Arabian Oryx (Oryx leucoryx) is an endangered animal that is being saved from extinction by the efforts of captive breeding programs, which have generally been considered a symbol of international conservation success. Long-term success of such programs, however, largely depends on the prudent use of molecular information for conservation management. More specifically, there is some concern that associated animal reintroduction programs might culminate in partial or total collapse of the Arabian Oryx. To maintain the genetic diversity of this endangered species, there is emphasis on implementing screening of different herds of Arabian Oryx for genuine selection of candidates for introduction in wild or for success of captive breeding programs.
Systems and methods for molecular fingerprinting to identify Inbreeding and Outbreeding Depression Factors (IODFs) in an animal are described. In one aspect, the systems and methods receive inputs such as information pertaining to a set of loci, allele quantity and size, genotype, and/or so on. The systems and methods calculate, based on at least a subset of inputs and a set of microsatellite markers, an IODF. The systems and methods evaluate the calculated IODF to determine if the animal is a suitable/good candidate for a breeding program.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
In the Figures, the left-most digit of a component reference number identifies the particular Figure in which the component first appears.
Long-term success of captive breeding programs depends on the prudent use of molecular information for conservation management. Mean D square (MDS) and observed heterozygosity (HO) are two absolute measures of determining the genetic makeup of a population. MDS is well suited to detect outbreeding depression whereas HO is especially important for detecting inbreeding depression. More particularly, MDS is based on a stepwise mutation model, characterized by an array of various microsatellite loci demonstrating allelic polymorphism (Valdes et al. 1993, Xu et al. 2000). The observation of MDS focuses on events deeper in the individual's ancestry that may simply not be obtained by HO. Generally, MDS and HO are regarded as independent predictors of outbreeding and inbreeding depressions respectively. A high MDS indicates an outbreeding depression and a low HO indicates an inbreeding depression.
In view of the above, both MDS and HO indices are mutually related to the outbreeding-inbreeding continuum, providing valuable information about suitability of individuals for captive breeding programs. However, an optimal degree of relatedness of mating individuals on the inbreeding-outbreeding continuum will maximize fitness of offspring. Breeding programs need accurate indices to measure the relatedness of parental lineages at both ends of the genomic divergence continuum.
In contrast to conventional techniques, the systems and methods described herein use an array of seven microsatellite markers to generate MDS and HO values automatically. In one implementation, a user inputs information including, but not limited to, a living being's number of loci, number of alleles, size of alleles, and genotypes to determine corresponding MDS and HO values. Then systems and methods compute a novel inbreeding-outbreeding depression factor (IODF) to identify corresponding genetic suitability for a breeding program. In one exemplary implementation, IODF values of <0.5 and >1.5 indicate significant inbreeding and outbreeding depressions, respectively. Since these values are arbitrary and depend on the number of samples, a larger sample size would favor better predictions from IODF values. As such, the novel systems and methods described herein provide a simple and authenticated tool for easy computation of indices of outbreeding-inbreeding continuum to assist in captive breeding, for example, of Arabian Oryx.
In one exemplary sample collection, blood samples were collected from twenty-four (24) Arabian Oryx; twenty-one (21) of these samples were obtained from Mahazat As-Sayd Protected Area (MSPA) and three (3) of the samples were from National Wildlife Research Center (NWRC), Saudi Arabia. For statistical evaluation, we considered all the twenty-four (24) samples as a single population due to three main reasons; (i) few samples from NWRC, (ii) NWRC being one of the sources of reintroduction in MSPA and (iii) genetic overlapping because of common founders at both the locations.
In one exemplary DNA extraction procedure, DNA was extracted from two hundred (200) μl blood sample using DNeasy Blood and Tissue Kit (Qiagen GmbH, Germany) according to manufacturer's instructions. The extracted DNA was finally dissolved in 200 μl of elution buffer and stored at −20° C.
This exemplary procedure amplified seven (7) microsatellite loci (RBP3, MCM38, MNS64, IOBT395, MCMAI, BM3501 and MB066) in all the samples. The primer sequences of these markers are shown in TABLE 1.
These particular loci of TABLE 1, although earlier utilized for cattle and Tibetan antelope, have not been utilized to measure inbreeding/outbreeding depression in Arabian Oryx. Zhou et al. (2007) used six of these loci to understand the genetic diversity and population structure of a single population of Tibetan antelopes. MacHugh et al. (1997) used one common locus (RBP3) together with other loci to study cross-species gene flow and phylogeographic pattern of different populations of cattle from Asia, Africa and Europe. Although both of these studies determined HO, they did not compute MDS values for evaluating inbreeding/outbreeding depressions. The forward primer for each marker was labeled with FAM (6-Carboxyfluorescein) whereas the reverse primer was unlabelled. The polymerase chain reactions (PCR) were performed in a total volume of 20 μl containing 2 μl 10×PCR Buffer, 2.5 mM MgCl2, 200 μM each dNTP, 25 nM of each primer, 25 ng template DNA and 0.5 U Taq DNA polymerase. After initial denaturation at 94° C. for 4 min, 25 cycles of 93° C. for 45 s, 55° C. for 45 s and 72° C. for 45 s were repeated followed by the final extension at 72° C. for 4 min. The aliquots of PCR products (0.25 μl) were mixed with 9.25 μl formamide and 0.25 μl ROX-500 standard. The contents were heated at 95° C. for 2 min and then rapidly cooled on ice before being electrophorsed on 3130XL genetic analyzer (Applied Biosystems, USA) for allele identification.
MDS is calculated using the following equation [1]:
where ni1 and ni2 are the number of repeats of the two alleles of the ith locus and N is the total number of microsatellite loci. MDS is based on the difference in the length of two alleles representing the length of time since the two alleles shared a common ancestor.
HO is calculated using the following equation [2]:
where NAH is the number of loci with heterozygous presentation and N is the total number of loci.
The systems and methods described herein utilize the following novel formula [3] to calculate IODF of an individual.
where HO is observed heterozygosity and N is total number of individual samples (different animals from the same population). IMDS, MeanMDS and MedianMDS are the individual, mean and median MDS values.
In one implementation, a user selects an icon (e.g., from a desktop or menu) associated with the CalcMDS computer program to instantiate/execute operations of the application. An exemplary representation of such a computer program module is “Molecular Fingerprinting Module 318” of
Referring to
At this point, the user inputs a respective number of alleles for each locus using drop-down combo boxes (e.g., see UI input controls 122). Note—according to this selection, the respective numbers of text boxes will appear for entering alleles' sizes (124).
To a user inputs respective sizes of all the alleles (allele size input(s) are shown as respective portion(s) of “other program data” 324 of
The user inputs the respective genotypes for each locus (e.g., please see UI input controls 126). In this exemplary implementation, capital letters are accepted in these text boxes.
The user selects the “Calculate HO, MDS” UI button 104 to compute the MDS and HO (shown as respective portions of “program data” 316 of
The user selects the “Calculate IODF” UI button 106 to calculate IODF values (i.e., “IODF Value(s)” 322 of
Exemplary Results: Allelic frequencies of microsatellite loci. In this example, and among the 7 microsatellite markers studies:
IOBT395 (90, 106, 110 and 174) and MCMAI (185, 187, 189 and 191) had four alleles each. Allele frequencies of different microsatellite markers are illustrated, for example, in TABLE 2. An array of 7 microsatellite markers used in this example implementation of the systems and methods clearly differentiated the individual animals.
Functional Evaluation: The functionality of various control tools of CalcMDS software (e.g., “Molecular Fingerprinting Module 318” of
The above computations (without “comments”) are also shown in the exemplary Report 2 (204) of
Implication for Captive Breeding
Both HO and MDS are suitable parameters for detecting inbreeding and outbreeding depressions respectively. Low individual heterozygosity is taken as an indicator of inbreeding whereas a high value of MDS reflects an outbreeding. The exemplary systems and methods illustrated a high level of heterozygosity in this population with an average heterozygosity of 0.601, which is comparable to a decade earlier heterozygosity of MSPA and Thumammah populations of Arabian Oryx. The results of MDS not only exhibited the allelic diversity but also revealed some sort of outbreeding mainly due to the presence of allele D of IOBT395 locus. There has been some evidence for outbreeding depression in Arabian Oryx however, its current intensity may not warrant any management action. Since the heterozygosity and allelic diversity are the reliable predictors of both the survival and adaptation, abilities of populations are important to maintain a high level of heterozygosity and allelic diversity, and thereby, substantially ensure success of captive breeding programs.
The novel Inbreeding and Outbreeding Depression Factor (IODF) of an individual provides a quick view of the individual's suitability for a breeding program based on inbreeding and outbreeding indices. In one exemplary implementation, an acceptable value is in the range of 0.5 and 1.5, whereas IODF<0.5 and >1.5 indicates, for example, inbreeding and outbreeding depressions respectively.
As illustrated, computing device 302 includes one or more processors 304 operatively coupled to system memory 306, mass storage devices 308, input/output (I/O) device(s) 310, and a display device 312. System memory 306 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) and random access memory (RAM). A basic input/output system (BIOS) containing the basic routines that help to transfer information between elements within computing device 302, such as during start-up, is typically stored in ROM. RAM typically contains program modules 314 and program data 316.
Computing device 302 may also include other removable/non-removable, volatile/nonvolatile computer storage media. By way of example only,
In this exemplary implementation program modules 312 includes molecular fingerprinting module 318 and “other program modules” 320 such as an operating system, device drivers, and/or so on. Molecular fingerprinting module 318 calculates an Inbreeding Outbreeding Depression Factor (IODF) 322 based on inputs represented by a respective portion of “other program data 324.” More particularly, molecular fingerprinting module 318 receives input data such as a number of loci, number of alleles and respective sizes, genotypes, and/or so on. A user may enter commands and information into the computing device 302 through input/output devices 310. I/O devices are a collection of interfaces that units of an operational system use to communicate with each other. Input devices 110 may include a keyboard, mouse, microphone, joystick, game pad, satellite dish, scanner, or the like. Output devices 110 may include but are not limited to network interface cards, printers, displays, sound systems, and/or so on.
After receiving input data such as that described above, operations of molecular fingerprinting module 318 calculate, using the input data, heterozygosity (HO), Mean D Square (MDS), and Inbreeding Outbreeding Depression Factor (IODF). These various values/results are shown as respective portions of other program data 324. Operations of module 318 evaluate the calculated information to identify suitability for a subject individual for a breeding program. Molecular fingerprinting module 318 then outputs information associated with one or more portions of the calculated information and breeding program evaluation. In one implementation, module to any presents information to a user via display device 112.
Exemplary computing system 300 is only an example of a suitable computing system and is not intended to suggest any limitation as to the scope of use or functionality of systems and methods described herein. For example, although only a single computing device 102 is illustrated, the system could utilize multiple computing devices, for example, in a distributed computing environment to implement the systems and methods described herein. Neither should computing system 300 be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in computing system 300.
Although the systems and methods for molecular fingerprinting to identify inbreeding and outbreeding depressions have been described in language specific to structural features and/or methodological operations or actions, it is understood that the implementations defined in the appended claims are not necessarily limited to the specific features or actions described. Rather, the specific features and operations of molecular fingerprinting to identify inbreeding and outbreeding depressions are disclosed as exemplary forms of implementing the claimed subject matter.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US10/52196 | 10/11/2010 | WO | 00 | 4/8/2013 |