465
based on genomic resources of model and non-model species (e.g. Arabidopsis
thaliana, Pinus taeda, Pinus pinaster, Cryptomeria japonica, Prosopis juliflora,
Eucalyptus) available in public databases [National Center for Biotechnological
Information (NCBI) and The Institute for Genomic Research (TIGR)]. In the last
decade, with the advent of HTS, scientific research has taken advantage of the new,
low-cost and fast-sequencing technologies to acquire genomic information of native
forest species from Argentina.
17.4.1 Transcriptome and Genome Sequencing
of Nothofagus Species
In Argentina, the genus Nothofagus is represented by six endemic species (N. alpina,
N. obliqua, N. antarctica, N. dombeyi, N. betuloides and N. pumilio) distributed on
the Patagonian Andes Range. Nothofagus alpina (Poepp. and Endl.) Oerst. (=
N. nervosa (Phil.) Dim. et Mil.) and N. obliqua (Mirb.) Oerst. are two closely related
species occurring in sympatry over approximately 18,000 ha (Sabatier et al. 2011),
but presenting ecological and physiological differences (Varela et al. 2010;
Azpilicueta et al. 2013) that suggest adaptive variation. Nothofagus pumilio is the
most abundant native tree of the Patagonian temperate forest. Its distribution spans
from 35° S to 56° S, occupying the high altitudes and building the tree line.
Photoperiod and temperature change considerably towards southern latitudes,
marking N. pumilio as a suitable model to test local adaptation and clinical variation
in genetic and adaptive traits. This species’ preference for colder environments indicates a particularly high sensitivity towards future changes in temperature as predicted in a Global Climate Change context (IPCC 2014). In spite of their ecological
and economical importance, little genomic resources were available for this species
(Azpilicueta et al. 2004; Marchelli et al. 2008; Soliani et al. 2010).
In the last decade, a first transcriptome sequencing of N. alpina was performed
on young leaf tissue using Roche/454 technology (Torales et al. 2012). The methodology included de novo assembly, functional annotation, and in silico discovery of
potential molecular markers (Fig. 17.2 and Table 17.1). The 82% of the identified
unigenes showed high similarity to unigenes from the Fagaceae Genomic database
(http://www.fagaceae.org/), while the others could be novel genes. Unigenes with
putative function (15,497) represented 50% of the N. alpina genes catalogue considering that the average number of genes encoded in a plant nuclear genome is about
30,000 (Logacheva et al. 2011). RNA-Seq on this species also allowed the identification of SSRs, with 55% of them located within gene coding regions (functional
SSR markers). Seventy-three (73) SSRs located in sequences with annotation function related to response to abiotic stress were selected, validated in N. alpina and
transferred to other South American Nothofagus species (N. obliqua, N. pumilio,
N. antarctica and N. dombeyi). A set of 27 SSRs was then validated for these species
providing a useful panel of markers for conservation and evolutionary studies and
17 Application of High-Throughput Sequencing Technologies in Native Forest Tree…
based on genomic resources of model and non-model species (e.g. Arabidopsis
thaliana, Pinus taeda, Pinus pinaster, Cryptomeria japonica, Prosopis juliflora,
Eucalyptus) available in public databases [National Center for Biotechnological
Information (NCBI) and The Institute for Genomic Research (TIGR)]. In the last
decade, with the advent of HTS, scientific research has taken advantage of the new,
low-cost and fast-sequencing technologies to acquire genomic information of native
forest species from Argentina.
17.4.1 Transcriptome and Genome Sequencing
of Nothofagus Species
In Argentina, the genus Nothofagus is represented by six endemic species (N. alpina,
N. obliqua, N. antarctica, N. dombeyi, N. betuloides and N. pumilio) distributed on
the Patagonian Andes Range. Nothofagus alpina (Poepp. and Endl.) Oerst. (=
N. nervosa (Phil.) Dim. et Mil.) and N. obliqua (Mirb.) Oerst. are two closely related
species occurring in sympatry over approximately 18,000 ha (Sabatier et al. 2011),
but presenting ecological and physiological differences (Varela et al. 2010;
Azpilicueta et al. 2013) that suggest adaptive variation. Nothofagus pumilio is the
most abundant native tree of the Patagonian temperate forest. Its distribution spans
from 35° S to 56° S, occupying the high altitudes and building the tree line.
Photoperiod and temperature change considerably towards southern latitudes,
marking N. pumilio as a suitable model to test local adaptation and clinical variation
in genetic and adaptive traits. This species’ preference for colder environments indicates a particularly high sensitivity towards future changes in temperature as predicted in a Global Climate Change context (IPCC 2014). In spite of their ecological
and economical importance, little genomic resources were available for this species
(Azpilicueta et al. 2004; Marchelli et al. 2008; Soliani et al. 2010).
In the last decade, a first transcriptome sequencing of N. alpina was performed
on young leaf tissue using Roche/454 technology (Torales et al. 2012). The methodology included de novo assembly, functional annotation, and in silico discovery of
potential molecular markers (Fig. 17.2 and Table 17.1). The 82% of the identified
unigenes showed high similarity to unigenes from the Fagaceae Genomic database
(http://www.fagaceae.org/), while the others could be novel genes. Unigenes with
putative function (15,497) represented 50% of the N. alpina genes catalogue considering that the average number of genes encoded in a plant nuclear genome is about
30,000 (Logacheva et al. 2011). RNA-Seq on this species also allowed the identification of SSRs, with 55% of them located within gene coding regions (functional
SSR markers). Seventy-three (73) SSRs located in sequences with annotation function related to response to abiotic stress were selected, validated in N. alpina and
transferred to other South American Nothofagus species (N. obliqua, N. pumilio,
N. antarctica and N. dombeyi). A set of 27 SSRs was then validated for these species
providing a useful panel of markers for conservation and evolutionary studies and
17 Application of High-Throughput Sequencing Technologies in Native Forest Tree…