Source: UNIVERSITY OF NEVADA submitted to
INTERPRETING CATTLE GENOME DATA: BIOLOGY, APPLICATIONS AND OUTREACH (NC-209)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0207937
Grant No.
(N/A)
Project No.
NEV05333
Proposal No.
(N/A)
Multistate No.
NC-1010
Program Code
(N/A)
Project Start Date
Jul 1, 2006
Project End Date
Sep 30, 2007
Grant Year
(N/A)
Project Director
Liu, W.
Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557
Performing Department
Ag Nutrition and Vet Sciences
Non Technical Summary
Beef production is one of the significant agricultural businesses in the western US including Nevada. The reproductive process is central to beef productivity. One of the most important components in the cattle reproductive process is bull fertility. However, bull fertility has not been studied at a molecular level because of the lack of molecular genetic markers and diagnostic tools. Extensive molecular genetic and functional studies from human and mouse have identified a candidate gene, Deleted in Azoospermia-Like (DAZL). Mutations and single nucleotide polymorphism (SNP) in this gene have been linked to azoospermia and infertility in several species including human, mouse, fly, and frog. This proposal initiates the first such study to address male infertility/subfertility in cattle. Our hypothesis is that the bovine DAZL gene plays an important role in male spermatogenesis and fertility in cattle and other farm animal species. Mutations identified in the bDAZL gene are potentially significant markers for selection for bull fertility. The goal of the project is to discover SNPs that originate either within or near the bDAZL gene and then to determine whether mutations in the bDAZL gene are associated with traits related to bull fertility. We expect that results from this research will improve our knowledge regarding the function of this gene in bull spermatogenesis and fertility, eventually leading to the use of a DNA-based test to assist in the selection of bulls at an early age in cattle breeding.
Animal Health Component
100%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013310108050%
3043310104050%
Knowledge Area
301 - Reproductive Performance of Animals; 304 - Animal Genome;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1080 - Genetics; 1040 - Molecular biology;
Goals / Objectives
Determine the location, structure, function and expression of genes affecting health, reproduction, production, and product quality in cattle.
Project Methods
1. Approaches to discovery of SNPs. Three different approaches will be applied. The first approach is an in silico bioinformatics approach. We plan to search bovine expressed sequence tags (ESTs) databases available online at TIGR and GenBank using the bDAZL cDNA sequence to identify all bDAZL related ESTs. These ESTs will be compared by multiple sequence alignment. Any sequence different in a particular base will be considered as a potential SNP. Our second approach is to pool DNA samples from four beef cattle breeds and use them as a template for PCR with bDAZL gene primers. Five to 10 bulls from each breed will be selected randomly. Sequencing the PCR fragments will allow us to identify SNPs, should a SNP occur in the amplicon. The third approach is to compare the top/bottom 10 bulls from one of the worlds largest AI companies (Semex Alliance, Canada) to identify SNPs. Similarly, DNA samples pooled from the top/bottom 10 bulls will be used in PCR and then in sequencing of the PCR fragments. In addition, we have collected a DNA sample from an infertile bull. We will add this DNA sample to the mix of top / bottom 10 pooled DNA. 2. Regions of bDAZL to focus on for SNP discovery. As both bDAZL transcripts are large, spanning ~ 33 kb in the genome, the discovery of SNPs will be initially focused on two region of the gene: the RRM and the 3-primer-UTR region that contains the sequence GUUC motifs. 3. Validation of bDAZL SNPs. Once a SNP is identified, we will verify the SNP by a PCR-RFLP method. 4. Association study of bDAZL SNPs with male fertility. Three experiments will be carried out for association studies. Experiment 1: Association analysis of SNPs with subfertile/infertile bulls. We will analyze all SNPs identified from bDAZL with DNA from infertile/subfertile bulls and compare the results with ones from normal fertile bulls, to study if any single SNP or a haplotype of a group of SNPs is the cause of infertility/subfertilty. Experiment 2: Association analysis of SNPs with top/bottom 10 bulls. In collaboration with Semex Alliance, we have selected the top / bottom 10 bulls in terms of their performance in NRR and semen quality. We plan to genotype these 20 animals to identify the trend in which SNP or haplotype is associated with higher or lower performance in bull fertility. Experiment 3: Design breeding strategy for bull selection in fertility. Once a bDAZL SNP is confirmed to associate with bull fertility performance, we will design a DNA-based assay to detect the SNP in the bDAZL gene for MAS to select bulls at an early age in beef breeding. We plan to select two beef ranches from Nevada. We will collect DNA samples for all newborn bull calves from the ranches, and provide them the bDAZL gene SNP genotype results, which will be incorporated into their bull selection as early as the results are obtained. Once these bDAZL-selected sires reach their breeding age, routine examination of semen quality will be carried out. The efficiency of the DNA-based assay in bull early selection for fertility will be evaluated

Progress 07/01/06 to 09/30/07

Outputs
OUTPUTS: Dr. Wansheng Liu is not longer at University of Nevada Reno and is therefore unavailable to submit a report. PARTICIPANTS: Dr. Wansheng Liu is not longer at University of Nevada Reno and is therefore unavailable to submit a report. TARGET AUDIENCES: Dr. Wansheng Liu is not longer at University of Nevada Reno and is therefore unavailable to submit a report. PROJECT MODIFICATIONS: Dr. Wansheng Liu is not longer at University of Nevada Reno and is therefore unavailable to submit a report.

Impacts
Dr. Wansheng Liu is not longer at University of Nevada Reno and is therefore unavailable to submit a report.

Publications

  • No publications reported this period


Progress 01/01/06 to 12/31/06

Outputs
Approaches to discover SNPs: Two different approaches were applied in this research. The first approach is an in silico bioinformatics approach. We searched the bovine expressed sequence tags (ESTs) databases available online at TIGR and GenBank using the bDAZL cDNA sequence and identified two ESTs related to bDAZL. These ESTs were compared by sequence alignment. Only one nucleotide substitution (C→G) in the bDAZL cDNA was identified and was considered as a potential SNP. When we realized that there were not enough bDAZL-related ESTs in the databases, a second approach was applied by pooling DNA samples from the high (10 animals) and low (10) non-return rate (NRR) bulls as well as four subfertile and one infertile bull to identify SNPs. DNA samples pooled from the 25 animals were used in PCR and then the PCR fragments were sequenced. Regions of bDAZL to focus on for SNP discovery: As both bDAZL transcripts (2996 bp and 1383 bp) are large, spanning 33 kb in the genome, the discovery of SNPs was initially focused on all exons and the 3'UTR region that contains the sequence 'GUUC' motifs. bDAZL SNPs: By the pooled-DNA-PCR-sequencing approach, we have identified 16 SNPs. Among all exons, only one silent SNP was identified from the exon 10. Six SNPs were identified from the 3'UTR region, and five SNPs were identified from the intron sequences adjacent to the exons. SNP genotyping and association study: To date, we have genotyped 10 SNPs among the 25 bulls. The SAS/JMP software was used to analyze the genotype, haplotype and trait association. The linkage disequilibrium (LD) test for allelic association indicated that there is a very significant LD among the 10 SNPs and their alleles. The marker-trait association test indicated that the SNPs from the bDAZL gene are significantly associated to the fertility trait (NRR). The test for haplotype-trait association indicated that different haplotypes are very significantly associated to the fertility trait. Our conclusion is that the bDAZL SNPs and their haplotypes can be used in MAS for sire early selection for the male fertility trait.

Impacts
Functional studies of the DAZ/DAZL gene in fly, frog, mouse and human all support a hypothesis that the bovine DAZL gene plays an important role in male spermatogenesis and fertility in cattle. Results from this study have tested this hypothesis and provided direct evidence in the function of this gene in bull spermatogenesis and fertility. The identification of SNPs from bDAZL and the confirmation of the SNP haplotype-fertility trait association will lead to the design of marker assisted selection (MAS) to select sires at an early age in cattle breeding. This, in turn, will significantly reduce the breeding cost, as bulls with subfertility or infertility are usually not identified until the age when they are expected to breed.

Publications

  • Liu, W.-S. and Ponce de Leon, F.A. (2006) Characterization and Mapping of the Bovine DAZL Gene. PAG-XIV, Conference Abstract, p
  • Liu ,W.-S., Wang, A., Uno, Y., Galtz, D., Beattie, C.W., Ponce de Leon, F.A. (2007) Genomic structure and transcript variants of the bovine DAZL gene. Cytogenetic and Genome Research 116 (1-2), 65-71.
  • Liu ,W.-S., Wang, A. (2007) Association of Single Nucleotide Polymorphisms (SNPs) of the Bovine Deleted in Azoospermia Like (bDAZL) Gene with Male Fertility. Provisional patent application.