Mapping of Quantitative Trait Loci Affecting Growth Traits in a Japanese Native Chicken Cross *

The Hinai-dori is a breed of chicken native to Akita Prefecture, Japan. An F2 resource population produced by crossing lowand high-growth lines of the Hinai-dori breed was analyzed to detect quantitative trait loci (QTL) for growth traits. Highly significant QTLs for body weight at 10 and 14 weeks of age and average daily gain between 4 and 10 weeks and between 10 and 14 weeks of age were accordingly mapped in a common region between ADL0198 and ABR0287 on chromosome 1 and between MCW0240 and ABR0622 on chromosome 4, respectively. A significant QTL for body weight at 4 weeks of age and a significant QTL for average daily gain between 0 and 4 weeks of age were mapped for the first time to the same region flanking ABR0204 and ABR0284 on chromosome 1. These QTLs are good candidates for application in the development of marker-assisted selection strategies for increasing growth efficiencies in the Hinai-dori breed and native breeds of chickens in Asia. (


INTRODUCTION
In poultry, many QTL mapping studies have been performed on breed crosses, e.g.broiler×layer (Sewalem et al., 2002), Red Junglefowl×White Leghorn (Kerje et al., 2003), White Leghorn×Rhode Island Red (Sasaki et al., 2004), and broiler×White Leghorn (Schreiweis et al., 2005).This approach has proved very successful in identifying QTLs that explain differences between these breeds; however, they provide no insight into whether these QTLs would be useful for breeds/lines other than the founder breeds used in the respective QTL studies.Therefore, studies using crosses between divergently selected lines within a breed are becoming popular for their potential contribution to the improvement of growth traits in broilers (Jacobsson et al., 2005;Ankra-Badu et al., 2009;Wahlberg et al., 2009) and eggshell traits in the White Leghorn (Takahashi et al., 2009(Takahashi et al., , 2010;;Yang et al., 2010).Moreover, there are few studies that described QTLs for growth traits within a native chicken breed.
The Hinai-dori is a slow-growing breed of chicken native to Akita Prefecture, in northern Honshu Island, Japan.Although Hinai-dori meat has a characteristic taste and the breed has been used for a long time, it has decreased in numbers in recent times owing to the introduction of exotic breeds, and for a while was at risk of extinction.The Hinaidori breed has been conserved by hobbyists who belong to the Preservation Society (PS) of the Hinai-dori Breed and is now mainly used for exhibition purposes.For effective use of the breed, selection experiments have been performed at the Livestock Experiment Station (LES), Akita Prefectural Agriculture, Forestry, and Fisheries Research Center (since 1973 when fertilized eggs were introduced to LES from PS) with a view towards increasing growth performance.At present, the average body weight of LES males at 300 days of age is approximately 1 kg heavier than that of PS males.F 1 chickens produced by crossing the improved LES Hinaidori sires with Rhode Island Red dams have been commercialized as the Hinai-jidori chicken, which is one of the most popular high-quality chickens on the Japanese market.The LES line was developed from PS chickens: hence, these individuals possessed genes that influence growth traits.In order to effectively detect QTLs affecting growth traits within the Hinai-dori breed, we developed an F 2 resource population by crossing PS sires with LES dams, and thereafter conducted QTL mapping.

F 2 resource population
F 1 chickens were produced by crossing three PS males with nine LES females, with one to three females randomly selected to mate with each male.The mapping population consisted of 359 F 2 individuals comprising 173 males and 186 females.The F 2 individuals were produced by full-sib mating of 17 F 1 males and 60 F 1 females.The F 2 chickens were hatched on the same day, raised in the same chicken house, and fed the same diet ad libitum for the duration of the experiment.Body weight was measured at day 0 (BW-0), 4 weeks (BW-4 wk), 10 weeks (BW-10 wk), and 14 weeks (BW-14 wk) of age.Average daily gain between 0 and 4 weeks of age (ADG 0-4 wk), between 4 and 10 weeks of age , and between 10 and 14 weeks of age (ADG 10-14 wk) was calculated from BW at each week of age.

Genotyping and QTL mapping
Chicken genomic DNA was extracted from blood using a DNA isolation kit (SepaGene, Sanko Junyaku, Tokyo, Japan).PCR amplifications were performed in a 6 μl reaction volume, which included 2.5 pmols of each primer, 200 μM each dNTP, 1.2 mM MgSO4, 0.125 units of KOD plus polymerase (KOD-201, Toyobo, Tokyo, Japan), 1× reaction buffer provided by the supplier and 30 ng genomic DNA in a 384-well plate on an iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA).PCR was performed as follows: hot start 75 s at 94°C followed by 10 cycles of 15 s at 94°C, 30 s at 60°C, and 60 s at 68°C, followed by 10 cycles with the same conditions except that the annealing temperature was 55°C, and then 30 cycles with an annealing temperature of 50°C, and finally with an elongation time of 9 min at 68°C.PCR products were electrophoresed on an ABI PRISMTM 3100 DNA Sequencer (Perkin-Elmer, Foster City, CA, USA) and analyzed using GeneScan (Ver.3.7) and GeneMapper (Ver.2.0) programs (Perkin-Elmer).One hundred and twenty-two microsatellite markers, which are informative in the F 2 resource population, were used (Table 1).Linkage analysis was performed using the program Map Manager QTX b18 (Manly et al., 2001) and linkage groups were compared with our linkage map (Takahashi et al., 2005).QTL analysis was performed using the QTL EXPRESS program (Seaton et al., 2002).A least-squares regression model was used for single-QTL analysis, including the fixed effects of sex, along with additive and dominance coefficients for the putative QTL.Detection of QTLs was based on an F-statistic that was computed from the sums of squares explained by the additive and dominance coefficients for the QTL.Significance thresholds of the F-statistic were derived at the experimental level on a single-trait basis using a permutation test, with 1,000 repetitions for each trait.The threshold levels of 5% and 1% at the experimental all fixed effects.A draft sequence of the chicken genome (May 2006 assembly), available on the University of California, Santa Cruz (UCSC) Genome Browser ( 2004) and the Ensembl Genome Browser (2004), was used in the present study.
It has been reported that there are many QTLs with minor additive effects in a broiler×broiler cross (Jacobsson et al., 2005;Wahlberg et al., 2009).These studies failed to detect any highly significant (p<0.01)QTLs at the experimental level, and each QTL accounted for a small proportion of the phenotypic variance, ranging from 1.5% to 4.4%; however, these authors used a resource population produced by crossing two broiler lines with significantly different body weights.Ankra-Badu et al. ( 2010) analyzed a similar broiler cross and detected a highly significant QTL on chromosome 4 flanking MCW0240 to LEI0073, which accounted for 4.92% of the phenotypic variance in body weight at 7 weeks, whereas the other QTLs accounted for small proportions of the phenotypic variance, ranging from 1.97% to 3.49%.We successfully detected two highly significant QTLs (HG2 and HG3) and one significant QTL (HG1), which accounted for a relatively large proportion of the phenotypic variance, ranging from 2.9% to 10.0%.In particular, HG2 plus HG3 accounted for 10.3% (HG2: 4.9% +HG3: 5.4%) and 18.1% (HG2: 9.2%+HG3: 7.9%) of the phenotypic variance of BW-10 wk and BW-14 wk, respectively.Moreover, HG1, HG2, and HG3 appear to act independently on each growth trait, since we could detect no QTL when we performed analysis using the F2 epistasis module in GridQTL software (Hernández-Sánchez et al., 2009).These data suggest that marker-assisted selection on the three QTLs could be effective in improving growth performance within the Hinai-dori breed without considering the epistatic interaction effects among the QTLs.
In conclusion, three QTL regions affecting growth traits were successfully detected in a resource population of the Hinai-dori breed.Of the three, a QTL in the middle of chromosome 1 affecting early development was detected for the first time.Since the resource population was developed from the same founder population, we believe that these QTLs contain key genes affecting growth traits.We plan to use the experience we gained in identifying a gene associated with eggshell traits from QTL analyses in the White Leghorn breed (Takahashi et al., 2009(Takahashi et al., , 2010) ) to approach to the candidate genes for the QTLs on chromosome 1 and 4 in future studies.

Table 1 .
Information for chicken microsatellite markers used in this study RMS is the residual MS from the reduced model, omitting the QTL but including all fixed effects, and FMS is the residual MS from the full model, including the QTL and where

Table 1 .
Information for chicken microsatellite markers used in this study(Continued)

Table 1 .
Information for chicken microsatellite markers used in this study(Continued)

Table 2 .
Phenotypic values of quantitative traits and quantitative traits loci affectiing body weight and average daily gain