論文の公開元へDevelopment and application of molecular genetic marker in the emu(Dromaius novaehollandiae)
概要
The emu (Dromaius novaehollandiae) is a ratite bird originally from Australia. In the recent years, emu have been expected to become a novel poultry producing low-fat meat, hypo-allergenic eggs, and oil containing high proportion of unsaturated fatty acids. Especially, emu oil is one of the most important products since it has anti-inflammatory properties and thus been utilized for skin care products. The domestication of emu began in the 1970s in Australia, and that were followed by America, China, Japan, and other countries. Unlike livestock such as cattle, pig, and chicken, genetic improvement for efficient production of the emu’s traits has not been progressed yet due to its short history of utilization. Therefore, genetic improvement of the emu is important challenge to its efficient production and development of the emu industry.
The aim of this study was development of DNA markers for simple and cost-effective sexing method and of novel microsatellite markers for estimation of genetic relationship among populations and for development of accurate parentage method.
Development of a simplified protocol for molecular sexing
The accurate sexing of emu chicks is essential for the success of plans to establish high-quality pedigrees. However, male and female emus have nearly identical appearance not only chicks but also adults. Although morphological differences of the internal genital tract can be used to distinguish the sexes, a high degree of diagnostic skill is required for accurate sexing. DNA-based sexing methods are highly accurate and can be used to diagnose sex without requiring a high degree of technical skill. Kloet (2001) first developed the molecular method for sexing the emu, and designated the diagnostic marker loci ESEXZ and ESEXW. ESEXZ and ESEXW can be distinguished by PCR-RFLP using BglII based on sequence differences identified by Kloet (2001). However, conventional PCR-RFLP is time consuming and costly, requiring the digestion of PCR products. In this study, the author attempted to simplify the protocol for sexing the emu by using multiplex PCR without restriction enzyme treatment. Multiplex PCR based on a W-specific primer, with the commonly designed primer set on both Z and W chromosomes, amplified both 197-bp and 272-bp bands in the female, and only the 272-bp band in the male. Sexing results obtained in this way were completely concordant with results obtained using the conventional PCR-RFLP method. Thus, development of simplified protocol for sexing the emu was succeeded, and this method might be useful tool to improve production efficiency by facilitating planned pairing and selection of individuals to establish high-quality pedigrees.
Genetic diversity of emu population in a Japanese farm based on microsatellite DNA analysis
To develop emu farming in Japan, its genetic improvement is one of the most important issues, and genetic diversity is the foremost parameters for efficient and sustainable breeding of these animals. In general, genetic diversity of livestock should be managed by selective mating while preventing inbreeding. Meanwhile, the genetic structure of emu populations in Japanese farms is scarcely known. Microsatellite sequences are useful genetic markers to evaluate genetic diversity in animal populations. The aim of this study was to determine the genetic diversity and population structure in the largest emu farm in Japan based on microsatellite markers.
The feather pulps of emu chicks (N = 131) were collected from 40, 20, 23, and 48 individuals hatched at 2013, 2014, 2015, and 2016, respectively, in the Okhotsk Emu farm (OEF) in Abashiri, Hokkaido, Japan. Using six microsatellite markers, genetic diversity and structure were investigated in this farmed emu population. The number of alleles (NA) were 4.83, 4.17, 4.17, and 7.17, in animals hatched in 2013, 2014, 2015, and 2016, respectively. Expected and observed heterozygosity (HE; HO, respectively) was 0.47/0.34, 0.43/0.33, 0.43/0.38, and 0.55/0.35, in each year, respectively. A high inbreeding coefficient (FIS) was observed in all tested generations (0.11-0.37). The Structure program and unrooted phylogenetic tree analysis showed that the OEF population is largely divided into three to five different clades. Our results suggested that the genetic diversity in the OEF population is low, and that it contains three to five genetic lineages. These data may help guide a more sustainable breeding of emus in Japan.
Development and characterization of ten novel microsatellite loci and genetic diversity of Japanese farm populations
The OEF population showed the low genetic diversity in previous section, indicating that there is necessity of genetic management via exchange of individuals among farms to increase genetic diversity. However, genetic structure and relationships among farmed emu populations in Japan are unknown and the number of microsatellite markers for genetic analysis of the emu is insufficient. In this section, the author aimed to develop novel microsatellite markers and to genetically characterize OEF, Japan Eco System, Co., Ltd. (JES) and Tohoku Safari Park (TFP) populations.
Total 102 microsatellite-enrichment genomic DNA libraries were isolated according to method provided by Glenn and Schable (2005), and 46 of those microsatellite regions could be amplified by PCR. Finally, 17 microsatellites were isolated from the 46 regions and developed 10 new polymorphic microsatellite markers. These microsatellite markers were used to characterize three farm emu populations in Japan. The number of alleles ranged from 3 to 13 and the expected (HE) and observed heterozygosity (HO) of these microsatellite loci was 0.19–0.80 and 0.18–0.65, respectively. The polymorphic information contents ranged from 0.18 to 0.79. Positive inbreeding coefficient (FIS) values were detected in all tested populations, and they ranged from 0.30 (TSP) to 0.13 (JES). Although these results suggest that farm populations of the emu in Japan resulted from inbreeding, JES population maintained a relatively higher genetic diversity compared with OEF and TSP populations. The fixation index (FST) values ranged from 0.03 to 0.06, and phylogenetic trees and population structure analysis confirmed no definitive genetic differentiation among the three populations. Therefore, these populations are at a relatively low level of genetic differentiation at present. Despite the lack of clear genetic differentiation at K=5, a slight deflection in allelic composition was observed among the populations. Taken together, the results presented here suggest that three major Japanese farmed emu populations can be largely divided into four genetic groups with slight deflection of allelic composition. The microsatellite markers developed in our study can be utilized for genetic analysis and preservation of genetic resources in the emu.
To more characterize genetic relationship among Japanese emu populations, composition of mitochondrial DNA (mtDNA) haplotypes was investigated. Nucleotide sequences of 348 bp on D-loop region were determined in 109 individuals derived from OEF, JES and TSP populations, and 4 substitution sites and total 3 haplotypes (Hap-a, -b and -c) were detected. The AMOVA indicated that 9% of total variance were “among population”, the FST value was 0.09 with significant genetic differentiation (P < 0.01). Therefore, low level of genetic differentiation among Japanese populations were found. Hap-a was the most frequently detected haplotype in all of tested populations. Hap-b was found in OEP (0.13) and JES (0.21), but not TSP. Although Hap-c was rarely found in OEF (0.03), its frequency was relatively higher in TSP (0.32). In conclusion, the mtDNA variations among Japanese farmed emu populations based on D-loop polymorphisms were revealed and a rare haplotype in many individuals derived from TSP were found. These results will contribute to efficient extending the genetic diversity of Japanese emu populations by the exchange of individuals among farms.
Development of 49 novel microsatellite markers from Next-generation sequencing data and a robust method for parentage tests
Pedigree information is essential for genetic improvement, however it is difficult to record because of complex reproduction system in the emu. The breeding system of the emu is generally polygamous, and multiple females lays eggs in one nest, which are then brooded by the males. To identify parent-offspring relationships in the emu, parentage test based on polymorphic DNA markers have to be developed. First, the author attempted parentage test using 9 microsatellite markers developed in previous section and confirmed that its accuracy was low. This fact indicated the necessity of many microsatellite markers for accurate parentage tests in the emu. In this section, the author aimed to extensively develop novel microsatellite markers based on the Next-generation sequencing (NGS) data and QDD pipeline and establish accurate parentage tests in the emu.
Feathers and eggshell membranes were collected from 28 adults and six chicks, respectively, at OEF. Although reproductive individuals at OEF are reared in mixed groups of more than 15 males and females, only two pairs were separated as a pilot study. Eggs laid from these pairs were collected, labeled and incubated in an incubator for approximately 50 days. Feathers collected from 24 non-parent individuals were used for negative control in the parentage test.
More than 25,000 microsatellite regions were isolated from NGS data via the QDD pipeline. The dinucleotide motifs, (AC)n, (AT)n and (AG)n, were the most frequently detected and were found on 10,167 (38.55%), 8,114 (30.76%) and 4,796 (18.18%) contigs, respectively. Novel 49 microsatellite markers were characterized in 20 individuals and showed NA ranged from 2 to 12, with an average of 4.2. HE / HO ranged from 0.39/0.07 to 0.70/1.00 with an average of 0.60/0.52. PIC value ranged from 0.06 to 0.89 with an average of 0.53, and 17 of 49 markers showed a higher polymorphism than 0.50. Thirty-four individuals were genotyped using 12 markers, and CERVUS simulations based on genotype showed that parents of all offspring were identified with 0.99-1.00 probability. Thus, the author succeeded to develop 49 novel polymorphic microsatellite markers and a robust method for parentage test for the Japanese emu.
Conclusion
This study succeeded to develop a simplified protocol for molecular sexing in the emu. In addition, the author revealed composition of microsatellites in emu genome and developed total 59 novel microsatellite markers. Finally, genetic structure and relationships were defined in Japanese major farm emu populations, and a system for robust parentage test was succeeded to established. These results predict to contribute efficient conservation of genetic resources, acceleration of genetic improvement and genomic analysis in the emu.
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