An advanced sheep (Ovis aries, 2n = 54) cytogenetic map and assignment of 88 new autosomal loci by fluorescence in situ hybridization and R-banding.

Presented herein is an updated sheep cytogenetic map that contains 452 loci (291 type I and 161 type II) assigned to specific chromosome bands or regions on standard R-banded ideograms. This map, which significantly extends our knowledge of the physical organization of the ovine genome, includes new assignments for 88 autosomal loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), by FISH-mapping and R-banding. Comparison of the ovine map to the cattle and goat cytogenetic maps showed that common loci were located within homologous chromosomes and chromosome bands, confirming the high level of conservation of autosomes among ruminant species. Eleven loci that were FISH-mapped in sheep (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) are unassigned in cattle and goat. Eleven other loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific ovine chromosome (OAR) bands but have only been assigned to chromosomes in cattle and goat.

Introduction

Cytogenetic maps, available for several domestic ruminants, are useful tools for studying complex animal genomes and chromosome evolution among bovid species (Piumi ; Robinson ; Di Meo ; 2002; 2005; Iannuzzi ,b; 2001a) and between bovid species and humans (Schibler ; Di Meo ; 2002; 2006; Iannuzzi ; 2001a). Assignment of individual genes and markers to the physical map allows the identification of rearrangements within conserved chromosome segments that have been designated using chromosome painting probes (Hayes 1995; Iannuzzi ; 1999), as well as defines the complex rearrangements that differentiate humans and bovids. Other practical applications of these maps are in clinical cytogenetics, to better define the chromosomal rearrangements and abnormalities that may be involved in abnormal phenotypes (Iannuzzi ; Pinton ). Cytogenetic maps are also essential for anchoring linkage and RH maps to specific chromosome regions and to define the order and orientation of linkage groups for which there is poor evidence from the RH and linkage mapping data (Gautier ).

Although many loci have been assigned to cattle, sheep and goat genomes by linkage and RH mapping, a relatively small percentage of loci have been physically located to single chromosomal regions or bands. Unfortunately, very few studies have used both RH and FISH data for confirmation of RH-map construction across whole chromosomes or specific regions (Gautier ).

The only cytogenetic map for sheep is available through SheepBase (http://www.thearkdb.org/species.html). This map includes a few well-positioned markers, but uses an old ideogram that differs from that reported in the latest standard chromosome nomenclature (ISCNDB 2001). A more detailed cytogenetic map covering all chromosome regions and constructed on the basis of the latest international chromosome nomenclature (ISCNDB 2001) is still lacking in this very important species.

In this study, a new and advanced cytogenetic map of sheep that contains 452 loci and covers almost all of the chromosome bands (mainly R bands) is presented. The map uses published data and the latest standard chromosome nomenclature (ISCNDB 2001). The map includes 88 loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), assigned by FISH and R-banding for the first time in sheep.

Materials and methods

Synchronized peripheral blood cell cultures and slide preparation steps were carried out as reported earlier (Di Meo ). Caprine BACs containing type I and type II loci were identified by PCR screening of the INRA goat BAC library (Schibler ) and have been previously used to build comparative maps between ruminants, pig, horse and humans (Schibler ; Di Meo ; 2002; 2006; Iannuzzi ; 2001a; Pinton ; Milenkovic ; Hayes ). Likewise, bovine BAC clones containing type I and type II loci were identified after PCR screening of the INRA bovine BAC library with appropriate primers as described by Eggen . This BAC library was used to construct a first draft of a physical map of the bovine genome and over 26 000 BAC clones of the library were end-sequenced and are thus available as BES (BAC-end sequences) in GenBank (Schibler ; 2006). Cattle and goat BAC libraries are available to the entire research community through the GADIE Biological Resources Center (http://www-crb.jouy.inra.fr/BRC/index.html). Table 1 summarizes information about all BAC probes used for this study.

Table 1

Autosomal loci mapped by FISH to ovine (OAR) chromosomal locations.

Locus symbol	Locus name	Clone	OAR	BTA	HSA
D3S32 (ILSTS096)	DNA segment	0337C071	1p13	3q	————-
TCHH (previous alias: THH)	trichohyalin	240D12	1p21	3q21	1q21-q23
CDC20	CDC20 cell division cycle 20 homolog (Saccharomyces cerevisiae)	13A62	1p35	3q35	1p34.1
CCT8	chaperonin containing TCP1, subunit 8 (theta)	325A10+ 802F111	1q12.2	1q12.2	21q21.3–21q22.1
CASR	calcium-sensing receptor (hypocalciuric hypercalcaemia 1, severe neonatal hyperparathyroidism)	139D121	1q31	1q31	3q21-q24
UMPS	uridine monophosphate synthetase (orotate phosphoribosyl transferase and orotidine-5′-decarboxylase)	296A82	1q31	1q31	3q13
AGTR1	angiotensin II receptor, type 1	361C82	1q41dist	1q42	3q21-q25
GYG1 (previous alias:GYG)	glycogenin 1	399A52	1q41dist	1q42	3q24-q25.1
TFDP2	transcription factor Dp-2 (E2F dimerization partner 2)	290G122	1q43	1q43prox	3q23
TF	transferrin	163H42	1q43dist	1q43dist	3q21
COL6A1	collagen, type VI, alpha 1	0133E091	1q45	1q45	21q22.3
D1S86 (BMS922)	DNA segment	0866A051	1q45	1q	————-
GALT	galactose-1-phosphate uridylyltransferase	112G92	2p13	8q13	9p13
VLDLR	very low density lipoprotein receptor	349B112	2p17	8q17	9p24
SFTPC	surfactant, pulmonary-associated protein C	0533D051	2p23prox	8q21dist	8p21
GSN	gelsolin (amyloidosis, Finnish type)	253E102	2p27dist	8q28	9q33
EN1	engrailed homolog 1	438B72	2q33	2q33	2q13-q21
SLC11A1 (old alias: NRAMP1)	solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1	264F42	2q43	2q43	2q35
PAX3	paired box gene 3 (Waardenburg syndrome 1)	337A32	2q43	2q43	2q35-q37
TMEM50A (old alias: SMP1)	transmembrane protein 50A	262H6+ 284H52	2q45	2q45prox	1p36.11
TGFA	transforming growth factor, alpha	211C112	3p14	11q14	2p13
BMS2621	DNAs segment	372H081	3p14	11q	————-
SFXN5	sideroflexin 5	0904C051	3p14	11	2p13
POMC	proopiomelanocortin (adrenocorticotropin/beta-lipotropin/ alpha-melanocyte stimulating hormone/beta-melanocyte stimulating hormone/beta-endorphin)	503E32	3p24	11q24dist	2p23
D5S3 (ETH10)	DNA segment	0356G021	3q21prox	5q	————-
D5S68 (BMS1658)	DNA segment	0771B051	3q33	5q	————-
HGF	hepatocyte growth factor (hepapoietin A; scatter factor)	217E92	4q22prox	4q15dist-21	7q21.1
NPY	neuropeptide Y	342C12	4q26 prox	4q25-q26	7p15.3
SSBP1	single-stranded DNA binding protein 1	264C102	4q34 dist	4q34dist	7q34
VAV1	vav 1 oncogene	72E32	5q15prox	7q15prox	19p13.2
GM2A	GM2 ganglioside activator	336E122	5q15	7q21	5
HSPA4	heat shock 70 kDa protein 4	544F111	5q22.1	7q22.1	5q31.1-q31.2
CSKB071	DNA segment	0078E011	5q22.1	7q	————-
D7S49 (BMS792-D0S246)	DNA segment	0478C121	5q22.3	7q	————-
IL12B	interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40)	0006B031	5q24	7q23-q24	5q31.1-q33.1
HADH (old alias:HADHSC)	L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain	232A92	6q15prox	6q15prox	4q22-q26
D6S29 (IDVGA65)	DNA segment	0980A051	6q17	6q22	————-
HMGCR	3-hydroxy-3-methylglutaryl- Coenzyme A reductase	39C12	7q13prox	10q12	5q13.3-q14
MYH7	myosin, heavy polypeptide 7, cardiac muscle, beta	86E22	7q15	10q15-q21	14q11.2-q13
MGAT2	mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyl- transferase	359E102	7q24	10q24	14q21
SORD	sorbitol dehydrogenase	201F12	7q32	10q32	15q15-q21.1
SPTB	spectrin, beta, erythrocytic (includes spherocytosis, clinical type I)	194B42	7q34prox	10q34prox	14q24.1-q24.2
TGM1	transglutaminase 1 (K polypeptide epidermal type I, protein-glutamine- gamma-glutamyltransferase)	265C82	7q34	10q34	14q11.2
TGFB3	transforming growth factor, beta 3	161F122	7q34dist	10q34dist	14q24
D9S15 (BM2504)	DNA segment	0006E021	8q14	9q	————-
B3GAT2	beta-1,3 glucuronyltransferase2 (glucuronosyltransferaseS)	60B091	8q14	————-	6q12
D9S16 (CSSM025)	DNA segment	016H121	8q16	9q17-q21	————-
ASCC3	activating signal cointegrator1 complex subunit3	914D121	8q21.2	————-	6q16
D9S55 (BMS345)	DNA segment	0163E121	8q22	9q	————-
RARSL	arginyl-tRNA synthetase-like	890B112	8q24	————-	6q16.1
CYP11B1	cytochrome P450, family 11, subfamily B, polypeptide 1	115F112	9q13	14q13	8q21-q22
D14S19 (RM180)	DNA segment	(0517E01)-517G011	9q15	14q	————-
D14S47 (BMS1941)	DNA segment	(0234A01)239A012	9q17	14q	————-
BRCA2	breast cancer 2, early onset	334F12	10q15	12q15	13q12-q13
ACACA	acetyl-Coenzyme A carboxylase alpha	42D122	11q13	19q13	17q21
MYH2	myosin, heavy polypeptide 2, skeletal muscle, adult	120C22	11q17	19q15-q16	17p13.1
LAMC2	laminin, gamma 2	191B72	12q23	16q23	1q25-q31
ITGB1	integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)	132H12	13q13dist	13q13dist	10p11.2
RAB18	RAB18, member RAS oncogene family	554C82	13q15prox	13q15prox	10
PSMA7	proteasome (prosome, macropain) subunit, alpha type, 7	946B22	13q22 prox	13q22 prox	20
DPEP1	dipeptidase 1 (renal)	262F42	14q13	18q13	16q24
MC1R	melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor)	132F32	14q13	18q13	16q24.3
GNAO1	guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O	527F2+657E32	14q15	18q15	16
PTGIR	prostaglandin I2 (prostacyclin) receptor (IP)	276E42	14q24dist	18q24dist	19q13.3
SLC6A3	solute carrier family 6 (neurotransmitter transporter, dopamine), member 3	72F42	16q24	20q24	5p15.3
IL2	interleukin 2	129G52	17q22	17q22dist	4q26-q27
NOS1	nitric oxide synthase 1 (neuronal)	208D82	17q24	17q25	12q14-qter
COMT	catechol-O-methyltransferase	475C72	17q26	17q26	22q11.21-q11.23
MITF	microphthalmia-associated transcription factor	52G52	19q22	22q22	3p14.1-p12.3
PBX2P1 (old alias: PBXP1)	pre-B-cell leukaemia transcription factor pseudogene 1	130G122	19q22	22q22	3q23-q24
BRD2	bromodomain containing2	948D011	20q13	————-	6p21.3
POLR1C	polymerase(RNA) I polypeptide C, 30 Kda	237C051	20q15dist	————-	6p21.1
PPP2R5D	protein phosphatase2, regulatory subunit B (B56),delta isoform	364A091	20q15dist	————-	6p21.1
TNRC5	trinucleotide repeat containing 5	364A091	20q15dist	————-	6pter-p12.1
BAT2	HLA-B associated transcript2	660D101	20q22prox	————-	6p21.3
BAT4	HLA-B associated transcript4	660D101	20q22prox	————-	6p21.3
C4B	complement component 4B	573A101	20q22	23q12d-q13p	6p21.3
HSPA1B (old alias:HSP70-2)	heat shock 70 kD protein 2	0573C021	20q22	23q22	6p21.3
CDC5L	CDC5 cell division cycle 5-like (S. pombe)	192C021	20q22	————-	6p
OLA-DRA2	major histocompatibility complex, class II, DR alpha	589B091	20q22	————-	6p21.3
D29S35 (BMS1112)	DNA Segment	0133G061	21q13	29	————-
LDHA	lactate dehydrogenase A	0039C071	21q22	29q22	11p15.1
DNTT	deoxynucleotidyltransferase, terminal	169D32	22q21	26q21	10q23-q24
PAX2	paired box gene 2	99A102	22q21dist	26q21	10q25
OAT	ornithine aminotransferase (gyrate atrophy)	84B52	22q23 dist	26q23prox	10q26
CYB5A (old alias: CYB5)	cytochrome b5 type A (microsomal)	369C22	23q12	24q12	18q23
DSG2	desmoglein 2	312D12	23q21	24q21-q22	18q12.1
F11	coagulation factor XI (coagulation factor 11) (plasma thromboplastin antecedent)	334A102	26q15	27q15	4q35

Bovine

caprine

BAC clones, as well as comparisons with both cattle (BTA) (BovMap; Hayes ) and human (HSA) (HUGO, known genes) chromosome locations are reported.

Labelling of probes was done with biotin or digoxigenin with BRL-Gibco and Roche kits respectively. Ethanol precipitation was carried out in the presence of bovine COT-1 DNA or caprine genomic DNA for bovine and caprine BAC clones respectively to suppress repetitive sequences. In situ hybridization, signal detection, chromosome staining, microscope observation and image processing were described before (Di Meo ). At least 20 metaphases were examined for each probe. Chromosome identification and band nomenclature for sheep chromosomes followed the R-banded standard ideogram reported in the latest international chromosome nomenclature (ISCNDB 2001). Only loci assigned to specific chromosome bands or regions in the present and previous studies, as well as those reported in SheepBase and references therein), were considered. Symbols of type I and type II loci followed HUGO (http://www.gene.ucl.ac.uk/nomenclature/) and BovMap (http://locus.jouy.inra.fr/cgi-bin/bovmap/intro2.pl) nomenclatures respectively. GoatMap data were fromhttp://locus.jouy.inra.fr/cgi-bin/lgbc/mapping/common/main.pl?BASE = goat.

Results and discussion

The frequency of hybridization signals on both chromosomes and chromatids, or on a single chromosome or chromatid, varied between 35% (ASCC3) and 81% (UMPS). All mapped loci were localized on homologous ovine chromosomes and chromosome bands when compared with cattle and goat positions. A few apparent differences between published and expected localizations were due to the banding techniques used in different studies. The data confirmed the high conservation of autosomal chromosomes among the bovid species. Loci FISH-mapped in the present study with locus name and symbol, clone identification and chromosome localization in sheep, cattle, goats and humans are listed in Table 1. Of these loci, 11 (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) have been FISH-mapped in sheep only. An additional 11 loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific sheep chromosome bands but only to whole chromosomes in cattle and goat (Table 1). Ten loci (BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, C4B, HSPA1B, CDC5L and OLA-DRA) were assigned to OAR20 extending the physical organization of this chromosome, which contains the major histocompatibility complex of sheep.

The revised sheep cytogenetic map, including loci previously mapped to specific chromosome bands or regions and the loci mapped in the present study on standard R-banded ideograms, is shown in Fig. 1. A total of 452 loci were assigned to specific chromosome bands or regions of sheep chromosomes, of which 291 are type I and 161 are type II, extending the cytogenetic map and density of markers available for this economically important species. These loci are also listed in, which includes the localization of all FISH-mapped loci in sheep and cattle and/or goat, the bovine syntenic groups and references.

Figure 1

The new comprehensive sheep cytogenetic map on the latest standard R-banded ideograms (ISCNDB 2001). The 452 loci include 291 type I loci (presented in normal characters) and 161 type II loci (in italics). Also see. Loci mapped in the present study are reported in bold.

Comparative mapping with human (Table 1) confirms previous comparative mapping data available from BovMap, SheepBase and GoatBase. Alignment of the cytogenetic map locations of loci between sheep, cattle and goats confirms the high degree of autosomal chromosome conservation among these bovid species, although some major discrepancies in the location of loci between OAR and BTA (or CHI) were observed: TNP1 (OAR2q33-q34, BTA2q42-q43), KRT1 (OAR3q21, CHI5q25), HEXA (OAR7q12, BTA10q15dist), ANK1 (OAR26q17, CHI27q19) and D9S6 (OAR9q24, CHI9q26). The assignment of D9S6 merits further investigation because OAR9 is currently designated as homologous to CHI14, not CHI9 (ISCNDB 2001). Other minor discrepancies were noted (designated as more than two bands of difference) for D6S29 (OAR6q17, BTA6q22) and C4B (OAR20q22, BTA23q12-q13).

Cattle, sheep and goat autosomes have been arranged using only one common chromosome banding system in the latest chromosome nomenclature of bovid species (ISCNDB 2001), as a result of their high chromosome banding similarities. In addition, all 31 bovine (and ovine/caprine) syntenic groups have been definitively assigned to specific chromosomes on the basis of official marker assignments performed with both G/Q and R-banded chromosomes of cattle (Hayes ), as well as in those of both sheep and goat chromosomes (Di Meo ). The use of a common chromosome banding system among chromosome of bovid species allows easier comparison of physical maps.

The new cytogenetic map presented here will be a useful tool for further studies on both molecular and clinical cytogenetics of this species. In addition it will allow a better anchoring of linkage (Maddox ) and future RH maps by providing independent evidence for the localization and orientation of markers on specific chromosome regions. With few exceptions, all sheep chromosomal bands have at least one locus in the ovine cytogenetic map described here. Use of this cytogenetic mapping data, along with linkage and RH-mapping information, will greatly advance our understanding of the physical organization of the sheep genome and provide a sound platform for local and complete sequencing of the sheep genome.