Title : ISSR-markers Assisted Genetic Diversity Assessment of Acid Lime [ Citrus aurantifolia 1 ( Christm . ) Swingle ] Germplasm of Eastern Nepal

Title: ISSR-markers Assisted Genetic Diversity Assessment of Acid Lime [Citrus aurantifolia 1 (Christm.) Swingle] Germplasm of Eastern Nepal 2 3 Nabin Narayan Munankarmi1,, Neesha Rana2, Tribikram Bhattarai1, Ram Lal Shrestha3, Bal 4 Krishna Joshi3, Bikash Baral4,, Sangita Shrestha2 5 6 1Central Department of Biotechnology, Tribhuvan University (TU), Kirtipur, Kathmandu, Nepal 7 2Molecular Biotechnology Unit, Faculty of Science, Nepal Academy of Science and Technology 8 (NAST), Khumaltar, Lalitpur, Nepal 9 3Nepal Agriculture Research council (NARC), Lalitpur, Nepal 10 4Department of Biochemistry, University of Turku, FIN20014 Turku, Finland 11 12 Corresponding Authors’ e-mail: 13 14 Nabin Narayan Munankarmi (e-mail: nabin2045@gmail.com) ORCID Number: 0000-0002-105015 0901 16 17


Introduction
Citrus, an important genus from Rutaceae family [1], is an ancient perennial crop more often cultivated in tropical and sub-tropical parts of the globe [2].Nepal is one of the countries in Asia where citrus is thought to have been originated [3], and has crucial role in the horticultural industry.
Acid lime [Citrus aurantifolia (Christm.)Swingle] and lemon [Citrus limon (L.) Burm.f.] are important fruit crops in Asia, and India is the largest producer of lime and lemon [6].Acid lime commonly known as "Kagati" in Nepal is enriched in vitamin 'C' with multi uses as for preparing juice, pickles, and salad.Its medicinal properties are attributed by its preventive and curative measures against various diseases of joints and bones, cold, influenza, dysentery, piles, scurvy, cold, and constipation [7].As Lemons and Limes juice have profound amount of citric acid comprising 1.38 and 1.44 g/oz resp.[8], beverages with citric acid are reported to reduce the content of calcium, and ultimately enhancing urinary citrate excretion.Hence, it could be a good dietary supplement for preventing and managing calcium Urolithiasis (kidney stone) [7,9].
Acid lime proves to be a crucial commercially cultivated fruit crop of Nepal, with a ranking of 3 rd after mandarin and sweet orange in terms of area coverage (2,731 ha).The cultivation of Acid lime is done in several districts of Terai (60 out of 75 ; low-lying land on the outer foothills of Himalayas) to the land lying on High-hill landscapes of Nepal, particularly concentrating in Eastern Nepal [10].
Unlike Mandarin and sweet orange, acid lime can be successfully raised from High-hill to Terai regions of Nepalese land [10].Three different cultivars of lime have been grown in Nepal, viz., Acid Lime, Eureka, and hybrids.Among them Acid lime bear high commercial value in the market due to its size, better aroma and enriched medicinal value [11].The favorable season for the production of lime in Nepal is from September -November, however, the demand of this fruit is throughout the year [12].
In Nepal, the production of acid lime is 8.3 ton/ha [13], which is itself very low as compared to the productivity of other countries like Argentina (19 ton/ha), and India (12.2 ton/ha) [14].The reason for this low production may be due to several stressors, like biotic stress (Pests and diseases) and abiotic stress (salinity, drought, and temperature).In addition, prevailing Climate Change is putting more pressure on gross crop productivity [15].Considering these scenarios, elite Acid Lime varieties with desirable qualities, like nematode resistance, resistance to disease, juice content, higher yield, stress tolerance etc., holds better promises.Development of such cultivars with desirable qualities can be achieved via breeding programs (conventional and non-conventional) viz., molecular markerassisted breeding, protoplast fusion, mutation breeding and genetic engineering [16][17][18].High level of variation in fruit quality, seasonality in flowering, harvesting time, productivity and disease resistance among acid lime accessions of different agro ecological zones have been made [19].
Therefore, the study of genetic diversity at the molecular level and conservation of acid lime germplasm of Nepal are crucial tasks that remain to be performed for its breeding and cultivar development program.
The major motive of plant breeders lies in improving the qualitative and quantitative traits of the existing cultivars.This has been achieved via conventional breeding involving the whole genomes followed by the selection of highest quality recombinants among several segregating individuals.
However, this is highly time-consuming and tedious that involves multiple crosses and several generations, vigilant linkage drag and phenotypic selection [20].Recently developed DNA-based molecular marker technologies have become immensely useful to plant breeders as complementary tools for conventional breeding.They have evolved as efficient tools for genetic diversity assessment, cultivar identification, marker-assisted selection and breeding, and recently to genomicsassisted breeding for crop improvement [21][22][23][24][25]. Genetic diversity estimation employing molecular marker tools is a fundamental task to be performed as it provides baseline information to plant breeders for detecting a unique germplasm that is required for the improvement and selection of horticultural traits and introduction of disease resistant cultivars to improve both the quality and quantity production of the fruit [26].genetic diversity assessment and cultivar identification; (v) genome organization and phylogenetics, etc. [29][30][31].
Of the various molecular-marker systems, PCR-based ISSR marker system have wide usage in studying genetics [32].These markers are polymorphic nature [33], abundance in the genome [34], and have the advantages of SSR markers, circumventing the major obstacle of the development of SSR markers, i.e. the requirement of flanking sequences for primer design and enjoying the advantages of random markers [35].ISSR technique combines the benefits of AFLP and SSR markers with RAPD's universality [22].The ISSR markers are informative for species where genome sequences are unavailable [36].The scoring is done as dominant markers, and inherited in Mendelian fashion [37].ISSR, a PCR-based marker has a capacity to rapidly screen and differentiate between closely related individuals [32].

Several molecular marker-based studies like RAPD and ISSR have been conducted in different
Citrus germplasm by [24,[38][39][40][41].Although the codominant SSR marker system based genetic study was carried out prior to this study using the similar samples [39], however, it was not based on advanced Capillary Electrophoresis (CE) and was carried out using conventional Agarose Electrophoretic system.Therefore, based on this as well as ISSR being more robust dominant marker system than RAPDs and condition where no prior genome sequence was known, our aim was to evaluate the genetic diversity and relationship among the acid lime germplasm of different localities of Eastern Nepal employing the ISSR markers and comparative analysis performed based on the results obtained from 3-different marker systems.An overall objective of this research was to set-up a baseline data to assist future breeding and conservation programs of Acid Lime in Nepal.In addition, our main research revolves around different questions, such as (i) the extent of genetic diversity in the Acid Lime cultivars of Eastern Nepal, and (ii) the agro-ecological zone which harbored most genetically diverse Acid Lime cultivars based on our present ISSR study.

Plant Materials
Altogether 60 young and healthy leaf samples (6 to 8 weeks old) of acid lime were harvested from farmer's plantation areas of Eastern-zone Nepal and were stored in an airtight zip-lock bags with silica gel.Random sampling was done from the selected trees of all agro-ecological zones, viz., Terai, Mid-hills, and High-hills (Suppl.Table 1).

DNA Extraction and PCR amplification
Dried leaf samples (100 mg) were ground to a fine powder using liquid nitrogen, and the genomic DNA was extracted according to the protocol of DNeasy plant DNA extraction mini-kit (QIAGEN).
The size of the obtained PCR products was analyzed by using Gene ruler TM 100 bp plus DNA ladder (Fermentas Life Sciences).

ISSR Profiling and Scoring of the Data
Using optimized ISSR-PCR reactions and cycling conditions, 49-different ISSR oligos were screened using fresh genomic DNA samples of acid lime.All experiments comprised 3-biological replicates and 3-technical replicates.Out of 49 oligos, 21 oligos that provide crispy, multiple, scorable, and reproducible bands were selected for further ISSR profiling.The ISSR profiles generated by each of the 21 oligos were used to score the bands, and the creation of binary data matrix.Scoring of all polymorphic and monomorphic bands was performed.Scoring of the markers as '0', '1' and '9' was performed for absence, presence and the failure of PCR amplification respectively [43][44][45].

Data Analysis
The binary data matrix was analyzed using  Genetic diversity in acid lime was computed using Numerical Taxonomy and Multivariate System (NTSYS, version 2.21i, New York, USA).Similarity indices were calculated applying a similarity to qualitative data.From these similarity indices, sequential, agglomerative, hierarchical and nested (SAHN) clustering was performed using the unweighted pair group method of arithmetic averages (UPGMA) algorithm [48].Similarity coefficients were computed-based on 3-different measures: Simple Matching Coefficient (SM) [49], Dice's Coefficient of Similarity (D) [43,50] and Jaccard's Coefficient (J) [44].The matrices of SM, J and D coefficients were compared by Mantel test [51] using MXCOMP option in NTSYS program.The cophenetic correlation test was applied for estimating the correlation between each of the similarity matrix and its corresponding phenogram.
The estimated correlation coefficient values show the goodness of fit of cluster analysis performed on the basis of each of SM, J and D. In order to evaluate trees constructed from UPGMA clustering by genetic similarity coefficients, Consensus Fork indices (CIC) were calculated using Strict Consensus method of NTSYS program for each combination of similarity coefficient and UPGMA clustering.CIc measures how resolved the tree is [47].The best-fitted similarity matrix coefficient was then employed for the genetic diversity assessment.
The number of scorable bands produced per primer ranged from 7-18 with variation in amplicon size ranging from 250-3,200 bp.The highest number of ISSR loci (18) was produced by primer UBC 857, whereas the lowest number of ISSR loci (7) was produced by primer C1 and UBC 834 in the total accessions (Suppl.

3.2.Genetic Diversity in Acid Lime Cultivars
The varied range of similarity indices were obtained, using Simple Matching (SM), Jaccard's (J) and Dice (D) coefficient i.e., SM (0.54 -0.94), J (0.42 -0.90) and D (0.57 -0.95) with an average similarity coefficient value of 0.79, 0.69 and 0.81 respectively.The Mantel test (Matrix comparison) result of original matrices showed a correlation value between J and D to be the highest and significant (0.99710) in comparison to SM and J (0.98143) and SM and D (0.98318) (Suppl.Table 3).
The highest CIC value (CIC = 1.00000) was observed for J and D coefficients (Suppl.Table 4).
Cophenetic correlation coefficient value (r) between the genetic similarity matrices and cophenetic matrices are presented in Suppl.Table 5. Unweighted Pair Group Method of Arithmetic Averages (UPGMA) distance for D coefficient gave the highest Cophenetic Correlation value (r = 0.90356) (Suppl.Table 5).Compared to J and SM coefficients, D coefficient was evaluated to be the best coefficient for deducing the genetic diversity and relationship among various acid lime cultivars as shown by the highest cophenetic correlation coefficient value (r = 0.90356) indicating very good fit for cluster analysis (Suppl.Table 5).
On comparative analysis made for the similarity coefficients, Dice coefficient was revealed to be the best, which was subjected further for interpreting genetic diversity, and the relationships among various accessions of acid lime representing different geographical gradients.Based on the Dice similarity coefficient, genetic similarity within 60 acid lime accessions ranged from 57% to 95% with an average of 81% (Suppl.7).

3.3.The genetic relationship based on UPGMA Cluster Analysis and PCoA
The 60 genotypes were separated into two major clusters (I and II) and three minor clusters (III, IV, and V) in the phenogram (Fig. 2).The accessions from High-hill, Mid-hill, and Terai zones were intermingled in different clusters.The cluster I comprised of 30 accessions from High-hill and Midhill agro-ecological zones.In this cluster, the highest genetic similarity coefficient was observed for the accession LT-20 and LT-21 (0.95), and the lowest similarity coefficient (i.e., highest genetic distance) was observed between LS-56 and LS-35 (0.571).Cluster II comprised of 25 accessions from High-hill, Mid-hill and Terai agro-ecological zones along with the exotic varieties of Vanarasi, Madrasi and Rampur (LKv-60, LKm-61, and LKr-62 respectively).In this cluster, accessions LS-37 and LS-39 had the highest similarity value of 0.993 followed by 0.940 (between LS-42 and LD-45), 0.927 (between LD-48 and LD-50) and so on.Cluster III and cluster IV consisted of single accession, LT-9, and LD-59 from High-hill and Mid-hill respectively.Cluster V comprised of three accessions (LS-56, LD-58, and LS-57) that belongs to Terai agro-ecological zone (Suppl.Table 8).
The cluster II has been separated from cluster I at a similarity coefficient of 0.803 and cluster IV separated from rest of the group at a similarity coefficient of 0.66.There was only a small genetic variation between cluster groups I and II (similarity %, 81.4 and 81.8 respectively) and clusters IV and V (similarity %, 73.7 and 73.9 respectively), whereas wider variation was observed between cluster IV and II (Fig. 2).
Two-dimensional plots of the principal coordinate analysis (PCoA) classified the 60 acid lime accessions based on ISSR allelic variation (Fig. 3).The first principal co-ordinate axis accounted for 14.51%) (Eigen value = 323.27;percentage of variance = 14.51%) and second accounted for 8.34% (Eigen value = 185.77;percentage of variance = 8.34%) of the total genetic variation with a cumulative variation of 22.85%.Therefore, groups were discriminated, with axes 1 and 2 expanding 22.85% of the total variation.Polymorphism reported in Citrus spp. is comparable with our present investigation (PP=87.18%)such as 89.4% in wild Citrus spp.[24], 87% in C. indica [52], and 100% of few commercially important Citrus spp.[53].The total amplification profiles generated by the 21 ISSR primers yielded 234 bands of which 204 were polymorphic and 30 were monomorphic, which gives us a clue about the existence of high level of genetic diversity among selected acid lime germplasm from 3-different ecological zones of Eastern Nepal.RAPD screens whole genome as revealed by 94.94% polymorphism in corresponding samples, much higher compared to present findings [41].However, ISSR amplicons correspond to specific inter SSR loci and ISSR-PCR is more stringent than RAPD because of the use of longer oligos (16-25 bp), that allows the use of high annealing temperatures [22].PIC value provides information about heterozygosity and is associated with the degree of polymorphism.Primers with comparably higher PIC values are useful in discriminating accessions [54].In our study, the PIC value ranged from 0.74 to 0.93 with an average of 0.89.The highest PIC value of 0.93 was observed for primer UBC 857 and lowest of 0.74 for UBC 807 with an average value of 0.85.PIC value of >0.80 indicates their usefulness for the assessment of genetic diversity of acid lime accessions (Suppl.Table 2).Interestingly, PIC value of the SSR based study of the similar germplasm revealed comparatively low values [39], which might be due to SSR markers being codominant and specific PCR-based marker system.Also, as the SSR-based study was conducted in conventional Agarose Gel Electrophoretic system (in contrast to Polyacrylamide or Capillary Electrophoresis), small allele size differences (varying in few bases) might not have been properly resolved.Capillary Electrophoresis has been shown to be the superior technique for SSR-based genetic analysis [55].However, the PIC value of the present study is comparable to that reported in our previous RAPD-based study (ranged from 0.78 to 0.88 with an average of 0.80) [41].Our current investigation revealed the primer (UBC 857) that had highest PIC value (0.93) to have highest Resolving power value (23.16) (Suppl.Table 2), which provides us a glimpse of quantitative data that allows us to make direct comparisons between the primers [47].

4.1.ISSR polymorphism and genetic diversity estimation in Nepalese Acid Lime
The clustering based on UPGMA analysis revealed the genetic diversity and relationship among acid lime accessions of three geographically diverse agro-ecological zones.No specific cluster was formed for accessions from different agro-ecological zones under study.Our result is congruent with others finding of being separated into two major and three minor clusters, apart from the distribution of different accessions in different clusters [39].Cluster I of present study comprised of accessions from High-hill and Mid-hill zones showing the close genetic relationship, whereas cluster II comprised of accessions from all three agro-ecological zones and the exotic varieties viz., Vanarasi, Madrasi and Rampur (LKv-60, LKm-61, and LKr-62 respectively).These exotic varieties were also clustered together on phenogram generated from SSR markers [39] and RAPD markers [41].
Accessions (LS-56, LS-57and LD-58) from Terai ecological zone are clustered together, similar to the results obtained in SSR-based phenogram by [39], indicating their genetic closeness.The intermixing of accessions that are grown in different agro-ecological domains in different clusters in the phenogram may be attributed to the genetic similarities among different accessions in various qualitative and quantitative traits.In order to improve varieties, the ideal parent for hybridization should be selected based on the level of genetic diversity estimated using molecular markers [56].
The high usage of morphological traits for the determination of genetic relationship among plants and its varieties exists [57].However, morphological markers do not often reflect genetic relationships because of their interaction with the environment and epistasis [58].On the basis of previous study on fruit diversity and vitamin C content, four elite accessions [two from high-hills, and one each from Terai and Mid-hills were confirmed to be of superior quality and recommend for conservation, breeding and various developmental purposes [59].In our present investigation, the first two accessions (LT-17 and LT-23) are clustered together in I group and remaining two (LD-49 and LM-44) are clustered in II group.
Dice similarity matrix based genetic diversity estimates within each of the three agro-ecological zones revealed wide genetic base in accessions from Terai agro-ecological zone (0.57-0.94) in comparison with Mid-hill (0.70-0.94%) and High-hill (0.75-0.95%).However, regarding between agro-ecological zones genetic base, the highest value was observed between High-hill and Terai accessions (57-95%) followed by Mid-hills and Terai accessions (57-94%).Terai accessions being most genetically diverse had a comparable genetic base to that of Mid-hill vs. Terai and High-hill vs.
Terai.The result is comparable with the result obtained using SSR markers where accessions from high and mid-hills have the high average genetic similarity (73% and 81%) in comparison to Terai (69%) [39].Our results showed that the collected accessions from different agro-ecological zones were not genetically distinct but highest diversity was observed in Terai accessions compared to in High and Mid-hills.In our investigation, the diversity indices like Shannon's information index (I) and Nei's gene diversity (H) were found to be 0.325 and 0.215 respectively in Terai agro-ecological zone which was highest among the three zones studied.This indicates Terai to have diverse gene pool, compared to Mid and High-hills.This discrepancy in diverse gene pool could be due to higher accessibility for the movement of germplasm in Terai within the country, and also from neighboring country India.In contrast, lower level of genetic variability as was observed in Mid-hills and Highhills might be due to acid lime trees being established in natural conditions in these zones [39].Our previous study using similar germplasm based on RAPD markers shows almost similar diversity indices values [41].

4.2.Application of ISSR-based Genetic Diversity Estimates in Acid Lime Breeding Program
In the present investigation, ISSR dominant markers were utilized for the genetic diversity analysis of acid lime cultivars of eastern Nepal.The advantage in the use of ISSR markers in plant breeding lies in their linkage to SSR loci.Although microsatellites themselves are probably non-functional and selectively neutral, they are linked to the coding regions, so that ISSRs mark the gene-rich regions [60].Also, because of quicker ISSR analysis procedure that detects over hundreds of bands per primer, this has gained increased attention to both the plant genetics and the breeders' alike [61].
To date, more polymorphism has been detected with the use of ISSRs than any other assay procedure [62][63][64].However, there are possibilities that the fragments with the same mobility in electrophoresis originate from non-homologous regions, which can contribute to some distortion in the estimates of genetic similarities [61,65].
Many qualitative and quantitative agronomic traits such as high juice content, fruit size, disease and insect resistance have the genetic basis of inheritance and can be enhanced by the use of molecular markers and marker-assisted selection (MAS) technique.Selection and Improvement of good qualitative and quantitative traits are important steps in the variety of developmental programs.
Moreover, breeding of good quality traits requires selection of parents with a wider genetic diversity [66].For this, sufficient knowledge about genetic diversity in the gene pool is required to adopt the efficient and valuable breeding approach.
Even though tremendous demand for Acid lime exists in Nepal, the country is not self-reliant in its sole production, thus import of bulk quantities is required to quench the thirst of growing population.
Nepal is enriched with a favorable geo-climatic condition for acid lime cultivation, however, its production per hectare is comparatively very low because of the lack of high yielding varieties, rapid diseases and pests' infestations, poor agronomic practices, not an introduction of disease-resistant cultivars, etc.Our current study assesses the genetic diversity of acid lime germplasm of eastern Nepal using ISSR marker technique and underscores the need for the conservation of these resources by the development of elite cultivars.
Using representative accessions of acid lime from three different agro-ecological zones, we investigated the genetic diversity of Nepalese Acid Lime using ISSR marker.Moderately overall Microsoft-Excel 2007.It estimates the banding characteristics; such as (i) Total number of bands obtained (TNB); (ii) Number of polymorphic bands (NPB); (iii) Percentage Polymorphism (PP); (iv) Polymorphic Information Content (PIC); (v) Band Informativeness (IB); and (vi) Resolving Power (RP) for each primer used {PP = NPB/TNB generated by each primer; PIC = 1-∑ (Pij)2 , where Pij is the frequency of the i th pattern revealed by j th primer summed across all patterns revealed by the primers[46]; IB = 1 -[2 × (0.5 -P)], where P represents the proportion of accessions comprising the band; Rp = ∑ IB[47]}.
The genetic diversity and relationship among the acid lime cultivars of different agro-ecological zones of Eastern Nepal were also studied through a Principal Coordinate Analysis (PCoA) using Multivariate Statistical Package (MVSP, version 3.21) and in terms of Percentage of Polymorphic Bands (PPB), Nei's Genetic Diversity (H) and Shannon's Information Index (I) using Popgene (Version 1.32).

Table 6
Considering these similarity indices, Terai accessions were shown to have wider genetic base followed by Mid-hill and High-hill.