A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome one (BBU1)

Abstract

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR₅₀₀₀. The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH₅₀₀₀) panel. The retention frequency of individual markers across the panel ranged from 17.8% to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.

Introduction

As livestock, the river buffalo (Bubalus bubalis) plays an important role in the livestock world economy by contributing high quality milk and meat for human consumption. Despite its economic importance, the river buffalo genome is not as intensively studied as other livestock species, such as domestic cattle. Thus, genome mapping of river buffalo remains important for identification of genes affecting economic traits.

The karyotypes of buffalo and domestic cattle appear very similar at the level of chromosome arms. While the cattle genome consists of 29 acrocentric autosomes and a pair, X/Y, of sexual chromosomes, the river buffalo genome has 5 biarmed and 19 acrocentric autosomes plus the X and Y chromosomes. According to previous studies and this latest map, all buffalo chromosomes arms have homology to single bovine acrocentric chromosomes. Buffalo (BBU) chromosome 1 appears to be a fusion of Bos Taurus (BTA) chromosome 1 and 27, BBU 2 equals BTA2 and 23, BBU3 equals BTA8 and 19, BBU4 equals BTA5 and 28, and BBU5 equals BTA16 and 29 at the cytogenetic level with state of the art banding (El Nahas, et al., 2001; Ianuzzi et al., 2003). All the other chromosomes have a one to one correspondence between the two species. Assignment of genes to these buffalo chromosomes to date is consistent with the cytogenetics prediction.

The late reports regarding the river buffalo genome mapping (Ianuzzi et al., 2003; Di Meo et al., 2006), describes a total of 302 loci (180 of type I and 122 of type II) physically assigned to its genome. Of the 302 loci, 256 were mapped by in situ hybridization (254 by FISH), 15 by both FISH and somatic cell hybrid analysis and 33 by using only somatic cell hybrid analysis. BBU1, the largest of the five biarmed chromosomes in the river buffalo genome, has only 13 genes and 10 microsatellites assigned by FISH or somatic cell panel mapping (Iannuzzi et al. 2003; Di Meo et al. 2006). In contrast, the third generation bovine RH map includes 67 markers and 175 markers assigned to BTA27 and to BTA1, respectively (Everts-van der Wind et al. 2005). BTA27 is known to contain economic trait loci (ETLs) influencing clinical mastitis (Goldammer et al. 2004), and both bovine chromosomes contain quantitative trait loci (QTLs) affecting milk yield, as well as milk fat and protein content (Polineni et al. 2006).

Taking advantage of buffalo-bovine homologies and the extensive resources now available as a result of the bovine genome sequencing project, the goal of this study was to construct the first radiation hybrid (RH) map of BBU1 by utilizing markers chosen from BTA1 and BTA27.

Materials and methods

Sixty-nine markers (including coding genes, ESTs and microsatellites) identified on BTA1 and BTA27 from the previous publications were typed on the RH panel as described elsewhere (Amaral et al. 2007). Most of these markers appeared on at least one of the genome-wide linkage and RH maps (Ihara et al. 2004; Everts-van der Wind et al. 2004; 2005); the original source for each marker is listed in Table 1.

Table1.

Cattle-derived markers mapped to BBU1 based on the BBURH₅₀₀₀ panel, along with their retention frequencies (RF), distances on the RH map, PCR primer sequences, annealing temperatures, GenBank Acessions and UniSTS ID.

Marker	Type	BBU1 linkage group	BTA chr	RF (%)	Marker position distance cR	Foward primer (5’-3’)	Reverse primer (3’-5’)	GenBank Acession	Reference/UNISTS ID	Tm (°C)
SH2D4A	gene	LG1	27	30.0	placed	TGCTGAAACAGATCCTGTCG	GGACTCCTGTTTTTCCATCG	XM_582664	new designed§	58
PLAT	gene	LG1	27	32.2	0.0	AGACTTTGTCTGCCAGTGCC	CTCTCTGCCGTGCTCCAC	X85800	251598	65
HOOK3	gene	LG1	27	30.0	placed	CTAGGGCAGCAGATCAATGAC	CAGCACAGCCTAAAATGAGC	XM_602978	new designed§	60
THAP1	gene	LG1	27	28.8	placed	AGAAGCTGAAGGAGGTGGTG	CACGCTGGGACTTCGACTAT	NM_001034648	new designed§	58
ADAM2	gene	LG1	27	28.8	20.5	CTCTGCACTCCAGCACCATA	GGGCAATGGCATTTGTTAAT	NM_174228	new designed§	58
BRF2	gene	LG1	27	31.1	44.6	TGAGCTCGTGGAAGACTCG	CGTCACTGAAGGTGGTCGTA	NM_001015582	new designed§	58
CSSM036	MS	LG1	27	34.4	52.4	GGATAACTCAACCACACGTCTCTG	AAGAAGTACTGGTTGCCAATCGTG	U03827	250971	65
INRA134	MS	LG1	27	34.4	placed	CCAGGTGGGAATAATGTCTCC	TTGGGAGCCTGTGGTTTATC	X73125	250829	59
WRN	gene	LG1	27	35.5	90.8	AACGCCATATATAGCAAGAC	GATCCAAAACAAAGCAAGAA	BE217410	279411	56
DCTN6	gene	LG1	27	32.2	113.4	CGTATGTGGGCAGAAATGTG	GCAGGCAGTCTCCACCATAG	NW_930571	new designed§	58
NRG1	gene	LG1	27	27.7	121.8	ACGGAAAAAGCTTCATGACC	ACCAGCTGCACGTTCTCG	XM_592564	new designed§	58
C8orf79	EST	LG1	27	33.3	139.0	TCTGAAAGTGAACAGCCAGGT	TGACGCCTATGGAGATGATG	AW632623	new designed§	58
CNOT7	gene	LG1	27	38.8	167.5	CCACCCAGTTACAGCATTGA	GGAATATGTGACCAGACCAGAG	AW289341	278215	64
RM209	MS	LG1	27	33.3	186.4	GTAGAAGTTAGTGACTGTCATCC	CCTCAGAGCCCCATACATTTCC	U32921	250931	65
MTNR1A	gene	LG1	27	34.4	placed	TTTCAACAGCTGCCTCAATG	GGAGAGGGTTTGCGTTTAAT	U73327	278796	61
SLC25A4	gene	LG1	27	34.4	199.6	CATTGATTGCGTGGTGAGAA	ATAATGATGCCCTGGACCGA	M24102	Li and Womack, 1997	65
BM1856	MS	LG1	27	38.8	215.2	GGCCTCAAGTTTCATCCATG	CATCAGCATGAAGCAACCC	G18402	53060	65
ODZ3	gene	LG1	27	33.3	233.8	TGACCAACGTGACATTTCCA	TTGAACCATGGTGTAGAACGA	XM_865074	new designed§	58
CARF	gene	LG1	27	27.7	257.5	AGCACTGCCTCTCAGGTGAC	CGATCCACTTTGTTGTCTGG	XM_590926	new designed§	58
DEFB1	gene	LG1	27	30.0	285.4	GGAAGACAGGAAGGCCTCTGG	CCTCACGTTTTCAGAACCAC	NW_001501821	251704	64
BM6526	MS	LG1	27	37.7	317.3	CATGCCAAACAATATCCAGC	TGAAGGTAGAGAGCAAGCAGC	G18454	28213	65
TGLA179	MS	LG1	27	40.0	placed	CTTTAATCAGCACACAGCTTCCCA	ATATGTGCTAGAAGTTTGGTCAACC	CM000203	251232	65
BMS1001	MS	LG1	27	42.2	327.1	GAGCCAATTCCTACAATTCTCTT	AGACATGGCTGAAATGACTGA	G18605	44386	65
DLGAP2	gene	LG1	27	42.2	359.4	CGGACCTCCATCCACTCC	CGTCCCTGCTGTCATAGTGG	XM_600714	new designed§	58
CLN8	gene	LG1	27	46.6	369.0	CCCTTCATGTGTCCAATGC	ATCCAGGAGATGCAGGTGAA	XM_609353	new designed§	58
IFNAR1	gene	LG1	1	37.7	441.6	TGAAGATAAGGCAATAATAC	TGAAGAGTTTTTCCAGATAA	BE217555	279374	54
AW267109	EST	LG1	1	45.5	462.7	ATGAAAGTCTTCTGTGCCATGC	TCAGTATTGCCACATGACATGC	AW267109	278038	60
IFNGR2	gene	LG1	1	51.1	472.2	AGACAAAGGCACAGCATTCCAC	GATTTCAAAGGGAGAGCTGGGT	AW289292	278555	60
BM6438	MS	LG1	1	52.2	placed	TTGAGCACAGACACAGACTGG	ACTGAATGCCTCCTTTGTGC	G18435	79562	63
ADAMTS1	gene	LG1	1	45.5	placed	CGACACAAGAGAGGAAAGATGG	CATCACTTTACCCGCTGCTATG	AW428596	277932	60
C21orf45	EST	LG1	1	47.7	placed	CCACCAAGGAAATCCAGCATAC	TATCCAAGTCACCTCCAGGTCA	AW463565	278143	60
KRTAP8	gene	LG1	1	46.6	503.2	TTGCTGAAATACCAGAGGCA	ATGACAAGAGTCATGAGCATGG		Harlizius et al, 1997	59
TGLA49	MS	LG1	1	50.0	520.3	GGCAGGACTTCACTCTTTTTCA	AGAAAAGGAATAATGAGACAGATTA	CM000177	239049	56
SOD1	gene	LG1	1	35.5	546.7	GTTTGGCCTGTGGTGTAATTGGAA	GGCCAAAATACAGAGATGAATGAA	NM_174615	Barendse et al, 1994	56
PRSS7	gene	LG1	1	34.4	619.0	CCAAGGTTCACAGAGTGGAT	GGCTTGTGACATGAGCTTAC	U09859	278971	65
RM095	MS	LG1	1	41.1	637.5	TCCATGGGGTCGCAAACAGTGG	ATCCCTCCATTTGTTGTGGAGTT	U32918	250928	58
STCH5	gene	LG1	1	38.8	652.8	TGATCTTCAGAACACGTCATAC	GTGAATGAATGTTTGGTGTCAG	AW267080	279153	56
ROBO2	gene	LG1	1	27.7	placed	GAATTGGCTGTCGATCTG	ACCTTTGTTCTGATCAGG	AW653888	279030	50
POU1F1	gene	LG1	1	34.4	710.1	GGCTGAAGAACTAAACCTGGAG	TAGGAGAGCCTATCTGCATTCG	X12657	278932	52
PROS1	gene	LG1	1	30.0	730.1	GTACAGGTGGATTTGGATGAAG	GAGAGCAACAGAGGTAAGAACA	X12891	278968	52
ESDN	gene	LG1	1	26.6	744.5	ATTGGGCTAGACCGTGCATA	GGAACCAGCACAGTTAAAGG	AW314878	278348	65
DOC1	gene	LG1	1	33.3	761.6	TTCACCAAGACAAGTTGCAG	CCCTTCCTTATCCGGTTTAT	AW289233	278315	58
ALCAM	gene	LG1	1	28.8	placed	CTGGAGAGCAGAACATGGGAAA	TATCCAGGCACTGATCCACTGA	X73801	277962	65
TGLA57	MS	LG1	1	30.0	802.2	GCTTTTTAATCCTCAGCTTGCTG	GCTTCCAAAACTTTACAATATGTAT	CM000177	250987	56
BBX	gene	LG1	1	34.4	812.9	CCAGTGAAGCGCCCTTTAAT	ACCACATGGACTCACTAGCATC	AW428450	278098	65
LOC151584	EST	LG1	1	30.0	823.6	ACCAATGCTGCCTGCTAACT	ACTCCAGGCCTTGCATGAAT	AW352867	278663	65
TACTILE	gene	LG1	1	20.0	849.5	TTGTAGACTGCACTGGTGGAAC	GGAGTCCATTGGAAGTGATCTG	AW289280	279172	65
BMS527	MS	LG1	1	25.5	904.2	TCAGTGAAAGCAAGAGAAATATCC	TCCATTCCCTTTGAATATCCC	G18871	65219	60
BM1312	MS	LG1	1	30.0	921.8	CCATGTGCTGCAACTCTGAC	GGAATGTTACTGAACCTCTCCG	G18434	30885	54
BMS4030	MS	LG1	1	30.0	938.8	TGTACCCAACACAGGAGCAC	TGACAGAGGGACCCATATCC	G19083	74568	65
CASR	gene	LG1	1	23.3	964.3	CGGTGTGCTTCTGTGGTTAGGT	CAGGCTGTCTGCAAAGTTCAGG	S67307	278154	65
PDIR	gene	LG1	1	24.4	983.6	AACTGTGGTTTGCGGAGAATAC	GAAAGTTGACCTGAGTGCAAAG	AW289373	278896	64
TRAD	gene	LG1	1	30.0	1004.7	GCGAGGAAAGGATAAAATC	ACATTTTTCCAGTTCACC	AW353439	279211	62
APOD	gene	LG1	1	26.6	1025.4	GAAGATCCCAGTGAGCTTTG	ATCAGCTCTCAGCTCCTTGT	AW357610	277980	51
PPP1R2	gene	LG1	1	26.6	1025.4	GCATCACTGCTGAATTTCAGAC	GTCAGTGTTCAGTTCTAGCCAA	AW315482	278944	64
BM6506	MS	LG1	1	26.6	1057.4	GCACGTGGTAAAGAGATGGC	AGCAACTTGAGCATGGCAC	G18455	47201	63
AHSG	gene	LG1	1	27.7	1065.9	GTCCCAACTTCTCATCCTTCCA	GGCAAGAGCACCTTTCAAAGTC	X16577	277947	60
TLOC1	gene	LG1	1	20.0	1118.9	CATAGCAGTTCTCCTGATCTGA	GTGATCTTTCTACAGCAGCTTC	AW289224	279200	65
IL12A	gene	LG2	1	22.2	placed	AAAGTCAAGCTCTGCATCCT	GTTATGAGAGACCTCAGCATTC	U14416	278561	58
SR140	gene	LG2	1	17.8	0.0	TACTACTGCCAGCAGATCCA	CTGCATCTGTAGACCTGTTG	AW289396	279143	58
RNF7	gene	LG2	1	24.4	39.6	CCTGTTCCCTGGTCCAAACTTA	GGAGTTATTGAAGCGGTTCCGT	AW428600	279028	65
FOXL2	gene	LG2	1	25.5	63.0	TCCTCCGGACGACACACTACAA	GAAATGTGAAACCCGGCAGCAG	AW267121	278443	65
CSSM019	MS	LG2	1	23.3	placed	TTGTCAGCAACTTCTTGTATCTTT	TGTTTTAAGCCACCCAATTATTTG	U03794	251074	59
NCK1	gene	LG2	1	25.5	75.6	CGCTCTTCTCTTGCTTCTAA	TGCAGAAGTAACTAAGGTGG	AW425735	278818	58
MX1	gene	LG2	1	20.0	109.2	CCGTAGTCTCTGCTGTCTCTTA	GGAATGACCCTTCTACAGTGCT	U88329	278800	65
CRYAA	gene	LG2	1	27.7	134.1	CCTAGAAAGTGGGGCATCCAT	GGTCACTCTGAGGTCTTTGCA	NM_174289	Barendse et al, 1994	64
HLCS	gene	LG2	1	27.7	152.7	TGAGCAGTGGCTGCGTTTAT	AACAGCTTCTCCTCCGTGAATC	AW354578	278525	61
CBR1	gene	LG2	1	22.2	168.9	CCCTGAACTGCAGCAGAAATTG	CCCGTTCTTTGTGTCTTCCA	AW461769	278157	61
BMS2263	MS	LG2	1	23.3	211.2	AACCCAGTCAACCAGCAAAG	CACCCCAGCCATCACTTC	G18934	2985	65

^*MS – Microsatellite

^**EST – Expressed Sequence Tag

^§New designed primers from known cattle ESTs.

Briefly, PCR reactions were performed in a MJ Research PTC-200 thermocycler with thermal gradient software. The markers were scored after amplification of DNA from the 90 radiation hybrid cell lines and control buffalo and hamster DNA. PCR mixtures included: 10mM Tri-HCl, 1.5 mM MgCl₂, 50mM KCl, pH 8.3 (20°C), 10 mM dNTPs, 0.2 mM each primer, 0.5 unit of AmpliTaq Gold polymerase (Perkin Elmer Applied Biosystems, Foster City, CA) and 50ng DNA in a 10μl-volume. The PCR conditions were as follows: initial denaturation at 94°C for 10 min, followed by 35 cycles at 94°C for 30 sec (denaturation), 50 to 65°C for 30 sec (annealing - according with the primer pair), extension at 72°C for 30 sec and a final extension at 72°C for 7 minutes.

The PCR products were electrophoresed through 2% agarose gels in 1.0X TBE buffer containing ethidium bromide, and photographed under UV light. PCR products were scored as 1 for present, 0 for absent, or 2 for ambiguous amplification. All primer sets were typed twice with the RH panel DNA and scored independently, in order to increase the accuracy of the results. Primer pairs that showed ambiguous results were typed a third time.

The BBU1 RH map construction was performed by using the software rh_tsp_map, version 3.0 (Schäffer et al. 2007) and CONCORDE (Applegate et al. 1998) linked to QSopt (http://www2.isye.gatech.edu/~wcook/qsopt/). We used the maximum likelihood criterion and our framework maps are called “MLE-consensus” maps because the markers are chosen so that the optimal order is the same for three variants of the MLE criterion that differ in the treatment of uncertain (coded as 2) entries in the RH vectors (Agarwala et al. 2000). The software distribution of rh_tsp_map tutorial (ftp://ftp.ncbi.nih.gov/pub/agarwala/rhmapping/rh_tsp_map.tar.gz) includes a tutorial describing the steps that can be used to construct a map for markers typed on a single or multiple panels. For the construction of the BBU1 map, we followed all the steps from “Preparing files” through “Placing additional markers” for making a map, but we did not proceed further to assign cR positions to the placed markers because this is a coarse map. Considering the number of markers, linkage groups were made using a pairwise LOD score threshold of 5.5.

Results and Discussion

The newly constructed BBU1 RH map (Figure 1) contained markers distributed within two linkage groups. Linkage group 1 (LG1; spanning 1118.9 cR) included a total of 58 markers (39 coding genes, 15 microsatellites and four ESTs) spanning the entire short arm and extending across much of the long arm, with 47 markers placed as framework and 11 markers placed in bins. The second linkage group (LG2; spanning 211.2 cR) included 11 markers (nine coding genes and two microsatellites) covering the remaining portion of the BBU1 long arm with 9 framework markers and two placed in bins.

Figure 1.

Comparison of BBU1 RH map (centre) with bubaline cytogenetic map (left) and cattle genome build 3.1 for BTA1 and BTA27 (right). The framework markers, whose order is better than the second best at least 0.50 LOD units, are in bold font. Placed markers assigned to the same MLE-consensus map interval are shown in boxes. Markers common to both BBU and BTA maps are joined by a solid line. A black line joins those markers on the BBU1 RH map that have been physically mapped by FISH to their location on the ideogram (Iannuzzi et al. 2003). Blue lines indicate markers on the BBU1 RH map with inverted order regarding the cattle maps and the river buffalo cytogenetic map. Comparison of SLC25A4 on the cytogenetic map with RH map is not shown as this marker is not localized to a band on 1p. The highlighted interval (4Mbp) of BTA1 map indicates the gene order based on Drogemüller et al. (2005) and Wunderlich et al (2006). *SH2D4A, DEFB1, DLGAP2, CLN8, and POU1F1 are assigned to contigs unplaced on BTA build 3.1. ^$Eighteen markers on RH MLE-consensus map and on Everts-van der Wind et al. 2005 map have a consistent marker order between the two maps. Since the Everts-van der Wind et al. (2005) map supports the marker order of RH map for PRSS7 and RM095, inversion for this pair with BTA 3.1 build is shown in green.

Of the 69 markers typed on the BBURH₅₀₀₀ panel, APOD and PPP1R2 had the same RH vector and were placed at the same position. Retention frequencies (RF) for all mapped markers ranged from 17.8% (SR140) to 52.2% (BM6438). Additional information about mapped markers, including their RF and cR position on the map is compiled in Table 1.

Because no other river buffalo linkage maps are currently available, we compared the mapped order of the markers from this new BBU1 RH map to the current bovine genome assembly (build 3.1) of chromosomes BTA1 and BTA27. The markers SLC25A4, PLAT, BM6526, DEFB1, KRTAP8, SOD1, AHSG from LG1 and the markers NCK1 and CRYAA from LG2 were also compared to their positions previously assigned by FISH (Iannuzzi et al. 2003), serving as anchor markers for the BBU1 RH map. As indicated on Figure 1, eleven inversions on the gene order were observed, including one disagreement with the order of FISH assigned markers and one inversion not supported by the map in (Everts-van der Wind et al. 2005).

This first RH map from BBU1, including 48 coding genes and 4 ESTs, is the starting point for the construction of a high resolution comparative map for this river buffalo chromosome. With our data we were able to generate a large linkage group (LG1) including markers from both bovine homologs (BTA27 and BTA1) spanning most of BBU1. The number of observed disagreements in the gene order positions among our RH map and the bovine sequence and the river buffalo cytogenetic assignment may contribute to improved maps for buffalo as well as maps for other members of the Bovidae family.