Analysis Of Microsatellite Loci Research Articles

Enemy release and genetic founder effects in invasive killer shrimp populations of Great Britain

The predatory “killer shrimp” Dikerogammarus villosus invaded Britain from mainland Europe in 2010. Originating in the Ponto-Caspian region, this invader has caused significant degradation of European freshwater ecosystems by predating and competitively excluding native invertebrate species. In contrast to continental Europe, in which invasions occurred through the migration of large numbers of individuals along rivers and canals, the invasion of Great Britain must have involved long distance dispersal across the sea. This makes the loss of genetic diversity and of debilitating parasites more likely. Analysis of nuclear microsatellite loci and mitochondrial DNA sequences of D. villosus samples from the four known populations in Britain reveal loss of rare alleles, in comparison to reference populations from the west coast of continental Europe. Screening of the British D. villosus populations by PCR detected no microsporidian parasites, in contrast with continental populations of D. villosus and native amphipod populations, most of which are infected with microsporidia. These findings suggest that the initial colonisation of Great Britain and subsequent long distance dispersal within Britain were associated with genetic founder effects and enemy release due to loss of parasites. Such effects are also likely to occur during future long-distance dispersal events of D. villosus to Ireland or North America.

Two new Salamanders of the genus Onychodactylus from Eastern Honshu, Japan (Amphibia, Caudata, Hynobiidae).

We describe two new species of hynobiid salamanders in the genus Onychodactylus from eastern Honshu, Japan, based on the morphological and genetic evidence. Onychodactylus intermedius sp. nov. is distributed in southern part of Tohoku District and northern Ibaraki and Niigata Prefectures, and was previously reported as S-Tohoku group. Onychodactylus intermedius belongs to the O. japonicus species complex, and differs from the other congeners in having relatively long tail, narrow head, short snout, 18 presacral vertebrae, and distinctly curved vomerine tooth series without gap. Onychodactylus fuscus sp. nov. is known from only four localities in Fukushima and Niigata Prefectures of Tohoku and Hokuriku Districts. It also belongs to the O. japonicus complex, but lacks the dorsal stripe, which is a diagnostic character of the species complex. In other characteristics, O. fuscus differs from the other congeners in having comparatively long tail, wide head and internarial space, shallowly curved vomerine tooth series with gap, and relatively few vomerine teeth. Both species described here breed in winter. Phylogenetically, the two new species are closely related to each other, forming a well-supported clade with O. tsukubaensis as their sister species. Onychodactylus intermedius sp. nov. is known to be parapatric with O. japonicus and O. nipponoborealis without hybridization, whereas O. fuscus sp. nov. is sympatric with O. japonicus at least in a single known locality, and analysis of microsatellite loci indicates they are reproductively isolated.

Genetic structure of wild boar (Sus scrofa) populations from East Asia based on microsatellite loci analyses.

BackgroundWild boar, Sus scrofa, is an extant wild ancestor of the domestic pig as an agro-economically important mammal. Wild boar has a worldwide distribution with its geographic origin in Southeast Asia, but genetic diversity and genetic structure of wild boar in East Asia are poorly understood. To characterize the pattern and amount of genetic variation and population structure of wild boar in East Asia, we genotyped and analyzed microsatellite loci for a total of 238 wild boar specimens from ten locations across six countries in East and Southeast Asia.ResultsOur data indicated that wild boar populations in East Asia are genetically diverse and structured, showing a significant correlation of genetic distance with geographic distance and implying a low level of gene flow at a regional scale. Bayesian-based clustering analysis was indicative of seven inferred genetic clusters in which wild boars in East Asia are geographically structured. The level of genetic diversity was relatively high in wild boars from Southeast Asia, compared with those from Northeast Asia. This gradient pattern of genetic diversity is consistent with an assumed ancestral population of wild boar in Southeast Asia. Genetic evidences from a relationship tree and structure analysis suggest that wild boar in Jeju Island, South Korea have a distinct genetic background from those in mainland Korea.ConclusionsOur results reveal a diverse pattern of genetic diversity and the existence of genetic differentiation among wild boar populations inhabiting East Asia. This study highlights the potential contribution of genetic variation of wild boar to the high genetic diversity of local domestic pigs during domestication in East Asia.

Reproductive variations in cownose rays (Rhinoptera bonasus) from Chesapeake Bay

The cownose ray, Rhinoptera bonasus, a K-selected species in Chesapeake Bay is subject to an unregulated fishery with no management plan in place. Understanding the reproductive potential for cownose ray is important for successful conservation and management. This study marks the first observation of multiple embryos and right uterus functionality. Eight instances of multiple embryos in cownose rays are reported; two sets of twin live births from captive rays and six separate in utero multiple embryos discovered during necropsy of fishery-dependent and -independent samples. All multiple embryos were in the left uterus. Live births in captivity were confirmed as two sets of twins through direct sequencing of a portion of the mitochondrial DNA and analysis of nuclear microsatellite loci from the newborn pups and putative mothers. In addition, first- and third- quarter term embryos were removed from the right uterus of two female rays during necropsy, marking the first reports of gestation in the right oviduct of cownose rays. The recovery of a three-quarter term albinistic cownose ray embryo through necropsy is also reported.

Triploid Induction in Atlantic Cod (Gadus morhua L.) by the Use of Different Pressure Levels

Different levels of pressure were investigated to see if it was possible to induce triploidy at a lower pressure than previously used (600 bar) for Atlantic cod (Gadus morhua). Newly fertilized eggs were exposed to different levels of pressure: 400, 500, and 600 bar, and one control group (no pressure). Induction of triploidy occurred at each pressure level used in this experiment. Mean weight and length at the termination of the trial period was significantly lower, in the 400 bar group, compared to the other treatment groups. Microsatellite loci analysis revealed over 90% triploid outcome in each experimental group. Lower incidence of deformities was seen in the 400 bar group compared to the 500 and 600 bar groups. Overall this study demonstrated successful triploid induction at both low (400 bar) and medium (500 bar) pressures.

Identification of European (Capreolus capreolus L.) and Siberian (C. pygargus Pall.) roe deer hybrids by microsatellite marker analysis

An analysis of 130 tissue specimens of the European (Capreolus capreolus) and the Siberian (C. pygargus) roe deer from nine regions of Russia and Ukraine using microsatellite loci analysis was conducted, aimed at the identification of hybrid animals. An optimized complex of 21 microsatellite loci was selected for the determination of species specificity and the search for interspecies hybrids. Hybrid animals were revealed in the Moscow and Volgograd regions. Their ratio in the total sample obtained in the European part of Russia was 11.9%.

Genetic diversity and population structure of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) as revealed by mitochondrial and microsatellite DNA.

Ecological surveys have indicated that the population of the critically endangered Yangtze finless porpoise (YFP, Neophocaena asiaeorientalis asiaeorientalis) is becoming increasingly small and fragmented, and will be at high risk of extinction in the near future. Genetic conservation of this population will be an important component of the long-term conservation effort. We used a 597 base pair mitochondrial DNA (mtDNA) control region and 11 microsatellite loci to analyze the genetic diversity and population structure of the YFP. The analysis of both mtDNA and microsatellite loci suggested that the genetic diversity of the YFP will possibly decrease in the future if the population keeps declining at a rapid rate, even though these two types of markers revealed different levels of genetic diversity. In addition, mtDNA revealed strong genetic differentiation between one local population, Xingchang–Shishou (XCSS), and the other five downstream local populations; furthermore, microsatellite DNA unveiled fine but significant genetic differentiation between three of the local populations (not only XCSS but also Poyang Lake (PY) and Tongling (TL)) and the other local populations. With an increasing number of distribution gaps appearing in the Yangtze main steam, the genetic differentiation of local populations will likely intensify in the future. The YFP is becoming a genetically fragmented population. Therefore, we recommend attention should be paid to the genetic conservation of the YFP.

Molecular phylogenetics and microsatellite analysis reveal cryptic species of speckled dace (Cyprinidae: Rhinichthys osculus) in Oregon’s Great Basin

Speckled dace (Rhinichthys osculus) is a small cyprinid that occurs throughout western North America and is the most commonly occurring fish in Oregon. Because of the high genetic and morphological variation in this species across its range, it has been referred to as a species complex; however, no revision to its taxonomy has occurred since 1984. Here, the phylogenetics and population genetics of speckled dace are examined throughout Oregon’s Great Basin to describe genetic variation and infer the geographic boundaries between distinct taxonomic entities and populations. We tested the validity of a putative subspecies, Foskett Spring speckled dace, that occurs in a single spring within Warner Valley in Southeast Oregon and is listed Federally as threatened. Dace were collected from Foskett Spring and all surrounding basins containing speckled dace (Warner, Goose Lake, Lake Abert, Silver Lake, and Malheur), as well as Stinking Lake Spring (located within Malheur), created phylogenetic trees from mitochondrial ND2 and nuclear S7 sequence data, and genotyped eight microsatellite loci for population-level analyses. Three highly divergent clades warrant species-level status: Malheur stream dace, Stinking Lake Spring dace, and dace from the other four basins combined. Although Foskett Spring dace were not monophyletic, substantial population structure occurs at the basin-level and separates Foskett Spring dace from other dace in the surrounding Warner Valley. Thus, we recommend ESU status for the isolated population of speckled dace in Foskett Spring. The high, previously unrecognized, taxonomic diversity within this region indicates a need for a range-wide phylogeographic study of speckled dace and an investigation of the morphological distinctiveness of the putative new species.

Significant population genetic structure of the Cameroonian fresh water snail, Bulinus globosus, (Gastropoda: Planorbidae) revealed by nuclear microsatellite loci analysis

In order to characterize the demographic traits and spatial structure of Cameroonians Bulinus globosus, intermediate host of Schistosoma haematobium, genetic structure of seven different populations, collected from the tropical zone, was studied using six polymorphic microsatellites. Intrapopulation genetic diversity ranged from 0.37 to 0.55. Interpopulation genetic diversity variation clearly illustrated their significant isolation due to distance with gene flow substantially limited to neighbouring populations. The effective population sizes (Ne) were relatively low (from 3.0 to 18.6), which supposes a high rate from which populations would lose their genetic diversity by drift. Analysis of genetic temporal variability indicated fluctuations of allelic frequencies (35 of 42 locus-population combinations, P<0.05) characteristic of stochastic demography, and this is reinforced by events of bottlenecks detected in all populations. These findings demonstrated that Cameroonian B. globosus were mixed-maters with some populations showing clear preference for outcrossing. These data also suggest that genetic drift and gene flow are the main factors shaping the genetic structure of studied populations.

The crayfish plague pathogen can infect freshwater‐inhabiting crabs

Summary The oomycete Aphanomyces astaci is generally considered a parasite specific to freshwater crayfish, and it has become known as the crayfish plague pathogen. Old experimental work that reported transmission of crayfish plague to the Chinese mitten crab Eriocheir sinensis, and the ability of A. astaci to grow in non‐decapod crustaceans, has never been tested properly. We re‐evaluated the host range of A. astaci by screening for the presence of A. astaci in two crab species cohabiting with infected crayfish in fresh waters, as well as in other higher crustaceans from such localities. The animals were tested with species‐specific quantitative PCR, and the pathogen determination was confirmed by sequencing of an amplified fragment of the nuclear internal transcribed spacer. Furthermore, we examined microscopically cuticle samples from presumably infected crab individuals for the presence of A. astaci‐like hyphae and checked for the presence of pathogen DNA in such samples. Screenings of benthopelagic mysids, amphipods and benthic isopods did not suggest infection by A. astaci in non‐decapod crustaceans. In contrast, both studied lake populations of crabs (a native semiterrestrial species Potamon potamios in Turkey, and an invasive catadromous E. sinensis in Sweden) were infected with this parasite according to both molecular and microscopic evidence. Analyses of polymorphic microsatellite loci demonstrated that A. astaci strains in the crabs and in cohabiting crayfish belonged to the same genotype group, suggesting crayfish as the source for crab infection. The potential for A. astaci transmission in the opposite direction, from crabs to crayfish, and potential impact of this pathogen on populations of freshwater crabs require further investigations, because of possible consequences for crayfish and freshwater crab conservation and aquaculture.

Pattern discovery for microsatellite genome analysis

Microsatellite loci comprise an important part of eukaryotic genomes. Their applications in biology as genetic markers are related to numerous fields ranging from paternity analyses to construction of genetic maps and linkage to human disease. Existing software solutions which offer pattern discovery algorithms for the correct identification and downstream analysis of microsatellites are scarce and are proving to be inefficient to analyze large, exponentially increasing, sequenced genomes. Moreover, such analyses can be very difficult for bioinformatically inexperienced biologists. In this paper we present Microsatellite Genome Analysis (MiGA) software for the detection of all microsatellite loci in genomic data through a user friendly interface. The algorithm searches exhaustively and rapidly for most microsatellites. Contrary to other applications, MiGA takes into consideration the following three most important aspects: the efficiency of the algorithm, the usability of the software and the plethora of offered summary statistics. All of the above, help biologists to obtain basic quantitative and qualitative information regarding the presence of microsatellites in genomic data as well as downstream processes, such as selection of specific microsatellite loci for primer design and comparative genome analysis.

Contrasting breeding systems revealed in the rainforest genus Davidsonia (Cunoniaceae): can polyembryony turn the tables on rarity?

Plant breeding systems can have a profound effect on a species ability to persist, colonise new areas and adapt to environmental change. Determining the breeding systems in rare and common congeners may shed light on factors influencing rarity. Endemic to Australia, the Davidsonia genus comprises three species of rainforest trees. The two sympatric, subtropical species, Davidsonia jerseyana (F.Muell. ex F.M.Bailey) G.Harden &amp; J.B.Williams, and D. johnsonii G.Harden &amp; J.B.Williams, are endangered whereas the tropical D. pruriens F.Muell. is widespread. Other than exclusive clonality in D. johnsonii, the reproductive systems in the genus are unknown. We used segregation analysis of microsatellite loci in open-pollinated progeny arrays to investigate the breeding systems in D. jerseyana and D. pruriens. Reproductive success, under glasshouse conditions, was measured by the proportion of viable seeds, germination rate and seedling growth and survival over 12 months. Davidsonia jerseyana appears to be predominantly selfing with high fecundity. In D. pruriens, polyembryony was common and 89% of the progeny were identical to the heterozygous maternal parent, implying likely apomixis. Overall, fecundity was significantly lower than for D. jerseyana, although survival was higher from D. pruriens polyembryonic than from monoembryonic seed. The high fecundity in D. jerseyana indicates a lack of inbreeding depression and also suggests that it would be less likely to be endangered than the less fecund D. pruriens. This raises the possibility that polyembryony and likely apomixis may provide a reproductive advantage to D. pruriens, which could otherwise share rarity with its congeners.

The effects of riverine impoundment on genetic structure and gene flow in two stream fishes in the Mobile River basin

Summary Riverine impoundments (reservoirs) are thought to impede natural migration in small‐stream‐inhabiting fishes, resulting in spatially and genetically fragmented populations. However, this hypothesis remains poorly tested, and the genetic consequences of riverine impoundment for stream fishes with differing dispersal capabilities are not well understood. This study utilised a combination of microsatellite DNA loci from 479 individuals and the mitochondrial (mt) DNA cytochrome b gene (810–1140 bp) from 83 individuals from eight streams to compare genetic structure and diversity between reservoir‐fragmented and non‐fragmented groups of two species of stream fishes with differing dispersal capabilities in the Mobile River basin of the south‐eastern United States. For both species, analysis of microsatellite loci revealed genetic discontinuities between neighbouring tributaries that have been fragmented by reservoir construction. This finding was in stark contrast to the high degree of continuity for both species between reference tributaries in a natural river setting. Results from mtDNA revealed no significant genetic structure within reservoir‐fragmented or non‐fragmented groups, indicating a lack of historical genetic structure (i.e. prior to reservoir construction). Microsatellite‐based estimates of genetic diversity and migration were differentially affected in the two species, indicating that stream fishes with relatively high dispersal abilities may be equally or more susceptible to reservoir fragmentation when compared to species with relatively low dispersal abilities. Collectively, our data revealed that riverine impoundment and reservoir‐induced habitat fragmentation adversely affect genetic characteristics in small‐stream‐inhabiting fishes. This is of particular interest in biodiversity hotspots such as the south‐eastern United States where hydroelectric and recreational reservoirs restrict connectivity in aquatic systems.

Sex-linked and autosomal microsatellites provide new insights into island populations of the tammar wallaby

The emerging availability of microsatellite markers from mammalian sex chromosomes provides opportunities to investigate both male- and female-mediated gene flow in wild populations, identifying patterns not apparent from the analysis of autosomal markers alone. Tammar wallabies (Macropus eugenii), once spread over the southern mainland, have been isolated on several islands off the Western Australian and South Australian coastlines for between 10,000 and 13,000 years. Here, we combine analyses of autosomal, Y-linked and X-linked microsatellite loci to investigate genetic variation in populations of this species on two islands (Kangaroo Island, South Australia and Garden Island, Western Australia). All measures of diversity were higher for the larger Kangaroo Island population, in which genetic variation was lowest at Y-linked markers and highest at autosomal markers (θ=3.291, 1.208 and 0.627 for autosomal, X-linked and Y-linked data, respectively). Greater relatedness among females than males provides evidence for male-biased dispersal in this population, while sex-linked markers identified genetic lineages not apparent from autosomal data alone. Overall genetic diversity in the Garden Island population was low, especially on the Y chromosome where most males shared a common haplotype, and we observed high levels of inbreeding and relatedness among individuals. Our findings highlight the utility of this approach for management actions, such as the selection of animals for translocation or captive breeding, and the ecological insights that may be gained by combining analyses of microsatellite markers on sex chromosomes with those derived from autosomes.

Genetic differentiation of spruce populations in northwest Russia according to the results of microsatellite loci analysis

Analysis of the genetic diversity of the spruce populations in the northwest of the European part of Russia using microsatellite markers revealed two relatively isolated genetic clusters. One of them is composed of trees that were identified as P. abies according to morphological traits. Another cluster unites spruces that were determined as P. obovata and P. fennica. The results of SSR analysis do not support the idea of the taxonomic independence of the hybridogeneous P. fennica species. The frequency of genotypes attributed to P. abies (according to the results of microsatellite markers) decreases significantly from south to north.

RELATION OF INBREEDING OF HORSES OF THOROUGHBRED BREED WITH DEGREE OF HOMOZYGOSITY OF MICROSATELLITE LOCI OF DNA

The degree of homozygosity of some 39 Thoroughbred horses was estimated from microsatellite analysis data. The power of inbreeding was detected towards horse pedigree. We suggested the use of genetic analysis of microsatellite loci of DNA for the determination of actual level of inbreeding. Keywords: inbreeding, degree of homozygosity, microsatellite loci.

Population Genetics and Bat Rabies: A Case Study ofEptesicus serotinusin Poland

The serotine bat, Eptesicus serotinus is the most frequently rabies-infected (European bat lyssavirus 1-type, EBLV-1) bat species in Europe. To confirm Lyssavirus infection of this bat in Poland, we tested for the presence of rabies virus RNA from oropharyngeal swabs using RT-PCR. There was a 0.9% (two out of 212 individuals) level of infection within the overall population of serotine bats studied. However, an appreciation of the potential for pathogen transmission and disease risk requires an understanding of the dispersal of the primary host, and any large-scale geographic barriers that may impede gene flow. Thus, we also studied the patterns of bat dispersal via population genetics using nuclear (seven microsatellite loci) and mitochondrial (mtDNA control region) markers, examined in 12 subpopulations distributed across the country. Molecular analyses of microsatellite loci indicated high genetic diversity at all sites (heterozygosity observed, H o = 0.53–0.78), and extremely weak genetic structure in the Polish population of the species. The overall F ST was 0.012 (95% confidence interval: 0.006–0.020), and pairwise values ranged from 0.00 to 0.05. Only 22% of individuals were assigned to the subpopulation from which they were sampled. The Bayesian approach implemented in STRUCTURE also confirmed that all examined subpopulations should be treated as a single group, indicating a high level of gene flow. There was some evidence for female philopatry (genetic differentiation was greater in maternally-inherited mtDNA than nuclear DNA) and male-biased dispersal, e.g., H o and the variance of mean assignment were significantly higher in males than in females. Twelve individuals (seven females and five males) were identified as potential first generation migrants. Their migration routes ranged from 60–283 km in females ( ± SE = 177.9 ± 29.37) to 27–385 km in males (206.4 ± 58.95); surprisingly, no sexual differences were observed and this finding suggests that female-mediated gene flow may occur. MtDNA also produced a strong genetic signal for the demographic expansion (Fu's F S statistics, F S = -26.30, P < 0.01 and a star-shaped haplotype network), which took place roughly 33,000 years BP, i.e., before the Last Glacial Maximum. The genetic uniformity of the Polish population implies that there is no migration barrier to EBLV-1, at least within the country, and the potential threat of rabies virus spreading via migration of infected animals may be higher than previously thought.

Phylogeography of the white-clawed crayfish (Austropotamobius italicus) in Spain: inferences from microsatellite markers

The white-clawed crayfish (Austropotamobius italicus), a cornerstone of Spain's aquatic ecosystems, was once widely distributed throughout much of the country. Unfortunately, its populations have suffered very strong declines over the last 40years due to the spread of introduced species (red swamp and signal crayfishes), diseases, habitat loss and other anthropogenic impacts. The present work examines the genetic variation in 23 Spanish and four Italian populations of white-clawed crayfish via the analysis of microsatellite loci. The data show genetic variation in the Spanish populations to be affected by drastic and successive bottlenecks. Notwithstanding, the diversity of these Spanish populations in terms of observed heterozygosity is similar to or even higher than that recorded for other European populations studied using these same markers. North-central Spanish populations are clearly differentiated from the country's remaining populations; they should be considered distinct management units. Processes occurred in historical and recent times, such as genetic drift and translocations, contribute greatly to this genetic structure. These data provide useful information for conservation of this species, since the preservation of its population structure and genetic variability should be goals for management decisions.

Origin of the chromosomal radiation of Madeiran house mice: a microsatellite analysis of metacentric chromosomes

Chromosome races of Mus musculus domesticus are characterised by particular sets of metacentric chromosomes formed by Robertsonian fusions and whole-arm reciprocal translocations. The Atlantic island of Madeira is inhabited by six chromosome races of house mice with 6-9 pairs of metacentric chromosomes. Three of these races are characterised by the metacentric 3.8 also found elsewhere in the distribution of M. m. domesticus, including Denmark and Spain. We investigated the possibility that metacentric 3.8 was introduced to Madeira during the initial colonisation, as this could have 'seeded' the cascade of chromosomal mutation that is the basis of the extraordinary chromosomal radiation observed on the island. Variation at 24 microsatellite loci mapping to three different chromosomal regions (proximal, interstitial and distal) of mouse chromosomes 3 and 8 was investigated in 179 mice from Madeira, Denmark, Portugal, Spain, Italy and Scotland. Analyses of microsatellite loci closely linked to the centromeres of these chromosomes ('proximal loci') do not support a common evolutionary origin of metacentric 3.8 among Madeiran, Danish and Spanish mouse populations. Our results suggest that Madeiran mice are genetically more similar to standard karyotype mice from Portugal than to metacentric mice from elsewhere. There is expected to be an interruption to gene flow between hybridising metacentric races on Madeira, particularly in the chromosomal regions close to the rearrangement breakpoints. Consistent with this, relating to differentiation involving chromosomes 3 and 8 on Madeira, we found greater genetic structure among races for proximal than interstitial or distal loci.

A standardized microsatellite marker panel for parentage and kinship analyses in channel catfish,Ictalurus punctatus

This research was designed to produce a standardized set of microsatellite loci for parentage and kinship analyses in channel catfish, the leading species of US aquaculture. Three panels of five to six markers each were developed that contained a total of two dinucleotide-, eight trinucleotide- and seven tetranucleotide-microsatellite loci respectively. The loci had a range of nine to 31 alleles per locus in an outbred population. Based on the allele frequencies measured in commercial randomly bred broodstock, the combined probability of non-exclusion of an unrelated candidate parent pair was 5.36e-18. The combined probability of non-exclusion of unrelated identical genotypes was 2.58e-08. The microsatellite panels were validated by parentage and kinship evaluation in three populations. A total of 697 spawns were collected from matings of outbred broodstock over three spawning seasons, and parents were determined unambiguously for all but three spawns. Genotype analysis also enabled the identification of half-sibling and full-sibling families produced by pond spawning. In a second experiment, parentage was unambiguously determined in nine spawns from a population consisting of broodstock derived from only four families. A third experiment demonstrated that all but one of 374 individuals from 10 full-sibling families could be assigned to a family after coculture in an earthen pond for 1year. The standardized microsatellite panels enable the development of pedigreed catfish populations and large-scale performance evaluations in common environments to support the genetic improvement of cultured catfish through selective breeding.