GENERATION AND MAPPING OF SWINE MICROSATELLITES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0183078
• Grant No.: 99-35205-8619
• Proposal No.: 1999-03297
• Project Start Date: Dec 15, 1999
• Project End Date: Sep 30, 2004
• Grant Year: 2000
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
VETERINARY PATHOBIOLOGY

Non Technical Summary
The current porcine genetic map is sufficient for identifying regions of the genome, i.e. chromosomal positions, containing genes contributing to traits of interest or quantitative trait loci (QTL) in diverse populations. However, when regions of interest are identified generally there are insufficient genetic markers available to narrow the region of interest. Moreover, when commercial populations are examined the number of gene variants (heterozygosity) declines when compared to the exotic breeds used to create the genetic maps. The levels of heterozygosity decline from 85% to approximately 50%. Thus there is a need for more genetic markers for studies in commercial populations to identify QTL and narrow these chromosomal regions. This project involves the isolation of additional porcine genomic fragments containing polymorphic sequences termed microsatellite and the generation of synthetic DNA pieces that can be used via the polymerase chain reaction (PCR) to identify differences in swine under study. These new markers will be physically mapped using a relatively new technique called radiation hybrid mapping. This technique will permit us to rapidly place, resolve and order these new markers more efficiently than the traditional genetic mapping techniques. This information will be made available to the public by publication in scientific journals and the World Wide Web. The synthetic DNA primers will be made available to researchers worldwide to investigate porcine QTL.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3599	1080	100%

Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1080 - Genetics;

Keywords

swine

animal genetics

genetic mapping

linkage (genetics)

quantitative analysis

traits

gene loci

genetic markers

polymerase chain reaction

recombinant dna

microsatellites

molecular genetics

livestock production

world wide web

information dissemination

gene cloning

dna sequences

comparative analysis

heterozygosity

dna fragments

polymorphism

Goals / Objectives
Develop additional swine genetic markers for the use in identification of economically important traits. These new genetic markers will be mapped on the I.N.R.A.- University of Minnesota porcine radiation hybrid panel. Once these markers are mapped results will be published and details made available via the World Wide Web. Oligonucleotide primers pairs will then be made available for other researcher to use.

Project Methods
A small insert porcine genomic DNA will be created by cleavage of DNA with the restriction enzyme Mbo I, size selected, and ligated into Bam HI digested M13mp18. This small insert library will be probed with radiolabeled oligonucleotide primers. Clones will then be plaque purified and sequenced. Sequences will be compared with existing porcine sequences and primer pairs developed from non-redundant sequences. Primer pairs will be optimized and mapped using the INRA-University of Minnesota porcine radiation hybrid panel. Primer sequences and radiation hybrid map positions will be reported in peer review journals and the World Wide Web. Oligonucleotide primer pairs will then be distributed to researchers requesting them.

Progress 12/15/99 to 09/30/04

Outputs
The objective of this research project was to rapidly, and cost effectively, generate and map 1,500 porcine microsatellite markers on a 7,000 Rad radiation hybrid panel (IMpRH7000). In the period that the project has been underway we have developed 1895 primer pairs and have mapped 1020 new microsatellite markers on the IMpRH panel. An example of some of our results are shown below. SSC refers to the chromosome that the marker was mapped to. SSC4 UMNp234 ATGACAGCTCCCCAGTTTTG GCTTTCCTGAACTCAGCACC SSC! UMNp271 AACAGACCTGGGGAAAACG GATCCAGTGGGCCTTGTCC SSC1 UMNp623 ACAAAGAATTACTGGTGCTCTTG ATTACATGGAAGTTGCTGAGAG SSC7 UMNp1350 ATCCTGGTGGGGAGGTATAC ACGCTGTATTGTTTCCGACC SSC3 UMNp1355 TTTTGCAGCTGAATAACATTGC ACTTGAACAGATATTCGCACCC SSC8 UMNp1358 CACTGGTCTCTTCTGAAATTTG TGAAACATGCAATCACCCC SSC15 UMNp1363 GCTGGAAAAGAAAACCATGG TGACCATTAGGCCATGGC SSC1 UMNp1365 TATTTTATTGCATGTGCACGC GTGGAGGAGTCATGCTTTAAGC SSC13 UMNp1376 TCAGGGGCTGAAGGACATAG TGCTCACAAAGCTATCATTTGC SSC7 UMNp1378 TCAGGCAACTGGTAGCACAG TTGGAAAATCTCAGCATCCC SSC2 UMNp1379 GCCATAAAGATGGAAACTCTGG ACCTGCCCCTGACTTTCC SSC18 UMNp1391 TCTTCTTTGCCAGGTAAGGC CTGCAACTATCTGAACCATTGG SSC13 UMNp1394 ATTGGACAACAGAATGCTTCC TAACCAGATATGACATGGTGGG SSC11 UMNp1408 CAATCTGAGAAGGCTAGCATCC TGCAGCCCTGTGTTAGTCTG SSC14 UMNp1410 AAAGAGTTTCCCCCTAAGAAAG ATTTCTCAAAGACCAAATGGTG SSC1 UMNp1412 GCGAGTGTAGCCATAAAAAG CAGCAGTGACATGCACACTG SSC1 UMNp1416 GCTGCAAATGGCACAATTTC ACCTAAGCGTCCACCAGATG SSC16 UMNp1417 GCAGCACGTACATGGCAG CCTCATCTGCCAGAGAAAGG SSC2 UMNp1429 GGTGTTGCTGTTCATTCTTTTC GACATGGGTTCATGAGCATG SSC2 UMNp1435 CCAAAAAATCCTGCCGTAAG TTTGTGCGTGTGAATGTGTG

Impacts
This project has developed tools that will enable and expand swine genetic research.

Publications

• No publications reported this period

Progress 01/01/02 to 12/31/02

Outputs
In the period that the project has been underway we have picked and frozen in excess of 11,000 M13 clones that were positive on a primary plaque screen. We expect that this should yield approximately 5,000 ms sequences. We already have 700 clones sequenced and have already mapped 400 new microsatellite markers on the IMpRH panel. An example of some of our results are shown below. SSC refers to the chromosome that the marker was mapped to. Marker SSC Forward Primer Reverse Primer UMNp18 12 AAGAGGAAAGCAGAGGGAGC TTCCCTACCCCTTCCAGC UMNp20 17 CTTCCTTATGCCGCGAGC TTTGTGTAATTGTTTCCATCCC UMNp27 5 GGTGGAGGTACAAATGCCC TCTGTGTCCTGCTGTAAACACC UMNp35 5 TGCACACCTCTTAGAGTGAACC GTTCCTCTTCTGAMCAGATTGC UMNp36 4 TTCTGACAGATAATCGTGGCC GAGTCTGAGAGGGGAGTTTGG UMNp37 TTTTGAGAGAAACTATAGCCACATC CCATGGATGGGACAGAATTC UMNp41 ACTCCACCAATACTTTCACTGC GGGTGTTGCCATTTTTGG UMNp42 3 GATCATACATCTTTCTAGAAGGCC GTTGCTCTGGAGTAGCCCAG UMNp43 0 ATTAGCCTCCCAGCCTGG AGCATCAGGAAAAGGAAAAGG

Impacts
We are extending the number of available markers for swine genomic research.

Publications

• Korwin-Kossakowska, A. , K. M. Reed, C. Pelak, E. Krause, L. Morrison, and L. J. Alexander. 2002 Radiation hybrid mapping of 118 new porcine microsatellites. Anim. Genet. 33: 220-223.
• Krause, E., Morrison, L. , Reed, K M. and Alexander, L J., 2002 Radiation hybrid mapping of 273 previously unreported porcine microsatellites. Anim. Genet. 33; 477-485.

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

Outputs
In the period that the project has been underway we have picked and frozen in excess of 11,000 M13 clones that were positive on a primary plaque screen. We expect that this should yield approximately 5,000 ms sequences. We already have 700 clones sequenced and have already mapped 400 new microsatellite markers on the IMpRH panel. An example of some of our results are shown below. SSC refers to the chromosome that the marker was mapped to. Marker SSC Forward Primer Reverse Primer UMNp18 12 AAGAGGAAAGCAGAGGGAGC TTCCCTACCCCTTCCAGC UMNp20 17 CTTCCTTATGCCGCGAGC TTTGTGTAATTGTTTCCATCCC UMNp27 5 GGTGGAGGTACAAATGCCC TCTGTGTCCTGCTGTAAACACC UMNp35 5 TGCACACCTCTTAGAGTGAACC GTTCCTCTTCTGAMCAGATTGC UMNp36 4 TTCTGACAGATAATCGTGGCC GAGTCTGAGAGGGGAGTTTGG UMNp37 TTTTGAGAGAAACTATAGCCACATC CCATGGATGGGACAGAATTC UMNp41 ACTCCACCAATACTTTCACTGC GGGTGTTGCCATTTTTGG UMNp42 3 GATCATACATCTTTCTAGAAGGCC GTTGCTCTGGAGTAGCCCAG UMNp43 0 ATTAGCCTCCCAGCCTGG AGCATCAGGAAAAGGAAAAGG

Impacts
We are extending the number of available markers for swine genomic research.

Publications

• Korwin-Kossakowska, A. , K. M. Reed, C. Pelak, E. Krause, L. Morrison, and L. J. Alexander. 2002 Radiation hybrid mapping of 118 new porcine microsatellites. Submitted to Anim. Genet.

Progress 01/01/00 to 12/31/00

Outputs
In the period that the project has been underway we have picked and frozen in excess of 11,000 M13 clones that were positive on a primary plaque screen. We expect that this should yield approximately 5,000 ms sequences. We already have 400 clones sequenced and have already mapped 160 new microsatellite markers on the IMpRH panel. An example of some of our results are shown below. SSC refers to the chromosome that the marker was mapped to. Marker SSC Forward Primer Reverse Primer UMNp18 12 AAGAGGAAAGCAGAGGGAGC TTCCCTACCCCTTCCAGC UMNp20 17 CTTCCTTATGCCGCGAGC TTTGTGTAATTGTTTCCATCCC UMNp27 5 GGTGGAGGTACAAATGCCC TCTGTGTCCTGCTGTAAACACC UMNp35 5 TGCACACCTCTTAGAGTGAACC GTTCCTCTTCTGAMCAGATTGC UMNp36 4 TTCTGACAGATAATCGTGGCC GAGTCTGAGAGGGGAGTTTGG UMNp37 TTTTGAGAGAAACTATAGCCACATC CCATGGATGGGACAGAATTC UMNp41 ACTCCACCAATACTTTCACTGC GGGTGTTGCCATTTTTGG UMNp42 3 GATCATACATCTTTCTAGAAGGCC GTTGCTCTGGAGTAGCCCAG UMNp43 0 ATTAGCCTCCCAGCCTGG AGCATCAGGAAAAGGAAAAGG

Impacts
We are extending the number of available markers for swine genomic research

Publications

• No publications reported this period