ABSTRACT

CONTENTS List of Abbreviations ................................................................ 220 6.1 Introduction ..................................................................... 220

6.1.1 Taxonomy and Importance of the Species .......... 220 6.1.2 Karyotype and Sex Chromosome System ...........221 6.1.3 Genome Sequencing Project and Genetic Map .. 223

6.2 Protocols ......................................................................... 225 6.2.1 Bombyx mori Culture .......................................... 225 6.2.2 Sex Discrimination of Bombyx mori Larvae

and Pupae by Outer Appearances ....................... 225 6.2.3 Tissue and Good Stage for Chromosome

Preparation .......................................................... 227 6.2.4 Chromosome Preparation ................................... 227 6.2.5 Types of Probes ................................................... 229

6.2.5.1 Bacterial Articial Chromosomes and Fosmids ......................................... 229

6.2.5.2 Genomic DNA ..................................... 232 6.2.5.3 Polymerase Chain Reaction Products .. 233 6.2.5.4 Telomeric Repeat ................................. 233 6.2.5.5 W Chromosome ................................... 234

6.2.6 Labeling .............................................................. 235 6.2.6.1 Nick Translation .................................. 235 6.2.6.2 Polymerase Chain Reaction ................. 236

6.2.7 Hybridization and Detection ............................... 237 6.2.7.1 Probe Purication and Denaturation .... 237 6.2.7.2 Slide Pretreatment and Denaturation ... 238 6.2.7.3 Hybridization ....................................... 238 6.2.7.4 Wash and Detection ............................. 238 6.2.7.5 Chromosome Staining ......................... 239 6.2.7.6 Reprobing ............................................ 239

6.2.8 Visualization and Mapping ................................. 240 6.2.9 Representative Results ........................................ 242

LIST OF ABBREVIATIONS AFLP, amplied fragment length polymorphism BAC, bacterial articial chromosome BSA, bovine serum albumin CGH, comparative genomic hybridization DABCO, 1,4-Diazabicyclo[2.2.2]octane DAPI, 4’,6-diamidino-2-phenylindole DOP-PCR, degenerate oligonucleotide primed-polymerase

chain reaction EDTA, 2,2’,2’’,2’’’-(Ethane-1,2-diyldinitrilo)tetraacetic acid EST, expressed sequence tag FISH, uorescence in situ hybridization GISH, genomic in situ hybridization HDR, high-density replica NGS, next-generation sequencing PCR, polymerase chain reaction RAPD, random amplied fragment polymorphism RFLP, restriction fragment length polymorphism SNP, single nucleotide polymorphism SSC, saline sodium citrate SSR, simple sequence repeats STS, sequence-tagged site

Lepidoptera, moths and butteries, is the second species-richest insect order, which comprise more than 130,000 species (Beccaloni et  al. 2003). The holokinetic chromosome nature of Lepidoptera as well as Trichoptera (cf., caddis ies: sister clade of Lepidoptera) is shared with Hemiptera (aphids and bugs) within Insecta (see

6.2.9.1 FISH Using Repetitive Sequences as Probes .................................................. 242

6.2.9.2 BAC-or Fosmid-FISH and Gene Mapping ............................................... 244

6.2.10 Troubleshooting .................................................. 248 6.3 Discussion ....................................................................... 249

6.3.1 Integration of Linkage Maps, Chromosomes, and Genomic Sequences ..................................... 249

6.3.2 Chromosome Organization and Evolution ......... 250 References ................................................................................ 252

Section 6.1.2). However, sex chromosome systems of Lepidoptera and Trichoptera are female heterogamety (WZ/ZZ, female/male), unlike male heterogametic system (XX/XY, female/male) of all others.