H. Jianlin,1,2 J.W. Ochieng,2 J.E.O. Rege3 and O. Hanotte2
1. International Yak Information Centre, Gansu Agricultural University, Lanzhou 730070, Gansu, P.R. China
2. International Livestock Research Institute (ILRI), P.O. Box 30709 Nairobi, Kenya
3. ILRI, P.O. Box 5689 Addis Ababa, Ethiopia
We have used three cattle Y-specific microsatellite loci (Edwards et al. 2000) and a mitochondrial DNA (mtDNA) specific marker (Ward et al. 1999) to assess the level of cattle introgression within one Bhutanese and four Chinese yak populations. Successful PCR amplifications both in female and male yak were obtained with microsatellite INRA126. Microsatellites BM861 and INRA189 were Y-specific in both cattle and yak with the presence of Bos taurus, B. indicus and B. grunniens diagnostic alleles. Moreover, marker INRA189 was shown to be polymorphic in yak with three alleles. Only one yak male (Gannan yak) had taurine cattle Y chromosome. Mitochondrial DNA (mtDNA) multiplex PCR reactions allowed us to assess the level of female cattle introgression. We only detected three female yak with a cattle mitochondrial DNA out of a total of 239 animals, one female in the Bhutanese and two females in the Tianzhu White yak population. Our results indicate that within the five yak populations studied the level of cattle introgression originating from the initial F1 crossbreeding female cattle with male yak is low.
Keywords: Cattle, diversity, introgression, microsatellite, mitochondrial DNA, yak
The yak (B. grunniens) and the cattle (B. taurus) are two separate species belonging to the same genus included within the family Bovidae. However, reproductive isolation between the two species is incomplete with the hybrids F1 female fertile and the hybrid F1 male sterile. Crossbreed males will only be fertile after the third generation (B3, Figure 1) of backcross with yak or cattle. F1 hybridisation between the two species is commonly practised as a very efficient way to improve the milk and meat production in intermediate altitudes (1000–3000 metres above sea level).
Redrawing according to Zhao Zenrong 1957, cited in Cai 1990; A and D show the two patterns of backcrossed hybridisation most frequently practised. The phenotypic appearance of B4 yak backcrosses in lineage A and D will often be indistinguishable from the parental yak. Similarly, the phenotypic appearance of B4 cattle backcrosses in lineage B and C will often be indistinguishable from the parental cattle. Lineage A and B will have a cattle mitochondrial DNA genome. Lineage C and D will have a yak mitochondrial DNA genome. Cattle introgression in lineage D and yak introgression in lineage B will only be detected using autosomal specific markers with cattle or yak diagnostic alleles.
Figure 1. Hybridisation patterns of yak with Bos taurus cattle.
The hybridisation of yak with B. taurus cattle in China can be traced back to 3000 years ago. According to written history, the ancient Qiang people started to practise hybridisation of yak with cattle during the Yin Dynasty (approximately 1100 B.C.) (Cai 1990). Additionally, it is thought that Wencheng, the Princess of Tang Dynasty, brought some Chinese B. taurus cattle to Tibet when she married Srong-Brtzan-Sgam-Po, the King of Tibet, in 641 A.D. (Zhang 1989).
The nomenclature of hybrids (yak × cattle crosses) is relatively complex and often the same cross will have different names in different regions or countries. There are two ways to produce hybrids (Figure 1). However, the desired F1 crossbreed will be more often obtained through the crossing of male cattle with female yak. Subsequent backcrossings are not very popular amongst farmers.
Intensive crossbreeding of yak with B. taurus cattle was first practised in China with cattle imported from The Netherlands in Xikang of Sichuan Province in the late 1930s by Prof Chen Zhichang, the pioneer in the field of animal science in China, but the programme was later abandoned for unknown reasons (Zhang 1989). Shortly after the foundation of People's Republic of China in 1949, similar large hybridisations breeding schemes were carried out in Qinghai, Sichuan, Gansu and Tibet. However, currently the crossbreeding of yak with cattle is not practised intensively in China in a controlled way.
To estimate the level of cattle introgression in today's yak populations of China and Bhutan, we used three cattle Y-specific microsatellite loci (Edwards et al. 2000) and one mitochondrial DNA (mtDNA) typing system (Ward et al. 1999) in four Chinese and one Bhutanese yak populations.
One Bhutanese (BHU) and four Chinese yak populations (Gannan yak (GY) in Luqu County, Tianzhu Black yak (TBY) in Tianzhu County, Tianzhu White yak (TWY) in the Tianzhu White yak Breeding Farm of Gansu Province, and a crossbreed of domestic yak with wild yak (QY) in the Datong Yak Farm of Qinghai Province) were studied. For mitochondrial DNA analysis two DNA samples were used as a positive control: a male F1 N'Dama × Kenyan Boran cross and a Siri cattle B. taurus sample both having a taurine mitochondrial DNA genome. For Y-specific microsatellite analysis only one positive control was used (a F1 N'Dama × Kenyan Boran cross with a B. indicus Y-chromosome). Information regarding the size of the Y-specific taurine and indicine alleles were obtained from Edwards et al. (2000) after correction to take into account the differences in allele size calling methods between the two studies.
Total genomic DNA was extracted from blood, following either the method of Sambrook et al. (1989) or the salting-out procedure of Montgomery and Sise (1990).
The control region cattle specific primers (mtD1: 5'– AGC TAA CAT AAC ACG CCC ATA C –3' and mtD2: 5' –CCT GAA GAA AGA ACC AGA TGC –3') were used in a multiplex PCR reactions with the highly conserved primers located on the 16S rRNA gene (mtR1: 5'– CCC GCC TGT TTA TCA AAA ACA T –3' and mtR2: 5–CCC TCC GGT TTG AAC TCA GAT –3'). PCR amplifications were performed in 15 µl containing 60–80 ng of DNA, 10 pmol of each primer, 0.5 units of Taq polymerase (Promega), 0.15 mM of each dNTP (Amersham), 1 × PCR buffer (10 mM Tris-HCl, pH 8.3) including 50 mM KCl, 0.001% gelatin (Sigma), 0.25% Nonidet P40 (BHD) and 1.3 mM MgCl2. The amplification programme—performed on a GeneAmp (Applied Biosystems) 9700 thermal cycler—was an initial denaturation step at 95°C for 3 min, then 30 cycles at 94°C for 30 sec, 55°C for 1 min and 74°C for 1 min. A final extension step at 74°C for 10 min was adopted for all amplifications. PCR products were analysed on 1.5% (w/v) ethidium bromide stained agarose gel for 1 hour at 150V in a 1 × TBE buffer. Results were viewed on a UV transilluminator and a photograph of the gel was taken using a Polaroid system camera (Kodak).
Two hundred thirty nine successful mitochondrial amplifications were obtained (GY, No. = 34; QY, No. = 53; TWY, No. = 59; TBY, No. = 33; and BHU, No. = 60).
Primer DNA sequences for INRA126, INRA189, BM861 can be found in Edwards et al. (2000). PCR amplifications for Y-specific primers were performed in 10µl containing 20–35 ng of DNA, 5 pmol of each primer, 1 unit of Taq polymerase (Promega), 0.125 mM of each dNTP (Amersham), 1× PCR buffer (10 mM Tris-HCl, pH 8.3) including 50 mM KCl, 0.001% gelatin (Sigma), 0.25% Nonidet P40 (BHD) and 2 mM MgCl2. The amplification programme—performed on a GeneAmp (Applied Biosystems) 9700 thermal cycler—was an initial denaturation step at 95°C for 3 min, then 30 cycles at 95°C for 30 sec, x°C for 1 min, (where x = 55–61°C), INRA126 = 55°C, INRA189, = 58°C, BM861 and 72°C for 1 min. A final extension step at 72°C for 7 min was adopted for all amplifications. PCR products were analysed on a 5% denaturing polyacrylamide gel using an ABI377 DNA sequencer and the internal size standard GENESCAN 350-TAMRA. Data were collected and analysed with the ABI PrismTM 377 (version 2.1) and GeneScanTM 672 (version 3.1) softwares. The third order least square method was used for size calling. Results were analysed using the GenotyperTM (version 2.0) software.
Data were obtained for 78 male and female samples for INRA126 (BHU, No. = 8; GY, No. = 28; and QY, No. = 42), 82 males for BM861 (GY, No. = 27; QY, No. = 23; TWY, No. = 6; TBY, No. = 2; and BHU, No. = 24) and 83 males for INRA189 (GY, No. = 29; QY, No. = 26; TWY, No. = 4; TBY, No. = 2; and BY, No. = 22).
One Y-specific cattle microsatellite, INRA126, was successfully amplified in both male and female yak (No. = 78). It confirms the previous observation by Edwards et al. (2000), based on six female yak samples, that this locus Y-specific in cattle is not Y-specific in yak. We observed two size alleles with all animals being monomorphic, a 182 bp allele in 77 animals and a 184 bp allele in one male yak (GY 31), the later being an allele of taurine origin (Edwards et al. 2000). It is possible that the yak X-chromosome has retained a homologous sequence to the Y chromosomal segment containing the INRA126 microsatellites (Edwards et al. 2000). However, it should be noted that in no animals we observed two alleles not even in the male yak showing the Y-cattle specific allele.
Microsatellites BM861 and INRA189 were both Y-specific in cattle and yak with B. taurus, B. indicus and B. grunniens diagnostic alleles. BM861 amplifies two different size alleles, 149 bp in 81 males (yak diagnostic allele) and a 159 bp (taurine diagnostic allele) in a unique male (GY 31), the same animal showing an allele of taurine origin at INRA126. Similarly, only one male, again GY 31, had a taurine Y-specific allele of 92 bp at INRA189. The other 82 males showing large size alleles (see below). The mtDNA of GY 31 is of yak's origin (data not shown).
The observation that one male yak had a cattle Y-chromosome was unexpected. Indeed the male F1 progeny between yak and cattle are known to be sterile. Most likely F1 males are present in the Gannan population and this male was sampled by mistake.
INRA189 is polymorphic in yak. Excluding the 92 bp taurine specific allele, we observed three other alleles of 98, 100 and 102 bp length (Figure 2). The frequencies of these alleles vary between yak populations. Only one allele (98 bp) is observed in the two males analysed of the Tianzhu Black yak (TBY), but at least two alleles are observed in the four other yak populations studied. The most common allele in the Bhutanese yak (21 males out of 22) is the 98 bp. On the contrary, the most common allele in the Gannan yak (21 males out of 29) and the Tianzhu White yak (3 males out of 4) is the 100 bp. The crossbreed population (QY) from three wild yak bulls with domestic female yak is the only one showing three alleles including the 102 bp allele not observed in any other populations. It supports that we may find in wild yak, genetic variation absent from domestic populations of yak.
Figure 2. Allelic variation observed at the Y-specific microsatellite INRA 189.
The results of the multiplex mtDNA PCR amplification of one Siri cattle, one N'Dama × Kenyan Boran cross and six yak samples are shown in Figure 3. The 16S band is present in both yak and cattle and it is used as an internal control. The PCR primer pair used to amplify the 357 bp of the mtDNA control region band is specific of cattle mtDNA (Ward et al. 1999) and it will not amplify the yak' mitochondrial DNA. Amongst 239 successful amplifications, only one female in the Bhutanese population (BHU) and two females in the Tianzhu White yak (TWY) populations have a cattle mtDNA. Following the possible crossbreeding schemes as described in Figure 1, these three animals would have inherited a cattle mitochondrial DNA through the A lineage with an initial crossbreeding with a female cattle (Figure 1).
Figure 3. Results of a multiplex mtDNA PCR amplification from one Siri cattle, one Gambian N’Dama × Kenyan Boran cross (NDKB), and 6 yak samples (TWY4, TWY1, BHU3, GY10, QY20, BHU7) using cattle mtDNA control region specific primers and 16S rRNA primers known to amplify both in yak and cattle (Warld et al. 1999). Samples with the 357bp length fragment of the mtDNA control region have the cattle mtDNA haplotype. It is detected in three yarks (TWY4, TWY1, BHU3).
Only one male (Gannan yak population) out of 82 to 83 males analysed had a cattle taurine Y-chromosome. It is not surprising, as the F1 crossbreed male cattle × female yak is known to be sterile. Our results suggest that a F1 male has been sampled by mistake in the Gannan herd. Interestingly, INRA189 is polymorphic in yak with at least three alleles. Further studies using a large number of populations and animals should clarify the geographic distribution and frequency of these alleles and may identify others.
Our study using cattle diagnostic mitochondrial DNA marker indicates that cattle introgression through the female lineages is either absent or low in the five yak populations studied. Indeed, out of 239 animals, only one female in the Bhutanese populations (BHU) and two females in the Tianzhu White yak (TWY) have the cattle mtDNA. However, as these results do not take into account possible cattle introgression through the male lineage (Figure 1, lineage D), it is possible that we are underestimating the level of cattle introgression in the populations. Such pattern of cattle introgression will only be detected using autosomal markers showing cattle diagnostic allele(s).
The authors thank Mr Qi Xuebin, Mr Tao Shixin, Mr Yang Xichao, Prof Lu Zhonglin and Mr Liang Yulin for providing invaluable assistance to blood sampling in the field and DNA extraction in the lab.
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