Genetic variation of Polish endangered Biłgoraj horses and two common horse breeds in microsatellite loci

Genetic variation of endangered Bi?goraj horses and two common Polish horse breeds was compared with the use of 12 microsatellite loci (AHT4, AHT5, ASB2, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, VHL20). Lower allelic diversity was detected in all investigated populations in comparison to other studies. Large differences in the frequencies of microsatellite alleles between Bi?goraj horses and two other horse breeds were discovered. In all polymorphic loci all investigated breeds were in the Hardy-Weinberg equilibrium. Mean Fis values and the results of a test for the presence of a recent bottleneck were non-significant in all studied populations. Comparable values of observed and expected gene diversity indicate no substantial loss of genetic variation in the Bi?goraj population and two other breeds. The lowest variability observed in the investigated group of Thoroughbred horses was confirmed. About 10% of genetic variation are explained by differences between breeds. Values of pairwise Fst and two measures of genetic distance demonstrated that Bi?goraj horses are distantly related to both common horse breeds.     

Genetic diversity and admixture analysis of Sanfratellano and three other Italian horse breeds assessed by microsatellite markers

Sanfratellano is a native Sicilian horse breed, mainly reared in the north east of the Island, developed in the 19th century from local dams and sires with a restricted introgression of Oriental, African and, more recently, Maremmano stallions. In this study, the genetic relationships and admixture among Sanfratellano, the other two Sicilian autochthonous breeds and Maremmano breed were assessed using a set of microsatellites. The main goals were to infer the impact of Maremmano breed in the current Sanfratellano horse and to provide genetic information useful to improve the selection strategies of the Sanfratellano horse. The whole sample included 384 horses (238 Sanfratellano, 50 Sicilian Oriental Purebred, 30 Sicilian Indigenous and 66 Maremmano), chosen avoiding closely related animals. A total of 111 alleles from 11 microsatellite loci were detected, from four at HTG7 to 15 at ASB2 locus. The mean number of alleles was the lowest in Oriental Purebred (6.7), the highest in Sanfratellano (8.3). All the breeds showed a high level of gene diversity (He) ranging from 0.71 ± 0.04 in Sicilian Oriental Purebred to 0.81 ± 0.02 in Sicilian Indigenous. The genetic differentiation index was low; only about 6% of the diversity was found among breeds. Nei's standards (DS) and Reynolds' (DR) genetic distances reproduced the same population ranking. Individual genetic distances and admixture analysis revealed that: (a) nowadays Maremmano breed does not significantly influence the current Sanfratellano breed; (b) within Sanfratellano breed, it is possible to distinguish two well-defined groups with different proportions of Indigenous blood.     

Comparison of Thoroughbred and Arabian horses using RAPD markers

We compared pools of DNA from 10 Thoroughbred horses and 10 Arabian horses for the presence of randomly amplified polymorphic DNA (RAPD) markers which might be useful in distinguishing between the breeds. Using 212 decamer oligonucleotides and our polymerase chain reaction (PCR) conditions, 173 of the primers produced scoreable bands. The number of bands ranged from 0 to 9 with an average of 3·6. In family studies using 11 arbitrarily selected primers, five of the 11 primers produced polymorphic bands which exhibited Mendelian inheritance as dominant markers. When comparing the pooled DNA from Thoroughbred and Arabian horses we found 10 primers which identified markers present in the pooled DNA from one breed but absent in the pool from the other breed. Testing individual horses revealed that only two markers were wholly absent for one group while being present among members of the other. Primer UBC-85 (5′-GTGCTCGTGC-3′) detected a pair of markers absent in Thoroughbred horses but present among 11 of 31 Arabian horses. These markers were 1500 and 1700 base pairs (bp) long and designated UBC-85^C and UBC-85^D, respectively. Primer UBC-126 (5′-CTTTCGTGCT-3′) detected a 1000 bp marker (designated UBC-126^C) absent in 20 of 20 Thoroughbred horses but present in 31 of 31 Arabian horses. UBC-126^C would be particularly effective for breed comparisons, especially if the DNA band were cloned, sequenced and an allelic marker present in Thoroughbred horses but rare or absent among Arabian horses was identified. The distribution of such markers among other horse breeds might be useful to infer relationships among breeds. These kinds of markers may also be useful in detecting unwanted crossbreeding between two horse breeds.     

Genetic relationships of five Indian horse breeds using microsatellite markers

The genetic relationships of five Indian horse breeds, namely Marwari, Spiti, Bhutia, Manipuri and Zanskari were studied using microsatellite markers. The DNA samples of 189 horses of these breeds were amplified by polymerase chain reaction using 25 microsatellite loci. The total number of alleles varied from five to 10 with a mean heterozygosity of 0.58 ± 0.05. Spiti and Zansakari were the most closely related breeds, whereas, Marwari and Manipuri were most distant apart with Nei's D_A genetic distance of 0.071 and 0.186, respectively. In a Nei's D_A genetic distances based neighbour joining dendrogram of these breeds and a Thoroughbred horse outgroup, the four pony breeds of Spiti, Bhutia, Manipuri and Zanskari clustered together and then with the Marwari breed. All the Indian breeds clustered independently from Thoroughbreds. The genetic relationships of Indian horse breeds to each other correspond to their geographical/environmental distribution.     

Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites

Ten (TG)_n positive clones, isolated from an equine genomic library and sequenced, contained 12–19 uninterrupted TG repeats. Primers for polymerase chain reaction (PCR) were synthesized and nine of these (TG)_n loci (HTG7-15) were successfully amplified and utilized in this study together with five previously reported equine microsatellite loci (HTG2-6). The PCR products were analysed by polyacrylamide gel electrophoresis followed by automated laser fluorescence detection or autoradiography. All microsatellites showed polymorphism and stable Mendelian inheritance. Differences in microsatellite variability between horse breeds were detected. A linkage analysis comprising HTG2-15, one coat colour gene and 16 genetic blood markers enabled addition of HTG2 to linkage group U2 and a new linkage group (U6) was established comprising the loci HTG7 and HTG12. Close linkage was excluded within a set of eight microsatellites. The estimated probability of exclusion in four breeds for a parentage test based on these eight loci varied between 0.96 and 0.99.     

Genetic diversity in German draught horse breeds compared with a group of primitive, riding and wild horses by means of microsatellite DNA markers

We compared the genetic diversity and distance among six German draught horse breeds to wild (Przewalski's Horse), primitive (Icelandic Horse, Sorraia Horse, Exmoor Pony) or riding horse breeds (Hanoverian Warmblood, Arabian) by means of genotypic information from 30 microsatellite loci. The draught horse breeds included the South German Coldblood, Rhenish German Draught Horse, Mecklenburg Coldblood, Saxon Thuringa Coldblood, Black Forest Horse and Schleswig Draught Horse. Despite large differences in population sizes, the average observed heterozygosity (H(o)) differed little among the heavy horse breeds (0.64-0.71), but was considerably lower than in the Hanoverian Warmblood or Icelandic Horse population. The mean number of alleles (N(A)) decreased more markedly with declining population sizes of German draught horse breeds (5.2-6.3) but did not reach the values of Hanoverian Warmblood (N(A) = 6.7). The coefficient of differentiation among the heavy horse breeds showed 11.6% of the diversity between the heavy horse breeds, as opposed to 21.2% between the other horse populations. The differentiation test revealed highly significant genetic differences among all draught horse breeds except the Mecklenburg and Saxon Thuringa Coldbloods. The Schleswig Draught Horse was the most distinct draught horse breed. In conclusion, the study demonstrated a clear distinction among the German draught horse breeds and even among breeds with a very short history of divergence like Rhenish German Draught Horse and its East German subpopulations Mecklenburg and Saxon Thuringa Coldblood.     

Microsatellite characterization of Cimarron Uruguayo dogs

Various genetic markers, including microsatellites, have been used to analyze the genetic polymorphism and heterozygosity in canine breeds. In this work, we used nine microsatellite markers to investigate the genetic variability in Cimarron Uruguayo dogs, the only officially recognized native canine breed in Uruguay. DNA from 30 Cimarron Uruguayo dogs from northeastern and southern Uruguay was analyzed. The allelic frequencies for each microsatellite, the genetic variability and the consanguinity were calculated, as were the polymorphic information content (PIC) and the probability of exclusion (PE). All of the microsatellites studied were polymorphic. FH 2361, FH 2305 and PEZ 03 were the most informative, with PIC values > 0.7, in agreement with results for other canine breeds. The PE values for the markers were within the ranges previously described and were generally greater for microsatellites with higher PIC values. The heterozygosity value (0.649) was considered high since only nine microsatellites were analyzed. Compared with data for other breeds, the results obtained here indicate that Cimarron Uruguayo dogs have high genetic diversity.     

Genetic diversity and bottleneck studies in endangered Bhutia and Manipuri pony breeds

For studying the genetic diversity and bottleneck problem in Bhutia and Manipuri pony breeds of India, we analysed DNA samples of 34 Bhutia and 50 Manipuri, true to breed, ponies using 47 polymorphic microsatellite markers. All the microsatellites were observed to be highly polymorphic in nature in both Bhutia and Manipuri breeds with mean no. of alleles as 8.702 ± 0.0493 and 8.416 ± 0.0548 respectively. Genetic diversity values in terms of heterozygosity values within individual breeds were also high with very low inbreeding (Fis 0.102 and 0.055 in Bhutia and Manipuri ponies, respectively). Number of alleles in both the populations together ranged from 3 to 18 with an average of 10.851 ± 1.583 per locus. The mean effective number of alleles was observed to 5.34 ± 0.253. All loci except ASB017 and HTG004 showed high values of allele richness (>5.0). The mean observed and expected heterozygosities were 0.7159 ± 0.022, 0.7986 ± 0.011 (Levene’s) and 0.7936 ± 0.011 (Nei’s), respectively. The high mean values of heterozygosity indicated the presence of high genetic diversity in both the pony populations. The overall mean value of within-population inbreeding estimates (Fis) was low (0.101 ± 0.023) indicating low to moderate level of inbreeding. Bottleneck studies revealed that no recent bottleneck problem has taken place in both the populations. Both pony populations were found to be in mutation drift equilibrium. The study reveals that both the pony breeds have high diversity and timely action needs to be taken to conserve them.     

A genetic analysis of the Italian Salernitano horse

Salernitano (SAL) is an ancient Italian horse breed developed over the course of the ages together with Napoletano and, during the 20th century, by crossing with Thoroughbred horse lines. Excellent in hurdle jumping, this breed is currently facing a concrete risk of extinction due to the lack of appropriate management strategies. This research is the first SAL genetic characterization that aims to set up the basic knowledge for a conservation plan. A representative sample of 61 SALs was analyzed by means of a set of 16 microsatellites markers (short tandem repeats (STRs)). The sequence of hypervariable D-loop mtDNA region was also performed on a subset of 24 mares in order to study the maternal diversity and obtain a complete picture of the internal genetic variation. All the molecular data were analyzed together with those obtained from three Sicilian horse breeds investigated in a previous research (Siciliano, Sanfratellano and Sicilian Oriental Purebred). STRs markers revealed a moderate level of genetic diversity in SAL (alleles/locus 5.1, He 0.67) and confirmed the hunch of genetic erosion. Autosomal variability highlighted a very light deficit of homozygotes (FIS=−0.067). Experimental D-loop sequences were compared by multiple alignments with those retrieved from biological databases and revealed two unreported haplotypes. The phylogenetic network, which was built on mtDNA sequences, included various cosmopolitan and European horses and showed SAL haplotypes distributed among different mtDNA lineages.     

Horse breed discrimination using machine learning methods

Genetic relationships and population structure of 8 horse breeds in the Czech and Slovak Republics were investigated using classification methods for breed discrimination. To demonstrate genetic differences among these breeds, we used genetic information — genotype data of microsatellite markers and classification algorithms — to perform a probabilistic prediction of an individual’s breed. In total, 932 unrelated animals were genotyped for 17 microsatellite markers recommended by the ISAG for parentage testing (AHT4, AHT5, ASB2, HMS3, HMS6, HMS7, HTG4, HTG10, VHL20, HTG6, HMS2, HTG7, ASB17, ASB23, CA425, HMS1, LEX3). Algorithms of classification methods — J48 (decision trees); Naive Bayes, Bayes Net (probability predictors); IB1, IB5 (instance-based machine learning methods); and JRip (decision rules) — were used for analysis of their classification performance and of results of classification on this genotype dataset. Selected classification methods (Naive Bayes, Bayes Net, IB1), based on machine learning and principles of artificial intelligence, appear usable for these tasks.     

Using molecular markers and multivariate methods to study the genetic diversity of local European and Asian chicken breeds

French and Asian subsets of chicken breeds were first analysed using 22 microsatellites and then compared to the AVIANDIV European set using 14 loci. Positive correlations were observed between F _IT or F _ST and typological values or variance of markers using the multivariate analysis mcoa . The first axis of the multivariate representation separated Asian from European breeds, revealing breeds with Asian ancestor. Using all or 14 loci, correct assignation rate was always higher than 93%. The Weitzman index and the aggregate diversity D were calculated using 22 loci within French and Asian breeds. The French breed Coucou de Rennes and the Hua-Tung breed seemed to contribute the most to the global diversity of each subset. This approach on French-only breeds and then on French with AVIANDIV domestic breeds (14 loci) showed that the Marans breed contributed the most. The AVIANDIV framework could be useful to evaluate the genetic diversity of local breeds and to help in connecting national and regional conservation policies.     

Genetic diversity and relationships of Portuguese and other horse breeds based on protein and microsatellite loci variation

There are three native Portuguese horse breeds: Lusitano, Sorraia and Garrano. This study compares diversity patterns of 17 protein and 12 microsatellite markers in these three as well as 30 other breeds to infer relationships among the breeds and to compare levels of polymorphism of these breeds for use in conservation efforts. The Garrano and the Lusitano showed a high level of genetic diversity, similar to that observed for most of the other analysed breeds, while the Sorraia and Friesian breeds showed low levels of variation for both genetic marker types. The combined protein and microsatellite data produced a tree that fit historical records well and with greater confidence levels than those for either data set alone. The combined genetic diversity and relationship information provides important baseline data for future breed conservation efforts, especially for a critically endangered breed such as the Sorraia.     

Molecular phylogeny of Indian horse breeds with special reference to Manipuri pony based on mitochondrial D-loop

Manipuri pony is the geographically distant breed of horse from the five recognized horse breeds found in the Indian subcontinent. The phylogenetic relationship of Manipuri pony with the other breeds is unknown. The diversity in the mitochondrial (mt) DNA D-loop region is employed as an important tool to understand the origin and genetic diversification of domestic horses and to examine genetic relationships among breeds around the world. This study was carried out to understand the maternal lineages of Manipuri pony using the 247 bp region of the mtDNA D-loop. The dataset comprised of eleven numbers of self developed sequences of Manipuri pony, 59 and 35 number of retrieved sequences of Indian horse breeds and other worldwide breeds respectively. A total of 35 haplotypes was identified with a high level of genetic diversity in the Indian breeds. A total of seven major mtDNA haplogroups (A–G) was identified in the Indian horse breeds that indicated the abundance of mtDNA diversity and multiple origins of maternal lineages in them. The majority of the studied sequences of Indian breeds (33.3 %) were grouped into haplogroup D and least (3.9 %) in haplogroup E. The Manipuri breed showed the least F_ST distance (0.03866) with the most diverged Indian breeds and with Thoroughbred horse among the worldwide. This study indicated a close association between Manipuri pony and Thoroughbred.     

Identification of Genetic Variation on the Horse Y Chromosome and the Tracing of Male Founder Lineages in Modern Breeds

The paternally inherited Y chromosome displays the population genetic history of males. While modern domestic horses (Equus caballus) exhibit abundant diversity within maternally inherited mitochondrial DNA, no significant Y-chromosomal sequence diversity has been detected. We used high throughput sequencing technology to identify the first polymorphic Y-chromosomal markers useful for tracing paternal lines. The nucleotide variability of the modern horse Y chromosome is extremely low, resulting in six haplotypes (HT), all clearly distinct from the Przewalski horse (E. przewalskii). The most widespread HT1 is ancestral and the other five haplotypes apparently arose on the background of HT1 by mutation or gene conversion after domestication. Two haplotypes (HT2 and HT3) are widely distributed at high frequencies among modern European horse breeds. Using pedigree information, we trace the distribution of Y-haplotype diversity to particular founders. The mutation leading to HT3 occurred in the germline of the famous English Thoroughbred stallion “Eclipse” or his son or grandson and its prevalence demonstrates the influence of this popular paternal line on modern sport horse breeds. The pervasive introgression of Thoroughbred stallions during the last 200 years to refine autochthonous breeds has strongly affected the distribution of Y-chromosomal variation in modern horse breeds and has led to the replacement of autochthonous Y chromosomes. Only a few northern European breeds bear unique variants at high frequencies or fixed within but not shared among breeds. Our Y-chromosomal data complement the well established mtDNA lineages and document the male side of the genetic history of modern horse breeds and breeding practices.     

An assessment of European pig diversity using molecular markers: Partitioning of diversity among breeds

Genetic diversity within and between breeds (and lines) of pigs was investigated. The sample comprised 68 European domestic breeds (and lines), including 29 local breeds, 18 varieties of major international breeds, namely Duroc, Hampshire, Landrace, Large White and Piétrain, and 21 commercial lines either purebred or synthetic, to which the Chinese Meishan and a sample of European wild pig were added. On average 46 animals per breed were sampled (range 12–68). The genetic markers were microsatellites (50 loci) and AFLP (amplified fragment length polymorphism, 148 loci). The analysis of diversity showed that the local breeds accounted for 56% of the total European between-breed microsatellite diversity, and slightly less for AFLP, followed by commercial lines and international breeds. Conversely, the group of international breeds contributed most to within-breed diversity, followed by commercial lines and local breeds. Individual breed contributions to the overall European between- and within-breed diversity were estimated. The range in between-breed diversity contributions among the 68 breeds was 0.04–3.94% for microsatellites and 0.24–2.94% for AFLP. The within-breed diversity contributions varied very little for both types of markers, but microsatellite contributions were negatively correlated with the between-breed contributions, so care is needed in balancing the two types of contribution when making conservation decisions. By taking into account the risks of extinction of the 29 local breeds, a cryopreservation potential (priority) was estimated for each of them.     

Genetic diversity of Cheju horses (Equus caballus) determined by using mitochondrial DNA D-loop polymorphism

We used sequence polymorphism of the mitochondrial DNA D-loop (968 bp excluding the tandem repeat region) to determine genetic diversity of horses inhabiting Cheju (a southern island of Korea). Seventeen haplotypes with frequencies from 1.5 to 21.5% were found among 65 Cheju horse samples. Genetic diversity (h) of the 17 haplotypes was calculated to be 0.91, indicating that the extant Cheju horse population consists of diverse genetic groups in their maternal lineage. Phylogenetic analysis showed that 17 types of Cheju (D-loop sequences determined), 5 Mongolian, 6 Arabian, 3 Belgian, 2 Tsushima, 2 Yunnan, 1 Przewalskii, and 3 Thoroughbred horses (published sequences for the latter seven breeds) showed that Cheju horses were distributed into many different clusters in the tree. Four Mongolian horses clustered with separate Cheju horse groups, showing that some Cheju horses are clearly of Mongolian origin. The analysis of partial sequences (284 bp) of the D-loop of 109 horses showed that Thoroughbred, Mongolian, Lipizzan, and Arabian breeds are as diverse as Cheju horses. Our data together with others' suggest that most horse breeds tested with reasonably sufficient numbers of samples are diverse in their maternal lineages and also are not uniquely different from each other.     

Genetic Diversity Among Turkish Native Chickens, Denizli and Gerze, Estimated by Microsatellite Markers

The genetic diversity of the Turkish native chicken breeds Denizli and Gerze was evaluated with 10 microsatellite markers. We genotyped a total of 125 individuals from five subpopulations. Among loci, the mean number of alleles was 7.5, expected heterozygosity (H (e)) was 0.665, PIC value was 0.610, and Wright's fixation index was 0.301. H (e) was higher in the Denizli breed (0.656) than in the Gerze breed (0.475). The PIC values were 0.599 and 0.426 for Denizli and Gerze, respectively. A phylogenetic tree was constructed using genetic distance and the neighbor-joining method. Its topology reflects the general pattern of genetic differentiation among the Denizli and Gerze breeds. The present study suggests that Denizli and Gerze subpopulations have a rich genetic diversity. The information about Denizli and Gerze breeds estimated by microsatellite analysis may also be useful as an initial guide in defining objectives for designing future investigations of genetic variation and developing conservation strategies.     

Population studies of 17 equine STR for forensic and phylogenetic analysis

As a consequence of the close integration of horses into human society, equine DNA analysis has become relevant for forensic purposes. However, the information content of the equine Short Tandem Repeat (STR) loci commonly used for the identification or paternity testing has so far not been fully characterized. Population studies were performed for 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) including 8641 horses representing 35 populations. The power of parental exclusion, polymorphic information content, expected and observed heterozygosity and probability of identity were calculated, showing that the set of 17 STRs has sufficient discriminating power for forensic analysis in almost all breeds. We also explored the reliability of individual assignment tests in identifying the correct breeds of origin for unknown samples. The overall proportion of individuals correctly assigned to a population was 97.2%. Finally, we demonstrate the phylogenetic signal of the 17 STR. We found three clusters of related breeds: (i) the cold-blooded draught breeds Haflinger, Dutch draft and Friesian; (ii) the pony breeds Shetland and Miniature horse with the Falabella, Appaloosa and Icelandic; and (iii) The Warmblood riding breeds, together with the hot-blooded Standard-bred, Thoroughbred and Arabian.     

Analysis of Genetic Diversity and Structure of Guanzhong Horse Using Microsatellite Markers

To determine the genetic diversity and validate the pedigree record of Chinese Guanzhong horse, 67 individuals were genotyped with eight microsatellite markers. In our study, the mean observed and expected heterozygosities were 0.51 and 0.66, respectively. The mean observed number of alleles for the Guanzhong horse was 3.88. Nonetheless, the total value of F_ST multiloci clearly indicates that about 0.5% of overall genetic variation is due to line founder differences, while differences among individuals are responsible for the remaining 99.5%. In addition, the polymorphic information content (PIC) result showed that five loci (HTG7, HMS7, HMS2, AHT4, and HMS6) were highly polymorphic (PIC?>?0.5) and three loci (HMS3, HTG6, and COR071) were moderate polymorphic (PIC?>?0.25). Genetic distances and cluster analysis showed that the genetic relationship among 67 Guanzhong horse was generally consistent with pedigree recorded. Our results not only evaluated the genetic diversity of Chinese Guanzhong horse, but also suggested that the eight microsatellite markers might be used as subservient markers for parentage verification and individual identification in the Guanzhong horse.     

Evaluation of the genetic diversity and population structure of Chinese indigenous horse breeds using 27 microsatellite markers

We determined the genetic diversity and evolutionary relationships among 26 Chinese indigenous horse breeds and two introduced horse breeds by genotyping these animals for 27 microsatellite loci. The 26 Chinese horse breeds come from 12 different provinces. Two introduced horse breeds were the Mongolia B Horse from Mongolia and the Thoroughbred Horse from the UK. A total of 330 alleles were detected, and the expected heterozygosity ranged from 0.719 (Elenchuns) to 0.780 (Dali). The mean number of alleles among the horse breeds ranged from 6.74 (Hequ) to 8.81 (Debao). Although there were abundant genetic variations found, the genetic differentiation was low between the Chinese horses, which displayed only 2.4% of the total genetic variance among the different breeds. However, genetic differentiation (pairwise FST) among Chinese horses, although moderate, was still apparent and varied from 0.001 for the Guizou–Luoping pair to 0.064 for the Jingjiang–Elenchuns pair. The genetic differentiation patterns and genetic relationships among Chinese horse breeds were also consistent with their geographical distribution. The Thoroughbred and Mongolia B breeds could be discerned as two distinct breeds, but the Mongolia B horse in particular suffered genetic admixture with Chinese horses. The Chinese breeds could be divided into five major groups, i.e. the south or along the Yangtze river group (Bose, Debao, Wenshan, Lichuan, Jianchang, Guizhou, Luoping, Jinjiang and Dali), the Qinghai‐Tibet Plateau group (Chaidamu, Hequ, Datong, Yushu, Tibet Grassland and Tibet Valley), the Northeast of China group (Elenchuns, Jilin and Heihe), the Northwest of China group (Kazakh, Yili and Yanqi) and the Inner Mongolia group (Mongolia A, Sanhe, Xinihe,Wuzhumuqin and Sengeng). This grouping pattern was further supported by principal component analysis and structure analysis.