**2. Adaptive evolution of sago palm**

*Genetic Variation*

adaptive evolution of plant.

gene of organisms.

heterozygous in segregated populations [1].

influenced by the surrounding factors is a molecular marker. Thereby, in expressing adaptive evolution and genetic characteristics, it is necessary to be based on molecular markers. Disclosure of the genetic characteristics of organism such as

Several DNA markers that can be used for accessing adaptive evolution of an organism are: Simple Sequence Repeat (SSR) in the nuclear genome and chloroplast genome (cpSSR), Random Amplified Polymorphism DNA (RAPD), functional gene such as Waxy gene in sago palm, 5S, Restriction Fragment Length Polymorphism (RFLP), and Amplified Fragment Length Polymorphism (AFLP), chloroplast DNA (cpDNA) such as *ma*tK gene, and mitochondrial DNA (mtDNA) such *nad* gene. These molecular markers are widely used as markers to express

SSR markers have been shown to have high polymorphisms in soybean and in apples [1–4], thereby, can be used for revealing the adaptive evolution of an organism. SSR is composed of 1–6 base pairs (bp) of repeated DNA sequences with varying amounts [5]. The polymorphic fragments (alleles) are produced from variations in the length of the SSR repeats which can be separated by electrophoresis to display the genetic profile of the genome and the organelle genome. SSR alleles are codominant monogenic inherited and can be distinguished between homozygous and

The advantages of SSR DNA markers or microsatellite markers in genome analysis are that SSR sequences are found in many eukaryotic genomes, high diversity, stable inheritance, co-dominant markers and high accuracy detection [6]. The RAPD marker is a technique that is widely used for genetic characterization because the RAPD technique is simpler than other techniques. Molecular markers related to the expression of certain genes are interesting molecular markers because it can be seen the variation of genes encoding certain characters, making it easier to trace genes that have specific expressions and are desired for the improvement of certain

The *Wx* gene molecular marker is a marker related to the starch biosynthesis process and amplifies the plant DNA sequences that linked to the starch formation. The Waxy (Wx) gene in cereals and *amf* in potato is called isoform gene, Granule-bound starch synthase I (GBSS I) that it encodes starch synthesis [7, 8]. Furthermore, starch synthesis process is regulated by one of the key genes, those the *Wx* gene [9]. Starch from rice plants consists of amylopectin and amylose [10]. Furthermore, it was stated that the *Wx* gene regulates the level of amylose content in starch-producing plants such as wheat and rice [10–12]. The motive structure of the *Wx* gene was reported that it has a very conservative sequence [8] so it fulfills the requirements to be used as a marker. The *Wx* gene marker have been used in various types of crops, i.e. rice [13], barley [9], wheat [14, 15], and sago palm

Large numbers of insertions and deletions in the genome can be detected using agaros gel separation techniques. A technique that is more suitable for small changes in DNA sequences, such as mutations or small deletions or insertions, is fragment analysis using sequencer tools. The technique can detect a change in the size of one base in a DNA fragment. The use of a separation technique that is able to distinguish the differences of one base pair makes it possible to detect the genetic diversity of sago palm that occur at the individual and population. The estimation of adaptive evolution that occurs over a long period of time (hundreds to thousands of years) can be determined based on the chloroplast Simple Sequence Repeat (cpSSR) marker and barcode *mat*K gene in the cpDNA genome. The barcode *mat*K gene was commonly use in the vascular plant, such as Dipterocarpaceae [18], Arecaceae [19]

plant in Indonesia will be better focused on molecular-based markers.

**28**

[16, 17].

Diversity is a reflection adaptive evolution in an organism. Variations within a population and inter species that are affected by the occurrence of adaptive evolution. Adaptive evolution of sago palm can be measured by using various markers. The characteristics of sago palm in Indonesia were shown widely varies in morphological phenotypic. It was reported that around Sentani, Jayapura there are 15 varieties [21]. These varieties show variation in a broad sense, not only in morphological characters, but also in their adaptation to the environment (tolerant to fire and waterlogging). Furthermore, the variation of sago palm in Papua is very large based on morphological phenotypic, there are 96 varieties based on vernacular name [22]. The variation base on morphological phenotypic may differences from another population and location because morphological characters are strongly influenced by environmental factors. Observing the variation of sago palm need a marker that are not influenced by the environment so that they can reflect the actual state of plant variation. Markers developed in a wide variety of organisms including plants, namely chloroplast genome molecular markers (cpDNA) and nuclear genome molecular markers (RAPD, Wx gene expression, and others).

The cpDNA molecular marker is a very conservative molecular marker, so it is very suitable to be used to estimate long-term adaptive evolution for a particular organism. The cpDNA locus mutation rates was estimated between 3.2 x 10–5 and 7.9 x 10–5 [23]. Apart from this, cpDNA sequences are conservative in comparison to nuclear genome because they do not undergo recombination in the genome and uniparental inherited [24, 25]. Based on the information found in the chloroplast genome, it is a difference that occurred hundreds or thousands of years ago.

The cpDNA markers were developed in plants showed that the cpDNA of sago palm varied, the total 10 haplotypes were found throughout Indonesia territorials [26]. Seven haplotypes were found on the island of Papua and three haplotypes were found apart from the island of Papua and two haplotypes were found on several islands (sharing haplotypes). Based on highly conservative cpDNA criteria, the variations in cpDNA detection were reflect conditions hundreds or thousands of years ago. It is hypothetically that gene flow of sago palm since ancient times moving from one island to another in various ways. It was found that only two haplotypes experienced displacement. This phenomenon was corresponded of *Pinus silvestris* L. and *Abies alba* Mill referred to as the refugee population [27, 28].

Base on the largest number of haplotypes were found on several islands where sago samples were taken, the island of Papua is the center of sago diversity because the island of Papua has the highest number of cpDNA haplotypes. Large amount of diversity is found in natural populations [29]. Based on this statement, it can be said that the sago palm in Papua is a natural population (not refers to a migrant population). When talking about the source of diversity, the islands of Papua, Sulawesi and Kalimantan are the sources of diversity of sago palm because it has a specific haplotype. Large number of haplotypes reflects the high variation or diversity in a population [28] and differences in cpDNA

haplotypes in each population reflect differences in genetic entities (sources of variation) [29].

Based on the developed molecular markers of the chloroplast genome (cpDNA) and nucleus genomes, it was revealed that individuals with different local names within and between populations were generally not different. This indicates that the environmental influence on the appearance of the morphological phenotype is very large because the local name given by the local community is based on morphological phenotypic and local language. In Papua alone, there are a lot of regional languages which make the local names for the sago palm too many. People in Jayapura (West, Central, and East Sentani) give local names for one type of sago palm which differs from one another [30]. If the grouping and naming of sago palm varieties is based on local names, there will be a very large number of vernacular names comparing from the real thing. It was documented that in Papua there are 96 vernacular name of sago palm [31]. Furthermore, the farmers indicated that there are 21 varieties in Sentani and Scientist only recognized 15 varieties out of 21 varieties based on morphological phenotypic [21]. Based on this information, it reflects confusion and there is an overlap in the naming of varieties, which makes the classification and number of varieties recorded larger than the real thing. Cases like these are make molecular markers play an important role for clarification as well as correction of varieties number.

Molecular markers of the chloroplast genome and nucleus genome developed on sago palm detected that sago palm in various islands in Indonesia experienced high diversities as seen from the varying values of genetic diversity: ∑H, HE, S, G, Ĥ, VĤ, π, πn, and P. This means that in a population there are individuals who are very different from one another. In general, it can be interpreted that the sago palm scattered in various islands in Indonesia, even though the samples from the island of Java with the Wx gene marker and samples from the islands of Ambon and Java with the nucleus genomic SSR markers are not differentiated. This is probably due to the discriminatory focus of each molecular marker that is different from one another. The Wx gene marker focuses its discrimination on genes encoding the biosynthesis of amylose. If the DNA sequence of the *Wx* gene in the population sample did not vary like the population sample from Bogor, then the amylose content did not vary either. Various *Wx* gene alleles determine the amylose content in starch-producing plants [10, 12, 32].

Based on the codominant molecular markers (*Wx* genes and nucleus genomic SSR) used, it shows that the level of heterozygosity of sago palm in various populations in Indonesia varies in terms of the ratio of heterozygous and homozygous values. Based on the *Wx* gene marker, it shows that the samples from the Palopo and Bogor populations are all heterozygous, in contrast the SSR markers of the nuclear genome of the individual samples from the Ambon and Bogor populations are all homozygous. This phenomenon reflects the degree of individual heterozygosity depending on the particular character observed. The heterozygous diversity of the *Wx* gene was relevant to the quality and quantity of plant starch production which also varied. Starch content of sago palm varied as well as the accumulated dry matter [21]. Variations in the *Wx* gene in wheat caused variations in the viscosity of the resulting starch production [15]. The heterozygosity values based on the nucleus genomic SSR markers also varied, although they were not as high as the heterozygosity values of the *Wx* gene markers [16]. SSR markers when designed based on SSR sequences information of the plant genome under study will produce high levels of polymorphism. Previous studies on various types of plants have shown that SSR markers are commonly used to measure adaptive evolution because of their high rates of polymorphism [33–36].

**31**

growth is shown on **Figure 2**.

*Adaptive Evolution and Addressing the Relevance for Genetic Improvement of Sago Palm…*

Genetic hierarchy and genetic differentiation based on chloroplast genome markers and nucleus genome indicate that sago samples with cpDNA markers and *Wx* genes differentiate at individual and population levels [16, 19, 26, 37]. Furthermore, samples with RAPD markers experience differentiation at the individual and population levels [16, 26]. The levels of genetic hierarchy observed were individual, population, and island levels [38]. On the other hand, the SSR marker of the nucleus genome was only a sample between populations from the island of Papua which experienced differentiation. This difference is strongly influnced by the nature and the degree of polymorphism of the genetic markers used. The conservative genetic markers such as *mat*K gene markers and mitochondrial *nad2* gene markers tend to show lower levels of polymorphism and only at lower levels of genetic hierarchy are significantly different [20, 37]. Low levels of polymorphism between populations and did not experience genetic differentiation in Pinaceae using the cpSSR marker, but with the RAPD marker, high polymorphism and genetic differentiation were found [39]. Furthermore, the cpDNA characters that

Genetic relatedness of the population based on phylogenetic constructs shows that the SSR molecular marker of the nucleus genome divides the sample into two groups, the cpDNA and RAPD molecular markers divide the samples into three groups, and the *Wx* gene molecular marker divides the sample into four groups [17]. The variations that occur may be due to the different nature and focus of discrimination for each molecular marker used. This case is something that is often encountered in various kinds of molecular markers. Previous studies have shown that different molecular markers infer variability, genetic relatedness, and adaptive evolution of individual or population variations [40–42]. Furthermore, the genetic variation in *Pseudotsuga menziesii* (Mirb.) Franco. using univarentally inherited (cpSSR) and biparental inherited (isozyme and RAPD) molecular markers concluded that the level of polymorphism and differentiation of cpSSR markers was

Based on molecular markers of cpDNA, RAPD, Wx genes, SSR nucleus genome, cpDNA *mat*K gene, and mitochondrial *nad*2 gene, it shows that sago palm in Indonesia are diverse [17, 19, 37]. The relevance of genetic diversity generated by molecular markers of the chloroplast genome and nucleus genome with the morphological diversity that has been revealed by sago plant researchers is that they both reveal that sago palm in Indonesia are diverse, but the level of diversity based on genetic markers is lower than that based on morphological markers [43]. The variation of sago palm in Papua is very large based on morphological phenotypics, namely that in total there are 96 varieties found from eight locations (Salawati, Waropen, Sentani, Kaureh, Wasior, Inanwatan, Onggari, and Windesi) in Papua and west Papua Province [22]. It was reported three varieties of sago palm in Kendari, Southeast Sulawesi [21]. Furthermore, it was documented 11 varieties of sago palm in Southeast Sulawesi, North Sulawesi and North Ambon based on morphological characteristics [44]. Genetic diversity based on the molecular markers that have been disclosed classifies sago palm in Indonesia from two to four groups. It was reported that sago palm is divided into two clusters and two sub-clusters [45]. The Morphological performance of Sago palm forest is shown on **Figure 1** and the morphological performance at the russet

Based on the molecular markers that have been used on sago palms, nothing has been associated with the morphological characters. The same thing was also that spineless and spiny of sago palm was not related to genetic distance based on RAPD markers [45]. It is believed that spine and spineless in sago palm is controlled

*DOI: http://dx.doi.org/10.5772/intechopen.94395*

evolved in the cotton genus were low [40].

lower than that of isozyme and RAPD markers [39].

#### *Adaptive Evolution and Addressing the Relevance for Genetic Improvement of Sago Palm… DOI: http://dx.doi.org/10.5772/intechopen.94395*

Genetic hierarchy and genetic differentiation based on chloroplast genome markers and nucleus genome indicate that sago samples with cpDNA markers and *Wx* genes differentiate at individual and population levels [16, 19, 26, 37]. Furthermore, samples with RAPD markers experience differentiation at the individual and population levels [16, 26]. The levels of genetic hierarchy observed were individual, population, and island levels [38]. On the other hand, the SSR marker of the nucleus genome was only a sample between populations from the island of Papua which experienced differentiation. This difference is strongly influnced by the nature and the degree of polymorphism of the genetic markers used. The conservative genetic markers such as *mat*K gene markers and mitochondrial *nad2* gene markers tend to show lower levels of polymorphism and only at lower levels of genetic hierarchy are significantly different [20, 37]. Low levels of polymorphism between populations and did not experience genetic differentiation in Pinaceae using the cpSSR marker, but with the RAPD marker, high polymorphism and genetic differentiation were found [39]. Furthermore, the cpDNA characters that evolved in the cotton genus were low [40].

Genetic relatedness of the population based on phylogenetic constructs shows that the SSR molecular marker of the nucleus genome divides the sample into two groups, the cpDNA and RAPD molecular markers divide the samples into three groups, and the *Wx* gene molecular marker divides the sample into four groups [17]. The variations that occur may be due to the different nature and focus of discrimination for each molecular marker used. This case is something that is often encountered in various kinds of molecular markers. Previous studies have shown that different molecular markers infer variability, genetic relatedness, and adaptive evolution of individual or population variations [40–42]. Furthermore, the genetic variation in *Pseudotsuga menziesii* (Mirb.) Franco. using univarentally inherited (cpSSR) and biparental inherited (isozyme and RAPD) molecular markers concluded that the level of polymorphism and differentiation of cpSSR markers was lower than that of isozyme and RAPD markers [39].

Based on molecular markers of cpDNA, RAPD, Wx genes, SSR nucleus genome, cpDNA *mat*K gene, and mitochondrial *nad*2 gene, it shows that sago palm in Indonesia are diverse [17, 19, 37]. The relevance of genetic diversity generated by molecular markers of the chloroplast genome and nucleus genome with the morphological diversity that has been revealed by sago plant researchers is that they both reveal that sago palm in Indonesia are diverse, but the level of diversity based on genetic markers is lower than that based on morphological markers [43]. The variation of sago palm in Papua is very large based on morphological phenotypics, namely that in total there are 96 varieties found from eight locations (Salawati, Waropen, Sentani, Kaureh, Wasior, Inanwatan, Onggari, and Windesi) in Papua and west Papua Province [22]. It was reported three varieties of sago palm in Kendari, Southeast Sulawesi [21]. Furthermore, it was documented 11 varieties of sago palm in Southeast Sulawesi, North Sulawesi and North Ambon based on morphological characteristics [44]. Genetic diversity based on the molecular markers that have been disclosed classifies sago palm in Indonesia from two to four groups. It was reported that sago palm is divided into two clusters and two sub-clusters [45]. The Morphological performance of Sago palm forest is shown on **Figure 1** and the morphological performance at the russet growth is shown on **Figure 2**.

Based on the molecular markers that have been used on sago palms, nothing has been associated with the morphological characters. The same thing was also that spineless and spiny of sago palm was not related to genetic distance based on RAPD markers [45]. It is believed that spine and spineless in sago palm is controlled

*Genetic Variation*

variation) [29].

well as correction of varieties number.

high rates of polymorphism [33–36].

plants [10, 12, 32].

haplotypes in each population reflect differences in genetic entities (sources of

Based on the developed molecular markers of the chloroplast genome (cpDNA) and nucleus genomes, it was revealed that individuals with different local names within and between populations were generally not different. This indicates that the environmental influence on the appearance of the morphological phenotype is very large because the local name given by the local community is based on morphological phenotypic and local language. In Papua alone, there are a lot of regional languages which make the local names for the sago palm too many. People in Jayapura (West, Central, and East Sentani) give local names for one type of sago palm which differs from one another [30]. If the grouping and naming of sago palm varieties is based on local names, there will be a very large number of vernacular names comparing from the real thing. It was documented that in Papua there are 96 vernacular name of sago palm [31]. Furthermore, the farmers indicated that there are 21 varieties in Sentani and Scientist only recognized 15 varieties out of 21 varieties based on morphological phenotypic [21]. Based on this information, it reflects confusion and there is an overlap in the naming of varieties, which makes the classification and number of varieties recorded larger than the real thing. Cases like these are make molecular markers play an important role for clarification as

Molecular markers of the chloroplast genome and nucleus genome developed on sago palm detected that sago palm in various islands in Indonesia experienced high diversities as seen from the varying values of genetic diversity: ∑H, HE, S, G, Ĥ, VĤ, π, πn, and P. This means that in a population there are individuals who are very different from one another. In general, it can be interpreted that the sago palm scattered in various islands in Indonesia, even though the samples from the island of Java with the Wx gene marker and samples from the islands of Ambon and Java with the nucleus genomic SSR markers are not differentiated. This is probably due to the discriminatory focus of each molecular marker that is different from one another. The Wx gene marker focuses its discrimination on genes encoding the biosynthesis of amylose. If the DNA sequence of the *Wx* gene in the population sample did not vary like the population sample from Bogor, then the amylose content did not vary either. Various *Wx* gene alleles determine the amylose content in starch-producing

Based on the codominant molecular markers (*Wx* genes and nucleus genomic SSR) used, it shows that the level of heterozygosity of sago palm in various populations in Indonesia varies in terms of the ratio of heterozygous and homozygous values. Based on the *Wx* gene marker, it shows that the samples from the Palopo and Bogor populations are all heterozygous, in contrast the SSR markers of the nuclear genome of the individual samples from the Ambon and Bogor populations are all homozygous. This phenomenon reflects the degree of individual heterozygosity depending on the particular character observed. The heterozygous diversity of the *Wx* gene was relevant to the quality and quantity of plant starch production which also varied. Starch content of sago palm varied as well as the accumulated dry matter [21]. Variations in the *Wx* gene in wheat caused variations in the viscosity of the resulting starch production [15]. The heterozygosity values based on the nucleus genomic SSR markers also varied, although they were not as high as the heterozygosity values of the *Wx* gene markers [16]. SSR markers when designed based on SSR sequences information of the plant genome under study will produce high levels of polymorphism. Previous studies on various types of plants have shown that SSR markers are commonly used to measure adaptive evolution because of their

**30**

**Figure 1.** *Morphological performance of sago palm forest.*

#### **Figure 2.**

*Morphological performance of sago palm in the russet growth. Spineless with purple color of the young leaf (A), spineless with green color of the young leaf (B), spiny with purple color of the young leaf (C), and spiny with green color of the young leaf (D).*

by certain genes, so that there are certain nucleotide sequences in the sago palm genome that undergo transcription and translation processes in the spine formation process. Molecular markers encoding the characteristics of sago palm can be designed if desired by reverse transcription of sequences encoding protein synthesis for spiny formation in sago.

Genetic relatedness based on the phylogenetic constructs of each tested molecular marker shows that the distribution of the level of sample similarity according to the size of genetic distance is not limited by location and geographical isolation because samples from one island to others islands blending with each other [17]. The blending of Stylosanthes sp. obsessions from various regions in the dendrogram construction indicates that the obsessions have geographic distribution [46]. It was reported that, if there is distance and geographic isolation in the long term, the population from one region to another will experience differentiation or adaptive evolution as happened in Brassicaceae [47]. Furthermore, Scientist documented that population differentiation of O. rifipogon affected by distance or geographic isolation [36].

**33**

*Adaptive Evolution and Addressing the Relevance for Genetic Improvement of Sago Palm…*

RAPD polymorphisms amplified on the PCR machine produced polymorphic fragments and the number of genotypes of each population. RAPD polymorphisms and high number of genotypes are a reflection of plant genetic diversity and adaptive evolution based on RAPD markers [38]. This result is in line with the diversities of sago palm revealed by using RAPD markers on several samples from Indonesia

Population genetic diversity shows that the population samples from Papua have the highest of polymorphic sites number (S), the moderate of pairwise differences values (π), and the highest percentage of haplotype polymorphic compared to other populations from several islands in Indonesia [38]. Genotype diversity equal to one means that no identical genotype is found in a population sample. The value of Ĥ of individual samples at the island level all shows number of one, which means that one sample of individuals with another sample of individuals differs from one another based on the RAPD markers. Sago progenies obtained from semi-cultivated sago populations showed genetic differences among the progeny tested [43]. The varying values of S, π, and Ĥ indicate the genetic variation values of sago palm population in Indonesia. In the previous studies of sago palm by using RAPD markers showed that the sago palm diversities among individual was recorded also high [48] and other scientist reported 15 varieties of sago palm based on morphological

The genetic hierarchy was estimated based on Analyses Molecular of Variance (AMOVA) calculations. The AMOVA calculation value shows that 89.35% of the total variety of samples is contributed by individuals with very significantly different with probability (P) values, 6.58% and 8.4% variance is contributed among populations [17]. The rates of diversities and adaptive evolution were detected in sago palm, those related to the genetic diversity of *M. sativa* L. by using RAPDs marker [47] as well as *Cynara scolymus* L. [49]. The statistical test method used to reveal the differentiation that occurs at the population and island level is also found to be used to reveal the differentiation that occurs at the population level in various types of plants, such as in *M. sativa* L [47], in *Acacia radiana* [50]*,* and in *Primula* 

Genetic relatedness among individual shows that the sago palm were classified into three groups based on the dendrogram construction. Group I include sago palm from all the populations as well as group III, while Group II includes sago palm from Jayapura, Serui, Manokwari and Ambon. This is related to the grouping of sago palm that it was reported in the previously study and divided sago palm from Indonesia and Malaysia into two groups and subgroups based on RAPD markers [48]. Previous sago genetic studies that focused on the Indonesian archipelago showed that sample individuals were divided into four groups based on RAPD markers [52]. Grouping of individuals in a dendrogram is largely determined by the genetic distance used, the method of grouping, and the desired bootstrap coefficient or rate. The differences between the groupings based on the cpDNA markers and the RAPD markers observed in previous studies are common in genetic related-

Genetic relatedness among population shows a clustering pattern similar among individual. Genetic relatedness based on the dendrogram sample construction at the island level shows that the samples from the island of Papua are more closely related to the samples from Sumatra and Kalimantan, the samples from the island of Sulawesi are closely related to the samples from Ambon, and the samples from

**3.1 Random amplified polymorphism DNA (RAPD) marker**

*DOI: http://dx.doi.org/10.5772/intechopen.94395*

**3. Genetic assessment of sago palm**

characters in around Sentani lake [21].

*elatior* (L.) Oxlip [51].

ness studies [24, 39, 41].

and Malaysia [48].

*Adaptive Evolution and Addressing the Relevance for Genetic Improvement of Sago Palm… DOI: http://dx.doi.org/10.5772/intechopen.94395*
