Selection methods for livestock breeding

Abstract

Disclosed herein are methods and compositions for identifying livestock with motility disorders by identifying MI haplotypes or genotypes in animals. In some embodiments, the methods are used in selective breeding and mating programs, and in some embodiments, the animals are Holstein cattle, Holstein crossbreds, or other breeds of cattle with Holstein ancestry.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 300,489, filed January 18, 2022, the entire contents of which are incorporated herein by reference.

[0002] Government support This invention was made with Government support under Hatch Act Project No. PEN04672 awarded by the U.S. Department of Agriculture. The Government has certain rights in this invention.

[0003] Sequence Listing This application contains an XML formatted Sequence Listing entitled "2023-01-17_900905.00047_WIPO Sequence listing.xml", which is 33,063 bytes in size and was created on January 17, 2023. The Sequence Listing has been submitted electronically herewith via the Patent Center, and is hereby incorporated by reference in its entirety.

[0004] The present disclosure relates to the fields of animal husbandry, animal breeding, and animal management, where genetic analysis is used to identify haplotypes and mutations associated with undesirable traits. [Background technology]

[0005] Selective breeding (also called artificial selection) is the process by which humans use animal and plant breeding to develop specific phenotypic traits (characteristics) by selecting which particular animals or plants, males and females, will reproduce sexually and produce offspring. Animal breeding utilizes techniques such as inbreeding, crossbreeding, and outbreeding. The deliberate use of selective breeding to produce desired traits has become very common in agriculture and experimental biology.

[0006] Dairy cows are a significant investment for dairy farmers, and enormous efforts, such as systematic animal breeding programs and artificial insemination, have been and continue to be invested in ensuring that the animals are highly productive and sustainable, that the milk produced is of high quality or has the desired composition, and that the dairy cows are healthy and fertile.

[0007] Traditional breeding techniques involve studying the progeny of a sire and evaluating their milk production, health, and fertility (transmitting ability) to manage further breeding. This standard technique takes several years and involves progeny testing each bull to assess true genetic worth. Many dairy cows must be bred to produce offspring. Cows must be kept and bred to determine phenotypic traits, calved, and ultimately allowed to milk for a period of time. Bulls with well-performing daughters are subsequently returned to active duty, at least five years of age. Bulls with poorly performing daughters are classified as having a significant financial loss and are culled, as there is no opportunity to recoup the investment of breeding the bull by selling semen.

[0008] Furthermore, selection based purely on phenotypic characteristics does not effectively account for genetic variation caused by complex gene actions and interactions, and the effects of environmental and developmental variants. Thus, there is a need for methods to genetically evaluate animals, both at the phenotypic and genetic levels, to enable breeders to select animals earlier and more accurately.

[0009] Marker assisted selection can reduce the high cost and long term commitment of progeny testing used to improve populations because young animals can be assessed immediately or even prenatally for the presence / absence of important markers and young animals such as bulls that are determined by genetic testing to have undesirable markers do not have to be progeny tested and bred. Thus, there is a need in the art for faster and more accurate genetic testing of livestock such as dairy cattle. Summary of the Invention [Means for solving the problem]

[0010] Disclosed herein are methods, systems, and compositions for identifying livestock, such as cattle (e.g., Bos taurus, Bos indicus), having a genetic trait associated with a dysmotility phenotype. In some embodiments, the method includes (a) obtaining a sample from a livestock cattle, wherein the sample comprises chromosomal DNA, (b) generating genetic data from the chromosomal DNA for position 78732954 to position 80748266 bp on chromosome 16 or a fragment thereof, (c) identifying a dysmotility (MI) haplotype in the genetic data, wherein the MI haplotype comprises (i) one or more of the alleles in the shaded portion of FIG. 5 and / or (ii) a T on the positive strand of DNA at position 79613592 bp on chromosome 16, and (d) selecting or removing the cattle as breeding stock based on the identification of (c). In some embodiments, the cattle is a heifer, a cow, an embryo, or a fetus. In some embodiments, the cattle is a bull. In some embodiments, the cattle is a Holstein. In some embodiments, the cattle is a cross between a Holstein and another breed. In some embodiments, the cattle is phenotypically normal for dysmotility. In some embodiments, the sample comprises one or more of semen, oocytes, tissue, cells, or blood.

[0011] In some embodiments, the genetic data comprises the DNA sequence of at least one of the bovine chromosome 16 positions 78732954-80748266 bp, or a fragment thereof. In some embodiments, the genetic data comprises the DNA sequence of both of the bovine chromosome 16 positions 78732954-80748266 bp, or a fragment thereof. In some embodiments, the MI haplotype comprises one or more SNPs associated with the MI phenotype at one or more of the bovine chromosome 16 positions 78732954-80748266 bp, or a fragment thereof, and genetic variants in linkage disequilibrium (LD) with those SNPs.

[0012] In some embodiments, the genetic data comprises RNA derived from positions 78732954-80748266 bp, or a fragment thereof, of bovine chromosome 16. In some embodiments, the RNA is derived from the voltage-gated calcium channel subunit α1S gene (CACNA1S).

[0013] In some embodiments, generating the genetic data comprises sequencing chromosomal DNA, such as by next generation sequencing. In some embodiments, generating the genetic data comprises an amplification process, such as polymerase chain reaction. In some embodiments, generating the genetic data comprises DNA microarrays, bead chips, or oligonucleotide binding.

[0014] Also disclosed herein are methods of selective breeding of livestock, such as cattle. In some embodiments, the methods include obtaining a genomic DNA sample from a bull, detecting a homozygous negative MI haplotype in the sample, and using semen from the bull to inseminate a female cow. In some embodiments, the female cow is in vitro fertilized. In some embodiments, the female cow is also homozygous negative for the MI haplotype. In some embodiments, the bull is a Holstein, Holstein cross, or another breed with Holstein ancestry, and the female cow is a cow or heifer of a Holstein, Holstein cross, or another breed with Holstein ancestry.

[0015] Also disclosed herein are methods of selective breeding, the methods comprising obtaining a genomic DNA sample from a female cow, detecting a homozygous negative MI haplotype in the sample, and using oocytes from the female cow for in vitro embryo production. In some embodiments, the female cow is a cow or heifer of a Holstein, a Holstein cross, or another breed with Holstein ancestry.

[0016] Also disclosed herein are methods of corrective breeding. In some embodiments, the methods include obtaining nucleic acid samples (e.g., genomic DNA samples or RNA samples) from a bull and a cow, ascertaining the respective MI haplotypes, and mating a cow (a cow) that is homozygous negative for the MI haplotype with a bull that is positive for the MI haplotype. In some embodiments, the methods include obtaining nucleic acid samples from a bull and a cow, ascertaining the MI haplotypes, and mating a bull that is homozygous negative for the MI haplotype with a cow that is positive for the MI haplotype.

[0017] Also disclosed herein are compositions, kits, and systems. In some embodiments, the compositions, kits, and systems include at least one nucleic acid molecule, the nucleic acid molecule includes SEQ ID NO:1, SEQ ID NO:2, or a fragment of SEQ ID NO:1 or SEQ ID NO:2, the fragment including an MI SNP. In some embodiments, the nucleic acid molecule is linked / attached to a solid support. In some embodiments, the compositions, kits, and systems further include an enzyme having polymerase activity, such as Taq polymerase. In some embodiments, the nucleic acid molecule includes one or more of a detectable marker, a linker (e.g., for attachment to a solid support), and / or a non-natural nucleotide base.

[0018] These and other embodiments, aspects, advantages and features of the present invention are described in part in the description that follows and will become apparent to those skilled in the art upon examination of the following description of the invention and the referenced drawings or by practice of the invention. The accompanying drawings depict one or more embodiments that do not necessarily represent the full scope of the invention.

[0019] The present invention will be better understood, and further features, aspects and advantages will become apparent, when considered in light of the following detailed description thereof, which refers to the following drawings, in which: [Brief description of the drawings]

[0020] [Figure 1] Manhattan plot of the significance levels of 101,917 DNA markers and movement disorder based on the manifestation model using the significant region on chromosome 16 (enlarged view). Markers above the genome-wide false discovery rate significance level lie on the solid black (main panel) and blue (inset) lines.

[0021] [Diagram 2] Possible inheritance pathways for locomotor dysfunction are shown for pedigrees with most pathways from a likely ancestral cow (Robust) common to the three affected calves, with genotypes represented by half-filled grey (high-likelihood carriers based on imputed genotype), half-filled black (carriers based on genotyping), half-filled grey (high-likelihood carriers based on offspring genotype), open with black outline (uncertain status), or filled (affected offspring), square (male) or circle (female).

[0022] [Figure 3-1] Figure 3 shows the inheritance pathway of locomotor dysfunction from a possible common ancestor cow (Robust) in eight pedigrees of affected calves from Farm 1 (a), Farm 2 (b, c), Farm 3 (d, e, f) and Farm 4 (g, h), with genotypes represented by half-filled grey (highly likely carriers based on one imputed genotype), half-filled black (carriers based on genotyping), half-filled grey (highly likely carriers based on offspring genotype), open with black outline (uncertain status), open with green outline (highly likely non-carriers based on imputed genotype) or filled (affected offspring), square (male) or circle (female). In (h), imputed genotypes were derived using findhapf90 using genotypes of affected calves, genotypes of controls, and genotypes from a population of 7622 Holsteins with genotype densities ranging from 3K to 150K. [Figure 3-2]As described for Figure 3-1. [Figure 3-3] As described for Figure 3-1. [Diagram 3-4] As described for Figure 3-1. [Figure 3-5] As described for Figure 3-1. [Diagram 3-6] As described for Figure 3-1. [Diagram 3-7] As described for Figure 3-1. [Diagram 3-8] As described for Figure 3-1.

[0023] [Figure 4] This is a photograph of a calf that is unable to stand and exhibits the MI (recumbent) phenotype.

[0024] [Figure 5-1] Figure 5 shows the DNA markers, their locations, forward assignment of alternative alleles (Allele1_F and Allele2_F), top assignment of alternative alleles (Allele1_Top and Allele2_Top), and assignment of alternative alleles A and B (Allele1_AB and Allele2_AB) present in the genotyped population; the number of affected calves with AA genotype (nAA), AB genotype (nAB), and nBB genotype (nBB); and the unadjusted p-value for the allelic model (p_Allelic), false discovery rate p-value for the allelic model (FDRp_Allelic), and false discovery rate p-value for the dominant model (FDRp_Dom) for the end of chromosome 16. The shaded region (starting at CHR16BP78732954 and continuing to CHR16BP8074266) represents the shared homozygous region (haplotype region) on chromosome 16. [Figure 5-2] As described for Figure 5-1. [Figure 5-3] As described for Figure 5-1. [Figure 5-4] As described for Figure 5-1. [Figure 5-5] As described for Figure 5-1. [Figure 5-6] As described for Figure 5-1. [Figure 5-7] As described for Figure 5-1. [Figure 5-8] As described for Figure 5-1. [Figure 5-9] As described for Figure 5-1. [Figure 5-10] As described for Figure 5-1.

[0025] [Figure 6] Sequence alignments for the affected calf, sire, and progenitor bull recumbency mutation at bp79613592 are shown. The top panel for each animal represents sequencing depth, with red indicating the mutation and blue indicating the reference genotype, and the bottom panel shows individual reads, with red indicating the mutant genotype and grey indicating the reference genotype. The affected calf reads are homozygous, and the sire and progenitor bull are heterozygous.

[0026] [Figure 7] The nucleotide and amino acid sequences flanking either side of and including the MI SNP are shown. The underlined sequences correspond to introns. SEQ ID NO:9 and SEQ ID NO:10 are also shown in Table 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention is described herein using several definitions, as set forth below and throughout the application. In addition, information describing certain attributes and embodiments is set forth in the accompanying Appendix, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0028] Unless otherwise specified or indicated by the context, the terms "a", "an", and "the" mean "one or more". Thus, references to "a", "an", and "the" generally include the plural of the respective terms. For example, a reference to a "SNP", "method", or "trait" includes a plurality of such "SNPs", "methods", or "traits". For example, a reference herein to an "association" includes a plurality of such associations, while a reference to a "chromosome" includes a single chromosome unless such interpretation is excluded from the context. Similarly, the terms "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. Similarly, "include", "including", and "or" are all to be interpreted as inclusive unless such construction is clearly prohibited by the context. As used herein, "examples," particularly when followed by a list of terms, are merely illustrative and should not be considered exclusive or comprehensive.

[0029] As used herein, the terms "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent in the context in which they are used. When there are uses of these terms that are not clear to those of ordinary skill in the art given the context in which they are used, "about" and "approximately" may mean within ±10% of the particular term, and "substantially" and "significantly" may mean more than ±10% of the particular term.

[0030] As used throughout, ranges in this specification are written in abbreviated form to avoid the need to be lengthy and to describe each and every value within the range. Any suitable value within the range may be selected as the upper, lower, or end value of the range, where appropriate. For example, the range of 0.1 to 1.0 represents the end values ​​or 0.1 to 1.0, as well as intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges subsumed within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, 0.7 to 1.0, etc.

[0031] As used herein, the terms "bovine" and "cattle" generally refer to members of the Bos genus, including, but not limited to, Bos taurus and Bos indicus (also called Bos taurus indicus). The disclosed compositions, systems, methods, kits, and platforms are not intended to be limited to members of the Bos genus, and may be used with other members of the Bovidae family. By way of example and not limitation, in some embodiments, the disclosed compositions, systems, methods, kits, and platforms are used to detect MI haplotypes in domestic cattle, such as Holsteins, Holstein crosses, or animals of another breed with Holstein ancestry. In the cattle industry, most breeds have "grade-up" programs that result in animals with, for example, Holstein genes present in a different breed, and vice versa.

[0032] The term "genotype" as used herein refers to the identity of the alleles present in an individual or sample. In the context of the present invention, genotype preferably refers to the description of the polymorphic alleles present in an individual or sample. The term "genotyping" a sample or individual for a polymorphic marker refers to determining the particular alleles or particular nucleotides carried by an individual at the polymorphic site.

[0033] As used herein, a "locus" or plural "locus" refers to the physical site or position of a particular gene or marker on a chromosome. Exemplary loci include positions 78732954-80748266 bp on chromosome 16 in cattle, such as Holsteins. In some embodiments, a locus is a single base pair (bp or BP), such as, for example, bp 79613592 on chromosome 16 in Holsteins.

[0034] As used herein, "linkage disequilibrium" (or "LD") refers to the association of alleles between particular alleles at two or more adjacent loci in a genome, for example, in a population. LD can be determined for a single marker or locus, or for multiple markers. Stated differently, LD is the correlation between nearby variants such that alleles at adjacent polymorphisms (observed on the same chromosome) are associated more frequently in a population than if they were unlinked.

[0035] As used herein, "allele" refers to one or more alternative forms of a particular nucleic acid sequence, where the differences may include, but are not limited to, one or more single nucleotide polymorphisms (SNPs), insertions, inversions, or deletions. The sequence may or may not be within a gene, and may be within a coding or non-coding region of a gene, for example, within a promoter or regulatory region, exon, or intron of a particular gene. As an example, the sequence of the DNA positive strand from bp 79613564 to 79613623 of Holstein, with the alleles (in this case SNPs) shown as [C] or [T], is [ka] (SEQ ID NO: 1 and SEQ ID NO: 2). The underlined sequences indicate introns.

[0036] The term "haplotype" refers to a combination of alleles or DNA markers on one chromosome. At the DNA level, a haplotype refers to a sequence of nucleotides found at two or more polymorphic sites in a locus on a single chromosome. There are multiple haplotypes on a given chromosomal region in a breeding population. As used herein, haplotype includes complete haplotypes and / or sub-haplotypes. A complete haplotype is the 5' to 3' sequence of nucleotides found at all polymorphic sites examined at a locus on a single chromosome from a single individual, and a sub-haplotype refers to the 5' to 3' sequence of nucleotides found at a subset of polymorphic sites examined at a locus on a single chromosome from a single individual. Relatedly, the term "haplotype pair" refers to two haplotypes found for a locus in a single individual. "Haplotyping" refers to the process for determining one or more haplotypes in an individual, and includes the use of pedigrees, molecular techniques, and / or statistical inference. By way of example and not limitation, the MI haplotype is represented by the shaded region in FIG. 5 encompassing bp 78732954 to 80748266 of bovine chromosome 16.

[0037] A "quantitative trait locus" (or "QTL"), as used herein, is a genomic sequence that is associated with a particular phenotypic trait. Multiple QTLs may be identified for a particular trait, and they are frequently found on different chromosomes. The number of QTLs significantly associated with a particular phenotypic trait may provide an indication of the genetic makeup of the trait, the number of genes that affect the trait, or the extent of the influence of one or more of these genes. One or more QTLs significantly associated with a trait may be candidate genes underlying such traits, which may be sequenced and identified. The significance of the degree of association of a given QTL with a particular trait may be statistically assessed, for example, through QTL mapping of alleles occurring at the locus and the phenotypes they produce. Statistical analysis is preferred to demonstrate whether the observed association with the trait is significant. The presence and location of QTLs identifies certain regions of the genome as potentially containing genes that are directly (e.g., structurally) or indirectly (e.g., regulatory) associated with the trait being analyzed. The associated probabilities can be plotted for various markers associated with the trait, located across chromosomes or throughout the genome. QTLs can reside in haplotypes associated with the phenotype.

[0038] "Polynucleotide" includes single- or multi-stranded nucleic acid molecules containing two or more consecutive bases, including parallel and anti-parallel strands of any single- or multi-stranded nucleic acid. Polynucleotides may be of any length, and thus include very large nucleic acids, as well as short nucleic acids such as oligonucleotides.

[0039] The term "oligonucleotide" generally refers to short polynucleotides, generally less than about 50 nucleotides. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that the corresponding RNA sequence (i.e., A, U, G, C, with "U" replaced by "T") is also included, unless otherwise specified or clearly distinguished by context or common sense.

[0040] As used herein, the term "dysmotility," which is used interchangeably with the term "recumbent" or "recumbency," refers to a condition in which an animal is unable to normally coordinate, control, or respond to voluntary muscle movements. By way of example, dysmotility may include traits described, such as inability to stand, inability to stand unassisted, inability to stand for the first 24 hours of life, loss of ability to stand, inability to stand followed by recovery of ability to stand, and poor growth and health, for example in calves. As used herein, dysmotility is associated with or caused by abnormal nutrition, injury, or metabolic product levels. Figure 4 provides an example of recumbency in a Holstein calf. Recumbent animals exhibit normal speech, appetite, and behavior despite being unable to sit up or maintain an upright position. Additionally, recumbency as defined herein is not due to abnormalities such as anemia, cholesterol, selenium or vitamin deficiencies, and disease or infection (e.g., Neospora, bovine viral diarrhea virus, rabies, and Listeria). Recumbent animals as disclosed herein exhibit unremarkable histological sections of the central nervous system, peripheral nerve, and muscle tissue.

[0041] As used herein, a "motility deficiency haplotype" (MI haplotype) refers to a haplotype of an animal, such as a Holstein cow, at positions 78732954-80748266 bp on chromosome 16. A positive MI haplotype is indicative of an animal that exhibits or is likely to exhibit, or that may transmit, a motility deficiency as defined above to its offspring. A negative MI haplotype is indicative of an animal that is unlikely to exhibit, and is unlikely to transmit, a motility deficiency to its offspring. By way of example, a positive MI haplotype is shown in FIG. 5. The alleles represented in the shaded region of FIG. 5 (78732954-80748266 bp on chromosome 16) are illustrative of MI haplotypes. In some embodiments, the alleles represented in the shaded region of FIG. 5 are evaluated for MI haplotyping. Conversely, a negative MI haplotype, indicative of an animal that does not have MI, is identified when the animal inherits alternative haplotypes across the chromosomal regions represented by different combinations of DNA markers.

[0042] The MI phenotype is associated with the MI haplotype, which is also associated with the SNP [C / T]. The sequence of the DNA plus strand from bp 79613564 to 79613623, which carries the SNP designated [C] or [T] (79613592 bp), is shown below.

[0043] [ka] (SEQ ID NO: 1 and SEQ ID NO: 2).

[0044] The underlined sequences represent introns.

[0045] The forward strand, reverse strand, reverse complement, and amino acid sequence of this region are shown in Figure 7. As used herein, [C] or [T] (or its complement or reverse complement), as shown in SEQ ID NO:1 or SEQ ID NO:2, respectively, may be referred to as an MI SNP.

[0046] The MI SNP is present in the voltage-gated calcium channel subunit α1S gene (CACNA1S) of Holstein cattle. The missense mutation changes a GGC codon to AGC, facilitating a glycine to serine amino acid substitution. The complete protein sequence of CACNA1S is shown below.

[0047] The amino acid sequence of CACNA1S (XP_024832342.1 CACNA1S [organism = Bos taurus] [gene ID = 100337204] [isoform = X1] is shown below (SEQ ID NO: 11 and SEQ ID NO: 12).

[0048] [ka]

[0049] The nucleic acid sequence encoding the CACNA1S amino acid sequence (XM_024976574.1 predicted: Bos taurus voltage-gated calcium channel subunit α1S (CACNA1S), transcript variant X1, mRNA) is shown below (SEQ ID NO: 13 and SEQ ID NO: 14).

[0050] [ka] [ka] [ka] [ka]

[0051] The exon containing the MI SNP and both flanking introns (CACNA1S / XM_024976575.1 / XP_024832343.1 79612962-79614080) are shown in the nucleic acid sequences below (SEQ ID NO: 15 and SEQ ID NO: 16). The underlined sequences correspond to the introns.

[0052] [ka]

[0053] The haplotypes shown in Figure 5 are based on previously documented polymorphic sites included in commercially available DNA marker testing arrays, panels, or chips. MI SNP 79613592 was not described or included in commercially available genotype panels prior to its discovery of association with MI.

[0054] MI haplotype determination

[0055] Disclosed herein are methods and compositions for identifying cattle, including young or adult bovine animals, embryos, semen samples, eggs, fertilized eggs, zygotes, fetuses, or other cell or tissue samples derived therefrom, to determine whether the cattle have a motility impairment (MI) haplotype. Determination of the MI haplotype, located at positions 78732954-80748266 bp on chromosome 16 in Holstein cattle, provides a rapid, accurate, and effective means of identifying whether an animal should be used for breeding stock, whether the animal is likely to develop motility impairment, and whether further progeny testing should be performed.

[0056] DNA markers have several advantages over phenotypic analysis, segregation is easy and unambiguous to measure. Also, DNA markers are co-dominant, i.e., heterozygous and homozygous animals can be distinctively distinguished. Once a marker system is established, selection decisions can be made very easily, since DNA markers can be assayed at any time after DNA-containing samples, such as blood or tissue samples, can be collected from individual young, or earlier by testing the embryos in vitro if very early embryos are collected. The use of marker-assisted genetic selection greatly facilitates and accelerates cattle breeding programs.

[0057] In one embodiment, the present invention provides a breeding method in which haplotype testing as described below is performed on bovine embryos, young adults, or adult cattle, and based on the results, certain animals are selected or removed for a breeding program. Preferably, individuals that are negative for the MI haplotype are selected. Preferably, these individuals are homozygous for the negative MI haplotype. For example, the individuals are homozygous for the negative MI haplotype.

[0058] The present disclosure relates to analyzing nucleic acid samples from cattle, including young or adult bovine animals, embryos, semen samples, eggs, fertilized eggs, zygotes, or other DNA-containing biological samples (e.g., their blood, cell, or tissue samples) to determine whether the cattle have one of the haplotypes indicative of dysmotility disclosed herein. The nucleic acid sample is representative of DNA from position 78732954 to 80748266 bp on chromosome 16 (the "MI region") or a fragment thereof. The nucleic acid may include genomic DNA, cDNA, or RNA.

[0059] In some embodiments, the method comprises haplotyping nucleic acid in bovine DNA at chromosome 16 positions 78732954-80748266 bp (the "MI region"), or a fragment thereof, for at least one copy of an MI haplotype, and optionally assigning a bovine MI haplotype score to the individual. This method may be used to identify the haplotypes of both copies of an animal's MI region, and assign a haplotype pair to the animal.

[0060] In some embodiments, haplotyping methods involve testing one copy of the MI region or a fragment thereof, identifying nucleotides at two or more polymorphic sites in that copy, and assigning a haplotype to the individual.

[0061] In some embodiments, the haplotyping method comprises testing one copy of the MI region or a fragment thereof and identifying one polymorphic site in that copy. In some embodiments, the single polymorphic site comprises the MI SNP, which is bp 79613592 of bovine chromosome 16, set forth in SEQ ID NO: 1 / 2: [ka] (SEQ ID NO:1 and SEQ ID NO:2), the underlines represent introns.

[0062] As can be easily understood by those skilled in the art, when the MI region is cloned and sequenced, any individual clone usually only provides haplotype information for one part of the two copies of the MI region present in an individual.If haplotype information is desired for both copies of the entire MI region of an individual, additional MI clones need to be tested.

[0063] Further provided is a method for genotyping the MI region of a bovine, which comprises determining the nucleotide pair identity at one or more polymorphic sites for two copies of the MI region present, wherein the one or more polymorphic sites (PS) have alternative alleles associated with the MI phenotype.

[0064] In some embodiments, the genotyping method includes testing both copies of the MI region or a fragment thereof, identifying nucleotide pairs at one or more polymorphisms associated with the MI phenotype in the two copies, and assigning a genotype to the individual. In some embodiments, "testing the region" may include testing one or more of the DNA containing the region, its mRNA transcript, or its cDNA. As will be readily understood by those skilled in the art, the two "copies" of a gene, mRNA, or cDNA, or fragments thereof, in an individual may be the same allele or may be different alleles. In another embodiment, the genotyping method of the present invention includes determining the identity of the nucleotide pairs at each of the polymorphic sites associated with the MI phenotype.

[0065] In some embodiments, genotyping involves evaluation of one or more of the alleles in the shaded region of Figure 5 (bp 78732954 to 80748266 of bovine chromosome 16). In some embodiments, genotyping involves evaluation of the following (underlined sequences represent intron sequences): [ka] The method includes evaluating a nucleic acid sample for the MI SNP at bp 79613592 of bovine chromosome 16, as shown in (SEQ ID NO: 1 and SEQ ID NO: 2).

[0066] The present disclosure further provides systems, platforms, and kits for genotyping and / or haplotyping bovine samples, the systems or kits comprising in a container one or more nucleic acid molecules designed to detect one or more polymorphisms associated with the MI phenotype, and optionally at least another component for performing such detection. In some embodiments, the kit comprises at least two oligonucleotides packaged in the same or separate containers. The kit may also contain other components, such as a hybridization buffer (where the oligonucleotides are used as probes), packaged in a separate container. Additionally or alternatively, if oligonucleotides are used to amplify the target region, the kit may contain a polymerase (preferably packaged in a separate container) and a reaction buffer optimized for polymerase-mediated primer extension, such as PCR.

[0067] In some embodiments, the system, platform, or kit may be configured to detect mutant proteins, such as mutant voltage-gated calcium channel subunit α1S gene (CACNA1S), in some embodiments, the mutation changes a GGC codon to AGC, facilitating an amino acid substitution of glycine to serine.

[0068] Thus, in some embodiments, the system, platform, or kit includes immunoassay reagents for detecting mutant proteins, including one or more antibodies and a buffer, where the primary or secondary antibody may have an attached enzyme that produces color when a substrate for the enzyme is added and reacts with the enzyme. The enzyme that produces color may be linked to either the antigen or the primary or secondary antibody.

[0069] In some embodiments, a breeding method is provided in which the above haplotyping and / or genotyping is performed on bovine embryos and, based on the results, certain bovines are selected for or dropped from a breeding program. In some embodiments, individuals with MI negative haplotypes are selected for continued and future breeding.

[0070] Through the use of linked marker loci, different haplotypes can be engineered in genetic improvement programs by a procedure called "marker-associated selection" (MAS) for genetic improvement within a breeding core, or "marker-associated introgression" to transfer beneficial alleles from the resource population for the breeding core (Soller 1990; Soller 1994, the entire contents of which are incorporated herein by reference).

[0071] Genotyping assays

[0072] I. Hybridization-Based Methods

[0073] A. Array

[0074] In some embodiments, the nucleic acid sequence containing the MI SNP can be incorporated into a genotyping platform, including a panel or a nucleic acid target array. As used herein, "panel" and "array" are used interchangeably and refer to a solid substrate, usually a glass, plastic, or silicon slide, used to assay and detect single nucleotide polymorphisms in parallel from a DNA sample, and output genotype data. Numerous panel / array platforms are commercially available and well known in the art. By way of example, bovine array genotyping platforms include, but are not limited to, Neogen's BOVUHDV03 and GeneSeek Genomic Profiler (GGP) Bovine 150K, Illumina's bovine array bead chips such as BovineSNP50 DNA Analysis BeadChip and BovineHD DNA Analysis Kit, and ThermoFisher's Axiom Bovine Genotyping 100K Array and Axiom Genome-Wide BOS 1 Bovine Array, Affymetrix platforms, among others. In some embodiments, the nucleic acid sequence containing the MI SNP can be incorporated into such commercially available genotyping platforms. Alternatively, the nucleic acid sequence containing MI SNPs can be incorporated into a custom-designed array designed to assay a specific pool of SNPs. Some of these SNPs may overlap with commercially available platforms. The incorporation of the nucleic acid sequence containing MI SNPs varies depending on the chemistry of the array platform, but follows the principles of array technology. For example, the nucleic acid sequence containing MI SNPs is used to design one or more complementary probe sequences. The probes are immobilized on the array.

[0075] By way of example, these array platforms represent a subset of the many commercially available array platforms that differ in SNP content, density (number), chemistry, throughput (number of samples that can be assayed simultaneously), or other characteristics. However, arrays generally rely on hybridization of fragmented, single-stranded sample DNA to short, single-stranded probes, also known as oligonucleotides, that are usually 25-50 bp in length. The probes are usually directly attached, printed, or synthesized onto the array surface in a grid pattern on a two-dimensional solid surface such as glass, plastic, or silicon. Probe sequences are designed using prior DNA sequence knowledge, which has been collected from a panel of individuals representing the genotypic diversity of the species, to artificially synthesize sequences that are complementary to the sample DNA fragment sequences. Hybridization depends on the binding of nucleotide bases to complementary bases, and hybridization occurs when the sequences of the probe and sample DNA are complementary. Even strands of DNA that are not perfectly matched can hybridize. Typically, the DNA sample sequence will be 100% identical to the probe, and in some embodiments 99%, 98%, 97%, 96%, 95%, etc. up to 80% identical.

[0076] Array platforms typically utilize fluorescent dyes that generate a signal when the dye is excited by a laser, and the emission spectrum is detected by a camera, whereby the signal reports the specific nucleotide identity at the assayed base pair position within the probe sequence. The resulting raw images are normalized to subtract background signal and converted to genotype data with confidence scores using computational algorithms (LaFramboise, Thomas, 2009). Variations in array technology exist, but the details provided herein are included as examples and not limitations.

[0077] B. Molecular Beacons

[0078] In some embodiments, nucleic acid sequences containing MI SNPs can be genotyped using probe oligonucleotides configured as molecular beacons. Molecular beacons are single-stranded oligonucleotide probes designed to have complementary sequences at each end of the probe sequence creating a stem-loop structure. A fluorophore is added to one end of the probe and a fluorescent quencher is added to the other end. When the molecules are in close proximity, as is achieved when in a stem-loop state, no fluorescence is emitted. However, when the probe sequence hybridizes to its target sequence (the complementary sample DNA sequence), the stem-loop denatures, thereby separating the fluorophore and quencher and allowing fluorescence. An individual's genotype at a particular location can be determined by designing one molecular beacon for one allele and another beacon for an alternative allele, but with different wavelengths of the probe for each beacon. Different alleles can be distinguished by the different wavelengths emitted by each fluorophore.

[0079] C. Dynamic Allele-Specific Hybridization (DASH)

[0080] In some embodiments, the nucleic acid sequence containing MI SNP can be genotyped using DASH genotyping, which is based on detecting the difference in melting temperature of double-stranded DNA when mismatched nucleotides exist.Fluorescent marker emits signal only when the molecule is bound to the dsDNA formed between allele-specific oligonucleotide and sample DNA.As the temperature of reaction increases, dsDNA is denatured into ssDNA, and marker no longer emits fluorescence.

[0081] II. Enzyme-Based Assays

[0082] Several enzymes can be used to develop SNP genotyping assays, including DNA polymerases, DNA ligases, and nucleases.

[0083] A. Polymerase Chain Reaction (PCR)-Based Methods

[0084] In some embodiments, the genotyping assay includes an amplification step, for example, to amplify a nucleic acid from a sample of a subject, the nucleic acid including the MI SNP. Those skilled in the art understand how to design primers and polymerase chain reaction (PCR) protocols to amplify a nucleic acid target of interest, such as a sequence that includes the MI SNP. Primers may be designed to be complementary to sequences adjacent to the MI SNP, such that the MI SNP and any sequence 3' of the forward primer and 5' of the reverse primer are amplified via PCR. Primer sequences may be variable in length, sequence composition, or position relative to the MI SNP, allowing variable length amplicons of different sequence composition to be amplified, the amplicons containing the MI SNP. PCR-based genotyping techniques include, but are not limited to, real-time PCR or quantitative PCR (qPCR), and end-point genotyping.

[0085] In some embodiments, nucleic acid sequences containing MI SNPs can be genotyped using real-time qPCR. Real-time qPCR approaches are well known in the art and generally rely on measuring PCR product accumulation over each PCR cycle (i.e., in real time) as opposed to after a certain number of PCR cycles as performed with PCR. qPCR typically relies on a reporter molecule / enzyme to determine the abundance of PCR product relative to a control in real time. SYBR Green is a type of non-specific dye that can be used to report on PCR product accumulation by binding to dsDNA and fluorescing upon binding. Hybridization probes may also be used, which consist of oligonucleotides that are complementary to sequences within the PCR product. The probes are tagged with different fluorescent dyes, one at the 3' end of one probe and the other at the 5' end of the second probe. As PCR products accumulate, the probes bind to the target DNA, which brings them both into close enough proximity to catalyze fluorescence resonance energy transfer (FRET), and energy is transferred from a shorter wavelength dye (donor) to a longer wavelength dye (acceptor). The amount of PCR product can be estimated based on the wavelength and intensity of the detected fluorescence.

[0086] In another embodiment, nucleic acid sequences containing MI SNPs can be genotyped using end-point genotyping, which involves measuring PCR amplification products after a certain number of PCR cycles. One example of an end-point PCR genotyping assay is Kompetitive Allele-Specific PCR (KASP) from LGC Biosearch, which is based on allele-specific PCR and fluorescence resonance energy transfer (FRET). Two allele-specific forward primers, each labeled with a distinct fluorescent dye, and a common reverse primer allow the amplification of sample DNA regardless of whether it contains that allele. SNP genotype is determined by the emission spectrum detected, with homozygous individuals being indicated by a signal consisting of one of two possible spectra depending on the allele, and heterozygous individuals being indicated by a signal containing both possible spectra.

[0087] B. Restriction fragment length polymorphism (RFLP)

[0088] In some embodiments, nucleic acid sequences containing MI SNPs can be genotyped using restriction fragment length polymorphism (RFLP). This assay involves digesting a DNA sample with one or more restriction enzymes into fragments, and the resulting fragments can be separated by gel electrophoresis. The banding pattern determines the composition of nuclease cleavage sites found within the DNA sample sequence, which ultimately reports on the original DNA sequence.

[0089] C.Flap endonuclease (FEN) (Invader assay)

[0090] In some embodiments, nucleic acid sequences containing MI SNPs can be genotyped using flap endonucleases (FENs). FENs are endonucleases that recognize and cleave specific molecular structures. One example of an assay that utilizes FENs, called Cleavase, is the Invader assay. Invader assays are well known in the art and basically involve designing an oligonucleotide Invader probe that is complementary to the 3' end of the variable SNP site. The last nucleotide of the Invader probe is a mismatched base that overlaps with the variable SNP site. A second allele-specific probe is designed to be complementary to the 5' end of the variable SNP site, but extends beyond the 3' side of the site. If the sequences of the sample DNA and the second allele-specific probe are complementary, they will hybridize and, in the presence of the Invader probe, form a three-dimensional complex that is recognized and cleaved by FENs. Fluorescence resonance energy transfer (FRET) technology can be used to detect the cleavage reaction by triggering a secondary cleavage reaction. A secondary cleavage reaction occurs between a quencher molecule attached to the 3' end of a second allele-specific probe and a fluorophore attached to the 5' end of the same probe. Primary cleavage by FEN separates the quencher from the fluorophore, which generates a detectable signal. The Invader assay is just one such genotyping assay and is intended to serve as an example. Variations of the Invader assay include, but are not limited to, the Serial Invasive Signal Amplification Reaction (SISAR), which allows both SNP alleles to be tested in a single reaction.

[0091] D. Primer Extension

[0092] In some embodiments, nucleic acid sequences containing MI SNPs can be genotyped using primer extension or single base extension, mini-sequencing techniques. A detection primer is designed to target the sequence immediately upstream of the SNP. The 3' end of the oligonucleotide is extended by a single base using dideoxynucleotide triphosphates (ddNTPs). Each ddNTP base corresponds to a different fluorescent dye. As a result, up to four alleles are detected, including differentiation of homozygous and heterozygous genotypes. Several detection platforms, such as mass spectrometry (matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF)), capillary electrophoresis, pyrosequencing, flow cytometry, or fluorescent plate readers, are capable of detecting SNPs.

[0093] E. 5'-nuclease (TaqMan)

[0094] In some embodiments, nucleic acid sequences containing MI SNPs can be genotyped using 5'-nuclease. TaqMan probes utilize a variation of fluorescence resonance energy transfer (FRET) technology in which a fluorophore is covalently attached to the 5' end of an oligonucleotide probe and a quencher is covalently attached to the 3' end of the same probe. As PCR products accumulate, the probe binds to the PCR products during denaturation cycles, and when Taq polymerase synthesizes a new strand, it encounters the hybridized probe. The exonuclease activity of Taq releases a fluorescently tagged nucleotide at the 3' end of the probe. The fluorophore is immediately released into solution where it fluoresces. This is because the fluorophore is no longer close enough to the quencher to perform FRET.

[0095] F. Oligonucleotide Ligation Assay

[0096] In some embodiments, nucleic acid sequences containing MI SNPs can be genotyped using an oligonucleotide ligation assay. This assay relies on DNA ligase, an enzyme that ligates two DNA probes at the SNP site, and ligation occurs when both probes have perfect base pair complementarity to the target sequence. The first probe sequence is an allele-specific probe designed to be complementary to the target DNA such that its 3' base corresponds to the target SNP, and the second probe sequence is designed to hybridize to the sequence directly adjacent to the SNP in the other orientation such that its 5' end is provided for the ligation reaction. Ligation can occur only if the probe matches and hybridizes to the target DNA. Any platform capable of detecting differences in sequence length can be used to determine whether the product is ligated or not, including gel electrophoresis, MALDI-TOF mass spectrometry, and capillary electrophoresis. Alternatively, oligonucleotides can be tagged and / or labeled to index the sample and allow high-throughput sequencing of the ligated product, and the genotype of the product can be determined.

[0097] IV. Nucleic Acid Sequencing

[0098] In some embodiments, MI SNPs can be detected by sequencing nucleic acid sequences that contain MI SNPs. Sequencing methodologies are broad, with several variations in platforms and chemistries. For example, first generation sequencing techniques utilize Sanger sequencing and rely on electrophoretic separation of chain termination products. Recently developed sequencing techniques, including next generation sequencing (NGS), are well known in the art and generally describe massively parallel sequencing via clonally amplified DNA templates in flow cells. Sequencing methods that can be utilized include, but are not limited to, whole genome sequencing (WGS), targeted resequencing, RNAseq, exome capture / sequencing, and genotyping by sequencing (GBS), where the nucleic acid sequenced can be DNA or RNA. These methods require the use of a sequencing platform, and factors such as chemistry, desired multiplexing level, desired amount of data output, and runtime must be considered when selecting a platform. Examples of sequencing platforms include, but are not limited to, Roche 454, Illumina MiSeq, Illumina HiSeq, Life Technologies SOLiD4, and Pacific Biosciences SMRT platforms.

[0099] V. Mass spectrometry

[0100] Mass spectrometry determines the mass of ionized molecules by measuring the rate of molecular migration in an electric field, where smaller molecules move faster. Some types of mass spectrometers separate fragments of different sizes and further fragment these to sequence oligonucleotides.

[0101] VI. Haplotype Assay

[0102] Detection of the haplotype is accomplished by detection of one or more polymorphisms comprising the haplotype, where the MI haplotype comprises 120 markers comprising the MI SNP in the range of 78,732,954-80,748,266 bp as shown in Table 2. The genotype of the MI haplotype markers may be determined using any of the genotyping methods described above, or a combination thereof. For example, one or more assays may be designed to detect the genotype of all or a portion of the 120 markers comprising the MI haplotype. The MI SNPs may be genotyped using the same assay as that used to detect the genotype of the MI haplotype markers, or may be genotyped using a different assay. In an embodiment, all or a portion of the 120 markers comprising the haplotype (optionally including the MI SNP) and additional markers in linkage with the MI SNP may be added to a commercially available array platform, such as those previously described.

[0103] Protein detection assays

[0104] An alternative method for detecting mutant individuals is by using immunoassays such as ELISA (enzyme-linked immunosorbent assay), which can detect the presence of a protein of interest or a mutant protein of interest. An antibody designed to bind to the protein of interest is added to a liquid sample, and this binding or that of a secondary antibody produces a detectable signal that can then be measured. In this way, the mutant protein can be detected by an antibody specific for the mutant versus the wild-type protein. Both wild-type and mutant forms of the protein encoded by the CACNA1S gene can be detected using this type of assay.

[0105] In some embodiments, the nucleic acid of the present disclosure comprising the MI SNP is added to an existing genotyping platform, panel, or array. In some embodiments, the nucleic acid is added to the platform, panel, or array in double-stranded form. In some embodiments, the nucleic acid is added to the platform, panel, or array in single-stranded form. In some embodiments, the nucleic acid is single-stranded and comprises a sense strand of DNA comprising the MI SNP. In some embodiments, the nucleic acid is single-stranded and comprises an antisense strand of DNA comprising the MI SNP. In some embodiments, the nucleic acid is single-stranded and comprises an RNA comprising the MI SNP.

[0106] In some embodiments, the nucleic acid comprises SEQ ID NO:1 and / or SEQ ID NO:2, a complement thereof, or a reverse complement thereof. In some embodiments, the nucleic acid comprises a fragment of SEQ ID NO:1 and / or SEQ ID NO:2, a complement thereof, or a reverse complement thereof, wherein the fragment comprises an MI SNP. In some embodiments, the fragment comprises 10 or more nucleotides 5' and 10 or more nucleotides 3' of the MI SNP position. In some embodiments, a nucleic acid comprising an MI SNP comprises about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, or about 100 or more nucleotides 5', 3', or both 5' and 3' of the MI SNP, or any combination of lengths on the 5' and 3' sides of the MI SNP. In some embodiments, a nucleic acid or fragment thereof comprising an MI SNP has about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 99% identity to SEQ ID NO:1, SEQ ID NO:2, their complements, their reverse complements, or any fragment thereof. In some embodiments, a nucleic acid comprising an MI SNP deviates from SEQ ID NO:1 or SEQ ID NO:2 due to variations in the genetic code. That is, although a nucleic acid sequence may differ from the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, their complements, or their reverse complements, a variant nucleic acid sequence (or fragment) encodes the same amino acid sequence.

[0107] In some embodiments, the nucleic acid containing the MI SNP also contains a detectable marker, which may embody several formats, such as a dye that binds to the double-stranded DNA of the nucleic acid containing the MI SNP, or a fluorescently labeled probe that hybridizes to the nucleic acid containing the MI SNP.

[0108] The following examples are intended to illustrate preferred embodiments of the invention and should not be construed as limiting the scope of the invention as defined in the claims. EXAMPLES

[0109] Example 1. Identification of haplotype-associated motility disorders in Holstein calves.

[0110] background

[0111] The Holstein genomic selection program screens for several recessive genes associated with stillbirth and late abortion (Agerholm et al., 2001; Charlier et al., 2012) and neonatal survival (Shuster et al., 1992; Schutz et al., 2016; Basiel et al., 2020). Genomic testing has also accelerated the discovery of recessive genes that result in embryonic loss (Fritz et al., 2013, 2018; McClure et al., 2014; Adams et al., 2016) by screening for regions where a given haplotype is not observed in the homozygous state when expected to be present based on haplotype frequency (VanRaden et al., 2011).

[0112] While genomic testing has facilitated rapid genetic improvement in addition to identifying genetic recessives, there has also been an acceleration of inbreeding when viewed on an annual and generational basis (Makanjuola et al., 2020). The recent increase has been accompanied by a period of contraction in male lines due to the widespread adoption of artificial insemination (Dechow et al., 2020). The rise in inbreeding and small effective population sizes have increased the likelihood that new genetic recessive conditions may become manifest in significant numbers of animals (Fritz et al., 2013). In recent years, motor disorders in newborn calves have been observed by veterinarians and farm staff in multiple states. Genetic abnormalities are the primary cause of neuromuscular defects in humans and other species (Laing, 2012) and may underlie the problems observed in calves. Therefore, the objective of this study was to perform a genome-wide association study of affected calves and normal pedigree members to determine if a genetic origin is possible and to establish the credibility of inheritance based on common ancestry.

[0113] Materials and Methods

[0114] Sample population. Table 1 provides details regarding the number of affected calves and the control population that were genotyped. Affected calves (34 total) were recorded on farms in New York (Farms 1 and 2), Florida (Farm 3), and Pennsylvania (Farm 4). The condition was first noted in December 2019, with the latest observation in November 2021. Affected calves had a range of severity, recovery, and relapse patterns, and were unable to stand unassisted again during the neonatal period. Some were unable to stand for the first 24 hours of life, while others were initially able to stand and then lost the ability to stand within the first month of life. Twenty-one calves were euthanized, seven calves were considered recovered, and six were stunted and thriving at the time of data collection. Table 1 shows the number of calves with locomotor impairment by farm and their recovery status, the number of impaired calves resulting from embryo transfer, the number of impaired calves that were genotyped, the number of control sibs that were genotyped, and the number of parents or other relatives for the genotype. [Table 1]

[0115] Necropsies of the calves were performed at the Cornell University College of Veterinary Medicine and the University of Florida College of Veterinary Medicine. Following histological examination of muscles and peripheral nerves, biopsies of the right biceps and right brachial plexus were performed in three calves on one farm. Blood samples were taken from four calves on farm 1 to determine whether cholesterol deficiency, anemia, or selenium deficiency contributed to the motor deficits, and blood samples were taken on farm 3 to evaluate potential selenium deficiency.

[0116] Calves were derived from 17 sire x dam combinations, with 22 calves derived from in vitro fertilization with 13 (farm 3), 3 (farms 3 and 4), and 2 (farm 2) full-sib pedigrees. All calves on Farm 3 were sired by a single bull mated to three dams.

[0117] Genomic and pedigree analyses. Tissue, hair, or blood samples were available for genotyping from 18 affected calves, ranging from 1 to 12 per farm; the remaining calves were culled before sample collection could occur. For the control population, 22 healthy full siblings (17 and 5 calves, respectively) from two dams on farm 3 were sampled. In addition, full sisters to the sires of the calves on farm 3 and the dams of the three calves on farm 4 were sampled. All samples were genotyped for 139,376 DNA markers (BOVUHDV03, Neogen, Lincoln, NE). Genotypes from the two dams on farm 3 were imputed using findhap.f90 (v3; Vanraden et al., 2011) and included in subsequent analyses.

[0118] Hypokinetic was scored as 1 for 26 unaffected animals, including control calves and genotyped relatives, or 2 for 18 affected calves. Association analyses were performed in PLINK (v1.9; Chang et al., 2015; www.cog-genomics.org / plink / 1.9 / ). A marker call rate of ≥99% was imposed, leaving 101,907 markers for analysis. The initial association analysis consisted of an allelic chi-square test assuming one degree of freedom (frequency of allele A against frequency of allele B) with p-values ​​generated using Fisher's exact test. This was followed by a dominance model (genotypes AA and AB against genotype BB) with a one-degree of freedom chi-square test. Multiple comparison tests were considered by implementing false discovery rate (FDR) p-values ​​to determine genome-wide significance at FDR P ≤ 0.05. Homozygosity screening was also performed to identify consecutive homozygosities.

[0119] Thirty-three affected calves had known sires and were included in the pedigree analysis. Two generations of sire identification (sire and maternal grandfather) were available for nine calves on Farm 1, whereas six or fewer generations (sires for calf sire + dam for 5 generations) were available for all remaining calves. All known ancestral cows were traced separately for the sire and dam of each affected calf to determine the number of calves that had ancestral cows in the sire and dam lineages and to identify plausible common ancestral cows. Imputed genotypes of potential ancestral cows were derived using findhapf90 using a population of 7622 Holsteins with genotype densities ranging from 3K to 150K, in addition to genotypes of affected calves and controls.

[0120] result

[0121] Clinical findings. Necropsy was unable to determine the cause of motor failure in any of the calves. There was no evidence of cholesterol deficiency, selenium deficiency, or anemia based on serum test results. Definitive disease diagnosis was not supported by histological examination of muscle and peripheral nerves, although intramuscular nerve branches of biceps biopsies showed moderate subneural edema with scattered macrophage infiltration of the endoneuria, endomysium, and perimysium. Similarly, moderate to severe edema was noted in the endoneuria and subperineurium with scattered mononuclear cell infiltration of the nerve bundles of the brachial plexus. Calves' behavior and appetite were normal, and there were no obvious clinical signs that would interfere with proper motor function.

[0122] Genome-wide association. A Manhattan plot demonstrating the significance level of each marker by the dominant model is shown in Figure 1, with a clear peak at the end of chromosome 16. In total, 78 markers in this region were significant (FDR P ≤ 0.05) in a 5.1 million bp region ranging from 75,332,437 to 80,448,457 bp (ARS-UCD1.2). The allelic model returned comparable results with more significant markers (88) over a slightly expanded range (5.7 million bp). The affected calves shared a homozygous common stretch of 2.1 million bp ranging from 78,732,954 to 80,748,266, with one control calf homozygous in the same region. Figure 5 contains a list of DNA markers in this region along with genotype frequencies in affected calves. Highlighted sequences represent allele positions in MI affected animals.

[0123] Haplotype associations of 120 markers ranging from 78,732,954 to 80,748,266 bp with the dysmotility phenotype are shown in Figure 2. All affected calves were homozygous and one of 26 controls was homozygous and located on Farm 3. Nine controls were homozygous and 16 were heterozygous for the alternative haplotype. The sire and both dams of calves on Farm 3 were heterozygous for markers in this region. [Table 2]

[0124] A second region ranging from 9,585,914 to 10,754,653 bp containing three significant markers was present on chromosome 20 with a dominant model (Figure 1), but the markers were not significant in the allelic model. Seventeen of the 18 affected calves were homozygous for these markers, while the remaining affected calves were heterozygous. Of the controls, six were homozygous for the same allele, 18 were heterozygous, and two were homozygous for alternative alleles. It is unclear whether this indicates that the condition is associated with multiple loci or was a spurious result. In contrast to the observations on chromosome 16, the affected calves were not all homozygous for the remaining markers spanning this region.

[0125] Pedigree analysis. The 33 affected calves with identified sires shared 63 known ancestral calves in their sire lineages born after 1990, most recently in 2008 and 2010. For the maternal lineage, all 24 affected calves with known maternal sires for at least five generations were traced to bulls born in 2008, compared with seven for bulls born in 2010. In addition, imputed genotypes suggested that the 2008 sire, and not the 2010 sire, was the carrier of the candidate region. Thus, bull ROYLANE SOCRA ROBUST-ET (Robust) was considered the most likely ancestral calf for the origin of these lineages. When expanded to include the remaining nine calves with known maternal sires, 30 of the calves could be traced to Robust. The pedigrees with the most pathways from Robust to affected offspring are shown in Figure 2, and the pedigrees with all calves genotyped or known sires for at least five generations are reported in Figure 3. On the paternal side of the pedigree, 30 of 33 affected calves could be traced to SEAGULL-BAY SUPERSIRE-ET (Supersire), whereas 23 of 24 on the maternal pedigree could be traced to Supersire. Genotyping indicated that Supersire was also a carrier of the suspect haplotype.

[0126] conclusion

[0127] Calves homozygous for the haplotype on chromosome 16 were at increased risk for motor impairment, characterized by the inability to stand. Although some calves recovered partially, the majority became so ill that they were euthanized due to the development of secondary symptoms, including pneumonia. Interestingly, apart from the motor impairment, the calves appeared healthy.

[0128] Despite the strong evidence for the candidate region, some uncertainty exists due to the inconsistent onset of the dyskinetic phenotype, the presence of homozygous calves that are able to stand unassisted, and additional regions with significant markers. This raises the possibility that the identified genomic region is spurious and the condition is non-genetic. The evidence for the region carrying the recessive allele was supported by high statistical significance, the presence of heterozygous haplotypes in the genotyped parents, common homozygous haplotypes across multiple affected families, and a plausible inheritance pathway.

[0129] Observations in humans and animal models support a genetic basis for the motor deficiency phenotype. Humans with defects in the CACNA1S gene are known to have muscle weakness (Fialho et al., 2018). In some instances, individuals suffer from hypokalemic periodic paralysis, which is associated with muscle paralysis that can last from minutes to days and can be triggered by diet and exercise. Similar to observations in calves with MI, CACNA1S mutations in humans result in variation in the frequency and duration of muscle weakness, as well as the age at which symptoms are first seen. Genetic conditions in genes other than CACNA1S can also cause MI. Most neuromuscular disorders have a genetic origin, and mutations in a wide range of genes can cause disease (Laing, 2012). Genetic recessive conditions and dominant de novo mutations have been associated with genetic pediatric neuromuscular diseases in humans (Herman et al., 2021). A hereditary juvenile-onset motor polyneuropathy has recently been described in cats and is characterized by mononuclear cell infiltration of intramuscular nerve branches ( Crawford et al., 2020 ). Mononuclear cell infiltration of skeletal muscle is a consistent feature of inflammatory myopathies ( Ascherman, 2012 ).

[0130] Most calves in this study were derived from IVF and embryo transfer. Developmental abnormalities, including epigenetic abnormalities and large-baby syndrome, have been associated with IVF in several species (Ventura-Junca et al., 2015). However, 11 calves derived from AI were affected, one resulting from natural mating. These observations in the context of strong genomic associations suggest that IVF is not a determining factor for the development of akinesia. Nevertheless, genotype × environment interactions with IVF, increasing the likelihood that a phenotype will be expressed if the genetic defect is inherited, cannot be discounted.

[0131] The rate of inbreeding on both bases and on an annual and generational basis has increased substantially since the introduction of genetic selection (Dechow et al., 2020; Makanjuola et al., 2020), which has implications for long-term selection responses and increases the likelihood of genetic recessiveness emerging (Fritz et al., 2013). The pedigrees in Figures 2 and 3 demonstrate how genetic recessiveness can spread rapidly given the current rate of inbreeding. All of the male ancestors in the pedigree are top AI sires, demonstrating how de novo or low frequency mutations can amplify rapidly. Supersire was listed as the most related progeny sire for Holsteins by the Dairy Cattle Breeding Council (CDCB, 2021) following a national genetic evaluation in December 2021. If bred randomly to the current Holstein population, the offspring would have an expected inbreeding of 11.9% based on pedigree and 11.5% based on genotype. The pedigree and genomic future inbreeding values ​​are 10.2% and 9.5%, respectively, for Robust.

[0132] Genomic testing has accelerated the discovery of genetic recessives that result in embryonic lethality (VanRaden et al., 2011). Unfortunately, haplotype screening methods are unlikely to identify conditions such as locomotor dysfunction, as calves appear normal at birth and can be genotyped as part of normal management routines. Genomic selection for heifer survival has been introduced into the National Genetic Selection Program by (Neupane et al., 2021), but heritability is low (less than 1%) and accuracy of genomic predictions is moderate. Furthermore, heifers have difficulty surviving for a variety of reasons, including mortality, injury, and reproductive failure. Genomic evaluation of quantitative traits with widespread underlying conditions is not clearly useful for detecting genetic recessives such as those observed herein, unless a large population of calves is affected by the specific condition.

[0133] The current inbreeding rate in the Holstein breed favors rapid amplification of the genetic defect. Support for a genetic defect on chromosome 16 conferring motility disorder includes genome-wide significance across multiple breeding combinations, heterozygous parental haplotypes, a plausible common ancestor for the affected pedigrees, and similar genetic conditions in other species. Despite strong evidence for the existence of the genetic condition, some caution is still warranted due to the inconsistent phenotypic presentation of calves. Future efforts will be required to identify the exact location and type of mutation to confirm the condition and facilitate genetic testing and selection.

[0134] Example 2. Sequence alignment and discovery of candidate mutations

[0135] One affected calf that was homozygous for the recumbency haplotype, the affected calf's sire, and a male progenitor that was phenotypically normal but known to be homozygous for the recumbency haplotype were sequenced at 30x depth using an Illumina (San Diego, CA) HiSeq paired-end platform. The homozygous progenitor was identified at the USDA Animal Genome Improvement Laboratory by tracing the haplotypes of the genotyped Holstein sire.

[0136] The sequence was aligned (Bowtie 2; https: / / bowtie-bio.sourceforge.net / bowtie2 / index.shtml) against bovine chromosome 16 (ARS-UCD1; https: / / www.ncbi.nlm.nih.gov / assembly / GCF_002263795.2). Variants from the reference genome were considered as candidate mutations if they were (1) homozygous in the affected calf, (2) heterozygous for the sire, or (3) not homozygous in the sire. bp79613592 (C to T in the forward strand, G to A in the reverse strand) was identified as meeting the genotype criteria. The mutation and surrounding variants were heterozygous for the sire. In the sire that was homozygous across the recumbency haplotype region, the mutation was heterozygous. See Figure 6.

[0137] The sequence of the DNA plus strand from bp 79613564 to 79613623, where the SNP is indicated as [C] or [T], is [ka] (Sequence number 1 / 2).

[0138] The mutation is present in the voltage-gated calcium channel subunit α1S gene (CACNA1S), which is encoded on the minus strand. The missense mutation changes a GGC codon to AGC, promoting an amino acid substitution of glycine to serine. Ensembl (https: / / useast.ensembl.org / index.html) predictions (Table 3) show nine potential transcripts spanning 39-44 exons, and the mutation is present in exons 30-32 of the transcript.

[0139] [Table 3]

[0140] The amino acid sequences corresponding to the mutated exons are shown for calf and the other five species in Table 4. The highlighted amino acids indicate that the amino acid sequence is conserved across many species.

[0141] [Table 4]

[0142] CACNA1S mutations

[0143] CACNA1S allows voltage-dependent activity of calcium channels and is highly expressed in skeletal muscle (https: / / www.ncbi.nlm.nih.gov / gene / 682930). Mutations are known to cause periodic paralysis in humans (https: / / www.ncbi.nlm.nih.gov / gene / 779) and mice (https: / / www.ncbi.nlm.nih.gov / gene / 12292), which corresponds to the calf recumbency phenotype observed in Holstein calves.

[0144] The mutation was submitted to the Ensembl Variant Effect Predictor (https: / / useast.ensembl.org / Homo_sapiens / Tools / VEP). The Sorting Intolerant From Tolerant (SIFT) score confirmed that the mutation was deleterious (SIFT score range 0–0.01) and predicted to have a moderate effect, corresponding to the partially penetrant nature of the recumbency in calves.

[0145] Example 3. Exemplary Assays and Results

[0146] Sixteen animals were genotyped (Figure 5) for the C to T SNP at bp 79613592 in the CACNA1S gene using a TaqMan assay. The genotyped animals included six recumbent calves that were homozygous for the MI haplotype (haplotype genotype = MI / MI); four normal animals that were previously determined to be carriers of the MI haplotype (haplotype genotype = + / MI); four recumbent calves that were not genomically tested (haplotype genotype unknown), and two normal animals that were not genotyped and were predicted not to contain the MI allele based on pedigree (haplotype genotype unknown). Of the heterozygous animals, two were bulls that were ancestors of the recumbent calves, and the remaining two were siblings of the recumbent calves. All recumbent calves were homozygous for the MI allele despite their prior genotype status. All animals heterozygous for the MI haplotype were heterozygous for the MI SNP, demonstrating that the MI genotype can be accurately ascertained by directly testing the SNP at bp79613592. All expected genotypes (MI / MI, + / MI, and + / +) were identifiable by the test, and the negative control did not demonstrate any detectable amplification.

[0147] [Table 5]

[0148] References

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[0150] Agerholm, J.S., C. Bendixen, O. Andersen, and J. Arnbjerg. 2001. Complex vertebral malformation in Holstein calves. J. Vet. Diagnostic Investig. 13. doi:10.1177 / 104063870101300401.

[0151] Ascherman, D.P. 2012. Animal models of inflammatory myopathy. Curr. Rheumatol. Rep. 14. doi:10.1007 / s11926-012-0245-7.

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[0153] CDCB (Council on Dairy Cattle Breeding). 2021. Sires Highly Related To Holstein Cows in December, 2021. https: / / queries.uscdcb.com / eval / summary / inbrd.cfm?R_Menu= HO.k#StartBody. Accessed December 30, 2021.

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[0172] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein may also be suitably implemented in the absence of any element or limitation not specifically disclosed herein. The terms and expressions used are used as terms of description rather than limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, although the invention has been illustrated by specific embodiments and optional features, it should be understood that modifications and / or variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0173] Several patent and non-patent publications are cited in this specification. The cited references are incorporated herein by reference in their entirety. If there is a discrepancy between a term definition in this specification as compared to a term definition in a cited reference, the term shall be interpreted according to the definition in this specification.

Claims

1. (a) obtaining a sample from a domestic bovine, wherein said sample comprises chromosomal DNA; (b) generating genetic data from the chromosomal DNA for position 78732954 to position 80748266 bp on chromosome 16, or a fragment thereof; (c) identifying a movement disorder (MI) haplotype in the genetic data, wherein the MI haplotype is (i) one or more of the alleles in the shaded areas of FIG. 5 ; and / or (ii) T on the DNA plus strand at position 79613592 bp on chromosome 16 Including, (d) selecting or removing said cattle as breeding stock based on the identification in (c). A method comprising:

2. 2. The method of claim 1, wherein the bovine is a heifer or a cow.

3. 2. The method of claim 1, wherein the bovine is a bull.

4. The method of claim 1 , wherein the bovine is phenotypically normal for dysmotility.

5. 10. The method of claim 1, wherein the bovine is a Holstein, a Holstein cross, or another breed of bovine with Holstein ancestry.

6. The method of claim 1 , wherein the sample comprises semen or oocytes.

7. The method of claim 1 , wherein the sample comprises a tissue sample or a blood sample.

8. The method of claim 1 , wherein the sample is from an embryo or fetus.

9. 2. The method of claim 1, wherein the genetic data comprises a DNA sequence of at least one of chromosome 16 positions 78732954-80748266 bp or a fragment thereof of the bovine.

10. 2. The method of claim 1, wherein said genetic data comprises the DNA sequence of positions 78732954-80748266 bp or a fragment thereof on chromosome 16 of both of said bovines.

11. 2. The method of claim 1, wherein the MI haplotype comprises one or more SNPs associated with the MI phenotype at one or more of positions 78732954-80748266 bp of bovine chromosome 16, or a fragment thereof, and a genetic variant in LD with the SNP.

12. The method of claim 1 , wherein generating genetic data comprises sequencing the chromosomal DNA or an amplified copy thereof.

13. The method of claim 12, wherein the sequencing comprises next generation sequencing.

14. The method of claim 1 , wherein generating the genetic data comprises an amplification step, such as a polymerase chain reaction.

15. A method of selective breeding comprising obtaining a genomic DNA sample from a bull, detecting a homozygous negative MI haplotype in said sample, and using semen from said bull to inseminate a cow, optionally wherein said cow is in vitro fertilized.

16. 16. The method of claim 15, wherein the cow is also homozygous negative for the MI haplotype.

17. 16. The method of claim 15, wherein the bull is a Holstein bull, a Holstein cross bull, or a bull of another breed with Holstein ancestry, and the cow is a Holstein cow or heifer, a Holstein cross bull or heifer, or a cow or heifer of another breed with Holstein ancestry.

18. A method of selective breeding comprising obtaining a genomic DNA sample from a female cow, detecting a homozygous negative MI haplotype in said sample, and using oocytes from said female cow for in vitro embryo production.

19. A method of corrective breeding comprising obtaining genomic DNA samples from bulls and cows, identifying the MI haplotype, and mating cows that are homozygous negative for the MI haplotype with bulls that are positive for the MI haplotype.

20. A method of corrective breeding comprising obtaining genomic DNA samples from bulls and cows, identifying the MI haplotype, and mating a bull that is homozygous negative for the MI haplotype with a cow that is positive for the MI haplotype.

21. A composition, kit, or system comprising at least one nucleic acid molecule, said nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:2, or a fragment of SEQ ID NO:1 or SEQ ID NO:2, said fragment comprising an MI SNP.

22. 22. The composition, kit or system of claim 21, wherein the nucleic acid molecule is attached to a solid support.

23. 22. The composition, kit, or system of claim 21, further comprising an enzyme having polymerase activity.

24. 24. The composition, kit, or system of claim 23, wherein the enzyme comprises Taq polymerase.

25. 22. The composition, kit, or system of claim 21, wherein the nucleic acid molecule comprises a detectable marker.

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