436
16.3 Handroanthus impetiginosus
16.3.1 Preliminary Characterization of Its Genetic Variability
The leaves of H. impetiginosus have high concentration of polyphenols (Jausoro
et al. 2010) which leads to an extraction of DNA of poor quality and failures in the
amplification of molecular markers. Three different dates of sampling (start of
sprouting, October 2014; 90 days later, January 2015; and 180 days from sprouting,
April 2015) were evaluated because it was suggested that the concentration of polyphenols in leaves decreases with increasing time since sprouting (Hernán Bach pers.
comm.). Three protocols of DNA extraction (i, Hoisington et al. 1994; ii, Doyle and
Doyle 1990 modified; iii, commercial Quiagen Kit) were assayed. Several features
of the collected leaves were considered, including their conservation until laboratory processing. The DNA extraction was evaluated by the color/appearance of the
pellet obtained and the quantity and integrity of DNA in agarose. In addition, DNA
extraction from flowers was attempted, but although DNA could be extracted,
amplification products could not be obtained.
Results indicated that 90 days from sprouting was the best date for sampling, and
Doyle and Doyle (1990) modified with two steps of chloroform-isoamyl was the
most appropriate protocol for DNA extraction. An additional proof of milling and
conservation of sampled leaf material was conducted. Milling with liquid N of previously dried leaves in silica gel and immediate DNA extraction was finally selected.
In fact, only for this protocol amplification products were obtained.
Transferability of ten SSRs (Tau 12, Tau 14, Tau 15, Tau 17, Tau 21, Tau 22, Tau
27, Tau 28, Tau 30, Tau 31) developed in Tabebuia aurea (Braga et al. 2007) to
H. impetiginosus was examined (Inza et al. 2019). The success of transferability as
well as the clearness of resolution patterns and the polymorphism levels was evaluated on a subset of individuals from different populations randomly chosen. Then,
those SSRs transferred successfully that showed a polymorphic pattern were evaluated across a greater number of samples to better examine the level of polymorphism (number of alleles).
A set of five markers (Tau 12, Tau 14, Tau 17, Tau 28, and Tau 31) transferred
from T. aurea (Braga et al. 2007) were finally selected to conduct population genetics studies. Conditions of polymerase chain reaction (PCR) were performed according to Braga et al. (2007). Molecular genetic variability of nine Argentine natural
populations of H. impetiginosus was assessed using the five SSRs transferred (Inza
et al. 2019). Young leaves from randomly chosen trees were collected considering a
minimum distance of 80–100 m between them in order to reduce the probability of
sampling-related individuals (Gillies et al. 1999). Collected leaves were dried in
silica gel (Chase and Hills 1991) and total genomic DNA extracted and stored at
−20 °C until its use.
Genetic diversity and differentiation parameters were estimated using the
GenAlEx 6.3 software (Peakall and Smouse 2006). A total of 54 alleles were amplified with a range of 23 (Tau17) to 2 (Tau 28) alleles per microsatellite. Genetic
L. F. Fornes et al.
16.3 Handroanthus impetiginosus
16.3.1 Preliminary Characterization of Its Genetic Variability
The leaves of H. impetiginosus have high concentration of polyphenols (Jausoro
et al. 2010) which leads to an extraction of DNA of poor quality and failures in the
amplification of molecular markers. Three different dates of sampling (start of
sprouting, October 2014; 90 days later, January 2015; and 180 days from sprouting,
April 2015) were evaluated because it was suggested that the concentration of polyphenols in leaves decreases with increasing time since sprouting (Hernán Bach pers.
comm.). Three protocols of DNA extraction (i, Hoisington et al. 1994; ii, Doyle and
Doyle 1990 modified; iii, commercial Quiagen Kit) were assayed. Several features
of the collected leaves were considered, including their conservation until laboratory processing. The DNA extraction was evaluated by the color/appearance of the
pellet obtained and the quantity and integrity of DNA in agarose. In addition, DNA
extraction from flowers was attempted, but although DNA could be extracted,
amplification products could not be obtained.
Results indicated that 90 days from sprouting was the best date for sampling, and
Doyle and Doyle (1990) modified with two steps of chloroform-isoamyl was the
most appropriate protocol for DNA extraction. An additional proof of milling and
conservation of sampled leaf material was conducted. Milling with liquid N of previously dried leaves in silica gel and immediate DNA extraction was finally selected.
In fact, only for this protocol amplification products were obtained.
Transferability of ten SSRs (Tau 12, Tau 14, Tau 15, Tau 17, Tau 21, Tau 22, Tau
27, Tau 28, Tau 30, Tau 31) developed in Tabebuia aurea (Braga et al. 2007) to
H. impetiginosus was examined (Inza et al. 2019). The success of transferability as
well as the clearness of resolution patterns and the polymorphism levels was evaluated on a subset of individuals from different populations randomly chosen. Then,
those SSRs transferred successfully that showed a polymorphic pattern were evaluated across a greater number of samples to better examine the level of polymorphism (number of alleles).
A set of five markers (Tau 12, Tau 14, Tau 17, Tau 28, and Tau 31) transferred
from T. aurea (Braga et al. 2007) were finally selected to conduct population genetics studies. Conditions of polymerase chain reaction (PCR) were performed according to Braga et al. (2007). Molecular genetic variability of nine Argentine natural
populations of H. impetiginosus was assessed using the five SSRs transferred (Inza
et al. 2019). Young leaves from randomly chosen trees were collected considering a
minimum distance of 80–100 m between them in order to reduce the probability of
sampling-related individuals (Gillies et al. 1999). Collected leaves were dried in
silica gel (Chase and Hills 1991) and total genomic DNA extracted and stored at
−20 °C until its use.
Genetic diversity and differentiation parameters were estimated using the
GenAlEx 6.3 software (Peakall and Smouse 2006). A total of 54 alleles were amplified with a range of 23 (Tau17) to 2 (Tau 28) alleles per microsatellite. Genetic
L. F. Fornes et al.