ABSTRACT
CONTENTS List of Abbreviations ................................................................ 382 11.1 Introduction ................................................................... 383
11.1.1 Taxonomy ......................................................... 383 11.1.2 Importance of Grasshopper Species ................. 384 11.1.3 Karyotype ......................................................... 385 11.1.4 Genome Size ..................................................... 386 11.1.5 Genome Sequencing Projects ........................... 387
11.2 Protocols ........................................................................ 387 11.2.1 Biological Materials and Grasshopper
Culture .............................................................. 387 11.2.2 Equipment, Materials, and Reagents ................ 389 11.2.3 Sources of Chromosomes ................................. 392 11.2.4 Tissue Extraction and Fixation ......................... 392
11.2.4.1 Mitotic Chromosomes in Adults....... 392 11.2.4.2 Mitotic Chromosomes in Embryos ... 393 11.2.4.3 Meiotic Chromosomes ...................... 394
11.2.5 Chromosome Preparations ................................ 395 11.2.5.1 Squashing ......................................... 395 11.2.5.2 Spreading .......................................... 395 11.2.5.3 Chromosome Microdissection .......... 396 11.2.5.4 Chromosomes for Fiber-FISH .......... 397
11.2.6 Staining Protocols ............................................. 398 11.2.6.1 Conventional Staining ...................... 398 11.2.6.2 C-Banding ........................................ 398 11.2.6.3 Silver Impregnation .......................... 400 11.2.6.4 Double Silver Impregnation and
Fluorescence In Situ Hybridization ... 401
LIST OF ABBREVIATIONS AFLP, amplied fragment length polymorphism BSA, bovine serum albumin CMA3, chromomycin A3 Cot, concentration of DNA and time DA, distamycin A DAPI, 4′,6 -diamidino-2-phenylindole dATP, deoxyadenosine triphosphate dCTP, deoxycytidine triphosphate dGTP, deoxyguanosine triphosphate DNA, deoxyribonucleic acid DNase, deoxyribonuclease dNTPs, deoxynucleotide triphosphates
11.2.6.5 Triple Fluorescent CMA3-DADAPI Staining .................................. 401
11.2.6.6 Acridine Orange Staining ................. 402 11.2.7 Molecular Techniques ....................................... 403
11.2.7.1 Genomic DNA Isolation ................... 403 11.2.7.2 DNA Amplication from
Microdissected Chromosomes ......... 404 11.2.8 Fluorescence In Situ Hybridization .................. 407
11.2.8.1 Probe Generation .............................. 408 11.2.8.2 Probe Labeling ..................................412 11.2.8.3 FISH with Direct Fluorophore
Labeling and Detection .....................414 11.2.8.4 FISH with Indirect Labeling and
Antibody Detection ...........................416 11.2.8.5 Chromosome Painting .......................418
