Source: PENNSYLVANIA STATE UNIVERSITY submitted to
GENOMIC STRUCTURE AND FUNCTION OF THE BOVINE Y-CHROMOSOME GENES IN THE MALE-SPECIFIC REGION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0220591
Grant No.
2010-65205-20362
Cumulative Award Amt.
$450,000.00
Proposal No.
2009-03306
Multistate No.
(N/A)
Project Start Date
Jan 1, 2010
Project End Date
Oct 31, 2014
Grant Year
2010
Program Code
[92120]- Animal Genome, Genetics and Breeding
Project Director
Liu, W.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Animal Science
Non Technical Summary
Infertility is a significant problem in the cattle industry due, in part, to lack of diagnostic tools for identifying low performance bulls until their breeding age. The completion of the human Y-chromosome sequencing project identified a total of 27 genes/families in the male-specific region (MSY), which are expressed extensively or predominantly in testis. These "testis genes" play vital roles in male fertility. Since the bovine genome sequence is from a cow, the bovine Y-chromosome (BTAY) coding and related sequences are still unavailable. With previous USDA-NRI grants, we have identified 23 BTAY genes/transcripts, nine of them are bovine homologs of the human Y chromosome genes and the remaining 14 are bovine-specific. These findings, together with the recent discoveries of species-specific transcripts in mouse, cat and horse, indicate that Y chromosome is poorly conserved among mammalian species. Therefore, we hypothesize that during evolution, the bovine MSY accumulated a set of X-degenerate and species-specific Y genes essential for maleness, spermatogenesis and male fertility. The goal of this proposal is to test our hypothesis with three specific aims: 1. Further discover and characterize the "gene content" of MSY by deep-sequencing the direct selected testis cDNAs; 2. Define the genomic structures of the bovine MSY genes and improve the MSY sequence map; 3. Identify polymorphisms (SNPs and indels) of MSY genes involved in spermatogenesis and male fertility. This project addresses an important problem in cattle, especially the beef industry. The outcomes of this project will not only increase our understanding of the basic biology of the bovine Y chromosome, but also have important implications for better understanding and improvement of bull fertility by using Y-polymorphic DNA markers in marker-assistant sire selection (MASS). This will allow early identification of infertile bulls and their elimination from genetic evaluations, resulting in significant time and financial savings to producers and the animal breeding industry.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043310108050%
3043410108050%
Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1080 - Genetics;
Goals / Objectives
The goals of our research are to define the genomic structure of the bovine Y chromosome (chr) male-specific region (MSY) and to identify MSY genes that impact bull fertility. The specific aims of this proposal are: 1. discover and characterize genes in the bovine MSY region; 2. define the genomic structure of the bovine MSY genes by improving the MSY sequence map; and 3. identify polymorphisms of MSY genes that are involved in spermatogenesis and male fertility. The expected outputs are: 1. Identification of the "gene content" of the bovine MSY (protein-coding genes and non-coding RNAs) expressed in testis; 2. Understand the genomic structure of all MSY genes identified in this project; 3. Development of a BAC-based Digital Minimum Tiling Path (DMTP) for the bovine Y-chromosome (BTAY) based on BACs deposited in GenBank; 4. Improved sequence map of MSY integrated with Y transcripts discovered in this project and BACs sequenced at Baylor College of Medicine (BCM); 5. Understand the expression profile for all bovine MSY genes/transcripts in mature animals and in five different developmental stages of testis; and 6. Identification of BTAY polymorphic markers including single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) associated with male fertility. The completion of this project will not only increase our understanding of the basic biology of the bovine Y chromosome, but also have important implications for better understanding and improvement of bull fertility through marker-assisted sire selection. Other outputs include the publication of at least three peer reviewed manuscripts and dissemination of the information to agricultural producers, scientists and to animal breeding and A.I. industry.
Project Methods
Our working hypothesis assumes that in the course of evolution, the bovine MSY has accumulated a set of X-chr degenerate and species-specific Y-chr genes essential for maleness, spermatogenesis and male fertility. Because almost all genes within MSY are expressed in testes, we believe that we can characterize the gene content of the bovine MSY region by determining the spectrum of BTAY transcripts present in testes. We will use a direct testis cDNA selection approach combine with next generation sequencing technology. Equal amount of total RNA extracted from bovine testes (at age 4 d, 20 d, 3 mon, 6 mon, 2 yr) will be pooled together for cDNA synthesis. Synthesized cDNAs will be ligated to an adaptor. After pre-hybridization with bovine Cot I DNA, the testis cDNAs will be hybridized with biotinylated whole genomic amplified BTAY DNA fragments. The hybrids will be extracted with streptavidin paramagnetic Dynabeads. Selected cDNAs will be PCR-amplified using the adaptor as priming site. The PCR products will be used for deep sequencing using Illumina GA2 with 36 bp short-insert paired-end (SIPE) sequencing, which will generate greater than 10 millions reads. The raw sequence data will be assembled and pre-analyzed using the Alpheus pipeline and bioinformatics tools. The assembled contigs will be BLASTed against all BTAY-BACs, the bovine genome sequence and all other non-redundant nucleotide sequences in NCBI to identify the known BTAY genes and the novel BTAY-specific transcripts, which will be further confirmed by a male-specific PCR. Full-length mRNA of BTAY genes/transcripts will be obtained by the 5'- and 3'-RACE methods. Normal expression pattern of the genes/transcripts in 11 different tissues/organs from adult bulls and in bovine ovarian tissue will be investigated using routine RT-PCR to discover where these genes/transcripts are expressed. QRT-PCR and testis section in situ hybridization (ISH) with cRNA probes will be used to study the spacial and temporal expression of these genes in testis. On the other hand, all the BTAY mapped transcripts as well as the singleton reads will be aligned with the BTAY-BACs that have been sequenced at BCM. Genomic structures of the BTAY genes will be analyzed on the basis of these alignments and genomic PCRs. We have established a BTAY assembly pipeline by developing several scripts in C++ and Vbscripts to incorporate the BLAST and EMBOSS programs for building a BAC-based Digital Minimum Tiling Path (DMTP) that will lead to the sequence assembly of MSY. If necessary, FISH will be applied to map the DMTPs on metaphase Y chrs. For SNP and indel identification, we will focus mainly on the BTAY protein-coding genes using two groups of bulls. The first group includes Holstein and Angus purebred bulls each with three subgroups of bulls evaluated as high, low fertility and subfertile/infertile. The second group includes five breeds, Hereford, Limousin, Jersey, Norwegian Red, and Wagyu. Gene-specific PCRs will be conducted to amplify the target genomic regions with a long PCR Kit. The PCR products will be sequenced to identify SNPs/indels, which will be validated in a group of ~ 650 AI bulls.

Progress 01/01/10 to 10/31/14

Outputs
Target Audience: Animal geneticists and breeders, AI industry, beef and dairy producers, as well as biologists in the fields of sex chromosomes, comparative genomics, molecular evolution, reproduction and cancer study. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? During the course of this study, a total of 2 Ph.D. students, 2 Master students, 3 post-docs and 5 visiting scholars were trained. In addition, we have provided an opportunity for over 10 undergraduate students to gain research experience in the PI's laboratory. The project also provided a flatform for national and international (China, Japan, France, Spain) collaborations and offerred resources for the newly developed graduate course, Animal Genomics. How have the results been disseminated to communities of interest? During the course of this study, a total of 16 peer-reviewed research articles, 3 book chapters, 1 technology disclosure, and 34 conference/meeting abstracts have been published. Furthermore, 24 invited oral presentations have been given since 2010 by the PI in the International Conference on the Status of Plant and Animal Genome Research, BioAsia, the International Conference on Beef Cattle Improvement and Industrialization in China, the International Society for Study of Reproduction (SSR) Conference, the International Ruminant Reproduction Symposium (IRRS) and different Universities in USA and abroad. These activities promote research and education in the sex chromosome and male reproduction and foster collaborative research, and will have an impact on biomedical and livestock industries. Although the Y chromosome sequence is available for humans, chimpanzees and macaques, little is known about the annotation and transcriptome of non-primate MSY. For the first time, we were able to comprehensively examine the bovine Y chromosome in terms of its structure, gene content, transcriptional activity and evolution. We found that the bovine MSY differs radically from the primate Ys with respect to its structure, gene content and density. Our results challenge the widely accepted hypothesis that MSY is gene-poor and transcriptionally inert. These results increase our understanding of the basic biology of the mammalian Y chromosome, and provide not only important comparative mapping information useful for studying the evolution of Y chromosome and bovid-lineages, but also details of the organization and function of these Y chromosome genes in bovine spermatogenesis and fertility. Most importantly, our results set up a stage for future research on the roles of bovid Y chromosome genes in spermatogenesis and male fertility, and on the potential to use Y-linked markers for bull fertility and semen quality selection, and cattle breeding and evolutionary studies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Gene content on the male-specific region (MSY) of the bovine Y chromosome (BTAY). Testis RNA-seq at three developmental stages: postnatal (20d), pubertal (8m) and mature (2y) revealed a physical coverage of ~81% of MSY on the BTAY draft assembly, indicating a large-scale transcriptional activity from BTAY. After annotation, we identified a total of 18 known genes/families, including 12 single-copy genes in the X-degenerate region and six multi-copy gene families in the ampliconic region. Comparative analysis identified six bovid-specific genes, including two single-copy genes (ZRSR2Y and RPL23AY) and four ampliconic gene families (ZNF280AY, ZNF280BY, PRAMEY and EGLY). In addition, a total of 46 novel single-copy and 339 novel multi-copy transcript units (TUs) were identified, which are bovid-specific. Ten of the multi-copy TUs, named BTY1-10, may have coding potential, while the remaining TUs were non-coding (nc) RNAs. Collectively, 1274 protein coding genes were identified on the bovine MSY, with a gene density of ~31.2 genes/Mb; this density is significantly higher than the ∼9.4 genes/Mb for BTAX and ∼10.2 genes/Mb for the entire genome. Hence, our results challenge the widely accepted hypothesis that MSY is gene-poor and transcriptionally inert. The gene content of the ovine MSY has also been analyzed. 2. The genomic structure of the bovine MSY. We identified three bovid Y-linked gene families, ZNF280AY, ZNF280BY and PRAMEY, that originated from the transposition of a gene block on the bovine autosome (BTA17) and subsequently amplified on the Y during evolution. These three gene families, together with TSPY and HSFY, form the basic structure of MSY. The bovine MSY is composed of ~80 repeat units. Each unit is ~420 Kb in size with palindrome-like sequences. 3. Transcriptomic activity in MSY. We found that over 95% of the genes and ncRNAs on BTAY are transcriptionally active in testis. These genes are pre-dominantly expressed and are differentially regulated during the testis development. One of these genes, PRAMEY, has been studied in details. The PRAMEY protein is involved in acrosome biogenesis, sperm motility, and fertilization. 4. Origin and evolution of the MSY gene families. Two major expansions of the BTAY ampliconic region occurred during evolution. An earlier expansion occurred ∼20 Mya during the Miocene, which coincided with the divergence time between cattle and sheep, and a later expansion occurred within 5 Mya during the Pliocene. These expansions may be involved in the diversification of the Bovidae. 5. Variation of BTAY. BTAY variations/polymorphisms including copy number variations (CNV), single nucleotide polymorphisms (SNP) and insertions and deletions (indel) have been analyzed in details. CNV of PRAMEY, ZNF280BY and HSFY have been investigated among 15 cattle breeds in the HapMap population. Surprisingly, only two Y-lineages are survived in the Holstein breed, raising alarm to the loss of genetic diversity in males. 6. Y chromosome variations and male fertility. Our results on Y-linked gene CNVs and SNPs indicated that the Y-linked variations are associated with testicular size, semen quality and male fertility.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Yue, X.-P. , Dechow, C., Liu, W.-S. (2015) A limited number of Y chromosome lineages present in North American Holsteins. J. Dairy Sci. (In press).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Maalouf, S.W., Liu, W.-S., Albert, I., Pate, J.L. (2014) Regulating life or death: Potential role of microRNA in rescue of the corpus luteum. Molecular and Cellular Endocrinology 398(1-2), 78-88. DOI: 10.1016/j.mce.2014.10.005
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Decker, J. E., McKay, S. D., Rolf, M. M., Kim, J. W., Alcal�, A. M., Sonstegard, T. S., Hanotte, O., G�therstr�m, A., Bradley, D. G., Seabury, C. M., Praharani, L., Babar, M. E., Yildiz, M. A., Michael P. Heaton, M. P., Liu, W.-S., Reecy, J.M., Saif-Ur-Rehman, M., Schnabel, R.D., Taylor, J.F. (2014) Worldwide patterns of divergence, migration and admixture in domesticated cattle. PLoS Genet. 10(3): e1004254. DOI: 10.1371/journal.pgen.1004254.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Liu, W.-S., Chang, T.-C. (2014) Y chromosome-linked genes implicated in spermatogenesis in cattle. Reproduction in Domestic Ruminants VIII. Ed: Jennifer Juengel, Akio Miyamoto, Christopher Price, Larry Raynolds, Mike Smith, and Robert Webb. Context Products Ltd., England. P239-255. DOI: 10.13140/2.1.1392.9922
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Liu, W.-S., Chang, T.-C. (2014) Y chromosome linked genes implicated in spermatogenesis in cattle. Conference Abstract, the 9th International Ruminant Reproduction Symposium (IRRS2014), August 25-29, Obihiro, Japan. P23.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Liu, Z.-H., Liu, W.-S., Ma, Y., Yue, X.P., Xiao, H.M., Li, J.-Q. (2014) Transcriptome comparison between the pubertal and adult testis in goat. Conference Abstract, the 10th World Congress on Genetics Applied to Livestock Production (WCGALP14), August17 - 22, Vancouver, Canada. P915.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Liu, W.-S. (2014) What have we learned from the bovine Y Chromosome? Conference Abstract, the 34th International Society for Animal Genetics Conference (ISAG2014), July 27-August 1, Xian, PR China.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zhao, Y.Q., Ning, G., Ocon-Grove, O.M., Diaz, F., Liu, W.-S. (2014) PRAMEY is a novel male germ cell-specific protein involved in acrosome biogenesis and blockage to polyspermy during fertilization in cattle. Conference Abstract, the 47th Annual Meeting of the Society for the Study of Reproduction (SSR2014), July 19-23, Grand Rapids, MI. No. 12.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Maalouf, S.W., Liu, W.-S., Albert, I., Pate, J.L. (2014) MicroRNA of the corpus luteum at maternal recognition of pregnancy. Conference Abstract, SSR2014, July 19-23, Grand Rapids, MI. P551.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Yue, X.-P., Dechow, C. D., Chang, T.-C., DeJarnette, J. M., Marshall, C. E., Lei, C. Z., Liu, W.-S. (2014) CNVs of two Y-linked genes, HSFY and ZNF280BY, in cattle and their association with male fertility in Holstein sires. Conference Abstract, International Plant and Animal Genome Conference (PAG)-XXII, January 11-15, San Diego, CA. P548.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Liu, W.-S., Yue, X.-P., Chang, T.-C., Krieger, K.B. (2014) Male fertility evaluation with a custom-made 384-SNP chip in cattle. Conference Abstract, PAG-XXII, January 11-15, San Diego, CA. P526.


Progress 01/01/13 to 12/31/13

Outputs
Target Audience: Animal geneticists and breeders, AI industry, beef and dairy producers, as well as biologists in the fields of sex chromosomes, comparative genomics, molecular evolution, reproduction and cancer study. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided a great opportunity for training post-doc (1), graduate students (1 MS, 1 PhD), undergraduate students (2) and visiting scholars (2). It also provided a flatform for national and international (China, Japan, Spain) collaborations and offerred resources for the newly developed graduate course, Animal Genomics. How have the results been disseminated to communities of interest? Although the Y chromosome sequence is available for humans, chimpanzees and macaques, little is known about the annotation and transcriptome of non-primate MSY. For the first time, we were able to comprehensively examine the bovine Y chromosome in terms of its structure, gene content, transcriptional activity and evolution. We found that the bovine MSY differs radically from the primate Ys with respect to its structure, gene content and density. Our results challenge the widely accepted hypothesis that MSY is gene-poor and transcriptionally inert. These results increase our understanding of the basic biology of the mammalian Y chromosome, and provide not only important comparative mapping information useful for studying the evolution of Y chromosome and bovid-lineages, but also details of the organization and function of these Y chromosome genes in bovine spermatogenesis and fertility. Most importantly, our results set up a stage for future research on the roles of bovid Y chromosome genes in spermatogenesis and male fertility, and on the potential to use Y-linked markers for bull fertility and semen quality selection, and cattle breeding and evolutionary studies. What do you plan to do during the next reporting period to accomplish the goals? 1. Continue to study the functional role(s) of Y-linked genes in spermatogenesis and male fertility. 2. Continue our work on the Y gene CNV and SNP variation. 3. Continue to work on the ovine and caprine Y chromosome.

Impacts
What was accomplished under these goals? 1. Novel transcripts identified in the bovine Y chromosome (BTAY). We have continued our research on the transcriptome of BTAY and published the results in PNAS (Chang et al. 2013). We identified a total of 386 novel transcripts in addition to the 17 known bovine MSY genes (families), including one gene family encoding for EGLY, ten potential protein-coding genes, termed BTY1-10, and 375 non-coding RNAs (ncRNAs). Temporal and spatial expression analyses revealed that the majority of the novel BTYs and ncRNAs were expressed predominantly in testes and were up-regulated during testis development. 2. A contig map of the ovine Y chromosome (OARY). We carried out the initial characterization of the ovine MSY (oMSY) by a combination of whole genome shotgun sequence (WGS) and BAC end sequence (BES). The WGS reads (~1 million pair-ends not assigned elsewhere in the genome assembly) were assembled into 4487 ovine Y-specific contigs (258 bp - 367 Kb) using a comparative assembly method based on the bovine MSY (bMSY) draft sequence. Alignment of the ovine BESs against the bMSY identified 605 Y chromosome BACs with one or both ends matched, of which 60 mapped in the X-degenerate and 545 in the ampliconic region. These BESs were used to orient the assembled contigs which resulted in a draft oMSY contig map that spans ~39 Mb (comparable to the 41.3 Mb of bMSY). The X-degenerate region contains genes well conserved with the bovine orthologs. The ampliconic region also contains a similar genetic composition to the bMSY, though its precise genomic organization requires further testing. This framework map of the oMSY, built with support from BESs and WGS assembly contigs, will be useful to study the genomic organization of the ovine Y chromosome. 3. The biological function of PRAMEY during spermatogenesis. We have made specific antibodies for the bovine PRAME16, PRAME17 and PYAMEY gene. We found that the PRAMEY protein is expressed predominantly in the acrosomal region of round, elongating and elongated spermatids and spermatozoa. Furthermore, the protein localization patterns of PRAMEY were coordinated with morphological alterations during acrosome formation in spermatids, and were significantly different in connecting piece, middle piece and principal piece of the sperm flagellum. We also analyzed the mouse ortholog of the bovine PRAMEY gene, namely Pramel1, which has a very similar expression pattern to the bovine gene. We concluded that the bovine PRAMEY and the mouse PRAMEL1 play a fundamental role in acrosome biogenesis and sperm motility. 4. Variation of the bMSY genes. We have continued the characterization of the copy number variations (CNVs) of the bMSY genes and their association with male fertility. We have examined 460 bulls from 15 cattle breeds. Significant difference in CNVs was observed within and among breeds. The median CN of PRAMEY, ZNF280BY, and HSFY was 13 (2-30), 223 (28-291), and 203 (55-353), respectively. We found that CNVs are associated with testis size, semen quality and male fertility traits. About 140 Y-linked SNPs were identified and arranged in a small SNP-chip, validation of the chip and A.I. bull genotyping are underway.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Yue, X.-P. , Chang, T.-C., DeJarnette, J. M., Marshall, C. E., Lei, C. Z., Liu, W.-S. (2013) Copy number variation of PRAMEY across breeds and its association with male fertility in Holstein sires. J Dairy Science 96, 80248034 (Featured article of the month by Editor-in-Chief).
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Chang, T.-C., Yang, Y., Retzel, E., Liu, W.-S. (2013) Male-specific region of the bovine Y chromosome is gene rich with a high transcriptomic activity in testis development. PNAS 110(30), 12373-12378.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Mistry, B.V., Chang, T.-C., Yasue, H., Chiba, M., Zhao, Y.-Q., Oatley, J., Diaz, F., Liu, W.-S. (2013) Differential expression of PRAMEL1, a cancer/testis antigen, during spermiogenesis in the mouse. PLoS ONE 8, e60611.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Vallimont, J.E., Dechow, C.D., Daubert, J.M., Dekleva, M.W., Bl�m, J., Barlieb, C.M., Liu, W.-S, Varga, G.A., Heinrichs, A.J., Baumrucker C.R. (2013) Feed utilization and its associations with fertility and productive life in 11 Commercial Pennsylvania Tie Stalls. J. Dairy Sci. 96, 1251-4.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Yue, X.-P., Chang, T.-C., Liu, W.-S. (2013) Copy Number Variation and Association Studies of PRAMEY in Cattle. Conference Abstract, PAG-XXI, Jan 11-16, 2013. San Diego, CA. P561.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Ingrid E. Lindquist, I.E., Cameron, C.T., Mudge, J., Retzel, E., Beattie, C.W., Liu, W.-S., Ford, S.P. (2013) De novo Transcriptome Assembly and Analysis of Placental Tissue from Undernourished, Control Fed and Obese Pregnant Ewes. Conference Abstract, PAG-XXI, Jan 11-16, 2013. San Diego, CA. P614.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Chang, T.-C., Jiang, Y., Dalrymple, B., Archibald, A., Talbot, R., Kijas, J., Worley, K.C., McEwan, J.C., Cockett, N., Liu, W.-S. (2013) A contig map of the ovine Y chromosome. Conference Abstract, PAG-XXI, Jan 11-16, 2013. San Diego, CA. P619.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Decker, J. E., McKay, S. D., Rolf, M. M., Kim, J. W., Alcal�, A. M., Sonstegard, T. S., Hanotte, O., G�therstr�m, A., Bradley, D. G., Seabury, C. M., Praharani, L., Babar, M. E., Yildiz, M. A., Michael P. Heaton, M. P., Liu, W.-S., Reecy, J.M., Saif-Ur-Rehman, M., Schnabel, R.D., Taylor, J.F. (2013) World-wide patterns of divergence, migration and admixture in domesticated cattle. Conference Abstract, PAG-XXI, January 12-16, 2013. San Diego, CA. W611.
  • Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Yue, X.-P. , Dechow, C. D., Chang, T.-C., DeJarnette, J. M., Marshall, C. E., Lei, C. Z., Liu, W.-S. (2013) Copy number variations of the extensively amplified Y-linked genes, HSFY and ZNF280BY, in cattle and their association with male reproductive traits in Holstein bulls. BMC Genomics.
  • Type: Theses/Dissertations Status: Other Year Published: 2013 Citation: Yaqi Zhao (2013) CHARACTERIZATION OF THE PRAME/PRAMEY GENE FAMILY DURING SPERMATOGENESIS. M.S. thesis, Penn State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhao, Y.-Q., Mistry, B.V., Diaz, F., Liu, W.-S. (2013) PRAME is involved in spermatogenesis. Conference Abstract, the 46th Annual Meeting of the Society for the Study of Reproduction (SSR2012), July 22-26, 2013. Montreal, Canada. P457.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Liu, W.-S., Chang, T.-C., Yang, Y., Retzel, E. (2013) Novel Transcripts Identified in the Bovine Y Chromosome. Conference Abstract, PAG-XXI, Jan 11-16, 2013. San Diego, CA. P542.


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

Outputs
OUTPUTS: The major achievements for the entire project (2010-2012) are: 1. Gene content of bMSY. There are a total of 28 protein coding genes/families on bMSY, including 12 single copy and 16 multicopy genes. The majority of bMSY genes (21/28) have not been studied before. Among the 12 single copy genes, eight are conserved in the other mammals, and two (UBE1Y and EIF2S3Y) are shared between cattle and non-primate, the remaining two (ZRSR2Y and RPL23AY) are lineage-specific in cattle. Out of the 16 gene families, 14 were first reported in this project and are lineage-specific in bovid, and remaining two (TSPY and HSFY) are present in some of the mammalian Ys studied to date. The ZNF280BY, ZNF280AY, and PRAMEY were derived from an autosome-to-Y gene transposition of a gene block containing ZNF280B-ZNF280A-PRAME on BTA17 during evolution. Collectively, 1274 protein coding loci were identified on bMSY, with a gene density of 31.2 genes/Mb, which is significantly higher than 9.4 for the X and 10.2 for the entire genome. In addition, 46 novel single-copy and 339 novel multi-copy non-coding (nc) RNAs were identified on bMSY. 2. Genomic structure and transcriptive activity of the bMSY genes. The genomic structure of the bMSY ampliconic region is significantly different from the human MSY. bMSY contains a total of ~80 palindrome-like repeat units (420 Kb/unit), each containing 4-12 loci of coding genes. This is the underlying mechanism for the massive amplification of the bMSY genes with copy number ranging from 2 to 300, suggesting that the genomic amplification of MSY is an essential mechanism shaping the Y evolution. The transcriptive activity of the bMSY genes in testes (20d, 8m and 2y) were determined by RNA-seq and RT-PCR. Over 95% of the transcripts on bMSY were transcriptionally active. The majority of the bMSY transcriptome, including 13/28 coding genes and 220/375 ncRNAs, were up-regulated during testis development. RT-PCR has been applied to validate the RNA-seq data. Gene Ontology analyses indicated that 61% of the differentially up-regulated genes were involved in reproduction and spermatogenesis. 3. Variation of bMSY. The copy number variations (CNVs) of the bMSY genes have been examined among 435 bulls from 15 cattle breeds. Significant difference in CNVs was observed within and among breeds. The median copy number of PRAMEY, ZNF280BY, and HSFY was 13 (2-30), 223 (28-291), and 203 (55-353), respectively. Association analysis indicated that CNVs are associated with testis size, semen quality and male fertility traits. Approximately 140 Y-linked SNPs were identified and arranged in a small SNP-chip (Illumina), validation of the chip and A.I. bull genotyping are underway. 4. Evolution of bMSY. Two major expansions of the bMSY genes occurred during the Miocene (~14-20 MYA) and Pliocene (5 MYA) periods. The amplification of the HSFY began 18.1-20.0 MYA, much later than the amplification of TSPY and ZNF280BY (35 MYA), which coincided with the divergence time between cattle and sheep (19.2 MYA). We propose that MSY may have played a critical role in the expansion of Bovidae lineage by serving as a genomic niche regulating male reproduction. PARTICIPANTS: PSU: Wansheng Liu (PI), Ti-Cheng Chang (Post-doc research associate), Ya-Qi-Zhao (Graduate student), Xiangpeng Yue (Graduate student), Blair Lewis (undergraduate student), and Emilia Sohn (undergraduate student). Cooperating Investigators: Hiroshi Yasue, Hideki Hiraiwa, Takeshi Shimogiri (National Institute of Agrobiological Sciences, Japan), Ernest Retzel, Andrew D. Farmer, John A. Crow (National Center for Genome Resources, Santa Fe), and Abel, F, Ponce de Leon (University of Minnesota). TARGET AUDIENCES: Animal geneticists and breeders, AI industry, beef and dairy producers, as well as biologists in the fields of sex chromosomes, comparative genomics, molecular evolution, reproduction and cancer study. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Although the Y chromosome sequence is available for humans, chimpanzees and macaques, little is known about the annotation and transcriptome of non-primate MSY. For the first time, we were able to comprehensively examine the bovine Y chromosome in terms of its structure, gene content, transcriptional activity and evolution. We found that the bovine MSY differs radically from the primate Ys with respect to its structure, gene content and density. Our results challenge the widely accepted hypothesis that MSY is gene-poor and transcriptionally inert. These results increase our understanding of the basic biology of the mammalian Y chromosome, and provide not only important comparative mapping information useful for studying the evolution of Y chromosome and bovid-lineages, but also details of the organization and function of these Y chromosome genes in bovine spermatogenesis and fertility. Most importantly, our results set up a stage for future research on the roles of bovid Y chromosome genes in spermatogenesis and male fertility, and on the potential to use Y-linked markers for bull fertility and semen quality selection, and cattle breeding and evolutionary studies. A total of 15 invited presentations on the bovine Y chromosome research have been given in the past three years in the International Conference on the Status of Plant and Animal Genome Research, the International Conference on Beef Cattle Improvement and Industrialization in China, the International Society for Study of Reproduction Conference, and six different Universities in China, India and USA. These activities promote research and education in the sex chromosome and male reproduction and foster collaborative research, and will have an impact on biomedical and livestock industries.

Publications

  • Brown, D. E., C. D. Dechow, W.-S. Liu, K. J. Harvatine, and T. Ott. 2012. Association of telomere length with age, herd and culling in lactating Holsteins. J. Dairy Sci. 95, 6384-6387.
  • Dekleva, M. W., C. D. Dechow, J. M. Daubert, W.-S. Liu, G. a. Varga, S. Bauck, and B. Woodward. 2012. Interactions of milk, fat and protein yield genotypes with herd feeding characteristics. J. Dairy Sci. 95(3),1559-1564.
  • Liu, W.-S., B. V. Mistry, T.-C. Chang, H. Yasue, M. Chiba, Y.-Q. Zhao, and J. Oatley. 2012. Differential expression of PRAMEL1, a cancer/testis antigen, during spermiogenesis in the mouse. The 45th Annual Meeting of the Society for the Study of Reproduction C State College, PA. August 12-15, 2012. SSR2012. (Abstract). P441.
  • Chang, T.-C. and W.-S. Liu. 2012. The male-specific region of the bovine Y chromosome is enriched for testis-expressed non-coding RNAs. State College, PA. August 12-15, 2012. SSR2012. (Abstract). P426.
  • Vallimont, J. E., C. D. Dechow, J. M. Daubert, M. W. Dekleva, J. Blum, C. M. Barlieb, W.-S. Liu, G. A. Varga, A. J. Heinrichs, and C. R. Baumrucker. 2012. Feed utilization and its associations with fertility and productive life in 11 Commercial Pennsylvania Tie Stalls. J. Dairy Sci. 95, 6801-4.
  • Yue, X.-P., T.-C. Chang, and W.-S. Liu. 2012. Copy number variations of PRAMEY are associated with bull fertility. State College, PA. August 12-15, 2012. SSR2012. (Abstract). P462.
  • Zhao, Y.-Q., B. V. Mistry, and W.-S. Liu. 2012. Expression analysis of the PRAME/PRAMEY gene family during bovine spermatogenesis. State College, PA. August 12-15, 2012. SSR2012. (Abstract). P428.
  • Liu, W.-S., T.-C. Chang, and E. Retzel. 2012. Differential transcriptome of the bovine Y-chromosome is associated with testis development in cattle. 33rd International Society for Animal Genetics Conference (ISAG2012), Cairns, Australia. July 15-18, 2012. (Abstract). P2032.
  • Liu, W.-S., and T.-C. Chang. 2012. Y-chromosome genomics: a comparison between the human, mouse, and bovine Y-chromosome. 3rd World DNA and Genome Day (WDD-2012), Xian, PR China. April 25-28, 2012. (Abstract). P50.
  • Chang, T.-C., Y. Yang, E. Retzel, and W.-S. Liu. 2012. The genomic organization and gene content of the male-specific region (MSY) of the bovine Y-chromosome. San Diego, CA. January 14-18, 2012. PAG-XX (Abstract). P573.
  • Liu, W.S. 2012. The heterochromatic region of the bovine Y chromosome is gene-rich. San Diego, CA. January 14-18, 2012. PAG-XX ( Abstract). W634.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The major accomplishments in 2011 are: 1. The discovery of the bovid-specific Y-chr genes ZNF280BY, ZNF280AY, and PRAMEY, which were derived from an autosome-to-Y transposition during evolution, has been published (Yang et al. 2011; Chang et al. 2011). 2. The bovine MSY contains three different types of sequences: X-degenerate (Xd), Y-amplified (Ya) and Y-transitional (Yt) sequence. The Ya is composed of about 80 repetitive units (RUs), each unit is about 420 Kb. All together, these RUs form the heterochromatic region (about 32 Mb). Our data indicated that the structure of BTAY is significantly different from the human Y. The most significant discovery is that the BTAY heterochromatic region is gene-rich. 3. The transcriptome of BTAY has been analyzed using a direct testis cDNA selection and Next-Gen sequencing approach. The selected cDNA was sequenced using Illumina GA2, resulting in about 13 million (36bp-pair-end) reads, which covered about 81% of the bovine MSY region and were de novo assembled into 4580 contigs (>=100bp). Seventy-nine contigs match to known Y-genes, while 36 Y-contigs match to ZNF280BY, ZNF280AY and PRAMEY. BLASTX search of the remaining contigs identified 222 matching to homologous proteins, which have never been reported on the Y. Additionally, 144 contigs are associated with transposons, suggesting transposition may play an essential role in the origin and evolution of BTAY. The remaining 2669 Y-contigs are novel, and most of them are intronless non-coding RNAs. 4. A new gene OFD1Y on BTAY and its orthologs in mammals have been characterized (Chang et al. 2011). The results indicated that: a) the eutherian OFD1X/Y were derived from the pair of ancestral autosomes during sex chr evolution; b) the autosomal OFD1 pseudogenes were derived from retropositions of OFD1X after the divergence of primates and rodents; and c) the presence of OFD1Y in the ampliconic region of the primate Y chr is an indication that the expansion of the ampliconic region may initiate from the Xd sequence. In addition, we found that different regions of OFD1/OFD1X/Y are under differential selection pressures. Our results provide insights into the molecular mechanism for why the OFD1X gene could cause two different syndromes, OFD1 and SGBS2 & JSRDs in humans. 5. We found that the mouse orthologs the bovine PRAMEY are expressed in late stage of spermatogenesis. The mouse Pramel1 protein is expressed specifically in the acrosome of post-meiotic germ cells and sperm tail, confirming our previous prediction that PRAMEY play a role in spermatogenesis. 6. Polymorphic markers on the BTAY have been identified, including 53 SNPs for 11 X-degenerate genes and 83 markers for 4 multi-copy Y-gene families. 7. We discovered, for the first time, a SNP and a polymorphic microsatellite marker for the horse Y-chr, which were genotyped across 1000 stallions worldwide. A total of 7 invited presentations on the bovine Y chromosome research have been given in 2011 in the International Conference on Beef Cattle Improvement and Industialization in China, the international Society for Study of Reproduction (SSR2011) conference and 5 different Universities in China. PARTICIPANTS: PD: Wansheng Liu Department of Dairy and Animal Sciences The Pennsylvania State University Email: wul12@psu.edu Website: http://www.das.psu.edu/directory/wul12 Collaborators/Contributors: Dr. Abel F. Ponce de Leon, University of Minnesota, MN Dr. Hiroshi Yasue, National Institute of Agrobiological Sciences, Japan Dr. Ernest Retzel, National Center for Genome Resources, Santa Fe, NM Dr. Andrew D. Farmer, National Center for Genome Resources, Santa Fe, NM Dr. John A. Crow, National Center for Genome Resources, Santa Fe, NM Dr. Jon Oatley, Penn State University, PA Dr. Bhavesh V. Mistry, Post-doc, Penn State University, PA Mr. Ti-Cheng Chang, graduate student, Penn State University, PA TARGET AUDIENCES: Animal geneticists and breeders, AI industry, beef and dairy producers, as well as biologists in the fields of comparative genomics, molecular evolution, reproduction and cancer study. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results on the bovine Y-chr increase our understanding of the basic biology of BTAY, and provide not only important comparative mapping information useful for studying the evolution of Y-chrs and specific Y-chr lineages, but also details of the organization and function of these Y-chr genes in bovine spermatogenesis and fertility. PRAME/PRAMEY protein family is one of the Cancer-Testis Antigens (CTAs) that are all (solely or predominantly) expressed in tumors and testes. Our results obtained from the bovine and mouse PRAMEY/Pramel1 set up a stage for future research on the roles of PRAME/PRAMEY in the crosstalk between the spermatogenesis and tumorigenesis, which will facilitate the understanding of both sperm and tumor developments. Our results on the OFD1/OFD1X/Y gene family reveal the origin and evolution of this family, and facilitate further clinical investigation of the OFD1-related syndromes. The identified BTAY polymorphic markers serve as an important resource for studying the association between Y-linked polymorphisms and spermatogenesis and bull fertility. In addition, these markers offer a basis for developing a marker-assisted selection (MAS) of fertility in bovine. The discovery and genotyping the SNP and the polymorphic microsatellite marker across 800 stallions (>30 breeds/populations) worldwide provide a means to study the equine domestication and evolution, in particular, to address the lost of diversity for the equine Y-chr and the molecular mechanism behind it.

Publications

  • Chang, T.-C., J. Klabnik, and W.-S. Liu. 2011. Regional selection acting on OFD1 genes. PLoS ONE 6(10):e26195.
  • Ma, J.-G., T.-C. Chang, H. Yasue, A. D. Farmer, J. A. Crow, K. Eyer, H. Hiraiwa, T. Shimogiri, S. N. Meyers, J. E. Beever, L. B. Schook, E. Retzel, C. W. Beattie, and W.-S. Liu. 2011. A high resolution comparative map of porcine chromosome 4 (SSC4). Animal Genetics 42(4):440-444.
  • Vallimont, J. E., C. D. Dechow, J. M. Daubert, M. W. Dekleva, J. W. Blum, C. M. Barlieb, W.-S. Liu, G. A. Varga, A. J. Heinrichs, and C. R. Baumrucker. 2011. Heritability of Gross Feed Efficiency and Associations with Yield, Intake, Residual Intake, Body Weight and Body Condition Score in 11 Commercial Pennsylvania Tie Stalls. Journal of Dairy Science 94(4):2108-2113.
  • Ponce de Leon, A. F. and W.-S. Liu. 2011. The bovine X and Y chromosomes. Chapter 7 in Bovine Genomics, Ed: James Womack, Wiley-Blackwell Publishing, USA. p75-100.
  • Liu, W.-S. 2011. The Y-linked testis genes and male fertility in cattle. Proceeding of the International Conference on Beef Cattle Improvement and Industialization in China. Yangling, Shaanxi, PR China. May 8-10, 2011. P4-8.
  • Liu, W.-S., Y. Yang, T.-C. Chang, H. Yasue, A. Bharti, and E. Retzel. 2011. Predominant expression of two bovid-specific Y-chromosome genes (ZNF280BY and PRAMEY) in spermatids. SSR 45th Annual Meeting. Portland, OR. Jul 31-Aug 4, 2011. P136. (Conference Abstract).
  • Mistry, B. V., T.-C. Chang, H. Yasue, A. Bharti, E. Retzel, D. Kim, J. Oatley, and W.-S. Liu. 2011. Roles of Prame in spermatogenesis. SSR 45th Annual Meeting . Portland, OR. Jul 31-Aug 4, 2011. P574. (Conference Abstract).
  • Toyokawa, K., W.-S. Liu, and J. L. Pate. 2011. Expression and regulation of PRAME in the bovine corpus luteum. SSR 45th Annual Meeting. Portland, OR. Jul 31-Aug 4, 2011. P221. (Conference Abstract).
  • Liu, W.-S., T.-C. Chang, Y. Yang, J. A. Crow, and E. Retzel. 2011. The transcriptome of the bovine Y-chromosome. International Plant and Animal Genome Conference (PAG)-XIX. San Diego, CA. January 15-19, 2011. P520. (Conference Abstract).
  • Chang, T.-C., Y. Yang, J. Klabnik, and W.-S. Liu. 2011. Regional selection acting on OFD1genes. Manuscript in preparation. Conference Abstract, PAG-XIX. San Diego, CA. January 15-19, 2011. P541. (Conference Abstract).
  • Lei, C. Z., T.-C. Chang, C. Penedo, T. Leeb, H. Haase, M. Bower, K. D. Makova, and W.-S. Liu. 2011. Chinese native stallions retain Y-chromosome diversity, signifying a new location for horse domestication. PAG-XIX. San Diego, CA. January 15-19, 2011. P620. (Conference Abstract).


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: i) The direct testis selected cDNA (with BTAY probe) was sequenced using Illumina GAIIx, and a total of 6,710,574 high-quality paired-end reads of 2x36 bp were generated. De novo sequence assembly resulted in ~4500 contigs, which are being annotated and confirmed for their presence on the Y chromosome; ii) We discovered three novel gene families, namely ZNF280BY, ZNF280AY, and PRAMEY, on BTAY, which were derived from an autosome-to-Y gene transposition of a gene block containing ZNF280B-ZNF280A-PRAME on BTA17 during evolution. This transposition is lineage-specific in bovid. The transposed Y genes were amplified differentially on BTAY after the transposition event; iii) We found that ~130 active ZNF280BY loci (and ~240 pseudogenes) and ~130 pseudogenized ZNF280AY copies are present over the majority of the male-specific region (MSY). Phylogenetic analysis indicated that ZNF280BY and ZNF280AY gene families fit with the "birth-and-death" model of evolution. The active ZNF280BY loci share high sequence similarity and comprise three major genomic structures, resulted from indels; iv) Assembly of a 1.2Mb BTAY sequence in the MSY ampliconic region demonstrated that ZNF280BY and ZNF280AY, together with HSFY and TSPY families, constitute the major elements within the repeat units; v) The ZNF280BY was found to express in different developmental stages of testis with sense RNA detected in all cell types of the seminiferous tubules while the antisense RNA detected only in the spermatids. Deep sequencing of the selected cDNAs revealed that different loci of ZNF280BY are differentially expressed up to 60-fold. Different copies of the ZNF280AY pseudogenes were also found to differentially express up to 10-fold. The temporal and spatial expression patterns of ZNF280BYs in testis suggest a role in spermatogenesis; vi) We found a total of 16 PRAMEY loci on BTAY, 8 of which were proven to be active transcriptionally. A phylogenetic analysis of the eutherian PRAME homologs revealed two major evolutionary clades. Positively selected sites were detected from one clade with amplified autosomal PRAME homologs, which were clustered in different regions on the PRAME proteins of different lineages. In contrast, the other clade containing the PRAMEY gene family is under stronger functional constraints. Compared to the bovine autosomal PRAME that is expressed predominantly in testis, PRAMEY is expressed exclusively in testis and up-regulated during testicular maturation. Furthermore, the sense RNA of PRAMEY was expressed specifically while the antisense RNA was expressed predominantly in spermatids. PRAME/PRAMEY gene family is a cancer-testis antigene (CTA), which is important in both tumorigenesis and spermatogenesis; vii) We characterized the bovine DDX3Y, DDX3X and PL10 genes and performed a detailed phylogenetic analysis. We found that the eutherian DDX3X/DDX3Y in the X/Y-added region originated from the translocation of the ancient PL10 ortholog on the ancestral autosome, whereas the eutherian PL10 was retroposed from DDX3X. In addition to the functional DDX3X/DDX3Y/PL10, conserved homologous regions on the autosomes and X chromosome are present. PARTICIPANTS: Wansheng Liu (PI), Ti-Cheng Chang (Graduate student - Bioinformatics and Genomics), Bhavesh Mistry (Post-doc research associate), Sarah Pierson (undergraduate student), and Jessica Klabnik-Bradford (undergraduate student). TARGET AUDIENCES: Animal geneticists and breeders, AI industry, beef and dairy producers, as well as biologists in the fields of comparative genomics, molecular evolution, reproduction and cancer study. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
These results increase our understanding of the basic biology of BTAY, and provide not only important comparative mapping information useful for studying the evolution of Y-chrs and specific Y-chr lineages, but also details of the organization and function of these Y-chr genes in bovine spermatogenesis and fertility. Most importantly, our results set up a stage for future research on the roles of bovid Y chromosome genes in spermatogenesis and male fertility.

Publications

  • Chang, T.-C., Y. Yang, H. Yasue, A. Bharti, E. Retzel, and W.-S. Liu. 2011. The expansion of the PRAME gene family in eutheria. PLoS ONE 6(2):e16867. http://dx.doi.org/10.1371/journal.pone.0016867.
  • Yang, Y., T.-C. Chang, H. Yasue, A. Bharti, E. Retzel, and W.-S. Liu. 2011. ZNF280BY and ZNF280AY: autosome derived Y chromosome gene families in Bovidae. BMC Genomics 12:13. http://dx.doi.org/10.1186/1471-2164-12-13.
  • Porada, C.D., C. Sanada, C. R. Long, J. A. Wood, J. Desai, N. Frederick, L. Millsap, C. Bormann, S. L. Menges, C. Hanna, G. Flores-Foxworth, T. Shin, M. E. Westhusin, W.-S. Liu, H. Glimp, E. D. Zanjani, J. N. Lozier, V. Pliska, G. Stranzinger, H. Joerg, D. C. Kraemer, and G. Almeida-Porada. 2010. Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A. Journal of Thrombosis and Haemostasis 8(2):276-85.
  • Liu, W.-S. 2010. Comparative genomics of Y chromosome and male fertility. A chapter in: Reproductive Genomics of Domestic Animals. Wiley-Blackwell Publishing, USA. Part 1, Chapter 6. pp. 129-146.
  • Liu, W.-S., T.-C. Chang, Y. Yang, H. Yasue, A. Bharti, and E. Retzel. 2010. Transcritional profile of the bovine Y-chromosome. ISAG2010. Edinburgh, Scottland. July 23-30, 2010. P1011. (Conference Abstract).
  • Chang, T.-C., Y. Yang, H. Yasue, and W.-S. Liu. 2010. ZNF280BY and PRAMEY: autosome derived Y chromosome gene families in cattle. PAG-XVIII. San Diego, CA. January 9-14, 2010. P555. (Conference Abstract).
  • Lei, C.Z., X. M. Zhang, X. P. Yue, X. Y. Lan, H. Chen, and W.-S. Liu. 2010. Y-Chromosome haplotype analysis revealing two paternal origins of the swamp buffalo in China. PAG-XVIII. San Diego, CA. January 9-14, 2010. P152. (Conference Abstract).
  • Liu, W.-S., T.-C. Chang, Y. Yang, H. Yasue, J. A. Crow, and R. Retzel. 2010. Functional genomics of the bovine Y-chromosome. PAG-XVIII. San Diego, CA. January 9-14, 2010. W548. (Conference Abstract).
  • Chang, T.-C, and W.-S. Liu. 2010. The molecular evolution of PL10 homologs. BMC Evolutionary Biology 10:127. http://dx.doi.org/10.1186/1471-2148-10-127.
  • Liu, W.-S., T.-C. Chang, Y. Yang, H. Yasue, J. A. Crow, and E. Retzel. 2010. Decoding the Bovine Y Chromosome. BioAsia2010. Hyderabad, India. Feb 3-6, 2010. P33. (Conference Abstract).