Cost-effective development of highly polymorphic microsatellite in Japanese quail facilitated by next-generation sequencing.

Next-generation sequencing technologies permit rapid and cost-effective identification of numerous putative microsatellite loci. Here, from the genome sequences of Japanese quail, we developed microsatellite markers containing dinucleotide repeats and employed these for characterisation of genetic diversity and population structure. A total of 385 individuals from 12 experimental and one wild-derived Japanese quail lines were genotyped with newly developed autosomal markers. The maximum number of alleles, expected heterozygosity and polymorphic information content (PIC) per locus were 10, 0.80 and 0.77 respectively. Approximately half of the markers were highly informative (PIC ≥ 0.50). The mean number of alleles per locus and observed heterozygosity within a line were in the range of 1.3-4.1 and 0.11-0.53 respectively. Compared with the wild-derived line, genetic diversity levels were low in the experimental lines. Genetic differentiation (FST ) between all pairs of the lines ranged from 0.13 to 0.83. Genetic clustering analyses based on multilocus genotypes of individuals showed that most individuals formed clearly defined clusters corresponding to the origins of the lines. These results suggest that Japanese quail experimental lines are highly structured. Microsatellite markers developed in this study may be effective for future genetic studies of Japanese quail.

Association between ovocalyxin-32 gene haplotypes and eggshell quality traits in an F2 intercross between two chicken lines divergently selected for eggshell strength.

Broken and cracked eggshells contribute significantly to economic losses in the egg production industry. We previously identified ovocalyxin-32 as a potential gene influencing eggshell traits, by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation (DEF) conducted over 14 generations. We determined the nucleotide sequences of six ovocalyxin-32 exons in the parent individuals and analysed the association between ovocalyxin-32 and eggshell traits in the F2 individuals. We identified three haplotypes (W, M and S) of ovocalyxin-32 in the parent individuals. A mismatch amplification mutation assay was performed to distinguish six diplotype individuals (WW, MM, SS, WM, MS and WS) inthe F2 population. The egg weight (EW) of SS-diplotype individuals was significantly higher than that of WW-, WM- and WS-diplotypes. Short length of the egg (SLE) of SS-diplotype individuals was significantly higher than that of WW-, WM- and MS-diplotypes. Long length of the egg (LLE) of SS-diplotype individuals was significantly higher than that of WM and WS-diplotypes. DEF of WW-diplotype individuals was significantly higher than that ofSS-, WM, MS and WM-diplotypes. Haplotypic effect analyses showed significant differences between the W-haplotype and the S-haplotypes in the EW, SLE, LLE and DEF. The DEF of M-haplotype was significantly lower than that of W- and S-haplotypes. These results suggest that S- and M-haplotypes are critical for high quality of eggshells in the F2 population. In conclusion, ovocalyxin-32 is a useful marker of eggshell traits and can be used to develop strategies for improving eggshell traits in commercial layer houses.

Mapping of quantitative trait loci affecting eggshell quality on chromosome 9 in an F(2) intercross between two chicken lines divergently selected for eggshell strength.

Broken and cracked eggshells are major causes of significant economic losses to the egg production industry. The quantitative trait loci (QTL) on chromosome 9 influencing the quality of eggshells were identified by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation conducted over 14 generations. Chromosome-wide highly significant (P < 0.01) QTL associated with egg weight (EW), short length of egg (SLE), long length of egg (LLE) and eggshell weight were mapped to the distal region of chromosome 9. Among the QTL affecting EW, SLE and LLE, ovocalyxin-32 was identified as a potential candidate gene influencing eggshell traits. Marker-assisted selection based on these QTL could be used to develop strategies for reducing the breakage and cracking of eggs in commercial layer houses.

Genetic mapping of quantitative trait loci affecting growth and carcass traits in F2 intercross chickens.

We constructed a chicken F(2) resource population to facilitate the genetic improvement of economically important traits, particularly growth and carcass traits. An F(2) population comprising 240 chickens obtained by crossing a Shamo (lean, lightweight Japanese native breed) male and White Plymouth Rock breed (fat, heavyweight broiler) females was measured for BW, carcass weight (CW), abdominal fat weight (AFW), breast muscle weight (BMW), and thigh muscle weight (TMW) and was used for genome-wide linkage and QTL analysis, using a total of 240 microsatellite markers. A total of 14 QTL were detected at a 5% chromosome-wide level, and 7 QTL were significant at a 5% experiment-wide level for the traits evaluated in the F(2) population. For growth traits, significant and suggestive QTL affecting BW (measured at 6 and 9 wk) and average daily gain were identified on similar regions of chromosomes 1 and 3. For carcass traits, the QTL effects on CW were detected on chromosomes 1 and 3, with the greatest F-ratio of 15.0 being obtained for CW on chromosome 3. Quantitative trait loci positions affecting BMW and TMW were not detected at the same loci as those detected for BMW percentage of CW and TMW percentage of CW. For AFW, QTL positions were detected at the same loci as those detected for AFW percentage of CW. The present study identified significant QTL affecting BW, CW, and AFW.

Migration and proliferation of primordial germ cells in the early chicken embryo.

In avian species, primordial germ cells (PGC) use the vascular system as a vehicle to transport them to the future gonadal region. The aim of this study was to elucidate the details of migration system and size of the PGC population in the early chicken embryo. We analyzed whole chicken embryos during stages X and 2 to 17 by immunohistochemical staining using specific antibody raised against chicken vasa homolog. At stage X, PGC were dense in the central zone of the area pellucida. Following the formation of the primitive streak, PGC moved anteriorly to the edge of the extraembryonic region. The size of the PGC population increased gradually during stages X (130.4 +/- 31.9) to 10 (439.3 +/- 93.6). At stage 10, PGC began to accumulate in the region anterior to the head, and then we could observe that PGC invaded into the vascular system in this region. At stage 11, the number of PGC decreased in the region anterior to the head (129.8 +/- 42.5 to 46.7 +/- 4.2) and increased in the blood vessels (194.0 +/- 41.6 to 285.0 +/- 7.5). No PGC could be recognized in the intermediate mesoderm, the future gonadal region, until stage 14, but they first appeared there at stage 15. The number of PGC recognized in the intermediate mesoderm increased from stage 15 to 17. Interestingly, the number of PGC between the left and right sides of this region was consistently and significantly different (P < 0.05) in females and males. The present study mainly clarified that chicken PGC continue to proliferate throughout early development, many PGC invaded into the vascular system from the region anterior to the head in stage 11, and PGC actively left the blood vessels and migrated to the intermediate mesoderm from stage 15.

Quantitative trait loci analysis for growth and carcass traits in a Meishan x Duroc F2 resource population.

We constructed a pig F2 resource population by crossing a Meishan sow and a Duroc boar to locate economically important trait loci. The F2 generation was composed of 865 animals (450 males and 415 females) from four F1 males and 24 F1 females and was genotyped for 180 informative microsatellite markers spanning 2,263.6 cM of the whole pig genome. Results of the genome scan showed evidence for significant quantitative trait loci (<1% genomewise error rate) affecting weight at 30 d and average daily gain on Sus scrofa chromosome (SSC) 6, carcass yield on SSC 7, backfat thickness on SSC 7 and SSC X, vertebra number on SSC 1 and SSC 7, loin muscle area on SSC 1 and SSC 7, moisture on SSC 13, intramuscular fat content on SSC 7, and testicular weight on SSC 3 and SSC X. Moreover, 5% genomewise significant QTL were found for birth weight on SSC 7, average daily gain on SSC 4, carcass length on SSC 6, SSC 7, and SSC X and lightness (L value) on SSC 3. We identified 38 QTL for 28 traits at the 5% genomewise level. Of the 38 QTL, 24 QTL for 17 traits were significant at the 1% genomewise level. Analysis of marker genotypes supported the breed of origin results and provided further evidence that a suggestive QTL for circumference of cannon bone also was segregating within the Meishan parent. We identified genomic regions related with growth and meat quality traits. Fine mapping will be required for their application in introgression programs and gene cloning.

Developmental ability of twin embryos produced by microinjection treatment into chick blastoderm.

Twins and triplets of chick embryos were produced by microinjection treatment of cell suspension into the blastoderm. One pair of the twin embryos had an ability to develop normally and survived up to Day 21 of incubation, but did not hatch. The other twins and triplets died within 7 days.

Bovine quantitative trait loci analysis for growth, carcass, and meat quality traits in an F2 population from a cross between Japanese Black and Limousin.

A genome-wide scan for QTL affecting economically important traits in beef production was performed using an F(2) resource family from a Japanese Black x Limousin cross, where 186 F(2) animals were measured for growth, carcass, and meat-quality traits. All family members were genotyped for 313 informative microsatellite markers that spanned 2,382 cM of bovine autosomes. The centromeric region of BTA2 contained significant QTL (i.e., exceeding the genome-wide 5% threshold) for 5 carcass grading traits [LM area, beef marbling standards (BMS) number, luster, quality grade, and firmness), 8 computer image analysis (CIA) traits [LM lean area, ratio of fat area (RFA) to LM area, LM area, RFA to musculus (M.) trapezius area, M. trapezius lean area, M. semispinalis lean area, RFA to M. semispinalis area, and RFA to M. semispinalis capitis area], and 5 meat quality traits (contents of CP, crude fat, moisture, C16:1, and C18:2 of LM). A significant QTL for withers height was detected at 80.3 cM on BTA5. We detected significant QTL for the C14:0 content in backfat and C14:0 and C14:1 content in intermuscular fat around the 62.3 to 71.0 cM region on BTA19 and for C14:0, C14:1, C18:1, and C16:0 content and ratio of total unsaturated fatty acid content to total SFA content in intramuscular fat at 2 different regions on BTA19 (41.1 cM for C14:1 and 62.3 cM for the other 4 traits). Overall, we identified 9 significant QTL regions controlling 27 traits with genome-wide significance of 5%; of these, 22 traits exceeded the 1% genome-wide threshold. Some of the QTL affecting meat quality traits detected in this study might be the same QTL as previously reported. The QTL we identified need to be validated in commercial Japanese Black cattle populations.

Identification of quantitative trait loci affecting corpora lutea and number of teats in a Meishan x Duroc F2 resource population.

Understanding of the genetic control of female reproductive performance in pigs would offer the opportunity to utilize natural variation and improve selective breeding programs through marker-assisted selection. The Chinese Meishan is one of the most prolific pig breeds known, farrowing 3 to 5 more viable piglets per litter than Western breeds. This difference in prolificacy is attributed to the Meishan's superior prenatal survival. Our study utilized a 3-generation resource population, in which the founder grandparental animals were purebred Meishan and Duroc pigs, in a genome scan for QTL. Grandparent, F1, and F2 animals were genotyped for 180 microsatellite markers. Reproductive traits, including number of corpora lutea (number of animals = 234), number of fetuses per animal (n = 226), number of teats (n = 801), and total number born (n = 288), were recorded for F2 females. Genome-wide significance level thresholds of 1, 5, and 10% were calculated using a permutation approach. We identified 9 QTL for 3 traits at a 10% genome-wise significance level. Parametric interval mapping analysis indicated evidence of a 1% genome-wise significant QTL for corpora lutea on SSC 3. Nonparametric interval mapping for number of teats found 4 significant QTL on chromosomes SSC3 (P < 0.01), SSC7 (P < 0.01), SSC8 (P < 0.01), and SSC12 (P < 0.05). Partial imprinting of a QTL affecting teat number (P < 0.10) was detected on SSC8. Using the likelihood-ratio test for a categorical trait, 2 QTL for pin nipples were detected on SSC2 and SSC16 (P < 0.01). Fine mapping of the QTL regions will be required for their application to introgression programs and gene cloning.

An in vitro culture method for chick embryos obtained from the anterior portion of the magnum of oviduct.

1. Chick embryos, obtained from the anterior portion of the magnum of the oviduct 60 to 80 min after the preceding egg had been laid, were cultured in vitro in small and large recipient eggshells until hatching. 2. Of 82 embryos cultured, 46.3% had survived to day 4 of incubation, and 19.5% survived to hatching. 3. The method for culturing embryos used in this experiment could facilitate research on the in vitro manipulation of early chick embryos.

Preservation of quail blastoderm cells in liquid nitrogen.

1. Quail blastoderm cells isolated from the yolk were preserved in liquid nitrogen. 2. Frozen-thawed blastoderm cells of the quail were viable and survived in vitro. By injecting the frozen-thawed cells into chick embryos, quail-chick chimaeras were produced.

Duration of egg formation in hens selected for increased rate of lay under 23 h and 24 h light-dark cycles.

1. The intervals from oviposition to the next ovulation and the times spent by the ovum in various parts of the oviduct were examined, in hens selected for increased rate of lay over 5 generations under a 23 h light-dark cycle (23HS line) and kept in a 22 h light-dark cycle, and in hens selected under a 24 h light-dark cycle (24HS line) and kept in a 24 h light-dark environment. 2. The intervals from oviposition to the next ovulation were 13 min and 25 min in the 23HS and 24HS lines respectively. Times from ovulation to entry of the ovum into the uterus were 4 h 44 min and 4 h 43 min in the 23HS and 24HS lines respectively, and times spent by the ovum in the uterus were 19 h and 19 h 24 min in the 23HS and 24HS lines respectively. 3. It is concluded that the reduced oviposition interval in the 23HS line compared to the 24HS line was caused by the reduction in the interval from oviposition to the next ovulation and the time spent by the ovum in the uterus.

Development in culture of the chick embryo from fertilized ovum to hatching.

An experiment was carried out to investigate whether thick albumen is essential for the normal development of the chick embryo. Fertilized ova recovered from the oviducts of hens were cultured in vitro and transferred to recipient eggshells with (method A) or without (method B) replacement of the thick albumen by thin albumen. Embryos from freshly laid eggs were transferred to recipient eggshells with (method C) or without (method E) replacement of the thick albumen by thin albumen or with replacement of the thick albumen by thin albumen diluted with solution of salts (method D). Embryos were then incubated until hatching. The rates of hatching of the cultured embryos were 34.4%, 16.2%, 50.0%, 6.9-26.7%, and 47.5% for methods A, B, C, D, and E, respectively. Thus the rate of hatching of cultured fertilized ova was increased by replacement of the thick albumen by thin albumen at the blastoderm stage. Chicks obtained by method A reached maturity and produced viable offspring, and this technique provides an improved method for the culture of fertilized ova to hatching.

Selection experiment for increased egg production under 23 H and 24 H light-dark cycles in the domestic fowl.

A selection experiment was conducted for increased rate of lay under 23-h (23 HS line) and 24-h (24 HS line) light-dark cycles over 5 generations. In generation 5, rate of lay was higher in the 23 HS line than in the 24 HS line, and the proportion of hens with mean intra-sequence intervals of less than 24 h in the 23 HS line increased to 88% in the generation 5 from 15% in the base generation. The realised heritability for rate of lay was 0.25 plus/minus 0.04 in the 23 HS line and 0.15 plus/minus 0.05 in the 24 HS line. Egg weight and shell weight decreased in both lines, and the decrease in the proportion of shell was larger in the 23 HS line than in the 24 line. It is suggested that a regimen of 23 h light and dark may be an effective environment for selection to improve the laying performance of a population approaching a plateau for egg production.

Production of quail-chick chimaeras by blastoderm cell transfer.

1. Quail-chick chimaeras were produced by injecting dissociated quail blastoderm cells into chick embryos. 2. Quail blastoderms were removed from the yolk and the cells were dispersed by trypsin treatment or pipetting. The cell suspension (1 to 5 microliters) was injected into the subgerminal cavity of unincubated chick embryos. The chick embryos were then cultured in recipient eggshells. 3. Quail blastoderm cells injected into the chick embryos adhered to the chick embryonic cells. The rates of hatching were 8.6% (38 chicks from 441 eggs) and 40.3% (48 chicks from 119 eggs) when the volumes of the cell suspension injected were 3 to 5 microliters and 1 microliter, respectively. 4. Seven out of 86 hatched birds were clearly identified as being chimaeric because part of the feather colouring was of quail specificity. In addition to these chimaeric birds, there were 8 chimaeric embryos which died before hatching. The distribution patterns of the quail feathers were varied among the chimaeric birds and embryos. 5. This technique provides a basis for the investigation of chick embryo cryopreservation, genetic transformation and analysis of cell lineage of chickens.

Genetic relationships among Japanese native breeds of chicken based on microsatellite DNA polymorphisms.

Genetic relationships among Japanese native breeds of chickens were studied on the basis of microsatellite DNA polymorphisms. DNA samples from 10 Japanese native breeds (Iwate-Jidori, Aizu-Jidori, Sadohige-Jidori, Siba-Tori, Onaga-Dori, Echigonankin, Hinai, Kinpa, Koeyoshi, and Tomaru) and one imported breed (White Leghorn) were analyzed using eight microsatellite markers that were isolated from a microsatellite DNA-enriched library of chickens (Takahashi et al. 1996). The PCR primers to detect (CA)n repeat length polymorphisms were synthesized based on the sequences of clones, and these markers were typed by PCR amplification and electrophoresis using a DNA sequencer. Since all eight microsatellite markers were polymorphic, genetic distance between the breeds could be calculated based on the frequencies of alleles of the microsatellites and phylogenetic relationships between the breeds could be estimated. Most Japanese native chickens were grouped into three groups that correspond to the origin breeds, Jidori, Shokoku, and Shamo. The results suggest that microsatellite DNA markers are a useful tool for studying the genetic relationships among chicken breeds.

Genetic mapping of quantitative trait loci affecting body weight, egg character and egg production in F2 intercross chickens.

Phenotypic measurements of chicken egg character and production traits are restricted to mature females only. Marker assisted selection of immature chickens using quantitative trait loci (QTL) has the potential to accelerate the genetic improvement of these traits in the chicken population. The QTL for 12 traits (i.e. body weight (BW), six for egg character, three for egg shell colour and two for egg production) of chickens were identified. An F2 population comprising 265 female chickens obtained by crossing White Leghorn and Rhode Island Red breeds and genotyped for 123 microsatellite markers was used for detecting QTL. Ninety-six markers were mapped on 25 autosomal linkage groups, and 13 markers were mapped on one Z chromosomal linkage group. Eight previous unmapped markers were assigned to their respective chromosomes in this study. Significant QTL were detected for BW on chromosomes 4 and 27, egg weight on chromosome 4, the short length of egg on chromosome 4, and redness of egg shell colour (using the L*a*b* colour system) on chromosome 11. A significant QTL on the Z chromosome was linked with age at first egg. Significant QTL could account for 6-19% of the phenotypic variance in the F2 population.