tóm tắt la ta (triệu tuấn): Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.

Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.Nghiên cứu phát triển chỉ thị phân tử phục vụ chọn giống tu hài (Lutraria rhynchaena, Jonas 1844) theo hướng tăng trưởng.MINISTRY OF EDUCATION AND TRAINING HA NOI NATIONAL UNIVERSITY OF EDUCATION TRIEU ANH TUAN RESEARCH AND DEVELOPMENT OF MOLECULAR MARKERS FOR BREED SELECTION OF OTTER CLAM (Lutraria rhynchaena Jonas, 18.

MINISTRY OF EDUCATION AND TRAINING

HA NOI NATIONAL UNIVERSITY OF EDUCATION

TRIEU ANH TUAN

RESEARCH AND DEVELOPMENT OF MOLECULAR MARKERS FOR BREED SELECTION OF OTTER CLAM

(Lutraria rhynchaena Jonas, 1844) IN THE DIRECTION OF

GROWTH Subject: Genetics Code: 9.42.01.21

SUMMARY OF THE DISSERTATION FOR THE DEGREE DOCTOR OF PHILOSOPHY (Ph.D) IN

BIOLOGY

Ha Noi – 2023

This dissertation is submitted to the Committee of the:

HA NOI NATIONAL UNIVERSITY OF EDUCATION

Supervisors:

Assoc Prof Dr Nguyen Xuan Viet Assoc Prof Dr Thai Thanh Binh

Referee 1: Assoc Prof Dr Nguyen Dang Ton

Viện Nghiên cứu hệ gen – Viện Hàn lâm KH&CN Việt Nam

Referee 2: Assoc Prof Dr Dang Thi Lua

Research Institute for Aquacultrure No.I

Referee 3: Assoc Prof Dr Nguyen Quang Huy

Hanoi University of Science, Vietnam National University

This research has been performed at the Ha Noi National University of Education Date of Oral Presentation:……,… /…… /2023

Copy of this dissertation is available at:

- The National Library of Viet Nam

- Library of Ha Noi National University of Education

PREAMBLE

1 Urgency of Dissertation Title

Otter Clam (Lutraria rhynchaena Jonas, 1844) is a bivalve mollusk,

preferring to live in warm waters, from 18-30oC and salinity ranging from 30‰ (Ha Duc Thang, 2010) Otter clam mainly feeds on organic humus, algae and plankton (Ha Duc Thang, 2010)

25-In Vietnam, otter clam is naturally distributed as well as widely farmed in the waters of Quang Ninh, Hai Phong and Khanh Hoa provinces with a total water surface area of about 1,000 ha, raising output of about 2,621.6 tons, and an estimated income of over 200 billion VND/year Otter clam has high nutritional value It’s rich in protein, minerals and amino acids in their body (Do Dang Khoa

et al., 2014)

Recently, the demand for otter clam is increasing, the economic efficiency brought from raising them is large, so the farming area of commercial otter clam has increased significantly As a result, there is a persistent demand for certified otter clam for breeding in huge quantities According to the report of the Directorate of Fisheries, it is estimated that each year, the commercial market for otter clam needs about 100 million grade I breeders (Directorate of Fisheries, 2020) Meanwhile, the advanced breeding programs in our country for commercial otter clam have not yet been applied, the production of seed by the traditional method both does not provide enough demand for qualified breeders and causes breed degradation due to inbreeding in breeder production Cultivated otter clam thus proves to suffer from slower, uneven growth and are vulnerable

to diseases (Van Don Department of Agriculture and Rural Development, 2019)

According to decision No 1664/QĐ-Tg dated October 4, 2021 on the development strategy for marine aquaculture to 2030 and a vision to 2045, for marine aquaculture in general and mollusk culture in particular, including otter clam, is towards sustainable production development, minimizing negative impacts on the environment and marine ecosystems (Government, 2021) To implement this strategy, it is necessary to focus on exploiting and developing indigenous breed sources with the application of scientific breeding programs to

improve survival rates, rapid growth and tolerance to environmental conditions

Breeding with the support of molecular markers is increasingly receiving attention and the applications of molecular markers in breeding in general and in

aquaculture in particular have achieved proud achievements (Gjedrem et al.,

2012) With the advent and application of new generation gene sequencing (NGS), many biological genomes in general and aquatic species in particular have been sequenced Exploiting genome sequences and transcriptomes, many molecular markers have been published that are reliable and effective tools for breeders in rapidly creating new varieties as well as improving genetics of

existing varieties (Hetzel et al., 2000)

Among them, the single nucleotide polymorphism (SNP) marker is considered to be one of the most accurate, effective and popular markers currently applied in breeding related to yield and quality (Liu, 2007) With outstanding strengths, the SNP indicator is being used as an effective selection tool for the traits of interest in breeding programs in many livestock species in general and aquatic species in particular (Liu, 2007) Importantly, SNPs can appear in the coding region, directly affecting the site of interest, and are very effective in determining the correlation between SNPs and certain traits (Beuzen

et al., 2000) Therefore, the application of molecular biology techniques to

accurately identify the species of interest and the successful use of generation sequencing techniques can provide genome sequencing documentation in order to develop a set of molecular markers in general, a set of molecular markers related to the growth trait of otter clam in particular will have both theoretical and practical significance for the culturing of otter clam farmers

next-Stemming from the above-mentioned theories and practices, we selected

and conducted the topic "Research and development of molecular markers

for breed selection of Otter clam (Lutraria rhynchaena, Jonas 1844) in the

direction of growth”

2 Research objective

The development of a set of molecular markers related to growth traits in Otter clam contributes to the development of its farming in particular and Vietnam's marine culture in general

- Research and development a number of SNP markers related to growth trait in Otter clam

4 Scientific and Practical Significance of Dissertation Title

Scientific significance:

- The thesis topic has provided some important data on the composition of natural Otter clam distributed in Van Don, the current status and potentials of Otter clam farming in Van Don district, Quang Ninh province

- The results of the study on the construction of the DNA barcode of the Otter clam is an important scientific basis for the accurate identification of the Otter clam species for sampling for genome sequencing and research and can be meaningful for the traceability of products from Vietnamese Otter clam

- The genome sequence data of the Otter clam built from the results of the thesis is valuable for genomics research, which is the basis for exploiting and developing a set of molecular markers for researchers to select and breed Otter clam

- Development of the number of SNP markers related to the growth trait of Otter clam provides a molecular biology tool that may be valuable for growth-oriented breeding in Vietnam

that are meaningful for growth-oriented selection and breeding by molecular markers in Otter clam species

5 New contributions of the dissertation

- The dissertation has evaluated the composition of the species of Otter clam distributed naturally in Van Don and the potential of its farming in Van Don, Quang Ninh

- Constructed DNA barcodes for Otter clam based on 16S rRNA and COI

gene sequences, providing additional tools that can accurately identify and classify Otter clam species

- For the first time, Otter clam genome was published on GenBank, contributing to providing reference genome data forthe research and exploitation

of Otter clam’s genomic data

CHAPTER 1 RESEARCH OVERVIEW 1.1 Classification position, distribution area of Otter Clam

1.1.1 Classification position

Mollusca: Mollusca Linnaeus, 1758

Bivalves: Bivalvia Linnaeus, 1758

Clam set (barrel gills): Veneroida Bieler R., 2010

Family: Mactridae Lamarck, 1799

Breed: Lutralia Lamarck, 1799

Species: Lutralia rhynchaena, Jonas 1844

(synonyms L philippinarum Reeve, L philippinarum Deshayes)

Vietnamese name: Otter Clam, English name: Snout Otter Clam, Otter Clam

Figure 1.1 Otter Clam (Lutralia rhynchaena) was collected in Van Don,

Quang Ninh 1.1.2 Natural distribution of Otter Clam

In the world, Otter Clam is distributed mainly in the waters west and south

of Australia, some Asian countries such as China, Thailand, Philippines and

North America (Beuzen et al., 2000)

In Vietnam, Otter Clam is distributed mainly in the North Sea, the salinity

is stable from 25 to 30 ‰, the bottom is sand, small gravel, or mollusk shells in the waters of Lan Ha Bay of Cat Ba island - Hai Phong City to Van Don island district - Quang Ninh (Pham Thuoc, 2006)

1.1.3 Economic value and situation of farming and exploiting Otter Clam

In the world, according to statistics, the global fishery production in 2018 reached nearly 80 million tons, in 2020 it will reach 94.6 million tons, of which mollusk production accounts for one third, otter clam production is estimated at

4 million tons (FAO, 2020)

1.2 DNA barcoding and its application in classification and species identification

1.2.1 DNA barcoding

DNA barcoding has become a popular, important and meaningful term for more than a decade all over the world In 2003, for the first time, the concept of DNA barcodes was introduced by Professor Paul Hebert to help identify samples, using short DNA fragments from specific gene segments or combining many genes

(Hebert et al., 2003) Using DNA barcoding techniques in research will help

taxonomists to accurately identify species and contribute to the assessment of

genetic diversity of organisms (Zalapa et al., 2012)

1.2.2 Application of DNA barcoding in the classification of creatures and mollusks

In classification, DNA barcoding has been applied to classify many species

of organisms Classification of fish classes (Lara et al., 2010), (Murgarella et al., 2016), (Weigt et al., 2012) Crustaceans were used to classify 4 lobster species

by sequencing the COI gene region, including one Vietnamese lobster and three

other species collected in Australia and Sri Lanka (Hieu et al., 2019)

On molluscs, DNA barcoding has been successfully applied to identify 14 bivalve species, the COI sequences have contributed to new findings related to

the high biodiversity of mollusks (Moira et al., 2021), DNA barcoding has been applied and proved very effective in classifying oysters (Trivedi et al., 2012), echinoderms (Ward et al., 2019), and sweet snails (Binh et al., 2017) In

barcoding analysis of clams, Liu (2018) used COI genomic DNA sequences of

56 species and 16S gene sequences of 19 species to determine the phylogenetic relationships of clams (Liu and Zhang, 2018)

1.3 Gene sequencing technology and molecular markers SNPs

1.3.1 Gene sequencing technology and applications

Next generation sequencing – NGS was born in that context, the birth of NGS technology based on the simultaneous development of sample preparation techniques (Template preparation, Sequencing and imaging), Genome aligment and assembly The use of NGS technology creates a huge amount of data (Solving from 8 Gb to 600 Gb), cheap price, low cost and outstanding advantages of fast and accurate sequencing Commonly used next-generation sequencing systems include Roche 454 (454 Life Sciences), HiSeq 2000/4000 (Illumina) and AB SOLiD (Life Technologies) sequencing

RAD- Sequencing (Restriction site Associated DNA Sequencing) is a genome sequencing technique that uses restriction enzymes to cut genomic DNA into short segments and attach adapters to both ends, a large number of genetic variants such as SNPs can be identified from the DNA sequence data by next-

generation sequencing (Baird et al., 2008)

Gene sequencing technology has been applied to successfully decode the

genomes of 13 mollusc species, including: Argopecten purpuratus (Li et al., 2018), Bathymodiolus platifrons (Sun et al., 2017), Chlamys farreri (Li et al., 2017), C gigas (Zhang et al., 2012), Limnoperna fortunei (Uliano-Silva et al., 2018), Modiolus philippinarum (Sun et al., 2017), Mytillus galloprovosystemis (Nguyen et al., 2014), Patinopecten yessoensis (Wang et al., 2017), Pinctada fucata (Takeuchi et al., 2012), (Du et al., 2017), Ruditapes philippinarum (Mun

et al., 2017), Saccostrea glomerata (Powell et al., 2018), Scapharca bringonii (Bai et al., 2019) and Venustaconcha ellipsiformis (Renaut et al., 2018)

1.3.2 SNPs Molecular markers and applications

Single nucleotide polymorphisms (SNPs) are DNA sequence modifications that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered in a population The distribution of SNPs in the genome is heterogeneous, with SNPs occurring at different frequencies in different chromosomal regions and in non-coding regions often higher than in coding regions (Liu and Cordes, 2004), most of the SNPs are in the form of two alleles and involve the substitution

of Cytosine (C) with Thymine (T), which is the most abundant variation in DNA sequences between individuals in a population

SNP markers have been applied in research in some important livestock,

the SNP density varies from 6.5K to 930K (Wall et al., 2014) In the

protein-coding region of the Pacific oyster, one SNP every 60 bp, in the non-protein-coding

region every 40 bp contains an SNP (Sauvage et al., 2007) Whereas in European

flat oysters, the SNP density was shown to be relatively high, in the coding region every 76 bp contains one SNP and the non-protein-coding region

protein-every 47 bp contains one SNP (Harrang et al., 2013)

1.4 Some genes related to growth trait in bivalve molluscs

Several studies have shown polymorphisms in growth-related genes in bivalve molluscs, the research group of Liying et al., (2014) has also studied IGFBP gene characterization (PyIGFBP) ) related to the growth trait of the

scallop Yesso (Liying et al., 2014) Two SNPs were associated with growth in

scallops, PyE2F3-1 and PyE2F3-2, in which the PyE2F3-1 gene was correlated

with growth in length, shell height, body weight and muscle weight (Xianhui et al., 2019)

On oyster subjects that had association and haplotype analysis of single nucleotide polymorphisms (SNPs), four SNPs (positions - 904, - 522, - 272, - 262 bp) and two haplotypes (CCCC and TCTC) is directly related to the growth rate

of C Gigas (Liting et al., 2021)

CHAPTER 2 RESEARCH MATERIALS AND METHODS

2.1 Research Materials

Research Materials

+ Samples were collected at Breeding Center of Brackish and Salty

Aquaculture, College of Economics, Technology and Fisheries The collection sample includes 01 Otter Clam muscle tissue sample collected for Otter Clam genome sequencing Muscle tissue samples of 30 fast growing Otter Clam individuals, 30 slow growing Otter Clam individuals for SNP screening sequencing Otter Clam digestive gland samples were collected for genomic sequencing

Research location: The extraction and purification of DNA products was carried out at the biotechnology department, College of Economics, Engineering and Fisheries, Tu Son city - Bac Ninh province Sequencing and reading were conducted at the Genomic Technology Center, Deakin University, Victoria - Australia

- Identification of comedian species by morphological method: Collected individuals are classified based on external morphological characteristics (shape, shell color, color of trunk, morphological classification criteria) according to the following criteria: Classification course on bivalve molluscs by Dang Ngoc Thanh (Thanh Dang Ngoc, 2007)

2.2.1.2 Evaluation of the current status and economic efficiency of Otter Clam farming in Van Don

The total number of survey samples was 400, the surveyed information included: farming area, farming method, stocking density, grow-out time, harvest

size, disease situation, survival rate, output and economic efficiency, compliance with the provisions of the law on the implementation of environmental protection measures at the establishment

2.2.2 DNA Barcoding Method for Vietnamese Otter Clam L rhynchaena

2.2.2.1 Comparing the similarity of 16S and COI gene sequences of Vietnamese Otter Clam and species in the family Lutraria

The DNA sequences of 16S gene regions, COI of Lutraria varieties were

queried and searched for sequences on the gene bank using BLASTP tool (Altschul et al., 1997)

2.2.2.2 Building a phylogenetic tree for the 16S and COI gene regions of Vietnamese Otter Clam and species of the Lutraria family

Evolution tree was built for 5 Otter Clam species in this study based on Maximum likelihood (ML) algorithm with K2P evolutionary model (Kumar et al., 2018), Bayesian inference (BI) algorithm (Ronquist and Huelsenbeck, 2003), BEAST v2.6.3 (Rambaut and Drummond, 2007)

2.2.2.3 Development of a DNA barcoding marker for 16S gene region and COI for Vietnamese Otter Clam

Lutraria's 16S, COI gene sequences were analyzed using BioEdit software

and MEGA X software

2.2.3 Genome sequencing and genomic sequencing for Otter Clam (L rhynchaena)

2.2.3.1 Library setup and sequencing for Vietnamese Otter Clam

*Set up Otter Clam genomic DNA library: Two Otter Clam muscle tissue

sample sequencing libraries were prepared (01 prepared with NuGen Celero DNA-Seq (Tecan Genomics, San Carlos, CA), 01 prepared with NEBNext Ultra DNA (New England Biolabs, Ipwich, MA) according to the manufacturer's instructions

*DNA sequencing: DNA sequencing of the Otter Clam genome was

performed jointly by an Illumina NovaSeq6000 sequencer and a MinION (ONT) sequencer, at the Genomic Center, Deakin University, Victoria - Australia

2.2.3.2 Library setup and genomic sequencing for Vietnamese Otter Clam

RNA was performed using the Zymo Quick-RNA Miniprep kit, an RNA library developed using the Nugen Universal Plus mRNA-Seq Kit (Tecan Genomics, San Carlos, CA) according to the manufacturer's instructions

Otter Clam transcriptome sequencing was performed using an Illumina NovaSeq6000 sequencer

2.3.4 ezRAD sequencing for SNP screening for Vietnamese Otter Clam to search for growth trait-associated SNPs

The fast- and slow-growing Otter Clam genomic DNA libraries were developed using the ezRAD technique (Toonen et al., 2013), using the TruSeq Nano HT Library Preparation kit (Illumina) The DNA library obtained by the ezRAD technique is a band of 350-550 bp in size

ezRAD Sequencing: Sequencing using the HiSeq6000 next-generation

sequencing system (Illumina), at the Center for Genomic Technology, Deakin University, Australia

2.2.5 Data analysis and processing methods

2.2.5.1 Processing of survey data on the species composition of the comedian and assessing the current status and potential of the tuftede farming profession:

Data analysis and processing methods: Survey data is processed and calculated according to (Institute of Marine Resources and Environment, 1994), survey data, interviews, rural rapid assessment (RRA) and questionnaire survey (QS) are carried out according to Groves' method (Groves et al., 2004) Combined one-factor ANOVA analysis using Minitab 16.4 software and using Microsoft Office Excel 2016

2.2.5.2 Data processing to build DNA barcodes to identify the remains of L rhynchaena:

The 16S rRNA genomic DNA sequences, COI of Lutraria species were aligned using Bioedit and MAFFT software (Katoh and Standley, 2013) Then conduct analysis using BioEdit software and MEGA X software

2.2.5.3 Otter Clam L rhynchaena genomic analysis group:

Genome analysis group of Otter Clam L rhynchaena:

Otter Clam Genome Sizing: Preprocessing Raw Datasets Using Fastp Tool

(v0.19.4) (Chen et al., 2018), dataset quality was preprocessed with the program Trimmomatic (v0.36) (Bolger et al., 2014) Evaluation of the assembly quality

of Otter Clam gene sequences using bowtie2 v2.3.3.1 software (Langmead and Salzberg, 2012) Estimate the size of the Otter Clam genome using the kmer value

in the program with Jellyfish v2.2.6 (Marçais and Kingsford, 2011) Using GenomeScope software to construct k-mer charts and estimate the genome size

of Otter Clam (Vurture et al., 2017)

Assemble the Otter Clam genome using MaSuRCA software (De novo)

(Zimin et al., 2013), evaluate the completeness of the assembly process using BUSCO v3.0.2 to (Simão et al., 2015) Editing of long reads and assemblies using minimap2 software (Li et al., 2018), error correction using Purge Haplotigs software (Renaut et al., 2018)

*Heterozygous estimation, repeat sequence and polymorphism search: Single

nucleotide polymorphism (SNP) detection by bowtie2 software (Langmead and

Salzberg et al., 2012), repeat sequence determination by RECON and RepeatScout software (Langmead and Salzberg, 2012), (Chen et al., 2018), (Price et al., 2005)

*Some features of the bivalve genome: Assembly size, length of the N50 gene region of the L rhynchaena genome and other characteristics will be compared with the genomes of 13 molluscs that have been announced in advance

*Assembly of the transcriptional genome: The RNA-seq library was

sequenced, the raw data set was preprocessed, and a transcriptome was created

to aid in genomic prediction De novo assembly was performed using Trinity software (Grabherr, 2011) Processing short read sequences using Bowtie2 software (Langmead and Salzberg, 2012) Align the transcriptome genome using

GMAP software (Zalapa et al., 2012)

*Gene prediction and annotation: Gene prediction was performed using

MAKER software (Holt and Yandell, 2011), major protein groups were aligned using MAFFT v7.394 software (Katoh and Standley, 2013), proteins were

determined by software IQ-TREE v1.5.5 (Nguyen et al., 2014)

Analysis of SNP molecular markers using BWA software (Li and Durbin, 2009) and detection of SNPs using SAMtools (Li, 2009) SNPs were aligned with contigs and nucleotide differences were detected on at least four read sequences