Research Article |
Corresponding author: Gennady I. Karlov ( karlovg@gmail.com ) Academic editor: Andrzej Joachimiak
© 2014 Nickolay Yakovin, Mikhail Divashuk, Olga Razumova, Alexander Soloviev, Gennady I. Karlov.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Yakovin N, Divashuk M, Razumova O, Siloviev A, Karlov G (2014) Use of laser microdissection for the construction of Humulus japonicus Siebold et Zuccarini, 1846 (Cannabaceae) sex chromosome-specific DNA library and cytogenetics analysis. Comparative Cytogenetics 8(4): 323-336. https://doi.org/10.3897/CompCytogen.v8i4.8473
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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.
Laser microdissection, plant sex chromosomes, fluorescence in situ hybridization, chromosome-specific DNA
Dioecy is relatively rare in the plant kingdom, in which only approximately 4% of angiosperm species are dioecious (
The most widespread method for the detection of new sex-specific DNA sites is to search for molecular markers that are linked to sex (
In plants,
Identification of specific chromosomes for microdissection is difficult in many plant species. It can be achieved by choosing a plant with chromosomes bearing a prominent morphological feature, for example, a large somatic chromosome such as the Y chromosome in Silene. In H. japonicus, sex chromosomes are difficult to distinguish from autosomes at the mitotic metaphase fig (
To investigate the structure of the sex chromosomes in H. japonicus, the XY1Y2 chromosomes were isolated by laser microdissection of the meiotic trivalent at the diakinesis and metaphase I stages and the DOP-PCR products were used for FISH and the creation of the DNA library.
The male H. japonicus plants (2n=17=14+XY1Y2) were grown in a greenhouse from seeds of cultivar “Samuray” (“Gavrish seeds”, Moscow, Russia) and were used to prepare the meiotic chromosomes. The one month old plants were exposed to a short day photoperiod (8 h day and 16 h night) to induce flowering.
For the preparation of H. japonicus meiotic diakinesis and metaphase I chromosomes, the significantly modified method of
For FISH experiments, the chromosome preparations were made as described above, except that conventional slides were used instead of the polyethylene naphthalate membrane-coated slides.
The P.A.L.M. MicroLaser system (P.A.L.M. GmbH) was used to dissect Y1-X-Y2 trivalent figures at diakinesis. The microscopic stage, micro-manipulator and laser micromanipulation procedures were computer controlled. All procedures for the dissection of chromosomes are adapted from experiments performed by
Chromosomes were used directly (without any enzymatic treatment) for amplification by DOP-PCR with regular primers designed by
A male-specific DNA marker (
For FISH experiments, the DOP-PCR products were labeled with dioxigenin-11-dUTP (Roche Diagnostics GmbH). One-half of a microliter of the primary PCR reaction was added as a temfig to 20 µl of DOP-labeling PCR mix. Cycling parameters were: 3 min at 95°C for initial denaturation; 30 cycles of 15 s at 94°C, 30 s at 56°C; and 2 min at 72°C, followed by a 5 min final extension at 72°C.
FISH was performed using a modified version of the method of
The FAST-FISH was performed as described by
The slides were counterstained with 4,6-diamino-2-phenylindole (DAPI, 0,5 µg/ml) in Vectashild (Vector). The hybridization signals were observed under a fluorescence microscope (Zeiss AxioImager.M1, Germany). Images were captured by a charge-coupled device (CCD) system (AxioCam MRm) and AXIOVISION software.
The DOP-PCR products were cloned into the pGEM®-T Easy Vector System (Promega, USA) as described by manufacturer. Clones were picked into 96 well figs, grown for 18 h, replicated and frozen at -80° C. One hundred randomly selected clones were tested by PCR with M13 primers on the insert present, and 24 randomly selected clones were sequenced using ABI Big Dye Mix v3.1 (Applied Biosystems Inc) with M13 primers, according to the manufacturer’s instructions. Products were resolved on an ABI 3130xl sequencer. BLAST analysis was performed according to the standard procedure. BLAT analysis was used to find homology of sequences against the C. sativa genome (http://genome.ccbr.utoronto.ca/index.html). BLAT on DNA is designed to quickly find sequences of 95% and greater similarity of length 25 bases or more.
The sex chromosomes from PMCs at meiotic diakinesis and metaphase I stages of H. japonicus can easily be distinguished from autosomes under a light microscope without any staining procedures, which allows for reliable identification and rapid isolation of pure chromosomes of interest (Fig.
To ensure that DOP-PCR product was obtained from sex chromosomes the male specific SCAR marker was used. The PCR product of expected size was obtained from DOP-PCR DNA and DNA from male plants only. No amplification was detected from female DNA and DOP-PCR product obtained after microdissection of autosomes (Fig.
To examine the quality of the DOP-PCR product, the standard FISH procedure was performed. DIG-labeled DOP-PCR products hybridized to the chromosomes of male plants in the absence of a competitor. Signals were observed uniformly on all chromosomes (data not shown).
The application of FAST-FISH, using lower concentrations of DIG-labeled DOP-PCR probe per slide and reducing the hybridization time from 16 h to 1 h, allowed for the differentiation of chromosomes by FISH signal (Fig.
The DOP-PCR product was used to construct a short-insert H. japonicus sex chromosomes-specific DNA library. Cloning of the DOP-PCR products resulted in 5 x 103 recombinant colonies per 100 µl PCR reaction mixture. The length of the cloned DNA fragments ranged from 450 to 3000 bp, with an average fragment length of 1000 bp. Twenty-four clones were randomly selected for sequencing. When we compared sequences with the NCBI database, using BLAST, 11 of them showed homology to sequences of plant retrotransposons (Table
Three sequences show homology to some sequences of H. lupulus and 13 sequences show homology to C. sativa. Two sequences show homology to hypothetical proteins or mRNA. Additionally, a database search of the recently sequenced C. sativa [14] using BLAT (http://genome.ccbr.utoronto.ca) showed homology in 21 of 24 sequences with the Cannabis genome (Table
Microdissection of H. japonicus sex chromosomes at meiotic diakinesis-metaphase I stage. a Selection of sex chromosomes (Y1-X-Y2 trivalent formation indicated by arrow) b Cutting out of the sex chromosomes c The gel electrophoresis of the microdissected sex chromosomes DOP-PCR product: 1 – negative control, 2 – 100 bp DNA ladder, 3 - DOP-PCR d The gel electrophoresis after PCR with the male sex specific marker on different DNA temfigs: 1 – 100 bp DNA ladder, 2 – DOP-PCR product from sex chromosomes, 3 – DOP-PCR product from autosomes, 4 – DNA of male plant, 5 – DNA of female plant.
Similarity of the sequenced Humulus japonicus sex chromosome specific clones to GenBank accessions, Cannabis sativa draft genome and RepBase database.
№ | Similarity to GenBank accessions | Tool | Similarity to Cannabis sativa *** |
1 | Humulus lupulus clone HlAT9 microsatellite sequence (AY588370.1) | blastn * | + |
gag-pol polyprotein [Phaseolus vulgaris] (AAR13317.1) | blastx * | ||
2 | Medicago truncatula DNA sequence from clone MTH2-46C14 on chromosome 3, complete sequence (CT962505.9) | blastn | + |
pol protein [Cucumis melo subsp. melo] (AAO45752.1) | blastx | ||
3 | No homology in GenBank and RepBase | + | |
4 | Medicago truncatula chromosome 5 clone mte1-70c24, COMPLETE SEQUENCE (CR932962.2) | blastn | + |
5 | retrotransposon gag protein [Cucumis melo subsp. melo] (ADN33993.1) | blastn | + |
integrase [Populus trichocarpa] (ABG37658.1) | blastn | ||
6 | Populus trichocarpa clone POP065-M23, complete sequence (AC209187.1) | blastn | + |
pol protein [Cucumis melo subsp. melo] (AAO45752.1) | blastx | ||
rve superfamily: Integrase core domain (pfam00665) | blastx | ||
7 | No homology in GenBank and RepBase | + | |
8 | Serratia proteamaculans 568, complete genome (CP000826.1) | blastn | - |
9 | No homology in GenBank and RepBase | - | |
10 | Nicotiana benthamiana mRNA for PME inhibitor (FN432042.1) | blastn | + |
11 | A family of autonomous Polinton DNA transposons (CR1-6_BF) | CENSOR ** | + |
12 | Gossypium raimondii clone GR__Ba0005I14-jfn, complete sequence (AC243106.1) | blastn | + |
Amphioxus CR1-6_BF autonomous Non-LTR Retrotransposon - consensus. | CENSOR | ||
13 | Lotus japonicus cDNA, clone: LjFL1-045-CB01, HTC (AK337120.1) | blastn | + |
integrase [Populus trichocarpa] (ABG37658.1) | blastx | ||
LTR retrotransposon from the western balsam poplar: internal portion. (Gypsy-39_PT-I) | CENSOR | ||
14 | No homology in GenBank and RepBase | - | |
15 | Humulus lupulus vps gene for valerophenone synthase, complete cds (AB047593.2) | tblastx * | + |
gag-pol polymerase [Arabidopsis lyrata subsp. lyrata] (ABW81018.1) | blastx | ||
16 | gag-protease polyprotein [Cucumis melo subsp. melo] (AAO45751.1) | blastx | + |
17 | hypothetical protein VITISV_026408 [Vitis vinifera] (CAN60970.1) | blastx | + |
18 | Humulus lupulus clone GT2-P16-8 microsatellite sequence (EU094990.1) | blastn | + |
HLUTR3CH_T3_051_H10_24JUL2006_066 HLUTR3CH Humulus lupulus cDNA, mRNA sequence (GD252950.1) | blastn | ||
19 | Cannabis sativa strain Purple Kush scaffold130939_1, whole genome shotgun sequence (AGQN01284755.1) | blastn (wgs) | + |
20 | No homology in GenBank and RepBase | + | |
21 | gag-protease polyprotein [Cucumis melo subsp. melo] (AAO45751.1) | blastx | + |
Vitis vinifera contig VV78X146750.38, whole genome shotgun sequence (AM458430.2) | tblastx | ||
22 | No homology in GenBank and RepBase | + | |
23 | No homology in GenBank and RepBase | + | |
24 | Daucus carota subsp. sativus clone BAC C235O6O genomic sequence (FJ148580.1) | blastn | + |
Retrotransposon gag protein [Asparagus officinalis] (ABD63156.1) | blastx |
To isolate sex chromosomes, we used a technique based on laser beam microdissection with the P.A.L.M. MicroLaser system. An accurate identification of the target chromosomes is the first step in microdissection and microcloning. Additionally, the quality of microdissected chromosomal DNA depends critically on the pretreatment, chromosome fixation and staining of the samples (
The results of standard FISH procedure with DIG-labeled DOP-PCR products is in agreement with previous observations showing that the DNA of microdissected plant chromosomes hybridized to all chromosomes as a result of widespread repetitive sequences contained in plant genomes (
The preferential, uneven distribution of DOP-PCR probes on the Y1 and Y2 sex chromosomes in FAST-FISH experiments is indicative of an abundance of dispersed repeats, such as retrotransposons, on Y chromosomes. These results agree with
The observation that about 12% of the sequences show significant homology to H. lupulus and 88% to C. sativa, whose genome is closely related to H. japonicus, indicates efficient amplification of DNA from H. japonicus chromosomes by DOP-PCR. Less apparent homology between H. japonicus and H. lupulus, compared to C. sativa, can be explained by the lack of sequence representation in the GenBank database. FISH with DOP-PCR probes led to a hybridization signal on all chromosomes, which suggests that a large amount of dispersed repeated DNA sequences are present in the genome of this species and in the DOP-PCR product. This was confirmed by sequencing, which showed that 44% of sequences were homologous to plant retroelements. The presence of multiple sequences with homology to plant retrotransposons is in agreement with FISH experiments in which a dispersed signal was seen on all chromosomes, given that retroelements are usually distributed throughout the genomes of plants (
It was concluded that laser microdissection is a useful tool for isolating the DNA of individual chromosomes, including the relatively small chromosomes of H. japonicus, and for the construction of chromosome-specific libraries for the study of the structure and evolution of the sex chromosomes. This is the first time a DNA library of the sex chromosomes Japanese hop has been constructed.
We thank the company “OPTEC” LLC for providing the P.A.L.M. MicroLaser system for this study. The authors acknowledge financial support from the Russian Foundation of Basic Research No.13-04-02116.