Use of laser microdissection for the construction of Humulus japonicus Siebold et Zuccarini, 1846 (Cannabaceae) sex chromosome-specific DNA library and cytogenetics analysis

By

Yakovin, Nickolay A.
Divashuk, Mikhail
Yakovin, Nickolay A.
Razumova, Olga
Siloviev, Alexander
Karlov, Gennady

Type

Article

Date of Publication

2014

Original Publication

Comparative cytogenetics : international journal of plant & animal cytogenetics, karyosystematics and molecular systematics

Volume

8

Series / Issue

Issue: 4

Pages

323-336

Subjects

chromosome-specific DNA , fluorescence in situ hybridization , Laser microdissection , plant sex chromosomes

Contributed by

Pensoft Publishers

Language

English

Published by

St. Petersburg, Russia [Published] with technical support of Zoological Institute RAS [2007]-

Identifiers

DOI: https://doi.org/10.3897/CompCytogen.v8i4.8473

 

Abstract

Dioecy is relatively rare among plant species, and distinguishable sex chromosomes have been reported in few dioecious species. The multiple sex chromosome system (XX/XY1Y2) of Humulus japonicus Siebold et Zuccarini, 1846 differs from that of other members of the family Cannabaceae, in which the XX/XY chromosome system is present. Sex chromosomes of H. japonicus were isolated from meiotic chromosome spreads of males by laser microdissection with the P.A.L.M. MicroLaser system. The chromosomal DNA was directly amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). Fast fluorescence in situ hybridization (FAST-FISH) using a labeled, chromosome-specific DOP-PCR product as a probe showed preferential hybridization to sex chromosomes. In addition, the DOP-PCR product was used to construct a short-insert, H. japonicus sex chromosomes-specific DNA library. The randomly sequenced clones showed that about 12% of them have significant homology to H. lupulus and 88% to Cannabis sativa Linnaeus, 1753 sequences from GenBank database. Forty-four percent of the sequences show homology to plant retroelements. It was concluded that laser microdissection is a useful tool for isolating the DNA of sex chromosomes of H. japonicus and for the construction of chromosome-specific DNA libraries for the study of the structure and evolution of sex chromosomes. The results provide the potential for identifying unique or sex chromosome-specific sequence elements in H. japonicus and could aid in the identification of sex chromosome-specific repeat and coding regions through chromosome isolation and genome complexity reduction.