Microsatellite DNA Markers and Their Correlation with Body Traits in Landrace, Duroc and Henan Western Black Pigs 

There are numerous local pig breeds in China (Li et al., 1999), Henan Western Black Pigs is a local breed originating from China that holds great potential for development due to its early sexual maturity, resistance to roughage, high adaptability, strong disease resistance and excellent quality of meat, which is delicate and has high nutritional value. Henan Western Black Pigs has the advantages of anti-rough feeding, strong disease resistance, good reproductive performance, good slaughter performance, good meat quality and rich muscle fat content, but it also has the disadvantage of slow growth. In order to obtain greater economic value, the peasants crossed Henan Western Black Pigs with Landrace and Duroc, resulting in a significant decline in number and quality of Henan Western Black Pigs.
 
The aim of this study was to explore the genetic relationship within Landrace, Duroc and Henan Western Black Pigs and to examine the genetic structure and relationship between microsatellite DNA and body traits. The physical appearance of breeding pigs was assessed, following the evaluation criteria proposed and correlated this with the body shape using the inductive descriptive method. (Phythian et al., 2012; Rátkyet_al2013; Vlot et al., 2015). In addition, microsatellite DNA of widely distributed and easily detectable was used to assess current genetic structure and genetic variation (Bai et al., 2016a; Kierepka et al., 2016).

Animal and DNA source
 
The Henan Western Black pigs and Duroc were sourced from the Luanchuan (Luoyang, China). The Landrace were sourced from the CHIA TAI GROUP (Luoyang, China). A total of thirty DNA samples of Henan Western Black Pigs were collected during the trial, fifteen DNA samples were obtained from Landrace, and fifteen DNA samples were also obtained from Duroc.
 
Microsatellite markers
 
Twelve primers were selected based on the research experiments (Wang et al., 2004; Session, 2011; Gélvezet_al2015; Montenegro et al., 2015; Sahoo et al., 2015a; Yu et al., 2015; Behl et al., 2017). Information on the twelve microsatellite primers is provided in Table 1.
 

 
Statistical analysis
 
Body measurement assessment data were statistically analyzed by EXCEL. The SPSS20.0 was used for variance analysis to study the genetic relationship between different genotypes at different sites and the body size traits of the black pig population in Western Henan. Genetic diversity data were analyzed with POPGENE32 (Guo and Elston, 1999; Kavitha et al., 2015; Jiménez-Menaet_al2016). Polymorphism information content value (PIC) for all the loci were estimated according to Deshmukh et al., (2015) and Bai et al., (2016b).

Genetic polymorphism
 
The experimental samples were divided into three groups: Black Pigs, Duroc and Landrace. The electrophoresis results of twelve microsatellites are shown in Fig 1. The three experimental groups detected a total of 150 alleles in twelve microsatellite loci (Table 2). From Fig 1 and Table 2, the twelve microsatellite loci polymorphism richer, for the average observed numbers of alleles, Black Pigs, Duroc and Landrace was 4.5833, 4.0000 and 3.9167. The average Shannon index of Black Pigs, Duroc and Landrace was 1.2738, 1.1836 and 1.1407. The average expected heterozygosity (He) of Black Pigs, Duroc and Landrace was 0.6802, 0.6857 and 0.6370. The highest polymorphic information content (PIC) of Black Pigs, Duroc and Landrace was 0.6192, 0.5919 and 0.5642.
 

 


Henan Western Black pigs are rich in genetic polymorphism. In our study, it had a more uniform allele distribution than Lanyu pigs (Chang et al., 2009). Henan Western Black pigs had a higher heterozygosity (0.6802) than several breeds including European pigs breeds (0.570) (Zhang and Graham, 2011), the Greek black pigs breeds (0.624) (Michailidou et al., 2014), the Criollo pigs breeds (0.622) (Revidatti et al., 2014), however, Henan Western Black Pigs were lower than Jeju Black Pig (0.706) (Oh et al., 2014). Thus, the Black Pig populations of Henan Western Black Pigs appear to have higher levels of genetic polymorphism than the experiment of Sahoo (2015b). The Shannon index reflects the uniformity of genetic variation. In our study, the Shannon index (I) ranged from 0.2449 to 1.7418, the average Shannon index of Black Pigs, Duroc and Landrace pigs was 1.2738, 1.1836 and 1.1407. This is different from the Landrace pigs Shannon Index (0.207) (Kim et al., 2002), which may be a difference in microsatellite sites and lead to differences in the Shannon index. The PIC is an important parameter in genetic diversity analysis as it reflects the amount of genetic information and the level of gene richness (Nagy et al., 2012). Among the twelve microsatellite loci in this study, SO155 was a low polymorphic marker, SO026, SO090 and SW911 were moderate polymorphic markers, and the remaining eight microsatellites were highly polymorphic markers.
 
Genetic distance reflects the phylogenetic evolution of the population studied, it is used to describe the genetic structure of the population and the differences between the varieties (Bai et al., 2015). It can be seen from Table 3 that the minimum genetic distance between the Black Pig and Duroc was 0.1414, while genetic distance between Landrace and Black Pig, Duroc were 0.1602 and 0.1864, respectively. According to the clustering diagram of Fig 2, the Black Pig with the Duroc were first clustered as a class, and then clustered with the Landrace.
 

 

 
The genetic resources of Henan Western Black Pigs had mutated. The inbreeding coefficient (Fis) and the genetic divergence (Fst) are two important indexes of the genetic differentiation and genetic distance between populations (Cortéset_al2016). When genetic divergence (Fst) >0.25, genetic variation was very serious; when 0.25 ≥ genetic divergence (Fst) >0.15, genetic variation was higher; when 0.15 ≥ genetic divergence (Fst) >0.05, genetic variation was moderate; when 0.05 ≥ genetic divergence (Fst) >0, genetic variation was smaller (Sahoo et al., 2016). In this study, at two loci (SO090, SO002) the Fis value deviated significantly from zero, at five loci of genetic variation was smaller, at six loci of genetic variation was moderate, only one microsatellite loci (SO155) had a higher genetic variation. Genetic differentiation analysis of Black Pigs, Duroc and Landrace pigs showed that the microsatellite loci SW24, SO090, SW1067, SO002, and SW857 had the least genetic differentiation, while the remaining seven loci were moderately differentiated (Table 4). The inbreeding coefficient (Fis) ranged from -0.2328 (SO002) to 0.3684 (SW1067). The genetic differentiation factor (Fst) also varied among loci, ranging from 0.0197 (SO155) to 0.1655 (SO090). The gene flow (Nm) at each locus was greater than 1, with an average of 3.8155.
 
Associations with microsatellite markers
 
In examining the associations between microsatellite loci and growth traits (height, back height, body length, chest width, chest depth, head length, ear area and forehead width) (Table 5). Microsatellite loci of SW72 were significantly associated with back height and chest depth (P<0.05), while SO002 were significantly associated with chest depth and head length (P<0.05). SW632 was only significantly associated with head length (P<0.05), SO090 was only significantly associated with body length (P<0.05) and SW1828 was only significantly associated with head length. There were no significant differences in other microsatellite loci, including SO026, SO155, SW24, SW240, SW1067, SW911 and SW875 (P>0.05).
 
Among them, there are three microsatellite markers affecting the head length: SW632, SW1828 and SO002; there are two microsatellite markers affecting chest depth: SW72 and SO002. In addition, SW72 has an impact on the back height and SO090 has an effect on body length. This indicates that certain traits may be controlled by multiple QTLs (or major genes).

This study found the Henan Western Black Pig has rich genetic polymorphism, with high levels of heterozygosity and genetic variation. Cluster analysis shows that the relationship between Black Pigs and Duroc Pigs is closer. There were significantly associated with body traits in five microsatellite loci, including SW72, SO002, SW632, SO090 and SW1828 (P<0.05). These results provide scientific reference for the further cultivation and utilization of black pigs.

This study was supported by the Joint Funds for Fostering Talents of National Natural Science Foundation of China and Henan province (project no. U1304324).