Research Article |
Corresponding author: Zhifeng Zhang ( zzfp107@ouc.edu.cn ) Academic editor: Kira Zadesenets
© 2019 Zhenkui Qin, Xueyu Li, Danwen Liu, Qing Wang, Li Lu, Zhifeng Zhang.
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:
Qin Z, Li X, Liu D, Wang Q, Lu L, Zhang Z (2019) Analysis of chromosome karyotype and genome size in echiuran Urechis unicinctus Drasche, 1880 (Polychaeta, Urechidae). Comparative Cytogenetics 13(1): 75-85. https://doi.org/10.3897/CompCytogen.v13i1.31448
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Karyotype and genome size are two primary cytogenetic characteristics of species, which are of great significance to the study of cytogenetics, taxonomy, phylogenesis, evolution as well as molecular biology. However, this basic cytogenetic information in echiurans is lacking. Therefore, we analyzed characteristics of karyotype and genome size in the echiuran worm Urechis unicinctus Drasche, 1880. In this study, coelomic cells of U. unicinctus were used for analyzing the genome size by a flow cytometry with chicken erythrocytes as DNA standard, and the 2C DNA content was determined to be 1.85 pg, which was corresponded to the genome size of 904.58 Mbp approximately. Furthermore, trochophores of U. unicinctus were dissociated and cells were utilized for preparing the chromosomes stained with DAPI, and the karyotype was determined as 2n = 30 (10m + 6sm + 6st + 8t), FN=52. Our data provided the basic cytogenetic information of U. unicinctus, which could be utilized in taxonomic study and whole-genome sequencing in future.
Urechis unicinctus, karyotype, genome size, flow cytometry
Echiurans (spoon worms) are a group of marine worms which are unsegmented, coelomate, bilaterally symmetrical and soft-bodied (
Karyotype, including chromosome number and composition, could reflect the taxonomic relationship between species and be used as a tool to explore biological diversity (
Genome size is the total DNA content within a single copy genome and is also referred to C-value, which is specific in every species and ranges from 0.02 pg (Pratylenchus coffeae Zimmermann, 1898, a plantparasitic nematode) to 132.83 pg (Protopterus aethiopicus Heckel, 1851, a marbled lungfish) in animals (
Urechis unicinctus, a commercial echiuran worm inhabiting the U-shaped burrows in the coastal mud flats, has unique roles in animal evolution, coastal sediment improvement and marine drug development (
U. unicinctus adults with 9.96 ± 0.42 cm in body length were purchased from an aquatic product market, which were collected from a coastal intertidal flat in Yantai, China.
Sampling
The mature sperms and oocytes were obtained by dissecting the nephridia of the healthy U. unicinctus, respectively. Artificial fertilization was conducted by mixing sperms and oocytes at a ratio of 10:1 in filter seawater (FSW), and then these fertilized eggs were cultivated until hatched in FSW (19.7 ± 0.3 °C, salinity 29 PSU, pH 8.29 ± 0.02). The hatched trochophores were collected using a 500 mesh sieve.
Chromosome preparation and karyotype analysis
Chromosomes of U. unicinctus trochophores were prepared as described by
The chromosome lengths, chromosome relative lengths and arm ratios from well-formed chromosomes in metaphase were measured and calculated using MICRO-MEASURE 3.3 software. Data were presented as mean ± SEM (n = 5). Chromosomes were classified according to the description of
Preparation of single cell samples
Coelomic fluids from 11 healthy worms were collected by syringe puncturing U. unicinctus body wall, respectively, and three duplicate samples were obtained from each individual. The coelomic cells were pelleted by centrifugation at 1000 g (4 °C) for 5 min, washed three times with PBS (pH 7.2), and then resuspended with PBS (pH 7.2). The suspension was added dropwise to the precooled 70% ethanol and fixed overnight at 4 °C. The next day, cell samples were collected through a 50 μm nylon mesh filtration, adjusted to 5×105 cells/ml, digested with 20 μg/μl RNase A for 10 min and then stained with 1 μg/μl propidium iodide (PI) for 30 min in the dark at room temperature.
Chicken erythrocytes (2C = 25 pg DNA) were used as an internal standard (
Flow cytometric analysis
Twelve samples, including a chicken erythrocyte, a U. unicinctus coelomic cell, and ten mixed samples containing 500 μl erythrocytes and 500 μl coelomic cells, were analyzed using a Coulter Cytomics FC500-MPL flow cytometer (Beckman, California, USA) equipped with a 488 nm laser source to detect the DNA content, and the output was processed in the software FLOWJO 7.6.1. Coefficients of variation (CV) were adjusted below 5% to ensure the reliability. The DNA content of U. unicinctus was then calculated according to
The well dispersed metaphase chromosomes from U. unicinctus trochophore cells are shown in Fig.
Metaphase chromosome and karyotype of U. unicinctus (2n = 30) stained with DAPI. m, metacentric chromosome; sm, submetacentric chromosome; st, subtelocentric chromosome; t, telocentric chromosome. Scale bar: 10 μm.
Chromosome idiograms of U. unicinctus. The dark regions showing short arms and the gray regions showing long arms.
Chr | p (μm) | q (μm) | Total (μm) | RL (%) | AR | Type |
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1 | 1.97 ± 0.01 | 2.20 ± 0.02 | 4.17 ± 0.03 | 7.09 ±0.04 | 1.12 ± 0.01 | m |
2 | 1.82 ± 0.05 | 2.15 ± 0.06 | 3.97 ± 0.09 | 6.75 ±0.11 | 1.18 ± 0.03 | m |
3 | 2.45 ± 0.04 | 3.48 ± 0.01 | 5.93 ± 0.01 | 10.1 ±0.01 | 1.42 ± 0.01 | m |
4 | 1.42 ± 0.04 | 2.21 ± 0.02 | 3.63 ± 0.04 | 6.18 ±0.05 | 1.56 ± 0.05 | m |
5 | 1.43 ± 0.01 | 2.40 ± 0.02 | 3.82 ± 0.03 | 6.50 ±0.04 | 1.68 ± 0.01 | m |
6 | 1.42 ± 0.02 | 2.94 ± 0.05 | 4.36 ± 0.07 | 7.42 ±0.08 | 2.08 ± 0.02 | sm |
7 | 1.24 ± 0.02 | 2.94 ± 0.05 | 4.17 ± 0.06 | 7.09 ±0.07 | 2.37 ± 0.04 | sm |
8 | 1.29 ± 0.03 | 3.29 ± 0.09 | 4.58 ± 0.08 | 7.79 ±0.10 | 2.55 ± 0.10 | sm |
9 | 0.92 ± 0.02 | 3.08 ± 0.07 | 4.01 ± 0.09 | 6.82 ±0.11 | 3.35 ± 0.04 | st |
10 | 0.59 ± 0.02 | 2.59 ± 0.04 | 3.18 ± 0.06 | 5.41 ±0.07 | 4.40 ± 0.08 | st |
11 | 0.53 ± 0.01 | 3.29 ± 0.07 | 3.82 ± 0.08 | 6.50 ±0.10 | 6.27 ± 0.09 | st |
12 | – | 3.74 ± 0.08 | 3.74 ± 0.08 | 6.36 ±0.10 | ∞ | t |
13 | – | 3.95 ± 0.07 | 3.95 ± 0.07 | 6.72 ±0.08 | ∞ | t |
14 | – | 3.09 ± 0.02 | 3.09 ± 0.02 | 5.26 ±0.02 | ∞ | t |
15 | – | 2.36 ± 0.02 | 2.36 ± 0.02 | 4.01 ±0.02 | ∞ | t |
The frequency histograms of DNA content from chicken erythrocytes and U. unicinctus coelomic cells were presented based on the flow cytometric analyses (Fig.
The 2C mean values of chicken erythrocytes (M) and U. unicinctus coelomic cells (N) in ten mixed samples and their ratios were presented in Table
Estimation of nuclear DNA contents in U. unicinctus using flow cytometer. A Chicken erythrocytes B U. unicinctus coelomic cells C one mixture sample of both. M, the 2C peak of chicken erythrocytes; N, the 2C peak U. unicinctus coelomic cells.
Summary of DNA content and genome size of U. unicinctus estimated using flow cytometry.
Sample | M | N | N/M | DNA content (pg) | Genome size (Mb) |
1 | 14.9 | 10.6 | 0.71 | 1.78 | 869.70 |
2 | 14.6 | 11.2 | 0.77 | 1.92 | 937.81 |
3 | 15.4 | 11.0 | 0.71 | 1.79 | 873.21 |
4 | 15.5 | 10.5 | 0.68 | 1.69 | 828.15 |
5 | 16.9 | 13.2 | 0.78 | 1.95 | 954.85 |
6 | 15.2 | 10.4 | 0.68 | 1.71 | 836.45 |
7 | 14.6 | 11.0 | 0.75 | 1.88 | 921.06 |
8 | 12.8 | 9.5 | 0.74 | 1.85 | 905.41 |
9 | 12.3 | 10.5 | 0.85 | 2.13 | 1043.60 |
10 | 14.8 | 10.6 | 0.72 | 1.79 | 875.57 |
Mean | 14.7 ± 0.4 | 10.8 ± 0.3 | 0.74 ± 0.02 | 1.85 ± 0.04 | 904.58 20.21 |
Studies on the chromosomes in echiurans are very limited, and all of them were conducted several decades ago (
Karyotypic information could be utilized to study the taxonomic relationships of species and biological diversity (
The karyotype asymmetry plot of echiurans, sipunculids and several annelids. a echiuran, U. unicinctus b echiuran, A. maculata c annelid, N. oligohalina d annelid, P. anderssoni e annelid, H. diversicolor f annelid, D. ghilarovi g annelid, E. balatonica h annelid, A. caliginosa i sipunculid, S. nudus j sipunculids, P. esculenta. symbols: circle, echiurans; square, sipunculids; triangle, polychaeta; hollow triangle, oligochaeta.
Species | Category | Karyotype | L/S1 | AR2 | Karyotype classification | Reference |
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Urechis unicinctus Drasche, 1880 | Echiura | 2n = 30 (10m + 6sm + 6st + 8t); FN = 52 | 2.51 | 2.54 | 3B | This study |
Achaetobonellia maculata Fisher, 1953 | Echiura | 2n = 20 (20m); FN = 40 | 3.96 | 1.03 | 1A |
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Sipunculus nudus Linnaeus, 1766 | Sipuncula | 2n = 34 (26m + 8sm); FN = 68 | 1.68 | 1.56 | 2A |
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Phasolosma esculenta Chen & Yeh, 1958 | Sipuncula | 2n = 20 (4m + 10sm + 6st); FN = 40 | 1.66 | 2.48 | 3A |
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Nereis oligohalina Rioja, 1946 | Annelida: polychaeta | 2n = 28 (14m + 2sm + 6st + 6t); FN = 50 | 2.54 | 2.01 | 4B |
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Perinereis anderssoni Kinberg, 1866 | Annelida: polychaeta | 2n = 38 (20m + 8sm); FN = 56 | 2.77 | 1.64 | 2B |
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Hediste diversicolor O.F. Müller, 1776 | Annelida: polychaeta | 2n = 28 (16m + 4sm + 8st); FN = 56 | 2.61 | 2.12 | 2B |
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Drawida ghilarovi Gates, 1969 | Annelida: oligochaeta | 2n = 20 (6m + 8sm + 6st); FN = 48 | 2.46 | 2.39 | 3B |
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Eisenia balatonica Pop, 1943 | Annelida: oligochaeta | 2n = 36 (10m + 20sm + 6st); FN =72 | 2.75 | 2.38 | 3B |
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Aporrectodea caliginosa Savigny, 1826 | Annelida: oligochaeta | 2n = 36 (12m + 18sm + 6st); FN =72 | 2.38 | 2.05 | 2B |
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In general, genetic information of higher organisms is more complex than that of lower organisms, so the genomic size of higher organisms is relatively greater. However, there is no inevitable correlation between genome size and organismal complexity, because genome often contains a large number of highly repetitive DNA sequences, resulting in the conflict of DNA content and its evolutionary level. Gregory and Hebert estimated genome sizes from 12 species of freshwater oligochaetes ranging from 0.8 to 7.6 pg, and 15 species of earthworms varied from 0.4 to 1.2 pg (
In the present study, the karyotype of an Urechidae animal, U. unicinctus, was discovered for the first time as 2n = 30 (10m + 6sm + 6st + 8t), FN=52. Meanwhile, the 2C DNA content was detected to be 1.85 pg and its genome size was estimated as 904.58 Mb. Our study provided effective cytogenetic information for taxonomic study and whole-genome sequencing of U. unicinctus.
This work was supported by the National Natural Science Foundation of China (31572601).