Genetic Diversity and Population Structure of Pinus kesiya through Trans-specific Amplification of Nuclear SSR Markers

De novo primer development is cost intensive and time-consuming, therefore using primers developed for other species on the target species is a more preferred alternative. In the present study, a total of 47 primer pairs from P. taeda, P. merkusii, P. resinosa and P. densiflora were used for trans-specific amplification of P. kesiya. It was observed that only 5 (10.6%) primer pairs out of 47 transferred in P. kesiya which may be due to the phylogenetic distance of the target species from the source species. The expected heterozygosity (HE) ranged from 0.490 to 0.603 with a mean of 0.540 and the observed heterozygosity (HO) ranged from 0.044 to 0.819 with a mean of 0.342. The study has shown that the nuclear SSR markers can be utilized for estimating the genetic structure of P. kesiya populations. Results of the present work will go a long way in implementing proper strategies for the better management and conservation of P. kesiya forests and initiating tree improvement programmes in this species.

Environmental changes are unpredictable and it is of utmost importance that sufficient genetic diversity is secured to permit the species to continuously evolve in response to environmental pressure. Cross-species amplification is a reasonable approach for assessing the genetic diversity of populations which eliminates the need for de novo development of polymorphic primers as it is time-consuming and cost intensive. Microsatellite or simple sequence repeats (SSRs) are highly informative markers that are co-dominantly inherited. They are highly polymorphic and their transferability between closely related species makes them useful for the genetic studies of related species (Weising et al. 2005). SSR discovery from genomic libraries has proven problematic in conifers, with a low return for effort (Rajora et al. 2001;Hodgetts et al. 2001) due to the large size and repetitive nature of the conifer genome. This is why microsatellite transfer across the species is seen as a valued methodology (Ginwal et al. 2011).
Pinus kesiya commonly known as Khasi pine belongs to the family Pinaceae. The genus Pinus section Diploxylon is characterized by hard timber and two xylem bundle. It is widely distributed between 30°N and 12°N in South East Asia. It occurs in Myanmar, India, Tibet, Laos, Vietnam, Thailand, the Philippines and the People's Republic of China (Hansen et al. 2003). In India, the species occur naturally in Khasi and Jaintia Hills and have been reported to grow at altitudes of between 800 and 2000 m amsl. Further to the east, it occurs throughout Manipur, Nagaland and Arunachal Print ISSN : 1974ISSN : -1712 Online ISSN : 2230-732X Pradesh at elevations ranging from 1200 to 2000 m amsl (Chaudhary and Bhattacharyya, 2002). It is among the principal species in Meghalaya and constitutes 8.29% of the total forested area (FSI, 2015).
P. kesiya is a commercially important species providing pulp, lumber and oleoresin. It is fast growing and has the capacity to adapt to various growing conditions and produces a high quality, long-fibered pulp (Hansen et al. 2003). In India, it is generally used for fuel wood and charcoal manufacture. The indigenous people of Meghalaya depend on P. kesiya for their traditional agricultural practices. They cultivate ginger (Zingiber officinale), turmeric (Curcuma domestica), paddy and vegetables under the Khasi pine based farming system. It also has medicinal properties and their young shoot is often used for treating children in case of a cough Jeeva et al. 2006).
The pine forest of Meghalaya is under immense pressure due to overexploitation and getting denuded as a result of unplanned developmental activities and massive tree felling. The pine forest has become fragmented and damaged and is at a risk of reduced genetic diversity. Although Khasi pine is an ecologically and economically important species of the Northeast, very few studies have been carried out with regard to their genetic diversity and population genetic structure. The existence of Khasi pine depends on the sound conservation and management practices for which understanding of the gene diversity estimates will be of immense help and importance. The focus of the study was to examine the cross-species transferability of nuclear microsatellite markers and to use these markers to find out the genetic diversity and population structure of 10 P. kesiya populations.

Plant material
A total of 250 individuals of P. kesiya from 10 geographical locations covering the entire distribution range in Northeast India were sampled for the study (Table 1). Samples were collected randomly and to avoid sampling individuals arising from the same parent, the distance between each individual was at least 100m.

DNA extraction and PCR amplification
The genomic DNA from the needles of the sample was extracted using the CTAB method with some modifications (Doyle and Doyle, 1990;Stange et al. 1998). The extracted DNA was qualified on 0.8% agarose gel and the concentration was measured using a Biophotometer. The samples of genomic DNA were then diluted to a final concentration of 15ng/µL as a working solution.
A total of 47 primers belonging to different Pinus species (Zhou et al. 2002;Change et al. 2004;Elsik et al. 2000;Nurtjahjaningsih et al. 2005;Watanabe et al. 2006;Boys et al. 2005) were tested for transferability in P. kesiya (Table 2). PCR amplification procedures were optimized on the basis of literature given by Vendramin with modifications such as the concentration of MgCl 2 and optimization of annealing temperatures, etc (Vendramin et al. 1996). The PCR reaction mixture consisted of 15 ng genomic DNA, 1x Taq buffer, 3.0 mM of MgCl 2 , 0.2 mM of dNTP, 0.2 µM of forward and reverse primer and 5 unit of Taq Polymerase (Bangalore Genei Ltd. India). Samples were amplified using the following profile: initial denaturation (94°C, 5 min), followed by 35 cycles of denaturation (94°C, 1 min), annealing (locus-specific temperature, 1 min), extension step (72°C, 1 min) and a final extension (72°C, 8 min). The DNA fragments were visualized in a gel documentation imaging system (GelDoc-It System, UVP Ltd).

Data analysis
The bands obtained were scored manually which were then prepared into Input files for further analysis using various software. The software POPGENE version 1.32 (Yeh et al. 1999) (Pritchard et al. 2000a and2000b). Ten independent STRUCTURE runs for each K (K = 1 to 10) were performed separately with 100,000 iterations and a burn-in period of 100,000. The value of K was detected by an ad hoc quantity based on the second order rate of change of the likelihood function with respect to K (ΔK) (Evanno et al. 2005).
The phylogenetic tree using Unweighted Pair-Group Method with Arithmetic Average (Sneath and Sokal, 1973) was constructed using the POPTREE2 program (Takezaki et al. 2010). The population genetic structure was inferred by an analysis of variance framework (AMOVA analysis) according to Excoffier et al. (1992), using the Arlequin software version 3.11 (Excoffier et al. 2005).

Trans-specific amplification and Genetic Diversity
A total of 47 primers were tested for cross-species amplification in P. kesiya out of which only 5 primers amplified positively and indicated 10.6% transfer rate, the rest showing non-specific or no amplification. The primers from P. merkusii and P. taeda exhibited a low transfer rate of 20% and 13% respectively while primers from P. densiflora and P. resinosa did not show any positive amplification in the target species (Fig. 1). This low rate of transfer was due to the phylogenetic distance between the species. According to Celinski et al. (2013), the rate of transfer of microsatellites successfully transferred between species depends directly on their divergence time. Previous studies have also shown that pines have low transferability of microsatellite loci between hard and soft pines which belong to different subgenera of the genus Pinus (Bérubé et al. 2003). The genetic diversity estimates of the 10 populations are given in Table 1. The observed number of alleles at the entire five loci ranged between 2 and 3 with a mean of 2.800 and a total of 14 alleles were detected. The observed heterozygosity (H O ) ranged from 0.044 to 0.819 with a mean of 0.342 and expected heterozygosity (H E ) ranged from 0.490 to 0.603 with a mean of 0.540 (Table 2). At the population level, expected heterozygosity ranged from 0.172 to 0.436 with a mean of 0.341 (Table 1) indicating that populations from LM and UR showed higher genetic diversity as compared to populations from UM and TM. Fixation index (F IS ) was positive in most populations which showed that there was inbreeding within populations. Often, outcrossing species passing through a genetic bottleneck may become inbred through mating among close relatives over several generations, and rapidly lose heterozygosity and allelic variation (Nei, 1975). Although it has been observed that the estimates of outcrossing in natural stands of pines is high (Muller et al. 1999) but instances of inbreeding are not uncommon which results in more homozygotes than expected under Hardy-Weinberg expectations.
The cluster dendrogram segregated all the 10 populations of P. kesiya into two major clusters (Fig. 2). The dendrogram did not show any clear demarcation of populations from different geographical locations.

Genetic structure and genetic differentiation
A strong correlation between genetic and geographical distances (Mantel test: r=0.433; P<0.05) revealed a pattern of isolation-by-distance (IBD) across the distribution range of P. kesiya (Fig. 3). This pattern suggested that the gene flow is more likely to occur between neighbouring populations as their dispersal might be constrained due to the distance between them (Hutchison and Templeton, 1999). As a result, more closely situated populations tend to be more genetically similar to one another (Wright, 1943). Partitioning of the molecular variance was done without assuming hierarchical structure as well as assuming hierarchical structure. Both revealed that most of the variation was within population (60.36% and 58.79%, respectively) as is clear from Table  3. This is in agreement with a number of studies where it has been observed that conifers show high levels of genetic variation within populations and relatively little differentiation among populations (Yeh and El-Kassaby, 1980;Wheeler and Guries, 1982;Hiebert and Hamrick, 1983;Loveless and Hamrick, 1984;Kim et al. 1994;Mueller-Starck, 1995;Agundez et al. 1997). Variance estimates were based on 1000 permutations. Accordingly, the difference between the individuals within the populations was statistically significant (P<0.001).

CONCLUSION
In the last decade, genetic analysis of populations has evolved as a result of the technical advancement of molecular markers (Wan et al. 2004). Using DNAbased markers such as microsatellite markers (SSRs) has made it possible to analyze the genetic diversity both at the population as well as the species level. The de novo development of polymorphic microsatellite markers is difficult in the large highly duplicated conifer genome (Mariette et al. 2001). Therefore, transfer of microsatellites among related species is a reasonable approach for genetic diversity analysis (Celinski et al. 2013).
Pine is the most widely distributed species of the world having high commercial value. Its ecological significance and utility have made it the prime focus for many molecular evolutionary studies across the world. In India, a few significant studies have emerged on Pine species using the molecular marker techniques. However, extensive studies on P. kesiya through this approach in India have not been reported so far. The study indentified some useful microsatellite markers for P. kesiya which can be used for assessing the population genetic structure and diversity of this species. The study also highlighted some of the high genetic diversity forests from among the populations studied which  can be used to establish seed production areas in these forests and the seed from such areas should be used for plantations and infusing diversity in less diverse populations. For maintaining the existence of the Khasi pine forests in the Northeastern region, sound conservation and management practices are required. Results of the present work will go a long way in implementing proper strategies for conservation and management of P. kesiya forests and initiating tree improvement programs in this species.