Genetic Diversity at the Microsatellite Loci
A TNA of 360 was observed for the six breed types in the present study. This was higher than that reported for five Cuban cattle breeds (n = 317; TNA = 299) screened for the same 30 microsatellite loci . This may be due to the fact that the Cuban cattle breeds were purebreds, while the present study included crossbred animals with a number of contributing breeds. The TNA was, however, lower than that reported for 27 indigenous Chinese cattle breeds (n = 1638; TNA = 480) for the same loci . This most likely is due to the big sample size and larger number of breeds from different parts of China used in the Chinese study. The range of the PIC values, however, was similar to that reported for seven of the native Chinese cattle breeds (PIC = 0.74 – 0.75)  and for the five Cuban breeds . In general, the high number of alleles and PIC values for the individual loci showed that the 30 microsatellite loci recommended by ISAG/FAO for genetic diversity evaluation of cattle are highly informative and suitable for the purpose.
Genetic Diversity within KK Cattle and KK Crossbred Types
The genetic diversity within the breed types was evaluated using the allelic variation. The MNA is consider as a good indicator of genetic variation. The Brahman breed exhibited the lowest MNA (7.7) among the six breed types. This may be due to the smaller sample size used (n = 32), but most likely due to founder effects and population bottlenecks as a result of breeding practices. The Brahman were introduced into Malaysia to be used for crossbreeding as well as to increase the local cattle population. The KK breed had lower MNA than the KK crossbred types. This may be attributed to crossbreeding incorporating the alleles of the parental breeds into the crossbred types ; the KK crossbred types would have the KK alleles as well as those of the other parental breeds, increasing the number of allele.
The MNA of KK and KK breed types were lower than that reported for four Chinese native cattle breeds (10.1-10.5) , but was higher than those reported for six Spanish native cattle breeds (4.9 - 6.7) . This may be attribute to the differences in population sizes and the sampling technique used. The sample size for the Spanish breeds were between 29 to 50 individuals. Moreover, the breeds occurred as isolated populations and four of them were considered as endanger. Isolated small populations lose genetic variability over time but they become distinctively different.
The average heterozygosity is the best general measure of genetic variation . The KK breed had the lowest Ho (0.54) and He (0.70) among the breed types studied, while the Charoke had the highest values (0.65 and 0.78, respectively). Low heterozygosity could be attributed to isolation and inbreeding, which if not addressed could eventually result in loss of unexploited genetic potential . High heterozygosity in Cheroke could be attributed to the mixed nature of this breed type. The rest of the breed types generally showed similar Ho values, ranging from 0.57 (KKX2) to 0.59 (KKX1). The mean values of Ho were lower than the mean values of He for all the breed types, indicating heterozygous deficiency.
The Ho estimation for KK and Brahman (0.58) were lower than that reported for 27 Chinese native breeds (0.61–0.76) , six Indian breeds (0.60- 0.72)  and ten Ethiopian breeds (0.64-0.70) . This may be due to the fact that China, India and Ethiopia are considered as cattle domestication centres, where it was believed there was contact between immigrant Asiatic indicine and taurine cattle . Therefore, the high heterozygosity values observed in the cattle breeds in these countries most probably are the consequence of the initial admixture of B. indicus and B. taurus cattle that formed the foundation stocks in the past.
FIS value indicates excess or deficit of homozygotes. In the present study, the mean values of FIS were positive for all six breed types, which indicates excess of homozygotes due to inbreeding in all the breed types. This may be due to non-random mating which may be expected in livestock herds. Selection and controlled mating were practised to a certain extent in the farms concerned, especially the nucleus farms. However, proper record keeping was generally lacking; records were often available for growth traits but pedigree records were often not available. The observed FIS values were higher than that reported for Chinese and Ugandan native cattle breeds [30, 37] screened using the same 30 microsatellite loci.
A population is said to be in Hardy-Weinberg equilibrium (HWE) when the gene and genotype frequencies remain constant from generation to generation, and the latter is of a definite proportion . In the present study the deviations from HWE was observed in all six breed types. Deviation from HWE could be attributed to many causes, among which are selection, assortative mating, migration and small population size, all of which could have influenced these populations. The overall numbers of loci that deviated from HWE were high compared to that reported for 10 Ethiopian and 10 Portuguese native cattle for the 30 microsatellite loci [35, 39].
In general, the low genetic diversity observed in the present study in terms of low mean number of the alleles, heterozygote deficiency and deviation from HWE could be attributed to many reasons, but the most probable reasons are inbreeding, small population sizes and assortative mating. The KK, Brakmas, Cheroke and Brahman animals used in the present study were from single nucleus herds. According to Phillips  there are many factors associated with establishing and managing nucleus cattle herd which lead to inbreeding. These include the nucleus herd size, whether the nucleus is open or close, the desired age structure of the nucleus, selection criteria and selection accuracy for the bulls and replacement cows, and completeness of the performance and pedigree records. When these factors were investigated, it was noticed that the records in these nucleus farms were limited and often incomplete. Vital pedigree information was often missing. Consequently, this would have affected the selection accuracy for the bulls and replacement cows. The record keeping at these farms has to be improved and the system reviewed regularly if genetic variability is to be maintained and herd performance is to be improved.
Genetic Variation and Relationship between the KK and KK Crossbred Breed Types
The genetic variation between KK and KK crossbred types were evaluated by estimation of F-statistics, gene flow, genetic admixture and genetic distance, as well as by phylogenetic analysis and principal component analysis (PCA). F-statistics (FIS, FST, and FIT) are measures of the deficit of hetrozygotes relative to expected HWE proportions in the specified population  For large, random mating populations, it is expected that the observed heterozygosity would be equal to the expected heterozygosity, and FIS would be equal or close to zero. In this case, FIT would be approximately equal to FST. However, when FIS is negative which implies no inbreeding, FST would generally exceed FIT. On the other hand, when FIS is positive, implying inbreeding in the population, FIT would exceed FST.
In the present study FIS was positive (0.198), and FIT (0.242) exceeded FST (0.054) indicating inbreeding in all breed types. The level of genetic differentiation among KK and its crossbred types measured in terms of FST (5.4%) was moderate. This mean that 5.4 % of the total genetic variation corresponded to between breed type differences, and 94.6% of the total genetic variation corresponded to within-breed type differences. This could be attributed to the fact that the most of the studied breed types were developed or originated from crosses with KK as the maternal line, and the Brahman breed too was involved in many of the crosses; thus the breeds types sharing some common alleles. The value of FST observed in the present study was lower than the FST values for the three Indian cattle breeds, Sahiwal, Hariana and Deoni (FST = 11.3 %), reported by  and 27 Chinese indigenous cattle breeds (FST = 8%) . This was probably due to the fact that the breeds used in these earlier studies originated from different parts of the respective countries. For example, in the Indian study the Sahiwal breed was native to Pakistan and found along the India-Pakistan border in the North, while the Hariana and Deoni were found in northern and western India respectively. In the study by Zhang  the 27 breeds were representatives from all parts of the vast land area of China, from north, south, east and west. The FST value in the present study was, however, higher than that reported by  for 10 Ethiopian cattle breeds (FST = 1.3 %). The most probable cause of this low level of genetic differentiation in these Ethiopian breeds is the fact that Ethiopian cattle breeds have common historical origins, and shared common grazing lands and watering points. Moreover, an uncontrolled mating practice, which is predominant in Ethiopia, increases the gene flow among the breeds.
It is very clear that the FST between KK and KKX1 (FST = 1.5%) and between KK and KKX2 (FST = 1.8%), were lower than the FST between KK and Brakmas (FST = 3%) and between KK and Charoke (FST = 4%). KKX1 and KKX2 represent unplanned breed types and the KK was probably the most common breed used in the mating, thus being a major gene contributor. The Brakmas and Charoke are synthetic breeds developed using planned breeding design, and, therefore they are more different from the KK than the other KK crosses. The genetic makeup of a synthetic breed is not easy to manage and monitor; it is influenced by inbreeding and selection for fitness and desired traits (fertility, fleshing ability, mature weight and coat colour, etc.) which may be bias to one of the parental breeds. In this study, it is clear that all breed types were bias towards the KK breed genes.
The degree between breed differentiations indicated a relatively moderate to high gene flow between the six cattle breeds (Nm = 4.38). The highest gene flow (16.84 %) was between the KK and KKX1 population. The gene flow between the KK and KKX1 populations (16.84%), between KKX1 and KKX2 populations (13.65 %), and between KK and KKX2 populations (13.39%) reflect the genetic similarity between these three breeds, supporting the findings based on the F statistics. High gene flow was also observed between the Brakmas and KKX2 (9.71%) and the Charoke and KKX1 (9.04%) also indicating their genetic closeness between these two pairs. The earlier association may be due to the fact that both Brakmas and KKX2 are crossbreds of KK and Brahman. As for the second pair, both Charoke and KKX1 shared the same ancestral KK population which was then kept as a nucleus herd at MARDI Station in Kluang, Johor. However, due to funding shortage this KK herd was not maintained and the animals were crossed with the different available breeds. Moreover, since both these breed types were from the same farm, there is a possibility that at times there was interbreeding between the two herds. Although the Brakmas and Charoke are crossbreds of KK they showed low inter breed gene flow (5.04%) compared to the others pairs. This could be attributed to the physical separation of the two breeds, and the breeding and selection programs practised in the respective farms; the latter may have been bias towards the genes of the exotic breeds.
The genetic admixture in the KK cattle and the KK breed types was estimated using structure analysis and frequency analysis of the zebu and taurine diagnostic alleles. The results of the structure analysis showed that the studied populations were split into three clusters: KK and KKX2 in the first cluster, Brakmas and Brahman in the second cluster and Charoke in the third cluster. KKX1 was distributed in all three inferred clusters. KK, Charoke and Brahman had more than 80 % membership coefficients in their respective inferred clusters. The genetically defined clusters agreed with the breeds’ histories. Although the KK and Brahman breeds are assumed to be pure breeds, the results showed that both these breeds had admixed (hybrid) individuals. There were genetic contributions from the Brahman (11%) and Charoke (5%) breeds to the KK cattle. The Charoke contribution may be due to a possible use of Charolais or Charoke semen for artificial insemination in this herd. It could also be attributed to the introduction of KK crosses from other farms (government and non government) which may have had Charoke as one of its ancestors into the KK herd. The results of the present study are in agreement with Payne  who stated that the majority of indigenous cattle breeds of Southeast and East Asia are subjected to crossbreeding, and so have genes from the Bos taurus and Bos indicus species.
The existence of admixed individuals in the Brahman breed may be attributed to the fact that the Brahman cattle in the present study were imported from Australia (Australian Brahman), which in turn originates from founder population imported from United States of America (USA). According to the American Brahman Breeders Association (ABBA) the Brahman breed in the USA was developed in the early 1900s from progeny of four Indian cattle breeds with some infusion of British-bred cattle .
The structure analysis also showed that there were contributions from KK to Brakmas (17%) and the Charoke (8%), though these were less compared to the contributions of KK to the composite crosses, 49% to KKX1 and 73% to KKX2. These results are concordance with the F-statistics and gene flow results. Once again this could be attributed to the effects of the breeding designs and selection programs for both Brakmas and Charoke that ensured that high proportion of the genes from the exotic breeds maintained in the synthetic breeds. The KKXI was identified as having a complicated genetic background. The animals of this breed type displayed membership in all the three clusters. This finding was consistent with the KKX1’s history, which revealed that its ancestors were crossed with different breeds, which included both zebu and Taurine breeds. This explained the high TNA, MNA and Ho. These genetic characteristics of the KKX1 may also be the result of a lack of breeding goals and controlled mating for this herd. Although the KKX2 is outcome of unplanned crossing of KK and Brahman animals, the results revealed higher genetic similarity between the KK and the KKX2 than the Brahman. This result corroborated the gene flow results between KKX2 and KK (13.4%) and between KKX2 and Brahman (4.9%). This is as expected in outcome of most crossbreeding activities at non-research farms. The mating beyond the initial crosses producing the F1 are not controlled to ensure the desirable proportion of the parental breeds. Often the crosses are backcrossed with the indigenous breed as these animals are more readily available and in larger numbers, thereby, the eventual population losing a large proportion of the exotic genes incorporated into the crosses.
Concerning Zebu and Taurine diagnostic alleles, the results show that Charoke had the highest proportion of African and European Taurine diagnostic alleles among the six breed types (7.4% and 4.7%, respectively). This was as expected as Charoke was a Charolais (B. taurus) cross, whereas the other crosses were B. indicus types. The introgression of Indian Zebu genes into the KK and the KK breed types (18.4 – 25.8%) was higher than African zebu genes (2.5 - 7.4%) and the European Taurine genes (1.6 – 5.2 %). The high frequency of the Indian Zebu diagnostic alleles is supported by the history of introduction of Zebu animals into Southeast Asia, where it was believed that the Indian Zebu cattle was spread from India through the human migrations and ancient sea trading routes . Similar levels of introgression of Indian zebu genes (17 − 26.3%) in seven indigenous cattle breeds in central and southern China have been reported by Zhang . Higher level of introgression of Indian zebu genes into indigenous cattle breeds from North Ethiopia (55.16 - 63.78%) was reported by Zerabruk  and among west-central African cattle breeds (58.1−74.0%) by Ibeagha-Awemu . This may be due to the fact that Ethiopia has been a gateway for cattle immigrations into Africa. It was believed that a major wave of B. indicus introgression may have started with the Arab settlements along the east coast of Africa . In general, the analysis of the diagnostic alleles produced results suggesting that the KK and the Brahman breeds in the present study was not genetically pure Zebu; they exhibited a proportion of Taurine backgrounds. This result was in agreement with that of the structure analysis. As stated earlier the taurine alleles are possibly the result of historical crossbreeding activities in the country using taurine breeds to improve production of the local cattle.
All phylogenetic trees reconstructed from the NJ method, based on the four genetic distance methods yielded trees, which were consistent with the historical information. Generally, the accuracy of the phylogenetic tree is confirmed by bootstrap values; nodes with high bootstrap values (above 0.70) are considered significant, whereas nodes with low values (below 0.50) were considered not significant. The tree topologies generated in the present study were confirmed by relatively high bootstrap values, ranging from 56 to 89. Concerning the four genetic measures, all trees showed similar results. However, the trees obtained using Cavalli-Sforza  and Nei’s DA genetic distances had high bootstrap values (80 – 89) compared to the Goldstein’s and Shriver’s trees (56 – 73).
The PCA revealed the relationship between KK and the other breed types. The central position of Brakmas between KK and Brahman, and of KKX1 between KK and Chaoke as revealed by PCA have been clarified by their admixed nature. The distant positioning of Brakmas and Charoke is evidence of high genetic divergence between these two breed types. The results indicate high frequency of KK genes in KKX1 and KKX2 higher than those of the exotic breeds that have been used in the initial crossing. In contrary, the Brakmas and Charoke have high frequencies of the genes of the exotic breeds. This may be attributed to these breed types being developed through planned crossing and selection for high performance traits of the Brahman and Charolais, respectively. These results are in agreement with those of the structure analysis and the phylogenetic analysis.