The initial gastrointestinal microbiota development plays an important role in host health and is related to several diseases throughout the human lifetime, because the microbiota and the human host are of mutualistic interactions[1-2]. Within the infant period, the microbiota is dynamic[3]. Many factors can influence the acquisition and establishment of infant gut microbiota, including delivery mode[4], feeding practices[5], antibiotic therapy[6], probiotics and prebiotics[7]and geography[8], etc.

Furthermore, the microbiota colonized in mother’s gut and derived milk microbiota is also one major factor that shapes gut microbiota of healthy breast-feeding have found that human milk may significantly influence infant gut microbiota colonization[9]and is also a source of commensal bacteria for the infant gut[10]. It has also been shown that there is a vertical transfer of gut lactic acid-producing bacteria from the mother’s gut to the derived milk, which can be further transferred through the milk to the infant’s gut[11-15]. Such microbial transmission from mother to infant through breast-feeding is further affected by ethnicity,which is important to shape the infant gut microbiota[16-17].For example, the composition of gut microbiome in African American was different from that of non-African Americans in both neonates and infants due to the difference of mothers’ethnicities[18].

It is necessary to explore the development process of infant gut microbiota and characterize the difference between ethnic groups[19]. Qinghai-Tibet plateau, the highest plateau on the world, is a typical high altitude region in China,with typical environmental conditions such as geographical locations of high altitude and climate conditions, including low temperature, low barometric pressure, low humidity, and high radiation[20]. Moreover, Tibetan ethnic group is one of the three largest traditional nomadic tribes in China keeping up until now, and they have particular cultural background and dietary habits such as Zanba, bitter tea. Tibetans generally have higher energy intakes like more meat and less vegetables than Chinese Han, the largest ethnic group in China. Previous studies showed that the Han adults had different gut microbial community composition and diversity from those of Tibetan adults[21-22]. However, it is not clear that whether the gut bacterial diversity of healthy breast-feeding infants is different among different ethnic groups in China,a multicultural society consisting of the major ethnic group Han and other ethnic minority groups.

In this study, we determined the gut microbial diversity of healthy breast-feeding infants in Tibetan and Han ethnic groups, aiming at comprehending two matters: firstly, to understand the microbial diversity of the Han and minority Tibetan infants; and secondly, to find out the general relationship between microbiota and ethnicity in infants by identifying whether there is compositional difference in gut microbiota between the two distinct ethnic groups.

1 Materials and Methods

1.1 Materials and reagents

QIAamp DNA Stool Mini Kitand QIAquick Gel Extraction kit were obtained from QIAGEN; PicoGreen dsDNA Assay Kit was obtained from Invitrogen; the primers of PCR was 338F: ACTCCTACGGGAGGCAGCA and 806R: GGACTACHVGGGTWTCTAAT[23]; and the PCR mixture contained 5 μL of Q5 reaction buffer (5 ×), 5 μL of Q5 High-Fidelity GC buffer (5 ×), 0.25 μL of Q5 High-Fidelity DNA Polymerase (5 U/μL), 2 μL (2.5 mmol/L)of dNTPs, 1 μL (10 μmol/L) of each Forward and Reverse primer, 2 μL of DNA Template, and 8.75 μL of ddH2O.

1.2 Instruments and equipment

NanoDrop ND-1000 spectrophotometer was obtained from Thermo Fisher Scientific (Waltham, MA, USA);GeneAmp PCR system 9700 was purchased from Applied biosystem (USA); DYY-10C electrophoresis apparatus was obtained from XX (Beijing, China), Illumina MiSeq platform was obtained from Personal Biotechnology Co. Ltd.(Shanghai, China).

1.3 Methods

1.3.1 Subject selection and fecal sample collection

We enrolled 20 healthy infants born and living in the Nyingchi, Tibet Autonomous Region (five males and five females) and Harbin, Heilongjiang province (five males and five females), China, respectively. All the infants were breastfeeding for 3-6 months without other supplements such as infant formula and any additional foods. There were no use of probiotics or antibiotics for these infants, nor the food allergies or gastrointestinal diseases in three months before collecting samples. Moreover, Tibet and Harbin infants’parents were indigenous residents and Tibetans’ parents are native nomadic tribes.

The fresh fecal samples were collected and put into sterile fecal collection tubes, and then stored at -80 ℃ until genomic DNA extraction. An informed consent was provided and confirmed by their legal guardians before participation.

1.3.2 DNA extraction and 16S rRNA gene amplification