Analysis of diversity and determination of duplicates among twenty one Dioscorea accessions through morphological and molecular characterization

Several accessions of Dioscorea, a tuber crop of the family Dioscoreaceae, have been conserved under field and in-vitro conditions at the Plant Genetic Resources Center, Sri Lanka. Therefore, identification of duplicates in the collection is necessary as they cause a considerable wastage of input during the conservation process and leads to confusion when using these accessions in plant variety improvement programs. In this study, 21 Dioscorea accessions belonging to Dioscorea alata, D. esculenta, D. bulbifera and D. pentaphylla were characterized by 75 morphological characters and 15 simple sequence repeat (SSR) markers. The morphological characters were distributed as; 31 leaf, 21 stem, five flower and 18 tuber characters. The dendrogram showed that there are four main clusters without duplication. Accession 107 recorded as D. pentaphylla was well separated from other accessions while four accessions (21, 36, 113 and 92) were clustered into non-related clusters with their species. The SSR analysis was conducted on 20 Dioscorea accessions (except for the accession 107) through Power Marker. Polymorphism was detected among all Dioscorea accessions with 2 to 5 alleles per marker and a genetic distance ranging from 0.1333 (among accessions 101, 102, 103 and 109) to 0.7529 (between accessions 83 and 133). The phenogram resulted in three major clusters, which is almost in agreement with the existing classification. A disagreement was observed with respect to the accession 36 recorded as D. alata and 127 recorded as D. bulbifera as both were clustering with D. esculenta. Duplicates, accession 113 and 101, were classified with D. bulbifera and accession 62, 26 were identified with D. esculenta, respectively.


Introduction
Root and tuber crops are vital for providing food for over two billion people, especially in the rural regions of Africa, Asia and the Caribbean.The most prominent root and tuber crops grown in the world are potato (Solanum tuberosum L.), cassava (Manihot esculenta Crantz), sweet potato [Ipomoea batatas (L.) Lam.], yams (Dioscorea spp.) and aroids (Colocasia spp.) (Sangakkara and Emmanuel, 2014).Those are efficient solar energy transferors, a good source of starch (Senanayake et al., 2011) while the productivity is 2.4 Mg dry matter ha −1 with 182 MJ of energy ha −1 day −1 when compared to Sri Lanka Journal of Food and Agriculture (SLJFA) ISSN: 2424-6913 Journal homepage: www.slcarp.lk1.9 Mg dry matter ha −1 and 151 MJ of energy ha −1 day −1 produced by rice (Sangakkara and Emmanuel, 2014).
Dioscorea is a genus consisting of 630 identified species including D. alata, D. esculenta, D. bulbifera, D. fenterfila, D. rotundata, D. trifida, D. composita, D. spicata and D. tomentosa.Nearly 40 Dioscorea varieties are grown in Sri Lanka (Senanayake et al., 2011) with many of D. alata and few D. esculenta and D. bulbifera varieties being edible.Because of many benefits such as being a cash crop and a food crop with health benefits, conservation of the Dioscorea has become important.There are about 85 Dioscorea accessions conserved under in vitro and field conditions at the Plant Genetic Resources Center (PGRC), Sri Lanka.Conservation is a high input process and having duplicates cause considerable wastage of inputs.Accession details derived mostly from ordinary community such as from farmers without scientific background are having a low credibility and therefore, incorrect identification is a barrier to deploy those accessions for further use.In addition, Dioscorea having high diversity with many beneficial characters are not thoroughly analyzed.Therefore, morphological and molecular characterization of Dioscorea accessions is a timely requirement.In this study, 21 Dioscorea accessions were characterized up to the species level with the objectives of identifying duplicates and the genetic diversity through morphological and molecular characterization.

Selection of site and plant material:
The study was conducted in the biotechnology division and an open field of the Plant Genetic Resources Center (PGRC) at Gannoruwa, Peradeniya in Sri Lanka.Twenty one Dioscorea accessions including four accessions of D. esculenta (accession 111, 83, 21 and 133), five of D. bulbifera (accession 127, 102, 103, 101 and 109), 11 of D. alata (accession 113, 92, 36, 30, 53, 65, 135, 128, 74, 26, 62) and one of D. pentaphylla (accession 107) were selected as shown (Table 1).Molecular characterization: The DNA was extracted from young Dioscorea leaves according to Mignouna et al. (2009).Fifteen SSR primers designed for microsatellites regions of Dioscorea genome were selected (Table 2) for the study.Otoo et al., 2015;Arnau et al., 2017;Siqueira et al., 2012;Tostain et al., 2006) The polymerase Chain Reaction (PCR) was done for 20 Dioscorea accessions (whole set PCR) for each 15 primers according to PCR Program shown in Table 3.The polyacrylamide gel images obtained from PCR products were manually scored for the presence or absence of alleles for each SSR marker and analyzed using Power Marker version 3.25.

Results and Discussion
The dendrogram of the morphological analysis revealed four clusters at 50% similarity level without duplicates (Figure 2).Accession 107 (Katuala cultivar) recorded as D. pentaphylla was well separated from the other accessions while four accessions (21, 36, 113 and 92) were clustered into non-related clusters with their species.In morphological characterization, some accessions were clustered away from main cluster of their species (accessions 113, 92 and 21) however, it was not so in the molecular characterization.Duplicates could be identified only by molecular characterization, which can be expected to provide more accurate clustering than the morphological characterization as morphology can be influenced by environmental factors (Carovic stanko et al., 2011).The genetic distance was minimum among the accessions belonging to the same species and maximum among different species.According to the cluster analysis, misidentifications such as with accession 36, which was recorded as D. alata in the PGRC records, was resolved and identified as D. esculenta according to both morphological and molecular clustering.Accordingly, identification of duplicates have helped minimizing high input requirement in conservation and to resolve issues with improper distribution of accessions from PGRC in future.The study also confirmed the highest divergence of D. pentaphylla (accession 107) and its unique species status.

Figure 2 .
Figure 2. Dendrogram of the morphological analysis

Figure 3 .
Figure 3. Phenogram of the molecular analysis

Table 1 .
Details of the selected Dioscorea accessions

Table 2 .
Details of the selected SSR primers

Table 3 .
Composition of the PCR Mixture (Promega ® USA)