agarose gel and transferred onto Positive™ mem- branes Appligene, France according to manufac-
turer’s instructions. Hybridizations were carried out as described
previously [3] at 65°C with randomly primed cDNA probes or at 50°C with oligonucleotide
probes.
2
.
4
. Gene mapping and sequencing YAC library screening was performed and yeast
DNA was prepared according to Ref. [32]. Se- quencing was performed either according to Ref.
[33] using the sequenase version 2.0 kit USB, USA, or by automated sequencing Company
ESGS, France.
Mapping data were processed by D. Bouchez INRA Versailles, France with respect to physical
mapping and Sean May Nothingham with re- spect to restriction fragment length polymorphism
RFLP mapping. Other routine DNA manipula- tions were as in Ref. [34].
2
.
5
. Bioinformatics Identification and search for LTP through data-
bases GenBank, The Arabidopsis Information Resource, TAIR was performed using both key-
word searching and the basic local alignment search tool BLAST BLASTX, BLASTN and
TBLASTN softwares [35]. Sequence comparisons and phylogenic analyses were performed using the
ClustalW software [36] and Phylip package [37] which is based on the neighbor joining method
validated by bootstrap statistical analysis. Mature amino acid sequences were aligned by ClustalW
using a PAM matrix. The results from the align- ment were used for constructing the tree using the
neighbor joining method. Those programs were accessed from the Infobiogen web server http:
www.infobiogen.fr, using the default options un- less otherwise indicated.
3. Results
3
.
1
. Identification of ltp-related cDNAs Expressed sequence tag EST database Univer-
sity of Minnesota was searched for files contain- ing the words lipid and transfer. More than 200
entries were found. The cDNAs were classed into families based on The Institute for Genomic Re-
search TIGR tentative consensus. The remaining sequences were compared to these consensus and
the ESTs that presented more than 93 identity over a 100 nucleotide stretch were considered as
being encoded by the same gene. Only ‘typical’ LTPs were retained, that is, proteins that con-
tained around 120 amino acid, with the typical LTP amino acid pattern [4]. Larger proteins 150
aa and more, and 7 kDa LTPs [38] were excluded. Six different classes were defined and the largest
representative from each class was sequenced and characterized. These cDNAs were designated ltp1
to 6; ltp1 and ltp2 corresponding to the ltp1 and ltp2 genes already described by Ref. [18] and ltp3
is likely to be identical to Clark and Bohnert ltp3 gene [39]. In addition, ltp4 is identical to ltp-a2
which was purified from a crude cell wall prepara- tion [25]. BLAST alignments were carried out
using these six genes and gene products as ‘probes’ and nine additional ltp genes were identified.
These genes were designated ltp7 to ltp15 Table 1.
3
.
2
. Sequence analysis All of the deduced proteins share a similar
hydrophobic profile, and contain a typical signal peptide [40]. The characteristics of the proteins
coded for by these cDNAs are summarized in Table 1. The number of amino acid ranges from
112 to 123 or from 89 to 98 if the signal peptide is excluded. The isoelectric point of the mature
proteins is usually close to 9, with the exceptions of ltp5 11.4, ltp6 and ltp12 7.7, ltp15 7.5.
Interestingly, ltp8, ltp9 and ltp14 are acidic, with pI of 4.9, 5.2 and 4.2, respectively. These are the
first acidic plant ltps ever reported. These proteins are rich in alanine, glycine and cysteine residues,
and devoid of glutamic acid except for ltp11 to ltp14, tryptophane except for ltp14 and histidine
except for ltp13, except for ltp9 and ltp15 which contain all 20 amino acids. The Arabidopsis
ATA7 protein [41], which appears to be related to ltps, has been added to Table 1. It is slightly larger
than ltps 114 amino acids plus 26 residues for the signal peptide.
When the nucleotide sequences are compared, the identity between two sequences is always lower
than 60, except for the couples ltp1ltp2 and
ltp4ltp3 which exhibit 79 and 77 homology, respectively. Because the sequence identity is re-
stricted to a smaller area, only ltp1 and ltp2 were found to cross hybridize when washed at moder-
ately stringent conditions 65°C, 0.3 M NaCl.
All genes for which both the gene and the cDNA sequences are available were found to con-
tain an intron 9 – 12 nucleotides upstream from the stop codon. Aside from ltp8 and ltp12 intron size
close to 450 nt, the size of this intron is always close to 100 nucleotides.
When compared to each other, the amino acid sequences deduced from the ltp genes exhibit from
22 to 79 identity and 55 to 90 similarity when conservative replacements are taken into account
Table 2. ATA7 shares 19 – 30 identity and 47 – 56 similarity to Arabidopsis LTPs. Cysteine
residues are conserved in all sequences Fig. 1. The central region of the protein is also strongly
conserved residues 42 – 57 and the residues val 7, leu 11, tyr 17, gly 21, gly 34, leu 38, asp 48, arg 49,
leu 77, pro 78 can be found in more than 80 of the sequences.
The comparison with other plant mature LTP proteins can be done through a phylogenetic tree
Fig. 2. A large homogenous group group 1 that contains only dicot genes comprises 32 members
from Nic.ta1 to Pha.vu. Within group 1, several clades can be distinguished that follows botanical
classification. This is obvious for Cruciferae and Solanaceae. A second group Group 2 appears
more heterogeneous: it comprises six Arabidopsis LTP proteins plus ATA7 [41], two castor bean,
two tobacco, two rice and one pine tree proteins. Monocotyledons can be considered to belong to a
third group Group 3, which includes a barley clade which seems to represent a transition of
some sort with group 1.
Table 1 Characteristics of mature Arabidopsis ltps deduced from cDNA sequences
a
MW kDa Amino acid number
Group TIGR Intron size
pI BAC
EST Gene
9.3 120
25+ 93 AC005499
118F16T7 ltp1
9.3 TC8103
TC9063 107
AC005499 124J8T7
9,4 ltp2
25+ 93 9,4
TC14044 TC14108
ltp3 23+ 92
86C11T7 9,2
9,1 AB016890
95 TC8698
ltp4 9,1
8,8 23+ 89
TC9488 108
AB016890 90B4T7
TC14119 111
TC10173 25+ 93
9.9 11,4
ltp5 124E14T7
AL133452 TC13850
ltp6 7.7
9,9 19+ 94
TC11002 88
AC012562 174N16T7
10.5 25+ 98
No 9.7
No intron? AC006439
No EST ltp7
ltp8 AI997024
AC005957 439
No 25+ 91
9 4.9
28+ 90 9.8
ltp9 5.2
No EST AC007267
No intron? No
TC53883 No BAC
22+ 94 9.9
ltp10 9.4
? AI998609
246G16T7 275H8T7
108 AL035678
No 8.5
ATTS5582 9.3
28+ 89 ltp11
7.7 No
9.7 25+ 93
464 AL133452
K2B3T7 ltp12
27+92 ltp13
No ?
AB011475 8.5
10.6 No EST
? AB016880
No EST No
ltp14 25+ 91
9.6 4.2
11 No EST
7.5 AF076275
? No
22+ 96 ltp15
26+ 114 12.7
6.5 ATA7
AF037589 AC006439
2 introns
a
For each gene, the expressed sequence tag EST sequenced is mentioned and the accession number of the bacterial artificial chromosome BAC indicated. The size of the intron typical of lipid transfer proteins LTPs — which is localized at the 3 end
of the open reading frame — is indicated in base pairs; question marks indicate the lack of either gene or cDNA sequence information which cannot allow formal identification of introns. The tentative consensus TC numbers given by The Institute
for Genomic Research, TIGR [44] is indicated. The number of amino acids corresponds to the mature protein after signal sequence number of amino acids in parentheses removal. The most probable cleavage site was determined according to [40].
Molecular weight MW and isoelectric point pI determinations were performed based on the mature protein sequences. Sequence data relative to ltp7 to ltp15 and ATA7 were retrieved from GenBank.
V .
Arondel et
al .
Plant Science
157 2000
1 –
12
5
Table 2 Comparison of amino acid sequences of Arabidopsis ltps
a
Ltp5 ltp1
ltp6 ltp7
ltp8 ltp9
ltp10 ltp11
ltp12 ltp13
ltp14 ltp15
ltp2 ltp3
ltp4 25.2 51.3
26.3 47.4 27.2 50.8
29.8 53.5 20 42.6
23.1 51.3 19 47.4
22.6 52.2 20.2 49.1
28.9 50 22.4 47.4
20.2 48.2 22.6 44.3
26.7 56 ATA7
23.9 52.9 39 66
55.6 76.8 33.3 69.9
26.3 58.9 42.6 79.8
37.6 63.4 44.8 76
64 81 25 55.9
ltp1 24 59.1
19.4 54.2 54 78
54 79 47 75
45.4 72.2 37.2 66
48 66 33 67
22.3 55.3 28.9 66
35.2 57.1 44.3 70.1
21.9 55.9 19.8 61.3
16.8 46.9 47.373.9
ltp2 49.574.7
46.3 78.7 48.5 78.8
41.3 79.3 28.7 60.6
46.9 77.1 42.4 67.4
52.6 84.2 54.679.4
24.5 54.8 79.390.2
25.3 62 22 53.1
ltp3 45.7 75.3
51.5 76.8 40.2 78.3
30.8 60.6 41.4 70.7
41.3 65.2 53.7 76.8
ltp4 27.1 57.6
24.5 57.1 22.4 49
49.576.3 36.7 67
48.5 69.7 34.4 64.5
28.1 56.2 41 71.6
36.6 60.2 43.8 70.8
24.2 56.4 ltp5
25 60.2 19.4 53.1
37 72 42.4 77.2
27.8 55.6 55.3 81.9
38.9 64.2 41.2 69.1
22.9 48.9 ltp6
26.8 62.8 20.4 53.1
32.7 68.4 26.5 57.1
38.4 72.7 36 60
42.6 68.3 24.2 51
24.2 53.1 18.4 48
ltp7 25 55.4
35.7 71.4 41.3 70.6
33.7 66.3 24.2 54.3
22.6 60.4 17.2 44.8
ltp8 25 54.2
31.5 57.6 25.8 54.6
26.9 56.5 18.5 58.2
22.5 51 ltp9
33 57.4 35.1 70.1
19.8 48.9 ltp10
27.8 60.6 16.5 51
ltp11 38.9 61
26.1 59.8 23.7 57.1
22.3 55.2 22.7 57
23.5 62.8 19.6 58.3
ltp12 27.7 56.5
ltp13 20 43.8
25.2 58.3 ltp14
a
The percentage of identical residues is indicated. The number of similar residues i.e. when conservative replacements are taken into account is indicated between parentheses. Ltp1 is from Thoma et al. [18] and ATA7 from Rubinelli et al. [41].
Fig. 1. Sequence alignment of ltp-deduced proteins of Arabidopsis. The probable cleavage site of the signal peptide was determined according to Ref. [40], and only the mature deduced amino acid sequences were aligned using the ClustalW software
[36], followed by processing with the EDITALN software. Amino acids that are strictly conserved are indicated by the corresponding symbol. , amino acids conserved in 80 of the sequences; + , in 60; :, in 40; and ., in 20.
3
.
3
. Southern blot hybridization analyses When hybridized and washed at high stringency
68°C, 15 mM NaCl, each probe reveals only one band on DNA restricted with most enzyme chosen
data not shown. At lower stringency hybridiza- tion at 50°C, 1.2 M NaCl, washes at 50°C, 0.3 M
NaCl, several additional bands can be detected, depending on the probe and the restriction enzyme
used. Ltp1 and ltp2 exhibit a similar pattern of hybridization. This is due to the strong nucleotide
similarities between the two genes combined with the fact that both genes are tandemly repeated on
chromosome 2 see below. The same holds true in the case of ltp3 and ltp4 which locate to chromo-
some 5. The ltp12 family comprises two strong bands plus up to five additional weak bands de-
pending on the enzyme and the ltp34 family one strong band plus up to four additional weak
bands. Ltp5 exhibits usually one to two bands while ltp6 detects one to two strong bands and up
to four additional weak ones. This suggests that ltp1 and ltp2 represent a small subfamily of two to
three genes, ltp3 and ltp4 a subfamily of two genes, ltp5 and ltp6 two subfamilies of one or two
genes each. The weakly hybridizing bands 10 – 12 might correspond to other ltps or to unrelated
genes Fig. 3.
3
.
4
. Gene expression studies Northern analyses where carried out on total
RNA isolated from different tissues. All genes were found to be highly expressed in flowers, and
the transcript can always be detected in siliques. Virtually no RNA can be detected in roots, while
the expression of the genes varies in leaves. Only ltp1, ltp2 and ltp5 are significantly expressed in
leaves. While the level of ltp5 mRNA seems to remain constant during the development of the
leaf up to its senescence, the expression of ltp1 is maximum in young leaves and decreases with time
after bolting. Ltp1 mRNA is barely detected at day 45, when the leaves start senescing. Ltp 6 is
slightly expressed during the first 24 days after germination Fig. 4.
ABA was found to induce strongly the expres- sion of ltp4 and ltp3 in fully expanded leaves,
while it does not alter significantly the expression of the other ltp genes.
Because there might be some cross reactions between the entire cDNA probes, the same experi-
ments were carried out using specific oligonucle- otides, and identical results were obtained data
not shown.
3
.
5
. Mapping experiments Ltp4 and ltp2 genes were found to detect an
RFLP between Columbia and Landsberg using EcoRV as restriction enzyme. They were mapped
using 90 recombinant imbred lines, and found to locate on chromosome 5, cosegregating with
marker m211A ltp4 and on chromosome 2, be- tween markers m323 2.2 cM and m529 3.4 cM
ltp2. Ltp1, ltp5 and ltp6 were found to hybridize to CIC YACs 3G1 and 2G9 for ltp1, 8E1, 7A4,
10B4, 9C9, 9D9, 10A11, 11G7 and 6F4 for ltp5, 1C12, 10B10 and 11D1 for ltp6. These data indi-
cate that ltp1 maps to chromosome 2 close to position 71cM, ltp5 to chromosome 3 close to
Fig. 2. Fig. 2. Phylogenetic tree for plant ltps. The phylogenetic tree
was built using the protein sequences indexed in GenBank, after removal of the signal peptide [40]. The neighbor joining
methodUPGMA version 3.573c from the PHYLIP package [37] was used, as indicated in Section 2. Sequences are men-
tioned by six first letters of the Latin name of the plant from which they were obtained followed by a number when there
were several lipid transfer protein LTP in a same species. Their accession numbers in GenBank are: Bras.na: Brassica
napus 1: X60318 2: U22175 3:U22105 4:U22174-Bras.ra: Brassica rapa L31938-Bras.ole: Brassica oleracea 1: L33904 2:
L33905 3: L33906 4: L33907-Hor.vu: Hordeum 6ulgare 1: U18127 2: Z37114 3: X68656 4: X68654 5: X96979 6: Z66529,
Z66528, U63993 7: U88090 8: X60292, X59253 9 Z3715- Tri.du:
Triticum durum X63669-Ory.sa: Oryza sati6a 1: U16721 2: U29176 3:D15364 4:D22795 5: D16036 6:U29176
7: U29176 8: X83433 9: X83434 10: Z23271 11: X83435 12: D15678- Zea: Zea mays 1: U66105 2: J04176-Sor.bi: Sorghum
bicolor 1: X71667 2: X71668 3: X71669-Nic.ta: Nicotiana tabacum 1: D13952, 2: U14167, 3:U14168, 4: X62395-Ric. co:
Ricinus communis 1: M86353 2: D11077-All.ce: Allium cepa S79815-Pha.vu: Phaseolus 6ulgaris: U72765-Pin.ta: Pinus
taeda U10432-Pru.du: Prunus dulcis 1: X96714 2:X96716- Ger.hy:
Gerbera hybrida Z31588-Hel. an: Helianthus annuus X92648-Dau.ca: Daucus carota M64746-Gos.hi: Gossypium
hirsutum 1: U64874 2:U15153-Lyc.pe: Lycopersicon pennellii 1:
U66466 2:
U66465-Lyc.es: Lycopersicon
esculentum
U81996-Spi.ol: Spinacia
oleracea M58635.
Arabidopsis
thaliana clones are ATA7 AF037589, ltp1, ltp4, ltp3, ltp4, ltp2, ltp6. Ltp 10 aminoacid sequence is deduced from ex-
pressed sequence tags ESTs and ltp7, ltp8, ltp9, ltp11, ltp12, ltp13, ltp14 and ltp15 are deduced from whole BAC se-
quences.
position 72 and ltp6 to chromosome 3. Ltp 1 and 2 appear to be close together on chromosome 2,
ltp4 and ltp3 on chromosome 5. Based on genomic sequencing and mapping data available from the
TAIR database, ltp7, ltp8 and ltp9 map to the same position 29 cM on chromosome 2, while
ltp12 is clustered with ltp5. Ltp11 and ltp15 lo- cates to chromosome 4 89 and 31 cM, respec-
tively, ltp13 and ltp14 to chromosome 5 94 and 117 cM, respectively and the map position of
ltp10 remains unknown Fig. 5. 4. Discussion
Lipid transfer proteins are an ubiquitous protein family in higher plants, whose biological function
remains unknown. One of the problems encoun- tered in studying LTPs is the number of isoformes
that can be detected. For instance, more than ten genes have been described in rice [17]. Arabidopsis
is the most suitable organism for obtaining an exhaustive collection of ltp isoformes, because the
small size of its genome suggests that genes families are likely to contain few members. The
main reason for using Arabidopsis for such a study is that an important part of its genome has
been already sequenced, and that many ESTs are available. ESTs are particularly well suited for
looking for LTPs since these proteins are short and almost half of the amino acid sequence is
included in an average EST. The other reason is that LTPs are frequently expressed at high levels,
and their mRNA represents usually a few percent of a plant total mRNAs. They are therefore well
represented in cDNA libraries. This was found to be the case for ltp1 to ltp6, while ltp8, ltp9 to ltp12
are represented by one to three ESTs. No EST could be found for the other genes.
A total of 14 ltp genes could be evidenced in Arabidopsis based on 80 of the genome sequence
and ten through EST analysis, for a total of 15 different genes. It is therefore likely that the Ara-
bidopsis 9 kDa ltp gene family contains about 15 – 20 genes. However, sequencing errors might
hamper the discovery of additional genes. For instance, a couple of nucleotide modifications
should be enough to detect another gene on bacte- rial
artificial chromosome
BAC T1N24
AF149413. The Southern blot hybridization data suggest that ltp genes consist in small subfamilies
of one to three genes which may, or may not, weakly cross hybridize at low stringency. Clark
and Bohnert [38] have recently characterized three cDNAs ltp1 to ltp3 In A. thaliana Wassilewskija
ecotype and the corresponding ltp1 and ltp2 genes [16,39]. These three members of the LTP
family are similar to those presented in the study although performed in a different ecotype. These
authors have also noted that there was very little crosshybridization, even between ltp1 and ltp2.
Therefore, Southern genomic DNA analyses are likely to underestimate the number of ltp genes.
Fig. 3. Southern blot hybridization analysis of ltp gene family in Arabidopsis. Columbia genomic DNA 1 mg per lane was
restricted during 4 h with 5 U of the restriction endonucleases BamHI 1, BglII 2, ClaI 3, EcoRI 4, EcoRV 5,
HindIII 6, XbaI 7 and XhoI 10. The DNA was fraction- ated on agarose gel, transferred to nylon membrane, and
probed with
32
P-labeled cDNA probes. The hybridizations were carried out at 50°C in 0.6 M NaCl, and the washes were
performed in 2 × SSC, 0.1 SDS at 50°C. Membranes where exposed to X-ray films between intensifying screens. The
molecular weight markers were Lambda DNA restricted by HindIII; they are indicated by stars from top to bottom, in
kilobase pairs: 23.1; 9.4; 6.5; 4.4; 2.3; 2.0; 0.56 on the left side of each membrane.
Fig. 4. Northern blot hybridization analysis of ltp gene expression. Total RNA 10 mg were fractionated on 1.5 formaldehyde- agarose gels, transferred to a nylon membrane and probed with cDNA inserts labeled by random priming using [
32
P]dCTP. Hybridizations were carried out at 45°C in 50 formamide. Washes were performed at 62°C in 2 × SSC 0.1 SDS. A Rab18
probe was used as a positive control for abscisic acid ABA induction, and a ribosomal DNA probe rib to assess for even loading of the RNA. RNAs were from 40-day-old plants for organ specific expression studies A, from 28-day-old leaves for
ABA induction C, and from leaves extracted at different stages of development B. The induction by ABA was carried out in 10
− 4
M for 24 or 48 h. L, leaves; S, siliques; F, flowers; R, roots.
Mapping data indicate that, although ltp genes can be found scattered through four chromosomes
of Arabidopsis, four clusters exist that comprise nine genes. Tandem repeats can be noted based on
genomic sequences
AC005499, AB016890,
AL133452 for ltp5-12, ltp4-3 and ltp1-2 [38]. The two last pairs exhibit more than 75 identity at
the nucleic acid level. Taken together, these data strongly suggest that these are duplicated genes.
Other pairs of tandemly repeated LTP genes have been characterized in various plants, such as ltp1
and ltp2 from Sorghum 6ulgare [42], Wax 9D and Wax 9C from Brassica oleracea [21] and LTP 4.2
and LTP4.3 in barley [43]. Another characteristic of those genes is the existence of an intron located
at the end of the open reading frame. This exis- tence is demonstrated for all the eight genes for
which both cDNA and gene sequences are avail- able. This intron appears to be a general feature of
ltp genes in plants.
The RNA hybridization analysis of all genes tested
indicate that
transcripts are
always present in flower and siliques. The main differ-
ences between the genes is their pattern of expres- sion in leaves, which varies especially with
regards to development. Clark and Bohnert [39] also noted that ltp1, ltp2 and ltp3 are highly
expressed in flowers and siliques. However, it has been found that ltp2 is also expressed in leaves. All
transcripts tested were undetectable in roots, which is also a general feature of plant ltps. It has
been observed that ltp4 and ltp3 from Arabidopsis are up-regulated by ABA. A similar result has
been obtained for LTP genes from other plants such as rice [17] and rapeseed [14]. Interestingly,
the promoter region of the ltp4 gene from barley [43] contains elements which could be linked to
ABA responsiveness. In Arabidopsis [16] the pres- ence of several expression-controlling motifs in
ltp1 gene, also found in ltp2 gene by [39], similar to those previously reported in plant genes in-
duced by various types of stress or by pathogen attack was observed.
The phylogenetic tree is very similar to those already published [17,39]. The group I defined by
Ref. [17] corresponds to the groups I and II, while group III corresponds to group II by Vignols et al.
[17]. As a control all rice sequences published by these authors have been included and their differ-
ent classes can be found at the same position in this tree. Concerning dicots it seems plausible that
two different ancestral genes exist: one corre- sponding to group I and the other one to group
III.
Interestingly, Arabidopsis
genes are
not far from being equally represented in both groups nine genes in group I versus six genes in
group III. Monocots and Gymnosperms are also represented in group III, so it is likely that the
putative ancestral gene to this group has started to differentiate early in higher plant evolution. Con-
cerning group II, the barley subgroup appears to be much more closely related to group I than to
group III. The situation appears to be different for the other monocots of group II. This discrepancy
might be due to the software used, and the fact that this tree is unrooted. In any case, the exis-
tence of at least two different groups appears clearly.
It has been suggested by Ref. [39] that the ancestor to Brassicaceae possessed already several
copies of ltp genes. The phyletic analysis suggest that this ancestor might have possessed no less
than five copies, A precursor to ltp9, 13 – 15, B precursor to ltp8, 11, C precursor to ltp6, 10, D
precursor to ltp1, 2, 5, 7 and E precursor to ltp3, 4, 12. The closeness of genes such as ltp3
and ltp4 suggests that this family of gene seems to continue to duplicate.
Fig. 5. Map position of ltp genes in Arabidopsis. Ltp1 and ltp2 were found to hybridize to yeast artificial chromosomes
YACs CIC 3G1 and 2G9, which map to chromosome 2 position 71 cM close to restriction fragment length polymor-
phism RFLP markers m429 and ve018. These two genes are tandemly repeated on bacterial artificial chromosome BAC
T6A23 AC005499. Ltp3 and ltp4 map to chromosome 5 position 111 cM close to RFLP markers hst and agp50.
They are tandemly repeated on bac MNC17 AB016890. Ltp5 hybridizes to YACs CIC 8E1, 7A4, 10B4, 9C9, 9D9,
10A11, 11G7 and 6F4. It locates to chromosome 3 position 71 cM, together with ltp12, close to markers AtEm1 and
m457. ltp6 maps to chromosome 3 position 29 cM, close to markers g4523 and mi357. Ltp7, ltp8 and ATA7 [41] are
contained in BACs T30D6 AC006439, T15J14 AC005957, ATF 20O9 AL021749, respectively, which map to chromo-
some 2 position 29.2 cM, chromosome 2 position 29.2 cM and chromosome 4 position 73.9, respectively. Ltp7 and ltp8
do not appear to be tandemly repeated. Ltp 9 is at the locus AC007267 chromosome 2 29.2 cM, ltp11 in BAC ATF17M5
AL035678, chromosome 4, 89cM, ltp13 in BAC K9L2 AB011475, chromosome 5, 94 cM, ltp14 in BAC MTG10
AB016880, chromosome 5, 117 cM and ltp15 in BAC T15F16 AF076275, chromosome 4, 31 cM.
5. Conclusion
