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Moderate to high resolution mapping of QTL affecting main milk production traits in RP1, RP2 RP4 and RP5, methodological and applied issues

High resolution QTL mapping (HRM) in dairy cattle is important for MAS, and for cloning quantitative trait genes. HRM in dairy cattle is enabled by the large sire half-sib daughter families in these populations. Accessing this information for a specific QTL containing region (QTLR) requires genotyping daughter families for a dense battery of markers spanning the QTLR.

Genotyping all daughters for all markers, requires enormous numbers of genotypes. Two approaches for HRM that reduce required genotyping were investigated: Selective Recombinant Genotyping (SRG); and selective DNA pooling using a dense marker map and a new "Fractionated Pooling Design (FPD)".

In SRG, daughters are identified that received a recombinant QTLR haplotype from their sire, since only these daughters carry information for mapping within the QTLR.

This requires that the daughters are "informative" for the markers flanking the QTLR, i.e., that the specific marker allele transmitted from the sire is identified. When the genotype of the dam of the daughters is unknown, the daughters are informative if their genotype is readable and differs from that of their sire. For Israel Holsteins, mean informativity over 168 markers was 0.69, and proportion of readable genotypes about 0.85. Thus, the expected proportion of daughters informative at both flanking markers of a typical QTLR will be 0.36. In actual analysis of a number of QTLR, this proportion ranged from 0.09 to 0.60; with a mean of 0.28.

Thus, to identify recombinant daughters, it would be essential to genotype multiple markers at each flank of the QTLR. Because of low marker informativity, therefore, SRG may not be useful in animal populations; but will remain useful for F2 and BC populations where informativity is high. Consequently, SRG was not pursued further in the BovMAS program. Instead, we turned to the FPD approach. The FPD is based on DNA analysis of multiple sub-pools made up of independent subsets of individuals taken randomly from the trait distribution tails in each family.

In contrast to standard selective DNA pooling, the FPD estimates QTL position and effect, with confidence intervals based on re-sampling techniques. In addition, the FPD provides QTL detection based on permutation tests rather than asymptotic distributions of test statistics; joint analysis of multiple families and markers (even if markers are not shared among families); estimation of family specific QTL effects; and analysis of multiple linked QTL.

Simulations showed that the FPD procedure was able to locate QTL with high accuracy. Application of the FPD to BTA13 involved 8 sires heterozygous for a QTL affecting PP, and 6 sires heterozygous for a QTL affecting MY. Pools were genotyped at 19 markers spanning the region 23 to 99cM. FPD analysis suggested three QTL located at 30cM, 55cM and 80cM, respectively, with confidence intervals about 10 cM for each QTL. Application to BTA20 involved 10 sires heterozygous for QTL affecting PP or MY. Pools were genotyped at 21 markers spanning the region 0 to 83 cM. FPD analysis identified a QTL affecting PP at position 48cM, with confidence interval of 12 cM.

Validation of the FPD for genome-wide association studies of quantitative variation will allow highly reliable and cost-efficient large-scale QTL analysis, providing results unattainable by standard selective DNA pooling analytical procedures. SRG and FPD were proposed in the original BovMAS Technical Annex. While BovMAS was being implemented, however, a new methodology for HRM based on the variance component methodology for QTL mapping was proposed elsewhere and proved highly effective in some instances. The LMU partner applied this methodology for HRM of BTA13 and BTA20 in the Fleckvieh breed. HRM for BTA13 was based on five families comprising 1835 daughters with extreme phenotypes, genotyped for 32 markers; and augmented by 238 sons from four families of a granddaughter design typed for 42 markers.

Two QTL were uncovered; affecting milk and protein yield at 63.82cM, and protein and fat percentage at 85.45cM, respectively. HRM for BAT20 was based on 1,365 sons in a granddaughter design genotyped for eight or 17 markers. The study confirmed a QTL affecting milk fat and protein percentage linked to GHR, and identified a new QTL for protein yield at 26cM. Best results were obtained by linkage analysis alone or by combined linkage disequilibrium and linkage analysis.

The contribution of linkage disequilibrium to the mapping results was small compared to literature reports. This could be due to lower LD for the Fleckvieh, compared to highly selected milk breeds. LMU also produced genotyping data at high density in the large complex pedigree of Fleckvieh and advanced backcross Fleckvieh x Red Holstein populations, which should enable fine mapping of QTL on BTA09, 18, 19, 28 and 29.


Ivica MEDUGORAC, (Person in Charge of Work)
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