Research Article |
Corresponding author: Xiaoting Huang ( xthuang@ouc.edu.cn ) Academic editor: Virmantas Stunžėnas
© 2016 Zujing Yang, Xuan Li, Huan Liao, Liping Hu, Zhengrui Zhang, Bosong Zhao, Xiaoting Huang, Zhenmin Bao.
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:
Yang Z, Li X, Liao H, Hu L, Zhang Z, Zhao B, Huang X, Bao Z (2016) Physical mapping of immune-related genes in Yesso scallop (Patinopecten yessoensis) using fluorescent in situ hybridization. Comparative Cytogenetics 10(4): 529-541. https://doi.org/10.3897/CompCytogen.v10i4.10047
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The innate immune system plays a pivotal role in defending invasion of microorganisms for scallops. Previous studies on immune-related genes in the Yesso scallop, Patinopecten yessoensis (Jay, 1857) have mainly focused on characterization and expression pattern in response to bacterial challenge, no research has been carried out on the cytogenetic level yet. In the present study, eight fosmid clones containing the sequences of key immune-related genes (PyNFkB, PyTRAF2, PyTRAF4, PyTRAF7, PyMyd88-1, PyMyd88-3, PyMKK-7 and PyTNFR) were isolated and seven of them were successfully mapped on chromosomes of P. yessoensis utilizing fluorescence in situ hybridization. Wherein, PyMyd88-1, PyMyd88-3 and PyMKK-7 located on the same chromosome pair with adjacent positions and the other genes were mapped on four non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of such genes of the Yesso scallop will lay a foundation for studies such as assignment of interested genes to chromosomes, construction cytogenetic maps and so on.
Cytogenetic map, immune-related genes, FISH, fosmid
Studying chromosomal distribution pattern of important functional genes at the cytogenetic level, which can be useful for discussion of gene evolution, chromosome rearrangement and so on, is a crucial part of genome research in any species. For example, by mapping Antennapedia (ANTP) class homeobox genes to the chromosome in a lophotrochozoan protostome,
However, limited researches on chromosomal localization of functional genes have been conducted in mollusk species as yet. This is mostly due to the short probe length and the technical limitations, genes and sequences with low copy numbers tend to be more difficult to obtain positive signals after fluorescence
The Yesso scallop, Patinopecten yessoensis (Jay, 1857), is a cold water bivalve and is naturally distributed along the coastline of northern Japan, the Far East of Russia and the northern Korean Peninsula (
Cytogenetic studies of the Yesso scallop showed it possessed 38 chromosomes with a karyotype formula of 3m+5sm+8st+3t (
In present study, to investigate the chromosome distribution of immune-related genes of the Yesso scallop, we selected 8 gene-containing fosmid clones as probes for FISH. As a result, we successfully mapped 7 immune-related genes to the chromosomes of the Yesso scallop. Fosmid clones were proved to have high efficiency in chromosome mapping of single or low copy genes in the Yesso scallop. Our results provided the first physical mapping of immune-related genes in P. yessoensis, aiding to understanding chromosomal assignment and evolution of these genes.
Trochophore larvae of P. yessoensis was treated for 2h at room temperature with 0.02% colchicine in sea water, the larvae were exposed to potassium chloride (KCl) solution (0.075M) for 30 min. Then it were fixed three times (15 min each) in fresh Carnoy’s fixative (ethanol:glacial acetic acid = 3:1 v/v). Chromosome spreads were obtained by dissociating fixed larvae in 50% acetic acid and dropping the cellular suspension onto slides heated to 56°C.
A fosmid library including 122, 880 clones of P. yessoensis has been constructed in our lab recently. The restriction fragments of two-dimensionally pooled fosmid clones were sequenced and generated sequences by a physical mapping technology based on next-generation sequencing, whole genome profiling (WGP) (
Plasmid DNA from gene-containing fosmid clones, with an average insert size of 30-45 kb, was extracted by standard laboratory method (
FISH experiments were performed following methods previously published (
Co-hybridization was conducted to investigate the relative chromosomal positions between each two fosmid clones. The protocol follows the same procedure of regular hybridization. And the hybridization mix with a total volume of 30µl contained 5-10ng/µl of each probe, 50% formamide, 10% dextran sulphate and 2×SSC.
The partial sequences of genes which were successfully located by FISH were amplified and sequenced to make sure those are gene-containing fosmid clones. Primers were published previously (
In total 19 genes were used for fosmid clones searching. Eight of them were managed to find matched fosmid clones (Table
Karyotype analysis results of mapped gene-containing fosmid clones: PF126M18 (A); PF123H24 (B); PF109F4 (G); PF106B20 (D); PF118H7 (E); PF123J11 (F); PF120E14 (G). Chromosome numbering is based on chromosome type and relative length. Wherein, chromosome pair 1, 2, 3 represent the metacentric (m) chromosomes. Chromosome pair 4, 5, 6, 7, 8 represent the submetacentric (sm) chromosomes. Chromosome pair 9, 10, 11, 12, 13, 14, 15, 16 represent the subtelocentric (st) chromosomes. Chromosome pair 17, 18, 19 represent the telocentric (t) chromosomes.
Clone name | Gene name | Chromosome type* | Location of signals |
---|---|---|---|
PF118H7 | PyMKK7 | st | On telomeric region of 14q |
PF123J11 | PyMyd88-1 | st | On middle region of 14q |
PF106B20 | PyMyd88-3 | st | On telomeric region of 11p, 13p and 14q |
PF123H24 | PyTRAF7 | sm | On centromeric region of 8q |
PF109F4 | PyNFkB | t | On telomeric region of 11p and 13p, centromeric region of 18q |
PF120E14 | PyTRAF4 | st | On telomeric region of 12q |
PF126M18 | PyTNFR | m | On middle region of 1q |
PF118E11 | PyTRAF2 | N/A | No positive signals were found |
In previous study, clusters of 18S-28S rDNA were localized on 2 different pairs of subtelocentric chromosomes of the Yesso scallop (
Co-hybridization results of fosmid clones with positive signals on mitotic metaphase chromosomes of P. yessoensis (A-I): Mapping of clone PF106B20 & 18S-28S rDNA (A), clone PF109F4 &18S-28S rDNA (B), clones PF123J11 & PF106B20 (C), clones PF118H7 & PF123J11 (D), clones PF126M18 & PF118H7 (E), clones PF123J11 & PF120E14 (F), clones PF123H24 & PF123J11 (G), clones PF123H24 & PF126M18 (H), clones PF126M18 & PF120E14 (I). The arrows and the open triangles indicate positive signals of different probes. Bars=10 µm.
In order to find out the chromosome position relations of the genes which were successfully mapped, karyotype analysis of FISH results were carried out firstly (Fig.
For those fosmid clones whose position relationship couldn’t justified by karyotype, co-hybridization was conducted (Fig.
The partial sequences of genes from the 7 mapped fosmid clones were amplified and products were successfully sequenced. A BLASTN analysis of the 7 sequences against the P. yessoensis genome sequencing data showed significant sequence matches as we expected and confirmed the existence of the genes in the fosmid clones (Table
Gene name (Clone name) | Primer | Primer sequence(5’-3’) | Identities |
---|---|---|---|
PyMyd88-1 (PF123J11) |
F- PF123J11 R- PF123J11 |
TTGCACATGCTCTGTCGCC GGACGCAGTTCGCTTTTGAT |
679/684 (99%) |
PyMyd88-3 (PF106B20) |
F- PF106B20 R- PF106B20 |
GAGTGTCGAGTGCGACTTCATG CGCCTTCAGTAGACGTTTCCAG |
944/946 (99%) |
PyTRAF7 (PF123H24) |
F- PF123H24 R- PF123H24 |
CCAGATTGTCACGCTGAAAGG CCAGATTGTCACGCTGAAAGG |
76/77 (99%) |
PyMKK7 (PF118H7) |
F- PF118H7 R- PF118H7 |
TCAAAGGCTAAGACGAGGAGTGC CAACCAATGTGATGCCCAGG |
773/779 (99%) |
PyNFkB (PF109F4) |
F- PF109F4 R- PF109F4 |
TGCCCGTGTTGTGGTAACCTTGG CGTGAGAGAGTTTTGTCCGCCCTT |
90/92 (98%) |
PyTNFR (PF126M18) |
F- PF126M18 R- PF126M18 |
AACAACCTACCTGAAACGGAACA CGGTTAGGATTTGGACAAGGAC |
54/56 (96%) |
PyTRAF4 (PF120E14) |
F- PF120E14 R- PF120E14 |
GGACTTATCGCTGTCAACC AAATGTGGCACCTTACTCG |
187/189 (99%) |
Particularly, because of the similarity between mapping results of clone PF106B20 and 18S-28S rDNA, PF109F4 and 18S-28S rDNA, plasmids of these two clones were used as template for amplification of 18S-28S rDNA. The obtained PCR products were successfully sequenced and the sequences were identical to the 18S-28S rDNA sequence of the Yesso scallop.
The innate immune system plays a vital role in scallop species defending against various invaders because of lacking of adaptive immunity (
In present study, PyNFkB, PyTRAF4, PyTRAF7, PyMyd88-1, PyMyd88-3, PyMKK-7 and PyTNFR genes were successfully mapped to chromosomes of the Yesso scallop. After co-hybridization, PyMKK-7, PyMyd88-1 and PyMyd88-3 were found to be located on the same chromosome pair with very adjacent positions. Previous studies have shown that genes with similar expression patterns tend to cluster more frequently than those with different expression patterns (
The genome-wide identification of five PyMyD88 duplication genes in the Yesso scallop showed that all five genes were located on the same linkage group. In present study, fosmid clones of two PyMyD88 genes (PyMyd88-1 and PyMyd88-3) were pinpointed and were successfully mapped to the same chromosome pair of the Yesso scallop. What is more, PyMKK7 gene was found to locate on the adjacent chromosomal position with that of PyMyD88 genes. It was thought that the higher number of PyMyD88 duplications in the Yesso scallop may be relevant to their specific and cooperative functions in the corresponding innate immune system and in PyMyD88-dependent and independent pathways against bacterial infection (
According to genome sequencing data (BioProject number PRJNA259405), PyMKK-7, PyMyd88-1 and PyMyd88-3 genes were contained in three different scaffold, which were scaffold7441 (PyMyd88-1), scaffold5789 (PyMyd88-3), scaffold11045 (PyMKK-7) respectively. Based on the established SNP linkage map (unpublished), scaffold7441 and scaffold5789 belonged to the linkage group 1. However, which linkage group was scaffold11045 distributed to was not clear. Through co-hybridization, the present study successfully mapped PyMKK-7 and PyMyd88-1 genes into the same chromosome pair and anchored scaffold11045 to the linkage group 1 just like scaffold7441 and scaffold5789 which helped genome assembly of the P. yessoensis.
Apart from the three genes mapped in the same chromosome pair mentioned above, no other genes were found to have the similar pattern in the present study. In the Zhikong scallop, five key TLR signaling pathway genes (CfTLR, CfMyd88, CfTRAF6, CfNFkB and CfIkB) were mapped in five non-homologous chromosome pairs which was similar with the FISH results of PyTRAF7, PyNFkB, PyTRAF4 and PyTNFR (
Chromosomal localization of immune-related genes in five model organisms.
Organism | NFkB | Myd88 | MKK | TNFR | TRAF4 | TRAF7 |
---|---|---|---|---|---|---|
Homo sapiens | NFkB2(4791*) Chr. 10 |
MyD88 (4615*) Chr. 3 |
MKK7(5609*) Chr. 19 |
TNFRSF18(8784*) Chr. 1 |
TRAF4(9618*) Chr. 17 |
TRAF7(84231*) Chr. 16 |
Drosophila melanogaster | Dorsal(35047*) Chr. 2L |
MyD88(35956*) Chr. 2R |
MKK7(32256*) Chr. X |
TNFR(32849*) Chr. X |
TRAF4(33638 *) Chr. 2L |
N/A |
Danio rerio | NFkB3(425099*) Chr. 7 |
MyD88(403145*) Chr. 24 |
MKK7(560913*) Chr. 1 |
N/A | TRAF4b(404035 *) Chr. 21 |
TRAF7(563746*) Chr. 3 |
Mus musculus | NFkB3(5970*) Chr. 11 |
MyD88(17874*) Chr. 9 |
MKK7(26400*) Chr. 8 |
TNFRSF1B(21938*) Chr. 4 |
TRAF4(22032 *) Chr.11 |
TRAF7(224619*) Chr. 17 |
Gallus gallus | NFkB2(386574*) Chr. 6 |
MyD88(420420*) Chr. 2 |
N/A | TNFRSF1B(395083*) Chr. 21 |
TRAF4(417577*) Chr. 19 |
TRAF7(416555*) Chr. 14 |
Moreover, fosmid clone PF109F4 and PF106B20 were mapped to three different chromosome pairs separately. The FISH mapping results of the two clones both showed two pair positive signals located on two subtelocentric chromosome pairs which were quite alike with the FISH mapping image of the 18S-28S rDNA. With PCR as well as sequencing results, it was proved that these two clones did contain partial 18S-28S rDNA sequence. The average insert fragment of fosmid clones applied within this ranged from 30kb to 45kb. As a result, beside covered the target sequences that needed for FISH mapping, it inevitably included other sequences from the Yesso scallop genome. Early study showed 18S-28S rDNA sequence was highly repetitive and also had quite high hybridization efficiency during FISH. As a result, possessing partial homologous sequences of 18S-28S rDNA was believed to be the reason of appearance of multiple positive signals using clone PF109F4 and PF106B20 as probes. The co-hybridization result of PF109F4 and 18S-28S rDNA, PF106B20 and 18S-28S rDNA also proved these phenomena.
FISH is a powerful tool for chromosomal localization of DNA sequences and have already been used for cytogenetic study in scallop species. Comprehensively speaking, published study were mostly focusing on the multi-copy sequencing mapping such as histone H3 gene , ribosomal genes and satellite DNA (
The present study revealed chromosomal distribution pattern of seven immune-related genes and enriched the number of chromosome markers in the Yesso scallop. These results will lay a foundation for the upcoming genome and cytogenetic research in P. yessoensis.
This research was funded by the National Natural Science Foundation of China (31270047), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-48) and Seed Improvement Project of Shandong Province.