Association analysis of mitochondrial genome polymorphisms with backfat thickness in pigs.

Mitochondrial DNA (mtDNA) variations and associated effects on economic traits have been widely reported in farm animals, as these genetic polymorphisms can affect the efficiency of energy production and cell metabolism. In studies related to metabolism, the deposition of fat was highly correlated with mitochondria. However, the effect of mtDNA polymorphisms on porcine backfat thickness (BFT) remained unclear. In this study, 243 pigs were collected to analyse the relationship between BFT and mtDNA polymorphisms. There were considerable differences in BFT, ranging from 5 mm to 18 mm. MtDNA D-loop sequencing discovered 48 polymorphic sites. Association analysis revealed that 30 variations were associated with BFT (P < 0.05). The polymorphism m.794A > G showed the maximum difference in BFT between A and G carriers, which differed at ∼2.5 mm (P < 0.001). The 48 polymorphic sites generated 22 haplotypes (H1-H22), which clustered into 4 haplogroups (HG1-HG4). HG1 had a lower BFT value than other three haplogroups (P < 0.01), whereas H4 in HG1 exhibited the lowest BFT of all haplotypes analyzed (P < 0.01). The results of this study highlight an association between mtDNA polymorphisms and BFT, and suggest the potential application of mtDNA in pig molecular breeding practices.

Mitochondrial DNA enrichment reduced NUMT contamination in porcine NGS analyses.

Genetic associations between mitochondrial DNA (mtDNA) and economic traits have been widely reported for pigs, which indicate the importance of mtDNA. However, studies on mtDNA heteroplasmy in pigs are rare. Next generation sequencing (NGS) methodologies have emerged as a promising genomic approach for detection of mitochondrial heteroplasmy. Due to the short reads, flexible bioinformatic analyses and the contamination of nuclear mitochondrial sequences (NUMTs), NGS was expected to increase false-positive detection of heteroplasmy. In this study, Sanger sequencing was performed as a gold standard to detect heteroplasmy with a detection sensitivity of 5% in pigs and then one whole-genome sequencing method (WGS) and two mtDNA enrichment sequencing methods (Capture and LongPCR) were carried out. The aim of this study was to determine whether mitochondrial heteroplasmy identification from NGS data was affected by NUMTs. We find that WGS generated more false intra-individual polymorphisms and less mapping specificity than the two enrichment sequencing methods, suggesting NUMTs indeed led to false-positive mitochondrial heteroplasmies from NGS data. In addition, to accurately detect mitochondrial diversity, three commonly used tools-SAMtools, VarScan and GATK-with different parameter values were compared. VarScan achieved the best specificity and sensitivity when considering the base alignment quality re-computation and the minimum variant frequency of 0.25. It also suggested bioinformatic workflow interfere in the identification of mtDNA SNPs. In conclusion, intra-individual polymorphism in pig mitochondria from NGS data was confused with NUMTs, and mtDNA-specific enrichment is essential before high-throughput sequencing in the detection of mitochondrial genome sequences.

Pregnancy outcomes after fresh versus vitrified-warmed embryo transfer in women with adenomyosis: a retrospective cohort study.

RESEARCH QUESTION: Is the singleton live birth rate superior for vitrified-warmed versus fresh embryo transfer in women with adenomyosis? DESIGN: This cohort study retrospectively analysed data from the Reproductive Hospital Affiliated to Shandong University between January 2013 and December 2018. A total of 612 women diagnosed with adenomyosis, with 322 fresh embryo transfer cycles and 290 vitrified-warmed embryo transfer cycles, were included in this study. The primary outcome was singleton live birth. Outcomes were adjusted using multivariable logistic regression analysis. RESULTS: Vitrified-warmed embryo transfer was associated with a higher rate of singleton live birth than fresh embryo transfer (25.9% versus 17.4%; P = 0.011). Although there was a trend towards a lower miscarriage rate after vitrified-warmed embryo transfer, the difference did not reach statistical significance (31.3% versus 40.6%; P = 0.111). The clinical pregnancy rate was comparable in the two groups (44.1% versus 44.4%; P = 0.946). Vitrified-warmed embryo transfer also resulted in a lower risk of preterm birth than fresh embryo transfer (7.0% versus 17.5%; P = 0.010). CONCLUSIONS: Vitrified-warmed embryo transfer may be associated with better pregnancy outcomes than fresh embryo transfer among women with adenomyosis. It seems that vitrified-warmed embryo transfer is more appropriate for specific populations.

Melatonin Inhibits 17ß-Estradiol-Induced Epithelial-Mesenchymal Transition in Endometrial Adenocarcinoma Cells via Upregulating Numb Expression.

OBJECTIVES: Melatonin (MLT) shows antitumor effects in various tumor types, including endometrial carcinoma. However, the molecular mechanism involved is unclear. In the current study, we investigated the effect of MLT on the estrogen-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells and explored the pathway that might be involved. DESIGN: Laboratory study was via cultured endometrial cancer cells. Design refers only to in vitro experiments. METHODS: In cell culture experiments, cell growth was examined using CCK-8 assays. The expression of Numb and EMT markers in Ishikawa cells was examined using Western blot analysis and real-time PCR. Cell invasion was examined using transwell assays. Cell migration was examined using wound-healing assays and transwell assays. Using immunohistochemistry analysis, the expression of Numb in human endometrial cancers was examined. RESULTS: In immunohistochemistry experiments, we found that 15.2% of atypical endometrial hyperplasia and 15.6% of endometrial carcinoma did not express Numb. In cell culture experiments, MLT inhibited cell proliferation, invasion, and migration induced by 17ß-estradiol (E2) in endometrial cancer cells. MLT decreased the expression of vimentin and Slug and increased the expression of Numb and E-cadherin in Ishikawa cells. Numb knockdown in cancer cells significantly increased cell proliferation, invasion, and migration. LIMITATIONS: No animal experiments were performed. CONCLUSIONS: MLT blocked E2-induced cell growth and EMT in endometrial cancer cells via upregulating Numb expression.

Breed-specific reference sequence optimized mapping accuracy of NGS analyses for pigs.

BACKGROUND: Reference sequences play a vital role in next-generation sequencing (NGS), impacting mapping quality during genome analyses. However, reference genomes usually do not represent the full range of genetic diversity of a species as a result of geographical divergence and independent demographic events of different populations. For the mitochondrial genome (mitogenome), which occurs in high copy numbers in cells and is strictly maternally inherited, an optimal reference sequence has the potential to make mitogenome alignment both more accurate and more efficient. In this study, we used three different types of reference sequences for mitogenome mapping, i.e., the commonly used reference sequence (CU-ref), the breed-specific reference sequence (BS-ref) and the sample-specific reference sequence (SS-ref), respectively, and compared the accuracy of mitogenome alignment and SNP calling among them, for the purpose of proposing the optimal reference sequence for mitochondrial DNA (mtDNA) analyses of specific populations RESULTS: Four pigs, representing three different breeds, were high-throughput sequenced, subsequently mapping reads to the reference sequences mentioned above, resulting in a largest mapping ratio and a deepest coverage without increased running time when aligning reads to a BS-ref. Next, single nucleotide polymorphism (SNP) calling was carried out by 18 detection strategies with the three tools SAMtools, VarScan and GATK with different parameters, using the bam results mapping to BS-ref. The results showed that all eighteen strategies achieved the same high specificity and sensitivity, which suggested a high accuracy of mitogenome alignment by the BS-ref because of a low requirement for SNP calling tools and parameter choices. CONCLUSIONS: This study showed that different reference sequences representing different genetic relationships to sample reads influenced mitogenome alignment, with the breed-specific reference sequences being optimal for mitogenome analyses, which provides a refined processing perspective for NGS data.

Mifepristone inhibited the expression of B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis.

BACKGROUND: The immune mechanism was shown to be involved in the development of adenomyosis. The aim of the current study was to evaluate the expression of the immune checkpoints B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints. METHODS: The expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis patient samples treated with or without mifepristone was determined by immunohistochemistry analysis. RESULTS: In adenomyosis patient samples, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phases. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phases. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria in both the proliferative and secretory phases. In the secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesions was significantly higher than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 was elevated in the proliferative phase compared with that in the secretory phase, while in the ectopic endometria, B7-H4 expression was decreased in the proliferative phase compared with the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis tissues after treatment with mifepristone. CONCLUSIONS: The expression of the immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is upregulated in adenomyosis tissues and is downregulated with mifepristone treatment. The data suggest that B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis.

Low-dose aspirin can downregulate progesterone resistance and increase the expression of LIF in endometriosis during the implantation window.

AIM: Study the effect of low-dose aspirin on the endometrial receptivity in endometriosis rat models. MATERIALS AND METHODS: This study is to explore the expressions of progesterone receptor and LIF among three groups of endometriosis rat models: control group (n = 12), EMs group (n = 15), and aspirin group (n = 17). The expressions of progesterone receptor (PR), PRA, PRB, and leukemia inhibitory factor receptor (LIFR) in eutopic endometrium were determined using immunohistochemistry technology, western blot, and qRT-PCR. The levels of LIF in eutopic endometrium and serum were detected by western blot, qRT-PCR, and ELISA. RESULTS: The expressions of PR, PRA, and PRB protein were significantly increased in the eutopic endometrium after low-dose aspirin treatment, and the level of PRB mRNA was also increased while the ratio of PRA/PRB mRNA was decreased in the eutopic endometrium. The levels of LIF in eutopic endometrium and serum were increased compared with the untreated endometriosis rats. However, the expression of LIFR was not statistically different among the three groups. CONCLUSIONS: The results suggest that the low-dose aspirin treatment could downregulate progesterone resistance and increase the expression of LIF of endometriosis rats during the implantation window, which could improve endometrial receptivity and enhance the pregnant rate of endometriosis. It may provide a potential treatment method for endometriosis-related infertility.

Relationship between mitochondrial DNA haplogroup and litter size in the pig.

Mitochondrial DNA (mtDNA) has been widely associated with complex traits in farm animals. The present study evaluated the effects of mtDNA on litter size in pigs. Mitogenome sequencing of 1017 sows distinguished 232 variations, including 229 single nucleotide polymorphisms and three indels, which constituted 11 haplotypes and further clustered into two haplogroups that differed significantly (P<0.05) in litter size. In order to explain the associations between the effect of haplogroup on litter size and different maternal origins, extant mitogenome sequences were used for phylogenetic or principal component analyses. The results of these analyses led to the identification of two groups, representing Chinese and European origins. The haplotypes corresponding to high litter size were all in the Chinese cluster, whereas haplotypes corresponding to low litter size were all in the European cluster. The results of this study suggest that the effect of haplogroup on litter size in the pig could be caused by diverse maternal origins, and that mtDNA haplogroup may be a marker for genetic selection for pig litter size.

Expression and significance of T-cell immunoglobulin mucin molecule 3 and its ligand galectin-9 in patients with adenomyosis.

The T cell immunoglobulin mucin molecule 3 (TIM-3), as a negative regulatory immune checkpoint, plays an important role in cellular immunity by binding to its ligand galectin-9 (Gal-9). Under abnormal conditions, this pathway can regulate the depletion of T cells and suppress the immune response. Abnormal expression of TIM-3 and Gal-9 has been confirmed in a variety of cancers, recurrent miscarriages, chronic infectious diseases and autoimmune diseases. More and more studies have shown that immune factors play an important role in the pathogenesis of adenomyosis. However, few studies have attempted to explore their expression in adenomyosis. The ectopic and eutopic endometrium were collected from 15 women with adenomyosis and 13 women without adenomyosis. TIM-3/Gal-9 expression in endometrial tissue was detected using immunohistochemistry, western blot analysis and real-time PCR. We observed TIM-3/Gal-9 expression in both eutopic and ectopic endometria, with elevated expression in adenomyosis. These data suggest that TIM-3/Gal-9 may be involved in the development and progression of adenomyosis.

Lnc-NA inhibits proliferation and metastasis in endometrioid endometrial carcinoma through regulation of NR4A1.

Endometrioid endometrial carcinoma (EEC) is the most common gynaecologic malignancy worldwide. Long non-coding RNAs have previously been demonstrated to play important roles in regulating human diseases, particularly cancer. However, the biological functions and molecular mechanisms of long non-coding RNAs in EEC have not been extensively studied. Here, we describe the discovery of Lnc-NA from the promoter of the transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) gene. The role and function of Lnc-NA in EEC remain unknown. In this study, we used quantitative real-time polymerase chain reactions to confirm that Lnc-NA expression was down-regulated in 30 EEC cases (90%) and in EEC cell lines compared with that in the paired adjacent tissues and normal endometrial cells. In vitro experiments further demonstrated that overexpressing Lnc-NA decreased EEC cell proliferation, migration and invasion and promoted apoptosis via inactivation of the apoptosis signalling pathway. Moreover, the results show that Lnc-NA expression was positively correlated with NR4A1. Furthermore, Lnc-NA regulated NR4A1 expression and activated the apoptosis signalling pathway to inhibit tumour progression. In summary, our results demonstrate that the Lnc-NA-NR4A1 axis could be a useful tumour suppressor and a promising therapeutic target for EEC.

Dynamic characteristics of the mitochondrial genome in SCNT pigs.

Most animals generated by somatic cell nuclear transfer (SCNT) are heteroplasmic; inheriting mitochondrial genetics from both donor cells and recipient oocytes. However, the mitochondrial genome and functional mitochondrial gene expression in SCNT animals are rarely studied. Here, we report the production of SCNT pigs to study introduction, segregation, persistence and heritability of mitochondrial DNA transfer during the SCNT process. Porcine embryonic fibroblast cells from male and female Xiang pigs were transferred into enucleated oocytes from Yorkshire or Landrace pigs. Ear biopsies and blood samples from SCNT-derived pigs were analyzed to characterize the mitochondrial genome haplotypes and the degree of mtDNA heteroplasmy. Presence of nuclear donor mtDNA was less than 5% or undetectable in ear biopsies and blood samples in the majority of SCNT-derived pigs. Yet, nuclear donor mtDNA abundance in 14 tissues in F0 boars was as high as 95%. Additionally, mtDNA haplotypes influenced mitochondrial respiration capacity in F0 fibroblast cells. Our results indicate that the haplotypes of recipient oocyte mtDNA can influence mitochondrial function. This leads us to hypothesize that subtle developmental influences from SCNT-derived heteroplasmy can be targeted when using donor and recipient mitochondrial populations from breeds of swine with limited evolutionary divergence.

Parton Distribution Functions from a Light Front Hamiltonian and QCD Evolution for Light Mesons.

We obtain the pion and the kaon parton distribution functions from the eigenstates of a light front effective Hamiltonian in the constituent quark-antiquark representation suitable for low-momentum scale applications. By taking these scales as the only free parameters, the valence quark distribution functions of the pion, after QCD evolution, are consistent with the data from the FNAL-E615 experiment. The ratio of the up quark distribution of the kaon to that of the pion also agrees with the CERN-NA3 experiment. Supplemented by known parton distribution functions for the nucleons, we further obtain the cross section consistent with experimental data for the π^{-}nucleus→µ^{+}µ^{-}X Drell-Yan process.

Knockdown of long noncoding RNA-taurine-upregulated gene 1 inhibits tumor angiogenesis in ovarian cancer by regulating leucine-rich α-2-glycoprotein-1.

To investigate the role of long noncoding RNA taurine-upregulated gene 1 (TUG1) on ovarian cancer-induced angiogenesis and to explore possible signaling pathways. Ovarian cancer cell line SKOV3 or CAOV3 was transfected with short hairpin-TUG1 to suppress TUG1 expression. Supernatant from cultured cancer cells was used as a condition medium to incubate endothelial cell line human umbilical vein endothelial cells, whose proliferation rate was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration and invasion of endothelial cells were examined by wound healing and Transwell assays, followed by in-vitro angiogenesis assay. One of the secretory factors mediating angiogenesis, leucine-rich α-2-glycoprotein-1 (LRG1), was measured in ovarian cancer cells. Signaling pathway mediating angiogenesis was further detected by western blotting. TUG1 was down-regulated in ovarian cancer cells by short hairpin RNA. Conditional medium originating from TUG1-knockdown cancer cells led to suppressed proliferation, migration, or invasion of endothelial cell line human umbilical vein endothelial cells. LRG1 expression and secretion was suppressed in ovarian cancer cells after TUG1 knockdown. Moreover, recombinant LRG1 rescued TUG1 knockdown-induced angiogenesis inhibition, and LRG1 probably mediated angiogenesis by tumor growth factor-ß signaling pathway in endothelial cells. Long noncoding RNA-TUG1 mediates angiogenesis of endothelial cells by regulating LRG1 secretion from ovarian cancer cells partially through tumor growth factor-ß pathway. Our results indicate the potency of TUG1 as a biomarker and therapeutic target for tumor-induced angiogenesis.

Association analysis of mitochondrial DNA polymorphisms with oocyte number in pigs.

In pigs, correlations between mitochondrial (mt) DNA polymorphisms and economic traits have been widely reported across and within swine breeds. In fecundity studies, the number of oocytes within ovaries was highly correlated with litter size. However, the effect of mitochondrial polymorphisms on porcine oocyte number remained unclear. In this study, 181 porcine ovaries were collected to analyse the relationship between oocyte number and mtDNA polymorphisms. There were considerable differences in oocyte numbers among different ovaries from commercial pig breeds, ranging from 2.7×105 to 1.3×106. Mitochondrial D-loop sequencing discovered 53 polymorphic sites. Association analysis revealed that 13 variations were associated with the number of oocytes (P<0.05). A C323T polymorphism showed the largest value between the C and T carriers, which differed at 105 oocytes (P<0.05). The 53 polymorphic sites generated 45 haplotypes, which clustered into two haplogroups, A and B. Haplogroup A had a higher number of oocytes than Haplogroup B (P<0.05), whereas Haplotype H6 in Haplogroup A had the highest number of oocytes (~7.5×105) of all haplotypes studied (P<0.05). The results of this study highlight a correlation between mtDNA polymorphisms and oocyte number, and suggest the potential application of mtDNA polymorphism analyses in pig selection and breeding practices.

Melatonin inhibits 17ß-estradiol-induced migration, invasion and epithelial-mesenchymal transition in normal and endometriotic endometrial epithelial cells.

BACKGROUND: Melatonin is a potential therapeutic agent for endometriosis, but its molecular mechanism is unclear. Here, we investigated the effect of melatonin on the epithelial-mesenchymal transition (EMT) in endometriotic endometrial epithelial cells and explored the pathway that might be involved. METHODS: This hospital-based study included 60 women of reproductive age using the endometrium for immunohistochemistry, 6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with endometriosis for isolation of endometrial epithelial cells or subsequent analysis, respectively. We examined the expression of Notch1/Numb signaling and EMT markers by immunohistochemistry analysis and western blot analysis, the invasion and migration of endometrial epithelial cells by transwell assays, and the cell proliferation by CCK8 assays. RESULTS: Compared with normal endometrium, the endometriotic eutopic endometrium showed increased expression of Notch1, Slug, Snail, and N-cadherin, and decreased expression of E-cadherin and Numb. Melatonin or Notch inhibition by specific inhibitor blocked 17ß-estradiol-induced cell proliferation, invasion, migration and EMT-related markers in both normal and endometriotic epithelial cells. CONCLUSIONS: Our data suggest that aberrant expression of Notch1/Numb signaling and the EMT is present in endometriotic endometrium. Melatonin may block 17ß-estradiol-induced migration, invasion and EMT in normal and endometriotic epithelial cells by upregulating Numb expression and decreasing the activity of the Notch signaling pathway.

Mifepristone Inhibits the Migration of Cervical Cancer Cells by Inhibiting Exocrine Secretion.

Cervical cancer (CC) is one of the most common gynecological malignancies, and metastasis limits the use of surgical resection. Metapristone (MIF) was reported to suppress the proliferation and migration of several cancer cells. Exosomes play a variety of roles in cellular biological processes. The relation of exosomes and CC is less studied. Cell viability, apoptosis assay and migration assay was conducted in HeLa cells treated by MIF by CCK-8 kit, staining by Annexin V-fluorescein isothiocyanate and propidium iodide, and wound test respectively. ISG15 expression level was examined in MIF-treated HeLa cells by Western blot. The migration of HeLa cells treated by MIF/GW4869 was measured by wound test. MIF suppressed the growth and migration, as well as induced apoptosis of CC cells. MIF inhibited the exocrine secretion of CC cells by upregulating ISG15, while treating CC cells by ISG15 stimulus, IFN, inhibited the secretion of exosomes. The inhibition of exocrine secretion by GW4869 enhanced the migration inhibition of MIF on CC cells. This study demonstrates that MIF suppresses the CC cell migration by inhibiting exocrine secretion through upregulating ISG1.

Identifying risk factors for cesarean scar pregnancy: a retrospective study of 79 cases.

OBJECTIVES: To explore the possible risk factors for cesarean scar pregnancy (CSP), the incidence of which is increasing rapidly in China. MATERIAL AND METHODS: 79 patients with CSP and 69 non-CSP expectant mothers with at least 1 previous cesarean section were employed in the study. The obstetric histories of the participants were collected and analyzed using Chi square test. RESULTS: We found that 77.2% CSP patients had ≥ 3 pregnancies and only 36.2% women had ≥ 3 pregnacies in non-CSP group. During the previous cesarean delivery, 21.5% of CSP patients had entered the first stage of labor, which was 43.5% in non-CSP group (P < 0.05). Cephalopelvic disproportion occurred in 51.9% of CSP patients, which was significantly higher than that (23.2%) in non-CSP group (P < 0.01). 11.4% of CSP patients had undergone cesarean section due to breech and shoulder presentation in the past, which was only 1.4% in non-CSP group. However, no significance was noted (P > 0.05). We did not find significant differences between the CSP and non-CSP patients in maternal age, multiple cesarean sections, gestational age, emergency or elective caesarean section. CONCLUSIONS: Multiple pregnancies, absence of the first stage of labor, and cephalopelvic disproportion might be the risk factors for the occurrence of CSP.

Normal endometrial stromal cells regulate 17ß-estradiol-induced epithelial-mesenchymal transition via slug and E-cadherin in endometrial adenocarcinoma cells in vitro.

Stroma-tumor communication participates in the pathogenesis of endometrial carcinomas. In previous studies, we found that normal stromal cells inhibited the growth of endometrial carcinoma cells. Here, we investigated the role of normal stromal cells in the epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells and explored the possible mechanism implied. We found that conditioned medium (CM) by normal endometrial stromal cells (NSC) reduced cell growth and induced cell apoptosis in Ishikawa cells. CM by NSC inhibited 17ß-estradiol-induced cell growth and apoptosis decrease in Ishikawa cells. Moreover, CM by NSC inhibited the migration and invasion, and 17ß-estradiol-induced migration and invasion in Ishikawa cells. Meanwhile, CM by NSC decreased Slug expression and 17ß-estradiol-induced Slug expression, increased E-cadherin expression and abolished 17ß-estradiol-induced E-cadherin reduction in Ishikawa cells. In conclusion, normal stromal factors can inhibit 17ß-estradiol-induced cell proliferation and apoptosis inhibition, and abolished 17ß-estradiol-induced EMT in endometrial cancer cell via regulating E-cadherin and Slug expression.

Early Holocene chicken domestication in northern China.

Chickens represent by far the most important poultry species, yet the number, locations, and timings of their domestication have remained controversial for more than a century. Here we report ancient mitochondrial DNA sequences from the earliest archaeological chicken bones from China, dating back to ∼ 10,000 B.P. The results clearly show that all investigated bones, including the oldest from the Nanzhuangtou site, are derived from the genus Gallus, rather than any other related genus, such as Phasianus. Our analyses also suggest that northern China represents one region of the earliest chicken domestication, possibly dating as early as 10,000 y B.P. Similar to the evidence from pig domestication, our results suggest that these early domesticated chickens contributed to the gene pool of modern chicken populations. Moreover, our results support the idea that multiple members of the genus Gallus, specifically Gallus gallus and Gallus sonneratii contributed to the gene pool of the modern domestic chicken. Our results provide further support for the growing evidence of an early mixed agricultural complex in northern China.