws onverte into highly verstile geneti mrkers PR prouts of ifferent lengths n be mplifie using primers flnking the vrible mirostellite region (Figure 1). ue to the vntges offere by mirostellites they hve been use to onstrut linkge mps 20. In ition to their ler utility for prtil pplitions, informtion bout the istribution n vribility of mirostellite sequenes in the genome of given speies n help eluite its geneti history from the stnpoint of evolution n rtifiil seletion. Mirostellite motifs in the silkworm genome In previous stuy 21, nlysis of mirostellite loi lone n hrterize from. mori sub-genomi librry ws reporte. The totl frtion of the genome nlyse (2810 kb), whih ounts for 0.53% of the ombyx genome (530 Mb) yiele 57 (A) n /(GT) n n URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST 2002

3 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY Alleles #1 AAAAAAAAAAA #2 AAAAAAAAAAAAA #3 AAAAAAAAAAAAAAAA Genotypes 1/1 2/2 3/3 1/2 1/3 2/3 Figure 1. Mirostellite lleles n their etetion. Three lleles of ifferent sizes t mirostellite lous ompose of (A) repet re shown. The rrows inite the lous-speifi primers esigne for the region flnking the (A) repet, use for PR mplifition. Illustrtions of the vrious gel ptterns tht woul be observe with ifferent llele ombintions re inite. Tble 1. Perentge of ifferent tegories of (GT) n n (T) n mirostellites in silkworm, honey bee (Estoup, A. et l. 42 ) n humn (Weber, 1990). Number in prenthesis is the number of (GT) n or (T) n mirostellites nlyse Silkworm Honey bee Humn Type of motif GT (21) T (7) GT (23) T (52) GT (57) Perfet Imperfet ompoun (GA) n /(T) n hrbouring lones. The frequeny of ourrene of inuleotie repets, ws estimte to be, on verge, one (GT) n every 49 kb, n one (T) n mirostellite every 104 kb. The frequenies of ourrene of these two types of mirostellite motifs ompre well with those reporte in honey bee, pig, rt n humn. The perfet (no interruption in the run of repets), imperfet (interruption in the run of repets) n ompoun ( mixture of repets of two or more motifs) repet mirostellites in the silkworm genome were on the orer of 62%, 29% n 7% respetively (Tble 1). This is in lose greement with 72% perfet, 28% imperfet, n 11% ompoun repets in humn 22 (Tble 1). The primer pirs evelope for the flnking sequenes of the mirostellite loi isolte from the silkworm genome suessfully mplifie the repets prouing vriety of o-ominnt mrkers. This inite tht the mirostellite loi were onserve n not generlly erive from trnsposble elements, whih re expete to be mobile, giving rise to null lleles. A summry of the mirostellite loi n PR mplifition onitions for sets of representtive mirostellite loi re given in Tble 2. The polymorphisms, whih mnifeste s size ifferenes in the PR prouts, oul be esily sore ross silkworm strins (Figure 2). For exmple, the 15 mirostellite loi, 11 (GT) n n 4 (A) n, revele totl of 113 lleles in 13 silkworm strins. The number of lleles sore t eh lous in these strins vrie from s few s 3 to s mny s 17. The (A) n motifs showe teneny to revel greter number of lleles ompre to (GA) n motifs. For exmple, 11 (GT) n loi revele totl of 85 lleles, rnging from 3 to 17, with n verge of 7.7 lleles per lous wheres the four (T) n loi revele totl of 28 lleles rnging from 5 to 9 with n verge of 7 lleles per lous. Heterozygosity vlues All 15 mirostellite loi revele lleli length polymorphism in the 13 silkworm strins nlyse. The verge heterozygosity vlue ws 0.79, rnging from 0.66 to The most polymorphi lous, st2763, whih revele 17 lleles, lso showe the highest heterozygosity URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST

4 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY Tble 2. Mirostellite loi, repet pttern, forwr n reverse flnking primer sequenes, number of lleles, lleli rnge, heterozygosity vlues n nneling temperture (T) in ombyx mori Primer sequene Lous Repet No. Allele size Hetero- Mgl 2 symbol motif length Forwr Reverse lleles rnge (bp) zygosity T ( ) (mm) St158 (A) 9(A) 4 5 -ttgttgt-3 5 -gtttttttt St211 (GT) gtgtgttg-3 5 -gtttttttt St256 (A) 5(TA) 7 5 -ttgtgggggtgt-3 5 -tgtggggggtt St346 (GA) 9 5 -ggggggtgg-3 5 -tggtttgtggtgt St892 (GT) ttgttggttt-3 5 -ttggtgttttgtt St951 (GA) ttgtgtttggg-3 5 -ttgtgttt St962 (GT) tttttt-3 5 -tgtgtgggtgtgtt St1013 (GT) 9 5 -gtgtgggtggt-3 5 -tgtttt St1411 (GT) 8(GT) 5 5 -gtgtttgtggtgg-3 5 -ttgttttttttttttg St1423 (A) tttgtggttt-3 5 -gtgtttttg St1893 (A) tggtgttttt-3 5 -tttggtg St2550 (GA) ggtttgtggt-3 5 -ggtgggttgtgtt St2604 (T) 9 5 -gtgttgtt-3 5 -gtttgtttttgtt St2763 (GT) 22(GT) 7 5 -ggttttt-3 5 -gtggtttgttg St3513 (GT) 5(TGG) 6(TA) 2 5 -gtttgtttgt-3 5 -tggtttttttttg bp 200 bp 100 bp M Hu 204 KA N 1 N 7 N 18 Figure 2. Alleli vritions etete by mirostellite lous (mestst10) in ifferent silkworm strins. N 4 2 of This lous hrboure two strethes of (GT) motifs, one with 22 repets n nother with 7 repets. The lous st892, with (GT) 10 motif tht revele only 3 lleles in the 13 silkworm popultions, showe the lowest heterozygosity vlues. There ws no ler eviene with regr to the reltionship between the repet length n egree of polymorphisms. For exmple, lous st951 (GA) 23, revele 5 lleles with heterozygosity vlue of 0.68, n the lous st2604 with (GA) 9 showe 8 lleles with heterozygosity vlue of The loi tht were similr in length lso iffere in their number of lleles n heterozygosity. The loi st211, st892, st962 n st1893, whih rry repet motifs of (GT) 10, showe 6, 3, 6, n 7 lleles respetively. These results re in ontrst to the reports on humn n other speies where longer repets hve been reporte to generte higher egree of polymorphism 22,23..nihi Gungnong Mori Nistri Pure Mysore izo Srupt Geneti iversity stuies A vriety of moleulr tehniques hve been employe suessfully in silkworm to etet genome-wie polymorphisms. These methos inlue Restrition Frgment Length Polymorphisms n vriety of PR-bse tehniques. In stuy reporte by Ngrju et l. 8 n Shrm et l. 9, bne krit ministellite, km(2)8 ontining 545 bp NA sequene onsisting of 66 opies of GATA repets intersperse with vrible number of inuleotie (TA) repets in severl lotions, ws use s probe. The probe revele intr- n inter-popultion geneti iversity when teste on 13 ivergent silkworm genotypes. The pheneti nlysis of RFLP t seprte the 13 strins into three groups: one onsiste of ll ipusing strins (exept HU 204 ); the seon onsiste of ll non-ipusing strins (exept Pure Mysore n Nistri); n the thir onsiste exlusively of Pure Mysore n Nistri (Figure 3). In nother stuy, six nonymous multilous probes obtine from PstI subgenomi silkworm librry, were use on the sme set of 13 silkworm strins 24. The geneti informtiveness expresse in terms of number of loi revele (effetive multiplex rtio, EMR) n mount of polymorphism etete (iversity inex, I) ws ompre with the three PR-bse tehniques, Rnom Amplifie Polymorphi NA (RAP), Inter-Simple Sequene Repet PR (ISSR- PR) n simple sequene repets (SSRs). The six multilous RFLP probes proue 180 prouts of whih 97% were polymorphi; the 15 SSR loi gve rise to n verge of 8 lleles eh, of whih 86% were polymorphi; n ISSR-PR proue 39 frgments of whih 77% were polymorphi (Tble 3). The highest iversity inex ws observe for ISSR-PR (0.957) n the lowest (0.744) for RAPs (Tble 4). The RAPs, ISSR-PR 418 URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST 2002

5 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY Hu 204 KA N 1 N 7 N Hu 204 KA N 7 N 1 N ISSR N 4 2 Gungnong Srupt Mori.nihi PureMysore Nistri izo N RAP N 4 2.nihi Mori Srupt Nistri PureMysore izo Gungnong N Hu 204 KA N 1 N 7 N Hu 204 KA N 1 N 7 N RFLP N 4 2.nihi Gungnong Mori Nistri PureMysore izo Srupt N 72 SSR N 4 2.nihi Gungnong Mori Srupt Nistri PureMysore izo N Figure 3. enrogrms generte from geneti similrity mtries lulte using Nei n Li oeffiients for ISSR-PR, RFLP, RAP n SSR mrker ssys., ipusing silkworm strins; N, nonipusing silkworm strins. Numbers on the noes inite the number of times prtiulr brnh ws reore per 100 bootstrp replitions following 1000 replitions. Tble 3. A summry of eh type of mrker ssy performe on 13 silkworm strins Totl number Totl no. Totl poly- Men no. prouts Perentge of polyof ssys prouts morphi prouts per ssy morphi prouts SSR 15 (primer pirs) Inter-SSR 6 (primers) RAP 40 (primers) RFLP 6 (probes) Tble 4. A summry of men I, EMR n MI for ifferent mrker ssys of silkworms Men effetive Mrker Men iversity multiplex rtio Mrker inex ssy inex (I) (EMR) (MI) SSR Inter-SSR RAP RFLP I = 1 pi 2, where pi is the llele frequeny of the ith llele. EMR = n p(n p/n), where n p is the number of polymorphi loi n n is the totl number of loi. MI = I EMR. n multilous RFLPs lerly seprte the ipusing n non-ipusing silkworm vrieties, s i the km(2)8 probe (Figure 3). The nlysis of the sme set of 13 silkworm strins using 5 ifferent mrker systems highlighte the utility of eh of the mrker systems in geneti nlysis of silkworm. These results show tht ISSR-PR n multilous RFLP mrkers re powerful tehniques for fingerprinting silkworm vrieties. Although most ISSR loi re ominnt rther thn o-ominnt, ISSR-PR mrkers offer severl vntges over RFLPs for genotyping, the mjor one being the rpi proution of lrge number of mrkers in ost-effetive mnner. euse of these vntges, the ISSR-PR tehnique hs potentil use in silkworm breeing, germplsm evlution, n geneti mpping. The reently utomte ISSR-PR tehnique by inluing fluoresent ye in the PR retion followe by nlysis on n AI utomte sequener 25 hs been shown to be iel for high-resolution mpping experiments n NA profiling (see the lter setion of this rtile). Although multilous RFLP probes offer fetures URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST

6 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY suh s high reprouibility n esy soring, the requirement of lrge quntity of templte NA, n Southern hybriiztion rener them less suitble for lrge-sle stuies. In the RAP tehnique, though wiely use, mny ftors suh s onentrtion of Mg 2+, qulity of the templte, therml yler n the soure of polymerse hve been reporte to ffet the repetbility of the results between ifferent lbortories. In this spet, RAP nlysis my not be metho of hoie for lrge-sle genotyping experiments. onsiering the reprouibility, geneti informtiveness n spee, SSRs re the most useful mrkers mong ll the mrker systems. Sine SSRs re o-ominntly inherite, multilleli in nture, n n be multiplexe (5 6 loi n be nlyse on single lne) n utomte, they re useful for geneti iversity, peigree evlution n geneti mpping stuies. Although the initil ost to evelop lous-speifi primers is high, the vntges tht they re robust n re menble to utomtion ultimtely inrese the ostbenefit rtio, prtiulrly onsiering the vlue of being ble to ompre t sets iretly. ross-mplifition of ombyx mirostellites in Sturnii silkmoths n Notui moths Avnes in lepioptern genomis hve been limite by generl lk of geneti informtion for iniviul speies beuse of lk of geneti tools with bro pplibility ross speies 26. An essible pproh to suh problem is to look for hromosome onservtion ross speies by using omprtive genome mpping. Reent results 27 (Ngrju et l., uner preprtion) show tht mny ombyx mirostellite loi re onserve ross Sturnii silkmoths whih inlue most of the ommerilly importnt wil silkmoths suh s Anthere ssm (Mug silkworm), A. mylitt (Tropil tsr silkworm), n A. pernyi (Temperte tsr silkworm), s well s Notui moth, Helioverp rmiger, serious griulturl pest. onsiering tht the genetis of these moths hs hrly been stuie, the mirostellite evelopment progrmme in. mori mkes it possible to rry out moleulr geneti nlyses of these moths without investing in mirostellite evelopment progrmme for eh speies. The use of heterologous primers will signifintly reue the ost of eveloping mrkers for the wil silkmoths n griulturl pest, Heliothis spp. esies, s mpping is lrey unerwy in Heliothis 28,29, omprtive mpping pproh will enble the preition of the presene of mirostellites ssoite with genes of interest. Suh onserve loi oul be potentilly use for ssessing the evolutionry reltionships within n ross speies with resonble egree of ury. Linkge mps The first ttempt to onstrut moleulr linkge mps for the silkworm utilize RFLPs generte by 15 hrter- NNNN(A)n (A)nNN A repet NNNNNNNNNNNNAAAAA NNNNNNNNNNNTGTGTGTGTG NNNNNNNNNNNN Genom i NA NNN A repet NN(A)n (A)n NNNN PR prout 3 -nhore prim er PR prout 5 -nhore prim er Figure 4. Inter-Simple Sequene Repet-PR (ISSR-PR). A single primer trgeting (A) n repet nhore either t the 3 (tel rrows) or t the 5 en (yellow rrows) of the repet, is use to mplify genomi sequene flnke by two inversely oriente (A) n repets (pte from Zietkiewiz et l. 41 ). 420 URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST 2002

7 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY Figure 5. The Menelin segregtion of Fluoresent-ISSR-PR (FISSR-PR) mrkers in silkworm. The FISSR-PR mrkers were generte using primer 5 RAY RAT R (GA) 7 3 on two prentl strins, p50 n 108, n their F 1 n F 2 offspring. The rrows n rrowhes inite mrkers speifi to p50 n 108, respetively. ize single opy sequenes, 36 nonymous sequenes erive from folliulr NA librry, n 10 loi orresponing to low opy number retrotrnsposon, mg 15. Reently, this work ws extene using s probes prtilly sequene NAs (ESTs) erive from n embryoni n mternl mrna librry. y using the lssi bkross strtegy to etet linkge (see below), smll number of segregnts (e.g., 15 nimls) from bkrosses of single heterozygous femles erive from strins p50 n J02 to inbre J02-erive mles hve been use to ssign more thn 200 oominnt mrkers to 27 linkge groups n one inepenent mrker. Integrtion of these linkge groups with the onventionl geneti mps of morphologil n biohemil mrkers is unerwy, together with the reiprol bkrosses esigne to ssign reombintion istnes within eh linkge group (W. Hr, H. nno, H. Fuji, pers. ommun.). An RAP linkge mp of silkworm hs been onstrute using 320 rnom primers, whih proue 243 polymorphi prouts between two prentl silkworm strins, 108 n P50. In the F 2 popultion of 101 iniviuls use for these mps, segregtion rtios of 168 bns were nerly 3 : 1 in hi squre tests, showing Menelin inheritne 12. The MAPMAKER progrm sorte 168 bns into 29 linkge groups. The sum of mp istnes is pproximtely 900 M. Reently, Ngrj 14 hs onstrute Z-hromosome RAP linkge mp using bkross popultion () erive from rossing n F 1 (mle) heterozygous for reessive sex-linke mrker, trnslusent lrvl skin (o) homozygous trnsluent (o) (femle). In silkworm, both F 2 (Figure 6) n bkross popultions (Figure 7) hve been use by ifferent groups. Four groups hve use n F 2 popultion erive from ross of the sme two referene prentl strins, 108 n P50. Although both strins re of hinese origin, they exhibit high phenotypi iversity for suh omplex hrters suh s size, growth rte, ipuse, nutritionl requirements, generl vigour n ooon properties suh s ooon weight, ooon shell weight, n silk fibre length, suggesting tht onsierble polymorphism exists t NA level. ifferent groups hve use ifferent number of F 2 offspring for mpping, ffeting the bility to etet linkge between istl mrkers. Shi et l. 15 hve use 52 F 2 progeny for RFLP mps, wheres Promboon et l. 12 n Ysukohi 13 hve use 101 n 166 F 2 progeny, respetively, for mps bse on RAPs. The SSR n Fluoresent ISSR (FISSR) mrkers hve been mppe reently using the sme 101 F 2 offspring use by Promboon et l. 12. (Muthulkshmi, Kthirvel, Selvenrn n Ngrju, uner preprtion). The FISSR mrkers re prtiulrly vntgeous for high throughput genotyping in mpping of genomes 25 (Figures 4 n 5). For onstruting high-ensity linkge mps, lrge number of F 2 iniviuls nee to be nlyse using number of mrkers. The templte requirement in the se of other onventionl PR methos is reltively high. The FISSR ssy provies lrge number of NA mrkers per primer n llows etetion of mrkers with s little s 2 5 ng of templte NA, on n utomte sequener obviting the neessity for using riotive isotopes. kross popultions hve been use for mpping by three other groups 14,16. Ngrj 14 hs use polyvoltine strin, Pure Mysore, whose lrvl skin is norml white (+/+), n bivoltine strin (o/o), whose lrvl skin is trnsluent, ontrolle by reessive gene, o, lote on the Z hromosome. Fifty five progeny were use for mpping. Tn et l. 16 hve use similr bk ross with 47 offspring. Similrly, Hr n oworkers hve use bkross strtegy to mke initil linkge ssignments of oominnt mrkers using smll number of segregnts (15 offspring), to be followe up with muh lrger number of progeny (on the orer of 100) to evelop reombintion mp (W. Hr, pers. ommun.). The use of bkross popultions for mpping is wellestblishe in ombyx genetis n reently in griulturl pests. It is vntgeous sine it vois omplitions rising from the use of ominnt mrkers n filittes biphsi pproh to stuy linkge ue to the ourrene of hismti oogenesis (Figure 7). This URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST

8 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY pproh ws elegntly emonstrte reently by Hekel et l. in the mpping of mjor gene onferring resistne to t toxin in the imonbk moth, Plutell xylostell 28, n in the tobo buworm, Heliothis viresens 29, using AFLP mrkers. In these stuies two bkross popultions were rete, one by rossing n F 1 femle (erive from the initil ross between suseptible strin n resistnt strin) with its suseptible prent n seon bkross popultion by rossing n F 1 mle with the suseptible femle prent. From the first, popultion informtion on the linkge group ontributing to the trit for resistne ws erive. This is possible beuse in the bsene of rossing over in the F 1 femles, ll of the genes present on the linkge group hrbouring the gene for t toxin resistne woul show bsolute linkge. The seon popultion ws use to etermine the lotion of the trit of interest in the orresponing linkge group beuse rossing over uring gmetogenesis in the F 1 mles results in reombintion of mrkers n thus llows one to etermine mp istne between mrkers. A similr strtegy bse on the moleulr mp oul be use to trk genes of eonomi interest in silkworm. Quntittive genetis The onerte tion of two or more iniviul genes s well s their intertions with eh other n with the environment etermine mny importnt phenotypi hrteristis. Suh hrteristis re referre to s polygeni or quntittive trits, n the iniviul gene lotions s quntittive trit loi (QTLs). In silkworm lmost ll of the importnt eonomi trits suh s feunity, silk ooon weight, silk fibre length, silk fibre thikness, n resistne to bulovirus re quntittive in nture. Very little is known bout the number of genes involve, their hromosoml lotions, or their gene prouts. The evelopment of high ensity geneti linkge mps tht re bse on NA mrker loi provies useful tool for the resolution of these omplex trits into their iniviul geneti omponents 30. riefly, iniviuls from geneti rosses suh s F 2 or bkross popultions, or reombinnt inbre lines or ner isogeni lines obtine by repete rossing of strins with ontrsting phenotypes, re evlute for the phenotype of interest n for their genotype lsses t NA-bse mrker loi t regu- F 2 interross strtegy Prent 1 x Prent 2 F 1 femle No rossing over x F 1 mle rossing over ours* A A b b gmetes b A A Type 1 progeny *Non-reombinnt gmetes not shown Type 2 progeny Figure 6. Shemti representtion of the behviour of mrkers in n F 2 interross. Ahismti oogenesis mkes it possible to test whether linkge groups re relly inepenent or not. As shown in the figure, ny F 2 iniviul nnot be homozygous for both mternl n pternl ominnt mrkers on the sme utosome. This is speilly true for ominnt mrkers where it is not possible to istinguish homozygotes from heterozygotes. The bsene of mternl mrker in ertin F 2 progeny mens tht the nonreombinnt utosome of the progeny is pternl (Type 2 progeny), n vie vers (Type 1 progeny). As result, eh F 2 iniviul n be type for eh linkge group s to whether its non-reombinnt hromosome is pternl or mternl in origin. 422 URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST 2002

9 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY A A Prent 1 b b x Prent 2 Prent 2 x F 1 mle F 1 femle Prent 2 x rossing over ours b A No rossing over A b gmetes A non-reombinnt progeny Reombinnt progeny non-reombinnt progeny Figure 7. iphsi nture of geneti linkge in silkworm. Any two loi on the sme hromosome re bsolutely linke in femles s there is no rossing over uring oogenesis n the loi nnot reombine. Linkge in mles, on the other hn, epens on the reombintion istne seprting the two loi, beuse rossing over ours uring spermtogenesis. This biphsi nture of linkge filittes sequentil pproh to mpping. In the first phse, bsolute linkge in offspring of femle-informtive rosses is use to ientify the linkge groups, eh of whih orrespons to hromosome, ontributing to the trit of interest. In the seon phse, the lotion of the gene ontributing to the trit of interest within these groups is nrrowe own further by nlysing reombinnt offspring of mle-informtive mtings. lr 10- to 20-M intervls ross the genome. The ifferenes between the men phenotype n mrker genotype lsses re then use to etermine the genome positions n the effets of QTLs tht ontribute to the phenotype. The NA mrkers tht re losely linke to the trits of interest in silkworm oul be use to selet the trit in question in segregting popultion of n F 2 ross or using bulke segregnt nlysis 31, or with reombinnt inbre lines 32 or ner isogeni lines proue in silkworm improvement progrmmes. Of ourse, the effetiveness of suh NA mrker-ssiste seletion (MAS) will epen on the ury of the phenotypi lssifition of the trit of interest n the egree of linkge between mrker(s) n trits of interest in silkworm. ross-breeing strtegies hve been extensively use s mens of hrnessing heterosis in the silkworm 33. In ft hybri silkworms hve ontribute signifintly to the rmti inrese in silk proution of the worl 34. An enormous mount of breeing effort hs been investe in the evelopment of silkworm hybris, n this hs resulte in the relese of mny hybri ombintions in Jpn, hin, Ini n other ountries. While onsierble suess hs been hieve in silkworm hybri progrmmes, experimentl t pertining to the geneti bsis of heterosis in silkworm hve remine sre. The reent vnes in genome reserh hve generte onsierble interest in preiting hybri performne using moleulr mrkers in rop breeing progrmmes. Most of the stuies hve been reporte in orn n rie. A lrge number of stuies onute in mize hve proue vrible results: high orreltions between moleulr mrker istnes of the prents n hybri performne were etete in some stuies 35, while low orreltions were observe in others 36,37. In rie lso, there re extensive stuies on the reltionship between moleulr mrker heterozygosity n hybri performne. As in mize, results re vrible 38. Seletion of genetilly pure n ivergent prentl strins is ritil to the suess of hybriiztion progrmme in silkworm. The use of NA mrkers to relte the geneti homozygosity n geneti istne of the prentl strins to the mnifesttion of hybri vigour, n unerstning the geneti bsis of heterosis, will be quite useful to selet suitble prentl lines for hybriiztion progrmmes. Reently, moleulr mrkers hve been genotype on set of 22 silkworm strins rosse in full-illel progrmme (Ro, hnrshekrih n Ngrju, uner preprtion) to eliit the reltionship between within n between strin geneti vribility n the bsis of mi- n better-prent heterosis. The results inite positive orreltion between geneti istne n mi-prentl heterosis; there is lso eviene to show tht prentl homozygosity hs is- URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST

10 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY tint influene on the egree of mnifesttion of heterosis. Most importnt, the stuies enble them to ientify the silkworm inbre lines whih proue highly heteroti hybris. onsiering tht lrge number of silkworm inbre lines re mintine in the silkworm breeing sttions in Ini n other silk-prouing ountries, the vilble moleulr mrkers shoul be use to ientify the prentl lines tht re most suitble for proution of heteroti hybris. The silkworm strin-speifi mrkers oul lso be use for uthentition of vrieties, protetion of breeers rights, n perioil monitoring of the purity of vrieties. Physil mpping of the silkworm genome Isoltion n hrteriztion of genes of interest using mp-bse loning methos n systemti sequening of the ombyx genome by the propose Interntionl onsortium requires high resolution physil mp. A lone-bse physil mp onsists of set of orere, overlpping inserts of lone genomi NA. Suh mp ffors unique resoure for stuying the struture n funtion of the genome. The mp filittes the moleulr ientifition of genes of interest, n the lones in the mp provie rey ess to substrtes for genome sequening. A lone-bse mp lso opens up new opportunities for stuies of genome evolution. lone-bse mps hve been ssemble for severl moel orgnisms n griulturlly importnt speies. In most orgnisms, sets of overlpping lones overing uninterrupte strethes of the genome (ontigs) re ssemble by eteting overlps by mens of shre restrition frgments in fingerprints or shre sequene tgge sites (STSs). The onstrution of omprehensive n stble NA librries lone in pproprite lrge-sle vetors is essentil to obtin high-resolution physil mps of omplex eukryoti genomes suh s silkworm. Tht the ury n effiieny of physil mpping re in iret reltion to the men size of lone frgments in genomi librries enourge the evelopment of severl lrge-sle vetor systems in reent yers. Yest rtifiil hromosomes (YA) were the first wiely use systems, but mny problems ssoite with lone stbility, himerism, n low yiel le to the evelopment of nother lrge-sle loning system bse on the E. oli F ftor whih hs lrgely superee YA-bse loning. These vetors re referre to s teril Artifiil hromosomes (A) 39. A librries for silkworm hve been onstrute in three lbortories 40 (Mit et l., uner preprtion) from two. mori referene strins, p50 n 108, using nuler NA from posterior silkglns p50. Altogether these librries ontin more thn 30 genome equivlents, with verge insert sizes rnging from kb. The onstrution of A ontigs using shre STSs, hromosome wlking, n the fingerprinting methos is 424 unerwy in the Ntionl Institute of Agrobiologil Sienes, Tsukub, Jpn (Mit, K., pers. ommun.). In erly stuies, the previously reporte lose linkge between the ombyx homologs of the invete (m in) n engrile (m en) genes ws onfirme by onstrution of A ontig tht ontine both sequenes 40. Initil sreening of A librries hs lso revele informtion bout the hromosoml orgniztion of two genes, seriin-1, whih oes for one of the mjor ooon proteins, n the gene enoing the preursor protein of ipuse hormone n pheromone biosynthesis-tivting neuropeptie (the H-PAN gene), whih re lote within 22-kb intervl n re ivergently oriente. In ition to their use to orient n orgnize known genes in fine-struture mpping of silkworm hromosomes, A ontigs will serve s primry tools for lrge-sle sequening of the silkworm genome. Initil efforts re unerwy to trget selete portions of the genome, inluing hromosomes 1 (Z), 2, n W, with long-term plns to rry out omplete genome sequene (Mit, K., pers. ommun.). onluing remrks The tools n triks re now in ple for lrge-sle genomis of silkworm. The ner future will bring n explosive growth in funtionl gene informtion from the iptern prigm rosophil, of whih muh will be useful for silkworm. Foresighte efforts re lrey unerwy to initite the Interntionl Silkworm Genome onsortium to sequene the ombyx genome. Tehnology trnsfer from other genome projets will mke importnt ontributions in this enevour. The next few yers shoul be exiting for silkworm genomis, with gret stries being me in the EST sequening projet, A librry preprtion, onstrution of linkge mps, onstrution of trnsgeni silkworms for refrtoriness to virl iseses, n nlysis of QTL. Eqully exiting is the potentil pplition of the results of silkworm genomis reserh to other lepiopterns, prtiulrly Heliothines n wil silkmoths, whih represent the estrutive n benefiil extremes of this lrge n iverse inset orer. 1. oir, H., Fujii, H., Kwguhi, Y., Kihr, H. n nno, Y., Geneti Stoks n Muttions of ombyx mori, Institute of Geneti Resoures, Kyushu University, Jpn, Gge, L. P., hromosom, 1974, 45, Interntionl Lepioptern Genome Sequening onsortium ( 4. Ams, M.. et l., Siene, 2000, 287, Interntionl Humn Genome Sequening onsortium, Nture, 2001, 409, Venter, J.. et l., Siene, 2001, 291, Ngrj, G. M. n Ngrju, J., Eletrophoresis, 1995, 16, URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST 2002

11 SPEIAL SETION: REENT AVANES IN SILKWORM IOLOGY 8. Ngrju, J., Shrm, A., Sethurmn,. N., Ro, G. V. n Singh, L., ibi, Shrm, A., Niphkr, M. P., Kthirvelu, P., Ngrju, J. n Singh, L., J. Here., 1999, 90, Tretjk, A. P., Ryskov, A. P., Sevstynov, G. A., Fillipovih, Y.. n Strunnikov, V. A., FES Lett., 1992, 303, Rey, K.., Ngrju, J. n Abrhm, E. G., Hereity (UK), 1999, 83, Promboon, A., Shim, T., Fujiwr, F. n Kobyshi, M., Genet. Res., 1995, 66, Ysukohi, Y., Genetis, 1998, 150, Ngrj, G. M., Ph thesis, Univ. of Mysore, Shi, J., Hekel,. G. n Golsmith, M. R., Genet. Res., 1995, 66, Tn, Y-., Wn,., Zhu, Y., Lu,., Xing, Z. n eng, H. W., Genetis, 2001, 157, Litt, M. n Luty, J. A., Am. J. Hum. Genet., 1989, 44, Tutz,., Nulei Ais Res., 1989, 17, Weber, J. L. n My, P. E., Am. J. Hum. Genet., 1989, 44, ietrih, W. F. et l., Nture, 1996, 380, Rey, K.., Abrhm, E. G. n Ngrju, J., Genome, 1999, 42, Weber, J. L., Genomis, 1990, 7, Wu, K. S. n Tnksley, S.., Mol. Gen. Genet., 1993, 241, Ngrju, J., Rey, K.., Ngrj, G. M. n Sethurmn,. N., Hereity (UK), 2001, 86, Ngrju, J., Kthirvel, M., Subbih, E. V., Muthulkshmi, M. n Kumr, L.., Mol. ell. Probes, 2002, 16, Hekel,. G., Annu. Rev. Entomol, 1993, 38, Ngrju, J. n Muthulkshmi, M., V Intl. ong. Ento. rzil, Hekel,. G., Ghn, L. J., Liu, Y.. n Tbshnik, E.., Pro. Ntl. A. Si. USA, 1999, 96, Ghn, L. J., Goul, F. n Hekel,. G., Siene, 2001, 293, Lner, E. S. n otstein,., Genetis, 1989, 121, Mihelmore, R. W., Prn, I., Kesseli, R. V., Pro. Ntl. A. Si. USA, 1991, 88, Reiter, R. S., Willims, J. G. K., Felmn, K. A., Rflski, J. A., Tingey, S. V. n Solnik, Pro. Ntl. A. Si. USA, 1992, 89, Ngrju, J., Urs, R. n tt, R. K., Seriologi, 1996, 36, Ngrju, J., Klimenko, V. n ouble, P., in Enylopei of Genetis (e. Reeve, E..), Fitzroy erborn Press, Lonon, 2001, pp Smith, O. S., Smith, J. S.., owen, S. L., rown, S. L., Tenborg, R. A. n Wll, S. J., Theor. Appl. Genet., 1990, 80, Goshlk, E.., Lee, M. n Lnkey, K. R., ibi, 1990, 80, uley, J. W., Sghi Mroof, M. A. n Rufener, G. K., rop Si., 1991, 31, Sghi Mroof, M. A., Yng, G. P., Zhng, Q. n Grvois, K. A., ibi, 1997, 37, Shizuy, H., irren,., Kim, U. J., Mnino, V., Slepk, T., Thiiri, Y. n Simon, M., Pro. Ntl. A. Si. USA, 1992, 29, Wu,., Kwski, S. n Ysukohi, Y., Mol. Gen. Genet., 1999, 261, Zietkiewiz, E., Rflski, A. n Lbu,., Genomis, 1994, 20, Estoup, A., Soligne, M., Hrry, M. n ornuet, S. M., Nul. Ais. Res., 1993, 21, AKNOWLEGEMENTS. We thnk M. Muthulkshmi, M. Kthirvel n M. Selvenrn for helpful isussions. Finnil support to JN by the ept. of iotehnology, Govt. of Ini, New elhi is grtefully knowlege. URRENT SIENE, VOL. 83, NO. 4, 25 AUGUST