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Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus.
PLoS One. 2018; 13(4):e0195829.Plos

Abstract

The co-existence of several ploidy types in natural populations makes the cyprinid loach Misgurnus anguillicaudatus an exciting model system to study the genetic and phenotypic consequences of ploidy variations. A first step in such effort is to identify the specific ploidy of an individual. Currently popular methods of karyotyping via cytological preparation or flow cytometry require a large amount of tissue (such as blood) samples, which can be damaging or fatal to the fishes. Here, we developed novel microsatellite markers (SSR markers) from M. anguillicaudatus and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple M. anguillicaudatus using the Illumina paired-end sequencing. Approximately 150 million raw reads were assembled into 76,544 non-redundant unigenes. A total of 8,194 potential SSR markers were identified. We selected 98 pairs with more than five tandem repeats for further assays. Out of 45 putative EST-SSR markers that successfully amplified and harbored polymorphism in diploids, 11 markers displayed high variability in tetraploids. We further demonstrate that a set of five EST-SSR markers selected from these are sufficient to distinguish ploidy levels, by first validating them on 69 reference specimens with known ploidy levels and then subsequently using fresh-collected 96 ploidy-unknown specimens. The results from EST-SSR markers are highly concordant with those from independent flow cytometry analysis. The novel EST-SSR markers developed here should facilitate genetic studies of polyploidy in the emerging model system M. anguillicaudatus.

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Authors+Show Affiliations

Feng B
College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University, Wuhan, P. R. China.
Yi SV
School of Biology, Georgia Institute of Technology, Atlanta, GA, United States of America.
Zhang M
College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University, Wuhan, P. R. China.
Zhou X
College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University, Wuhan, P. R. China.

MeSH

AnimalsComputational BiologyCypriniformesExpressed Sequence TagsGene Expression ProfilingMicrosatellite RepeatsPloidiesReproducibility of ResultsTranscriptome

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

29649332

Citation

Feng, Bing, et al. "Development of Novel EST-SSR Markers for Ploidy Identification Based On De Novo Transcriptome Assembly for Misgurnus Anguillicaudatus." PloS One, vol. 13, no. 4, 2018, pp. e0195829.
Feng B, Yi SV, Zhang M, et al. Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus. PLoS One. 2018;13(4):e0195829.
Feng, B., Yi, S. V., Zhang, M., & Zhou, X. (2018). Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus. PloS One, 13(4), e0195829. https://doi.org/10.1371/journal.pone.0195829
Feng B, et al. Development of Novel EST-SSR Markers for Ploidy Identification Based On De Novo Transcriptome Assembly for Misgurnus Anguillicaudatus. PLoS One. 2018;13(4):e0195829. PubMed PMID: 29649332.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus. AU - Feng,Bing, AU - Yi,Soojin V, AU - Zhang,Manman, AU - Zhou,Xiaoyun, Y1 - 2018/04/12/ PY - 2017/12/28/received PY - 2018/04/01/accepted PY - 2018/4/13/entrez PY - 2018/4/13/pubmed PY - 2018/7/28/medline SP - e0195829 EP - e0195829 JF - PloS one JO - PLoS One VL - 13 IS - 4 N2 - The co-existence of several ploidy types in natural populations makes the cyprinid loach Misgurnus anguillicaudatus an exciting model system to study the genetic and phenotypic consequences of ploidy variations. A first step in such effort is to identify the specific ploidy of an individual. Currently popular methods of karyotyping via cytological preparation or flow cytometry require a large amount of tissue (such as blood) samples, which can be damaging or fatal to the fishes. Here, we developed novel microsatellite markers (SSR markers) from M. anguillicaudatus and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple M. anguillicaudatus using the Illumina paired-end sequencing. Approximately 150 million raw reads were assembled into 76,544 non-redundant unigenes. A total of 8,194 potential SSR markers were identified. We selected 98 pairs with more than five tandem repeats for further assays. Out of 45 putative EST-SSR markers that successfully amplified and harbored polymorphism in diploids, 11 markers displayed high variability in tetraploids. We further demonstrate that a set of five EST-SSR markers selected from these are sufficient to distinguish ploidy levels, by first validating them on 69 reference specimens with known ploidy levels and then subsequently using fresh-collected 96 ploidy-unknown specimens. The results from EST-SSR markers are highly concordant with those from independent flow cytometry analysis. The novel EST-SSR markers developed here should facilitate genetic studies of polyploidy in the emerging model system M. anguillicaudatus. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/29649332/Development_of_novel_EST_SSR_markers_for_ploidy_identification_based_on_de_novo_transcriptome_assembly_for_Misgurnus_anguillicaudatus_ DB - PRIME DP - Unbound Medicine ER -