Construction of a high-resolution genetic linkage map and comparative genome analysis for the reef-building coral Acropora millepora Shi Wang, Lingling Zhang, Eli Meyer and Mikhail V Ma
Trang 1Construction of a high-resolution genetic linkage map and
comparative genome analysis for the reef-building coral Acropora
millepora
Shi Wang, Lingling Zhang, Eli Meyer and Mikhail V Matz
Address: Section of Integrative Biology, School of Biological Sciences, University of Texas at Austin, 1 University Station C0930, Austin, TX
78712, USA
Correspondence: Shi Wang Email: swang@mail.utexas.edu
© 2009 Wang et al.; licensee BioMed Central Ltd
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Coral genetic map
<p>A high-resolution genetic linkage map for the coral Acropora millepora is constructed and compared with other metazoan genomes, revealing syntenic blocks.</p>
Abstract
Background: Worldwide, coral reefs are in decline due to a range of anthropogenic disturbances,
and are now also under threat from global climate change Virtually nothing is currently known
about the genetic factors that might determine whether corals adapt to the changing climate or
continue to decline Quantitative genetics studies aiming to identify the adaptively important
genomic loci will require a high-resolution genetic linkage map The phylogenetic position of corals
also suggests important applications for a coral genetic map in studies of ancestral metazoan
genome architecture
Results: We constructed a high-resolution genetic linkage map for the reef-building coral Acropora
millepora, the first genetic map reported for any coral, or any non-Bilaterian animal More than 500
single nucleotide polymorphism (SNP) markers were developed, most of which are transferable in
populations from Orpheus Island and Great Keppel Island The map contains 429 markers (393
gene-based SNPs and 36 microsatellites) distributed in 14 linkage groups, and spans 1,493 cM with
an average marker interval of 3.4 cM Sex differences in recombination were observed in a few
linkage groups, which may be caused by haploid selection Comparison of the coral map with other
metazoan genomes (human, nematode, fly, anemone and placozoan) revealed synteny regions
Conclusions: Our study develops a framework that will be essential for future studies of
adaptation in coral and it also provides an important resource for future genome sequence
assembly and for comparative genomics studies on the evolution of metazoan genome structure
Background
Although substantial effort is being devoted to understand
physiological mechanisms of coral stress tolerance and
accli-mation [1-3], virtually nothing is currently known about the
mechanisms that might enable their adaptation to the
chang-that the coral Acropora millepora shows considerable
genet-ically determined variation in thermal tolerance and respon-siveness of the larvae to the settlement cue, which may be the raw evolutionary material for future local thermal adaptation
or modification of the larval dispersal strategy in response to
Published: 10 November 2009
Genome Biology 2009, 10:R126 (doi:10.1186/gb-2009-10-11-r126)
Received: 30 July 2009 Revised: 12 October 2009 Accepted: 10 November 2009 The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2009/10/11/R126
Trang 2map would enable identification of the quantitative trait loci
(QTLs) associated with these and other adaptation-relevant
physiological traits [5,6] To date, however, no genetic map
has been constructed for any coral species, mainly due to lack
of genetic resources for most corals
The coral A millepora, like the majority of hermatypic (algal
symbiont-hosting) corals of the order Scleractinia, is a diploid
hermaphrodite with 2n = 28 chromosomes [7] A millepora
is common across the Indo-Pacific As a representative of the
most speciose and ecologically important coral genus
Acro-pora, A millepora is becoming the leading coral model in
terms of molecular groundwork Currently, 50 microsatellite
markers are available for this species [8,9] Although these
markers are obviously not enough for linkage mapping, they
are already the largest marker collection available for any
reef-building coral Single nucleotide polymorphisms (SNPs)
are the most abundant type of genetic variation in eukaryotic
genomes, and are the preferred genetic markers for a variety
of applications such as high-resolution linkage mapping, QTL
mapping of complex traits, and for combining these results
with population genomics, which is arguably the most
power-ful way of detecting and understanding the process of natural
adaptation [10] Previously, our group has released a large
body of sequence data for A millepora obtained by 454
sequencing of the larval transcriptome [11] More than
33,000 putative SNPs have been identified in these data
Since the detected SNPs reside in or immediately next to the
protein-coding sequences ('gene-based SNPs'), they are
par-ticularly useful for QTL mapping and population genomics
studies because they have the potential for quickly identifying
causal genes underlying complex traits [12,13]
A genetic linkage map, especially gene-based, is also an
excel-lent platform for comparative genome studies Recent
com-parative genome analyses based on genetic maps have
already provided new insights into genome organization,
evo-lution, and function across different organisms [14-20] For
example, comparison of the Caenorhabditis briggsae genetic
map and the Caenorhabditis elegans genome reveals
exten-sive conservation of chromosome organization and synteny
despite a very long divergence time (80 to 110 million years),
suggesting that natural selection operates at the level of
chro-mosomal organization [14] In another study, a genetic
link-age map of the blind Mexican cavefish Astyanax mexicanus
has been successfully applied to predict candidate
quantita-tive trait genes relating to rib number and eye size by
anchor-ing cavefish QTLs to the zebrafish genome [16] The phylum
Cnidaria is the sister group of the Bilateria Anthozoan
cni-darians such as corals are phylogenetically basal in the
phy-lum Cnidaria, and have proven to be particularly informative
for understanding the evolution of metazoan genetic and
developmental complexity [21,22] Identification of
con-served synteny blocks across coral and other metazoan
genomes would help to unravel ancestral metazoan genome
architecture
Here, we report the first high-resolution genetic linkage map
for a reef-building coral, Acropora millepora, which was
con-structed based on a family of larvae from a cross between two naturally heterozygous coral individuals from Magnetic Island, Australia (an outbred full-sib cross design) An inves-tigation of SNP transferability was carried out in two more populations Sex differences in recombination were observed
in the coral linkage map Comparison of the coral map with other metazoan genomes (human, nematode, fly, anemone and placozoan) was conducted to identify syntenic regions This coral genetic map should lay a solid foundation for a variety of future genetic and genomic studies such as QTL mapping of adaptive traits, population genomics, compara-tive genomics, and assembly of the coral genome
Results SNP marker development
For SNP marker development, we designed PCR primers for 1,033 candidate SNPs, which were previously identified in the
A millepora larval transcriptome by 454-FLX sequencing
[11] After PCR amplification, 603 produced single strong bands with expected sizes, of which 427 SNPs were hetero-zygous in at least one parent of the mapping family, 91 were homozygous in both parents but for two different alleles, and
85 showed no genetic variations in two parents Although we restricted the expected amplicon length to about 100 bp in primer design, 208 primer pairs still produced single strong bands but of larger than expected sizes, indicating potential introns in the vicinity of the SNPs Longer amplicons greatly diminish the precision of high-resolution melting (HRM) SNP analysis, so most of these intron-containing amplicons were discarded Only four SNP markers developed based on intron sequences were included in this study The remaining
222 attempted SNP assays resulted in poor amplification (very little or no product) or bad melting curves, suggesting non-specific amplification
In order to evaluate the transferability of our markers in other
populations of A millepora, we randomly selected 48 SNP
markers to test their applicability on 7 colonies from 2 Aus-tralian Great Barrier Reef locations, Orpheus Island (n = 4) and Great Keppel Island (n = 3), which are 80 km and 570 km away from Magnetic Island, respectively All the 48 SNP markers could be successfully amplified in the assayed sam-ples Notably, 36 (75%) and 31 (65%) of them were still poly-morphic in the Orpheus Island and Great Keppel Island populations, respectively, despite the fact that only a few indi-viduals were assayed
Linkage mapping
Linkage analysis was carried out using JoinMap 4.0 software [23] In total, 469 markers (431 SNPs and 38 microsatellites) were heterozygous in at least one parent of the mapping fam-ily, and were therefore included in the linkage analysis Seg-regation analysis showed that 380 markers conform to the
Trang 3expected Mendelian ratios at P ≥ 0.05 level More than half of
the distorted markers depart only slightly from expected
Mendelian ratios (0.01 <P < 0.05).
At the initial logarithm of the odds (LOD) threshold of 4.5,
293 markers were grouped into 14 linkage groups, which
cor-responds to the known haploid chromosome number for this
species Then 124 markers were added to the established
groups at LOD = 3, and 14 additional markers were added at
LOD = 2.5 After data partitioning by the Joinmap 4.0
pro-gram, the maternal (1:1 female type) and paternal (1:1 male
type) datasets contained 167 and 155 markers, respectively,
which were subsequently used for constructing sex-specific
maps based on the two-way pseudo-testcross strategy [24]
The female map contains 152 markers and spans 1,185.8 cM,
while the male map contains 149 markers and spans 945.4 cM
(Figures 1, 2, 3 and 4) The female map is 240.4 cM (30%)
longer than the male map, even discounting L8 and L14
where recombination information is missing for one parent
Large differences between recombination rates in the male
and female parents were observed for linkage groups L4, L5,
L6, L10 and L11 (Table 1) Notably, we found that the
poly-morphism level revealed by markers in L8 was significantly
lower than the average in the male parent (chi-square test, P
< 0.0001) More interestingly, we found that more than half
of the annotated genes in this linkage group were putatively
involved in sexual reproduction (Table 2)
The consensus map contains 429 markers (393 SNPs and 36
microsatellites) in 14 linkage groups (Figures 1, 2, 3 and 4),
and spans 1,391 cM with an average marker interval of 3.4 cM
The length of each linkage group ranges from 46 cM to 161.5
cM Marker density varies dramatically across linkage groups (Table 1) For example, both L1 and L14 are approximately 95
cM in length, but L1 contains 59 markers whereas L14 con-tains only 12 markers Nine putative stress-related genes were identified in the consensus map (Figures 1, 2 and 3; EM and MVM, unpublished) [25,26] These genes are involved in cytoskeleton formation, heat shock, oxidative stress, protein degradation, and vesicular transport
Genome lengths estimated by two different methods [27,28]
are similar at 1,484.8 cM (G e1 ) and 1,501.9 cM (G e2), respec-tively The average of two estimates was taken as the expected genome length - 1493.4 cM Given an estimated genome size
of 200 Mbp for A millepora [1], the average recombination
rate across all linkage groups is approximately 7.5 cM/Mbp The genome coverage of the current map was estimated as 93.1%
Comparative genome analysis
Comparison of the markers mapped in this study with the previously annotated coral larval transcriptome [11] allowed the assignment of nearly all markers (97%) to longer cDNA sequences, which included all markers derived from 454 tran-scriptome sequences Of the 416 sequences so identified, 286 (69%) corresponded to known genes based on the previously described transcriptome annotation [11]; 280 genes mapped
by this process were each associated with a single marker, with 6 genes containing two markers each The accession numbers, gene annotation, and synteny information for all mapped markers are shown in Additional data file 1
Table 1
Summary of the coral genetic linkage map
Linkage group Number of
markers
Length (cM) Average marker
interval (cM)
Length in female map (cM)
Length in male map (cM)
Ratio of female/
male recombination rate
*Not available (NA) due to the lack of recombination information for one of the parents
Trang 4Comparison of the mapped sequences with assembled
genomes from other metazoans identified putative homologs
for between 48% (nematode) and 80% (sea anemone) of the
mapped coral genes, and a similar comparison with the yeast
genome identified putative homologs for 29% of mapped
coral genes These pairs of putative homologs allowed for
comparison of the coral genetic map with assembled genome
sequences of other metazoans, identifying conserved synteny
blocks in 11 of the 14 coral linkage groups, each of which
tained from 3 to 12 markers The largest synteny block
con-served between coral and another metazoan was found in
linkage group 4, with 12 markers spanning 69 cM in the coral
linkage group and their best matches spanning 5 Mb in
scaf-fold 5 of the Trichoplax adhaerens genome (Figure 5) An
overlapping set of markers within this same linkage group
also showed conserved synteny with the anemone
Nemato-stella vectensis (Figure 5) Synteny blocks were identified in
each of the metazoan comparisons; each comparison
identi-fied 4 to 13 blocks, with each block containing 3 to 12 markers
(Table 3) Most of the conserved synteny blocks identified
here involved intra-chromosomal rearrangements, in which
linkage was conserved but gene order was not (for example,
the synteny block conserved between coral and placozoan in
Figure 5) Notably, a parallel comparison between the coral
map and the yeast genome found no evidence of conserved
synteny, even though the small genome size of yeast
(approx-imately 12 Mb) would be expected to relax one of the
opera-tional criteria for determining synteny (the requirement that
matches occur within ≤10 Mb in the yeast chromosome)
We tested for the significance of synteny blocks using
ran-domly shuffled permutations of the original data, which
revealed that a non-trivial number of synteny blocks could be
expected to emerge in these comparisons by random chance
(Table 3) Although numerous synteny blocks were detected
in comparisons between coral and Drosophila melanogaster
or C elegans, the number of blocks detected was not
signifi-cantly higher than expected by chance for either comparison
(P = 0.68 and P = 0.39, respectively) In contrast, the other
three metazoan genomes we investigated each showed
signif-icantly more synteny than expected by chance (anemone, P < 0.001; placozoan, P = 0.002; human, P = 0.002) Obviously the comparison with yeast (Saccharomyces cerevisiae),
which found no conserved synteny, was unaffected by these statistical tests Each of the metazoan genome comparisons identified at least one synteny block that contained more markers (n = 6 to 12) than expected by chance These signifi-cant blocks of conserved synteny are depicted in Figure 5, and the syntenic markers in each block are described in more detail in Additional data file 1
Discussion SNP marker development in coral
Molecular markers are useful tools for assessing important ecological and evolutionary issues such as connectivity, local adaptation, range shifts, biodiversity depletion, speciation, and invasion Despite widespread concerns about the future
of reef-building corals in the changing climate, genetic resources for corals remain scarce The traditional ways of developing microsatellites or SNP markers are quite costly and time-consuming Moreover, due to technical problems and low abundance in the genome, it has been shown that development of a large number of microsatellite markers in acroporid corals is particularly difficult based on the tradi-tional microsatellite-enriched genomic library method [29] Despite the advantages of SNP markers for a variety of tasks [30], their use in non-model organisms such as corals has been hampered primarily due to the costs of high-throughput SNP discovery and genotyping With the introduction of the next-generation 454 sequencing technology, high-through-put SNP discovery is now feasible for any non-model organ-ism Our previous study [11], as well as others recently published [31-33], demonstrates a cost-effective way to
pro-Table 2
A list of genes from linkage group 8 that are putatively involved in sexual reproduction
C20407S208 20.6 Death-associated protein kinase 3 (Dapk3) Spermatogenesis [95]
C19470S311 23.3 RNA-binding protein MEX3C (Mex3c) Regulation of germ cell mitosis [96]
C12216S415 49.9 Translocon-associated protein subunit beta (Ssr2) Spermatogenesis [99]
C43885S203 52.9 Chromodomain-helicase-DNA-binding protein 1 (CHD1) Gametogenesis [100]
C12479S421 62.2 Putative tyrosinase-like protein tyr-1 (tyr-1) Spermatogenesis [101]
C6250S141 68.1 Zinc finger CCHC domain-containing protein 9 (ZCCHC9) Spermatogenesis [102]
C25187S178 76.8 SNARE-associated protein Snapin (Snapin) Spermatogenesis [104]
C63883S448 101.1 WD repeat-containing protein 47 (Wdr47) Spermatogenesis [101]
Trang 5A genetic linkage map (L1 to L4) of the reef-building coral A millepora
Figure 1
A genetic linkage map (L1 to L4) of the reef-building coral Female (F) and male (M) maps are shown on the left and right, respectively, and the consensus map is shown in the center Homologous loci are connected with solid lines Putative stress-related markers are shown in red Distorted loci are indicated
by asterisks (*0.01 <P < 0.05, ** P < 0.01; *** P < 0.001).
C13288S189
0.0
C11422S292
6.1
WGS211
13.0
C19740S286
20.4
C1024S157
21.1
C17912S202
22.4
EST254 **
28.0
C19263S650
32.1
C19862S335
34.0
C11959S269 *
36.0
C21833S285
40.9
C22405S305
43.6
C14455S306
53.4
C22875S709 *
62.2
EST181
63.9
C10466S190
75.0
C52176S400 *
75.1
C18841S310
78.2
C11099S398
82.7
C15620S247
84.7
C13698S442
90.0
C841S459
95.6
WGS079
96.5
C21349S456 0.0
C1328S290 1.9
C11524S150 * 4.7
C13288S189 5.8
C20570S83 7.3
C11422S292 9.4
WGS211 11.1 C25302S260 15.8
C24856S313 19.7
C19740S286 22.1
C1024S157 23.1
C17622S201 * 23.5
C12140S96 24.6
C17828S268
26.7 C21470S842 29.7
C3633S408 30.8
C17438S197 33.6
C17912S202 34.4
C15044S328 35.4
C45199S349 36.6
EST254 **
36.7 C19263S650 38.4
C19862S335 39.1
C35020S147 39.8
C18397S183 40.1
C11959S269 * 42.5
C22545S1379 43.0
C22826S366 44.5
C13905S483 * 47.4
C26852S307 48.2
C21833S285 49.2
C21186S526 49.9
C22405S305 51.1
C20625S210 53.0
C14455S306 54.5
C20763S245 56.2
C3729S182 59.2
C22875S709 * 60.3
C12606S270 60.7
EST181 62.1 C23083S345 62.5
C17741S312 65.0
C25628S525 68.5
WGS051 70.1 C10466S190 70.8
C52176S400 * 70.9
C18443S396 * 73.1
C18841S310 73.9
C22993S160 ***
76.2 C11099S398 77.7
C31833S405 *
78.6 C15620S247 79.2
C22973S285 81.8
C11470S398 84.7
C13698S442 86.9
C841S459 91.8
WGS079 **
92.6 C15873S711 94.5
C14269S102 * 94.7
C11524S150 * 0.0
C20570S83 6.4
WGS211 15.6
C17622S201 * 25.0
C45199S349 37.4
C35020S147 41.5
C26852S307 49.5
C20625S210 51.5
C20763S245 58.1
C23083S345 65.6
C25628S525 72.1
C18443S396 * 79.8
C22993S160 ***
87.5
C31833S405 *
88.9 WGS079 * 97.7
C15873S711 98.9
C36218S165 0.0
C45380S826 15.6
C24159S323 35.2
C20274S537 * 36.7
C12902S674 50.3
C237S473 58.6
C24096S618 **
60.0
C6659S249 * 71.4
C17077S225 0.0
C16387S343 23.4
C22900S198 **
38.9
C36218S165 0.0
C18580S230 13.2
C23375S174 * 15.8
C45380S826 16.1
C18366S189 24.1
EST164 26.5 C14319S510 * 35.9
C24159S323 36.4
C14226S523 * 38.1
C20274S537 * 40.6
C21618S209 42.8
C19944S225 44.3
C13648S225 44.5
C19364S520 47.0
C22643S340 ***
47.2 C20821S413 50.4
EST165 ***
54.0 C20399S426 55.4
C12902S674 56.2
C13142S250 EST062 **
57.5 C237S473 61.7
C22821S388 62.7
C24096S618 **
64.5 Apam3_166 66.0
C10697S175 66.7
C25444S173
68.4 C14487S191 ***
71.9 C13354S446 **
72.6 C6659S249 * 73.9
C20442S307 75.4
C26831S450 76.0
C13486S116 **
76.9 C24129S242 78.9
C14357S360 79.3
C2435S173 81.7
C25234S280 82.6
C25652S324 84.0
C23734S391 84.9
C15351S256 * 85.1
C17077S225 86.7
C16387S343 87.3
C15493S507 89.1
C17287S307 90.7
C12507S635 90.8
C18231S140 91.3
C22109S391 * 93.7
C25536S620 * 95.6
C22900S198 **
96.0 C15056S244 99.0
C54074S403 ***
101.5 C14474S185 101.8
C26329S310 105.5
C25946S829 105.7
C11020S415 108.6
C25425S128 112.8
C14242S316 114.0
C18580S230 0.0
C23375S174 * 2.0
C18366S189 8.1
EST164 12.9 C14319S510 * 22.6
C14226S523 * 25.5
C13648S225 31.5
C22643S340 ***
34.1 C20821S413 37.5
C20399S426 43.0
C12902S674 46.6
Apam3_166 53.3
C25444S173 54.0
C14487S191 ***
55.0 C13354S446 **
55.2 C13486S116 **
58.1 C25234S280 62.0
C23734S391 66.6
C15351S256 * 69.0
C15493S507 75.1
C22109S391 * 77.5
C25536S620 * 82.1
C15056S244 86.4
C14474S185 91.2
C14242S316 104.8
C188S318 *
0.0
C28595S225
19.9
C15111S282
23.4
C34124S511
26.0
C19002S323
38.6
C16956S551
46.7
WGS131
55.9
C1136S272
62.3
C11110S247 *
67.6
C21244S233
72.4
C29060S309
81.7
C15670S505
94.2
C26271S403 0.0
C188S318 * 1.6
C10862S253 9.4
C17498S226 **
16.0 C28595S225 16.4
C38503S228 18.5
C15111S282 28.0
C22427S223 29.6
C27925S129 32.2
C15176S465 34.2
C34124S511 36.3
C16912S265 37.8
C19002S323 39.5
C19713S134 42.0
C20998S134 44.0
C18165S232 44.2
C12093S318 44.8
C10565S307 **
46.2 C16956S551 49.2
C13265S200 49.6
C23489S194 53.9
WGS131 55.9
C20581S243 58.4
C24932S258 61.1
C23738S719 62.5
C11110S247 * C1136S272 63.7
C16621S398 64.7
C22425S453 68.0
C24216S175 69.0
EST016 70.4 C166S563 72.5
C21244S233 73.2
C12174S605 75.1
C13535S196 **
75.4 C11242S364 78.0
C28868S363 * 79.5
C29060S309 84.6
C22138S164 86.5
C15670S505 90.9
WGS035 91.9
C18064S518 98.1
C23209S177 **
99.6 C60613S230 105.8
C10810S897 112.3
C26271S403 0.0
C10862S253 12.0
C17498S226 **
24.9 C22427S223 29.2
C15176S465 30.6
C16912S265 37.4
C19713S134 40.4
C12093S318 43.2
C10565S307 **
44.6 C13265S200 48.0
C23489S194 52.4
WGS131 56.2
C20581S243 57.0
C23738S719 60.6
C16621S398 63.6
C22425S453 66.1
C24216S175 67.8
EST016 69.7 C166S563 71.6
C12174S605 74.0
C11242S364 76.8
C16965S252 83.3
WGS035 85.7
C17479S262 0.0
C15084S136 16.7
C24438S225 24.0
C14364S490 34.2
C3724S507 35.8
C14018S197 42.2
C27153S258 43.6
C29226S281 60.1
C18185S479 62.8
C7889S263 **
C18487S1302 ***
70.7 C22633S340 72.5
C1063S181 78.1
WGS116 ***
86.8 C13992S181 **
90.4 C26116S342 96.6
C48806 102.8 C17914S739 104.5
C11759S946 113.3
C12464S260 120.1
C11999S90 124.5
C13550S341 141.1
C17479S262 0.0
C15084S136 16.7
C24438S225 24.0
C14364S490 34.2
C3724S507 35.8
EST007 37.3 C14018S197 42.2
C19797S331 43.6
C27153S258 44.1
C18363S421 50.2
C7134S210 56.4
C13990S341 57.9
C29226S281 59.8
C18920S453 60.4
C18185S479 62.6
C5239S208 * 65.2
EST149 68.1 C10773S305 70.0
C7889S263 **
72.3 C18487S1302 ***
72.4 C11797S545 72.8
C22633S340 73.7
C10625S161 * 74.0
C76S562 76.4 C13301S439 77.1
C1063S181 77.8
C19928S437 80.4
C23327S599 84.1
WGS116 **
85.8 C20443S297 * 89.4
C13992S181 **
89.9 C20163S412 92.9
C63602S197 95.1
C26116S342 96.6
C14848S1085
99.4 C11461S560 100.9
C48806 102.7 C17914S739 104.1
C14404S340 ***
108.7 C11759S946 112.5
C12464S260 119.7
C11999S90 124.3
C13550S341 141.0
EST007 0.0
C18363S421 11.8
C7134S210 18.2
C17330S121 22.8
C5239S208 * 26.9
C10773S305 31.5
C11797S545 34.5
C10625S161 * 35.4
C19928S437 41.2
WGS116 45.5 C20443S297 * 51.1
C20163S412 55.2
C63602S197 57.1
C14848S1085
61.9 C14404S340 ***
70.6 L1-F L1 L1-M L2-F L2 L2-M
L3-F L3 L3-M L4-F L4 L4-M
Trang 6A genetic linkage map (L5 to L8) of the reef-building coral A millepora
Figure 2
A genetic linkage map (L5 to L8) of the reef-building coral A millepora Female (F) and male (M) maps are shown on the left and right, respectively, and the
consensus map is shown in the center Homologous loci are connected with solid lines Putative stress-related markers are shown in red Distorted loci
are indicated by asterisks (*0.01 <P < 0.05, ** P < 0.01; *** P < 0.001).
C26311S424 **
0.0
C25225S451
16.7
C11329S180 *
31.0
WGS189
42.1
C18576S293
66.1
C18603S149
79.3
C29080S200
91.7
C15741S475 ***
105.6
C14154S231
122.4
C22820S193 **
0.0 C26311S424 **
6.8 Amil2_010 * 14.4
C15891S454 ***
19.5
C8136S163 *
20.8 C59049S135 23.8
C25225S451 24.8
EST032 27.8
C6723S318 28.7
C12395S564 32.0
C15021S282 * 34.9
C70S236 35.3
C15238S417 38.7
C11329S180 * 40.3
C23525S293 40.9
C11670S169 42.8
WGS152 44.7
C21844S313 **
47.6 WGS189 51.2
C25713S318 52.6
C16442S295 54.7
C10924S223 59.1
C29432S370 61.8
C24388S705 63.0
C26140S243 66.2
C11439S315 70.5
C15985S312 70.6
C18576S293 72.6
C22761S360 75.4
EST121 76.6
C18603S149 79.5
C29080S200 90.1
C15741S475 ***
102.1 C14154S231 118.2
Amil2_010 * 0.0
C15891S454 ***
2.4 C59049S135 4.4
EST032 C6723S318 11.7
C12395S564 11.9
C70S236 15.2
C15238S417 17.4
C11670S169 21.7
WGS152 24.1
C21844S313 **
26.3 WGS189 27.9
C25713S318 32.3
C10924S223 39.8
C29432S370 42.1
C26140S243 45.7
EST121 56.7
C23978S544 * 0.0
C31340S160 10.3
C3255S483 19.0
C15113S204 29.7
C915S149 38.9
C10475S502 45.7
C15522S127 * 53.5
C1023S218 63.0
WGS134 72.6
C29463S468 82.1
C11520S633 94.1
C16774S791
99.3
C21914S231 112.9
C13394S333 142.8
C23978S544 * 0.0
C31340S160 11.3
C3255S483 20.2
C15113S204 30.7
C915S149 39.8
C16279S643 43.2
C10475S502 46.5
C19478S130 52.4
C20167S379 54.6
C288S173 56.9
C15522S127 * 58.7
C26478S226 66.1
C1023S218 70.2
C23950S250 74.2
C10005S217 75.3
WGS134 81.8
WGS205 * 84.7
C29463S468 86.4
C27026S472
91.4 C11520S633 92.6
C16774S791
95.3 C23085S183 98.6
C19533S241 100.8
C21914S231 106.8
C11535S517 108.7
C19178S536 116.6
C1114S124 119.7
C13394S333 130.2
C15415S232 133.9
C16634S406 134.2
C4134S257 142.0
C52394S280
144.4 C22526S224 148.7
C1379 161.5
C20167S379 0.0
WGS134 26.0
WGS205 * 31.7
C27026S472
38.7 C19533S241 47.8
C11535S517 55.7
C1114S124 68.2
C15415S232 84.2
C22526S224 100.3
C8085S432
0.0
EST122
12.5
C15286S686
22.1
C11076S81
33.7
C50281S478 *
50.9
C27337
57.5
WGS153 **
62.7
C12987S419
81.1
C23566S420 0.0
C26794S214 5.5
C45851S374 15.5
C15318S250 18.1
C49697S354 C19092S284 23.7
C8085S432 26.7
C19982S400 34.4
EST122 35.9
C27071S243 40.3
C17050S589 43.3
C15286S686 45.8
C24897S240 48.2
C20102S582 48.8
WGS145 49.3
C20479S292 53.1
C11463S192 55.9
C16449S173 58.0
C11076S81 59.6
C10050S780 67.2
C14161S301 72.7
C50281S478 * 73.2
C24813S193 73.5
C27337 78.7
WGS153 **
83.0 C14532S618 84.4
C23508S203 92.4
C12987S419 100.3
C26794S214 0.0
C45851S374 10.7
C49697S354 C19092S284 19.1
C19982S400 30.1
EST122 31.8
C27071S243 36.0
C20102S582 45.4
C16449S173 50.3
C10050S780 67.1
WGS153 * 74.6
C2348S700 0.0
C28447S501 1.2
C18084S286 9.8
C18442S324 13.5
C22464S266 * 18.6
C20407S208 21.2
C19470S311 * 23.7
C11715S299 * 27.2
C55647S531 32.8
C25725S230 * 37.2
C25677S330 43.3
C21253S536 ***
46.3 C12216S415 50.0
C17151S285 56.9
C12479S421 63.3
C969S127 67.9
C6250S141 68.2
C15011S233 71.3
C25187S178 75.3
C17471S281 84.7
C19916S128 90.2
C63883S448 99.8
C2348S700 0.0
C28447S501 1.1
C18084S286 9.4
C18442S324 13.0
C22162S248 17.2
C22464S266 * 18.6
C20407S208 20.6
C19470S311 * 23.3
C16549S511 24.5
C11715S299 * 27.5
C55647S531 32.5
C24321S173 35.3
C25725S230 * 37.8
C25677S330 43.6
C21253S536 ***
46.4 C12216S415 49.9
C43885S203 52.9
C17151S285 56.0
C12479S421 62.2
C969S127 67.7
C6250S141 68.1
C15011S233 73.0
C25187S178 76.8
C17471S281 85.2
C19916S128 92.1
C15269S273 92.8
C63883S448 101.1
L5-F L5 L5-M L6-F L6 L6-M
L7-F L7 L7-M L8-F L8
Trang 7A genetic linkage map (L9 to L12) of the reef-building coral A millepora
Figure 3
A genetic linkage map (L9 to L12) of the reef-building coral A millepora Female (F) and male (M) maps are shown on the left and right, respectively, and
the consensus map is shown in the center Homologous loci are connected with solid lines Putative stress-related markers are shown in red Distorted
loci are indicated by asterisks (*0.01 <P < 0.05, ** P < 0.01; *** P < 0.001).
C26997S204
0.0
WGS112
8.5
WGS227
22.1
C14723S141
31.5
C14246S887
36.8
C9608S288
45.1
C16176S198 ***
68.0
C17475S294 0.0
C16716S153 * 4.8
WGS092 6.7
C49658S304 11.1
C14641S195 25.2
C17299S143 27.3
C26997S204 31.1
C63538S709 31.7
C20768S189 37.0
C16181S885 38.1
WGS112 38.6
C21135S139 49.2
WGS227 53.1
C16127S174 60.1
C14723S141 62.8
C14246S887 68.3
C25192S305 68.7
C22982S334 71.8
WGS217 **
74.9 C9608S288 76.4
C16176S198 ***
84.6
C17475S294 0.0
WGS092 2.0
C14641S195 19.3
C20768S189 33.0
WGS112 * 45.1
C22982S334 65.7
C12097S324 0.0
WGS101 13.3
C490S693 28.0
EST014 0.0
C13861S511 32.2
C12097S324 0.0
WGS101 13.1
C25351S196 15.2
WGS005 17.3
C490S693 29.5
C25688S405 29.8
EST014 35.4
C23210S557 41.5
C16458S418 45.4
C5145S66
46.7 C22489S363 52.4
C11638S270 53.6
C22100S336 56.6
C24238S242 60.3
C15774S399 68.3
C13861S511 69.3
C12550S536 82.1
C989S461 89.4
C25688S405 0.0
EST014 6.0
C23210S557 12.9
C5145S66
17.9
C24238S242 29.3
C15774S399 39.7
C14259S283
0.0
C18993S556
16.8
C19881S196
33.5
C1419S315 *
0.0
C12729S314
28.6
C14259S283 0.0
C16096S170 5.2
C12118S364 **
11.0 C16269S320 11.5
C18993S556 20.2
C49448S110 20.7
C14755S556 22.8
C1166 * 27.2
C55644S292 27.3
C30854S314 33.1
C19881S196 35.2
C15355S114 **
39.8 C1419S315 * 45.4
C16867S473 48.2
C986S247 49.6
C12677S188 58.5
C24058S463 63.8
C12729S314 67.0
C14755S556 0.0
C15355S114 **
16.7
C16867S473 25.6
C24058S463 45.0
C19560S178 0.0
C22182S205 8.7
C15150S931 15.0
C16136S488 19.0
Amil2_002 25.2
C25131S634 **
31.9
C1405S258 42.3
C52436S128 **
58.5
C23019S237 0.0
C19560S178 7.0
WGS107 11.3
C22182S205 15.7
C12219S331 16.0
C15150S931 21.7
C40003S97 23.7
C16136S488 26.0
C45133S676 * 29.4
Amil2_002 31.6
C22306S240 35.7
C25131S634 **
38.5 C50909S225 42.3
C2365S347 47.2
C1405S258 48.9
C6267S266 52.9
C52436S128 **
66.0
C23019S237 0.0
C12219S331 19.7
C45133S676 * 35.2
C2365S347 55.1
L9-F L9 L9-M L10-F L10 L10-M
L11-F L11 L11-M L12-F L12 L12-M
Trang 8duce a large number of gene-associated SNPs from
transcrip-tome data obtained by 454 sequencing Such gene-derived
SNPs are particularly useful for non-model organisms, since
they stand a better chance of identifying causal genes
under-lying complex traits in these organisms in the absence of
genome sequence data [12,13] The criteria that we used for
SNP mining (at least 3× occurrence of the minority allele and
at least 6× read coverage) are more stringent than those typi-cally used (2× occurrence of the minority allele, and 4× or 5× read coverage) [11,31,32] In our experience, the use of these stringent criteria enhances the success rate of marker devel-opment from 454 sequencing data
A genetic linkage map (L13 and L14) of the reef-building coral A millepora
Figure 4
A genetic linkage map (L13 and L14) of the reef-building coral A millepora Female (F) and male (M) maps are shown on the left and right, respectively, and the consensus map is shown in the center Homologous loci are connected with solid lines Distorted loci are indicated by asterisks (*0.01 <P < 0.05, ** P
< 0.01; *** P < 0.001).
C10890S256 *
0.0
WGS196
17.2
C23502S311
21.4
C26043S200 *
22.1
C11040S312
37.9
C23126S678
48.8
C10890S256 * 0.0
WGS196 1.7
C24140S397 2.9
C26275S382 **
14.9 C23502S311 18.9
C24582S267 **
20.9 C26043S200 * 22.1
C25568S279 23.4
C13315S616 29.8
C19552S147 31.4
C11040S312 35.4
C16499S363 37.0
C12260S193 * 44.1
C23126S678 46.0
WGS196 0.0
C26275S382 **
16.3
C25568S279 25.4
C13315S616 32.3
C16499S363 39.5
C12260S193 * 48.5
C1723S422 0.0
Amil2_022 15.4
C22110S143 29.0
C25285S214 41.3
C16637S215 44.4
C22687S231 53.4
C13965S176 61.1
C19502S541 63.7
C19168S356 68.4
C294S372 76.1
C21164S307 80.0
C17723S124 95.2
C1723S422 0.0
Amil2_022 18.3
C22110S143 33.1
C16637S215 57.4
C22687S231 74.5
C19168S356 89.9
C294S372 99.8
L13-F L13 L13-M L14 L14-M
Table 3
Synteny blocks between A millepora and other eukaryotic genomes and their significance
Comparison Blocks (n) Markers per block Overall significance Blocks (n) Markers per block
Nematostella vectensis 6 3-6 < 0.001 1 6
Drosophila melanogaster 13 3-10 0.679 2 9-10
Overview of synteny blocks identified by comparisons between the genetic map of A millepora and other eukaryotic genome sequences, with
permutation tests to evaluate significance of synteny blocks Probabilities were based on permutation tests, as described in Materials and methods
The P-value reported for overall significance reflects the likelihood that the observed number of conserved synteny blocks would be expected by
random chance Significance of synteny block sizes was based on the likelihood that a block containing at least that many markers would be expected
by chance
Trang 9Conserved synteny blocks
Figure 5
Conserved synteny blocks Each synteny block represents a set of mapped coral markers and their best matches in another metazoan genome Synteny blocks were defined as groups of at least three markers, each of which was ≤10 cM from its nearest neighbor within a linkage group in the coral map, and for which the best matches in another genome were also each ≤10 Mb from their nearest neighbors in the same chromosome or scaffold All blocks
shown here contain more markers than expected from chance (P < 0.05) based on permutation analysis (n = 1,000) For each block, the coral linkage
group is shown as a white horizontal bar, with syntenic marker positions (in cM) indicated on the bar For each linkage group containing a synteny block, each comparison with the other genome is shown as a horizontal grey bar, with marker positions (in Mb) indicated on the bar Relationships between
coral markers and other genomes, based on sequence similarity (tblastx, bit-score ≥50), are indicated by diagonal lines connecting each coral marker with its best match.
LG1
|
LG2
1.9 Mb 15.8 Mb
|
| | | || || |
| |
|
| | | | | | ||| | | | | | | ||| | ||
LG4
5.6 Mb 0.6 Mb
|| || | |||| || |
| ||| | | | | | || | | | | | | |
LG5
6 Mb 14.7 Mb
|
|
| | | | | |
49.9 Mb 77.1 Mb
| | |
| || | | || | || | | | | | |
Drosophila melanogaster Ch3R
Drosophila melanogaster Ch3L
Trichoplax adhaerens Sc1
Nematostella vectensis Sc2
Trichoplax adhaerens Sc5
Homo sapiens Ch14 Caenorhabditis elegans Ch5
Trang 10SNP genotyping via high resolution melting analysis
Among the methods available for high-throughput SNP
gen-otyping, the simple, fast and cost-effective HRM method is
especially suitable for non-model organisms The original
HRM method requires one fluorescently labeled probe for
each assay [34,35] Later, this method was simplified by using
an unlabeled probe in the fluorescent dye solution, but the 3'
end of the probe still required costly chemical modification to
prevent extension of the probe [36,37] In this study, we
fur-ther decrease HRM genotyping cost simply by adding two
mismatched bases to the 3' end of an unlabeled probe instead
of chemical modification
SNP marker transferability between populations
Transferability of the assays to different populations is
argu-ably the most important problem that may arise when trying
to apply SNP markers to broad-scale population studies The
markers developed for one population may turn out to be
appreciably polymorphic only in populations well connected
to the original one, while being essentially homozygous in
other, more isolated populations The degree of connectivity
between A millepora populations between three reefs in the
Great Barrier Reef (representing northern, middle, and
southern regions) has been previously evaluated using
alloz-yme markers [38] Similar to nearly all coral species in that
analysis, A millepora demonstrated genetic subdivision
among sampled sites (high Fst values), although not without
some connectivity (an estimated 5 to 30 exchanged migrants
per generation) Oliver and Palumbi [39], on the other hand,
detected strong barriers to connectivity over longer spatial
scales (across Pacific archipelagoes) in two closely related
species, A cytherea and Acropora hyacinthus, using several
intron- and mitochondrial DNA-derived markers that were
developed for phylogeography applications The study of the
natural genotypic diversity and connectivity between A.
millepora populations is of great interest for understanding
the evolutionary responses of reef-building corals to ongoing
climate change, and is among our high-priority research
areas for the future This emphasizes the importance of
deter-mining whether our SNP markers are polymorphic in other
populations, or mostly represent 'private alleles' specific to
the Magnetic Island (and perhaps even more specifically,
Nelly Bay) population Fortunately, in our interpopulation
transferability test, most (65 to 75%) of the SNP markers we
tested were polymorphic in just seven A millepora colonies
from Orpheus Island and Great Keppel Island, which are 80
km and 570 km away from Magnetic Island, respectively
Although this result suggests that the detected SNPs
repre-sent relatively common alleles in these populations, the
dis-tance between these populations is just a fraction of what was
assayed in the Ayre and Hughes study [38], and so it remains
to be seen how far this allele sharing extends Still, this result
is quite promising and suggests the potential for application
of these SNP markers to inter-population studies of local
adaptation in A millepora.
Mapping population
For animals and plants with short generation times, very effi-cient mapping populations (second generation (F2), back-cross, recombinant inbred lines, double haploid, and so on) can be generated from the crosses among homozygous pater-nal strains or recombinant inbred lines, which usually requires multiple generations of sib-mating or self-fertiliza-tion Despite several advantages of those methods, it would be very difficult, if not impossible, to produce such mapping populations in corals because most corals have long genera-tion times (approximately 5 to 10 years in some corals, and 3
to 5 years in most acroporids), and the adult colonies are rather difficult to maintain Last but not least, to our knowl-edge, synchronized coral mass spawning, an essential requirement for making genetic crosses, has never been rec-reated in laboratory-raised corals In short, corals make poor laboratory models; however, this does not diminish the value
of ecological and evolutionary questions pertaining to these
organisms Fortunately, previous studies have shown that A millepora, like many other corals, is a highly heterozygous
species [8,9] Because of this, an outbred full-sib family would
be a suitable mapping population for constructing a linkage map [40-45] Although marker configurations are more com-plicated in such a family, they can be deduced after analyzing the parental origin and genetic segregation of the markers in the progeny (for a review, see [46]) In particular, coral larvae offer several key advantages over adult colonies for linkage mapping in that they are easy to obtain in great numbers, and,
in this species, they do not contain algal symbionts, which would be a potential source of DNA contamination
Map density and recombination rate
In the consensus map, marker density is dramatically varia-ble across linkage groups, indicating that the protein-coding
genes in A millepora, like in human [47], are distributed very
unevenly among chromosomes This also suggests that including anonymous genetic makers into the current map will likely increase marker density in less populated linkage
groups The current genetic map covers 93% of the A mille-pora genome and has a resolution of 3.4 cM, which should be
sufficient for QTL mapping [48,49] The average recombina-tion rate across all linkage groups is approximately 7.5 cM/
Mb in A millepora, which is much higher than human (1.20 cM/Mb [50]), mouse (0.5 cM/Mb [50]), D melanogaster (2 cM/Mb [51]), and even the plant Arabidopsis thaliana (5 cM/
Mb; calculated based on data from The Arabidopsis Informa-tion Resource website [52]) This suggests that QTLs, if iden-tified, can be narrowed down to rather small genomic regions
in this coral species Nine putative stress-related genes were mapped in the consensus map (markers colored red in Fig-ures 1, 2 and 3), and it would be interesting to see whether any
of these are highlighted in future QTL mapping of adaptive physiology traits, such as heat tolerance Moreover, SNPs in these genes might also prove useful for the study of allele-spe-cific gene expression [53] Last but not least, the high-resolu-tion genetic linkage map would be invaluable for assembling