Genetic diversity of Miyamasukashi‐yuri (Lilium maculatum Thunb. var. bukosanense), an endemic and endangered species at Mount Buko, Saitama, Japan

Abstract

Lilium maculatum var. bukosanense, Miyamasukashi-yuri in Japanese, grows endemically in Saitama Prefecture, Japan, and is listed in the Saitama Prefecture Red Data Book as a critically endangered plant. Articles about its genetic diversity and population genetic structure have not been reported. Before it is lost, population genetic studies can provide information for efficient in situ conservation as well as for possible future use in breeding programs. Therefore, we carried out a population genetic study of natural L. maculatum var. bukosanense using chloroplast DNA (cpDNA) and inter-simple sequence repeat (ISSR) markers. In this study, fresh leaves of 46 L. maculatum var. bukosanense individuals were collected from two populations in Mount Buko. By polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of cpDNA, we estimated the maternal origin of individuals sampled. Sixteen cpDNA primer pairs were tested, and only four of them produced clear bands. Twenty-four combinations from these PCR fragments combined with six restriction enzymes were assayed; however, only 18 of them could be digested, producing two to four fragments. All individuals of L. maculatum var bukosanense analyzed were virtually identical for all primer-enzyme combinations. These findings suggest that the maternal origin of the 46 individuals analyzed is close. In addition, we analyzed ISSR markers to provide mainly biparental inheritance. PCR products of five anchored simple sequence repeat primers showed polymorphism, indicating that the individuals sampled were not clones, and thus it appears that they could have been reproduced from seeds. The primers yielded a total of 30 polymorphic bands (i.e. an average of six polymorphic bands per primer). The mean genetic distance and genetic identity values between the populations were 0.032 and 0.97, respectively. These findings suggest that all individuals from both populations are genetically similar. The individuals growing at 1000 m have greater genetic variation than those individuals from 650 m and this could possibly be attributed to altitude. A significant correlation between gene diversity and population size is also reported. From the results presented here some strategies for its in situ and ex situ conservation might be considered.

Marker Assisted Characterization in Tigridia pavonia (L.f) DC

“…The percentage of polymorphic fragments varied from 66.6 to 100%, with an average of 87.5% (Table 4). These results are similar with those reported by Yamagishi et al (2002) and Arzate-Fernández et al (2005b), who used these ASSR primers and proved their efficiency for detecting high polymorphism percentage in different species of the genus Lilium.…”

“…In each primer, the anchor consisted of a triplicate of distinct sequence (Table 4). The amplification conditions for the primers 3'-ASSR02 and 3' -ASSR15 were those described by Arzate-Fernández et al (2005b), and consisted of an initial cycle of 9 min at 94 °C, 1 min at 46 °C and 1 min at 72 °C and a final cycle of 9 min at 94 °C, 1 min at 46 °C and 10 min at 72 °C. For primers 3'-ASSR20, 3'-ASSR29 and 3'-ASSR35, the amplification cycles were those used by Yamagishi et al (2002) and consisted of an initial cycle of 9 min at 94 °C, 1 min at 46 °C and 1 min at 72 °C and a final cycle of 10 min at 72 °C.…”

Genetic diversity of Cosmos species revealed by RAPD and ISSR markers

“…This suggests that the ISSR markers can be a highly informative, fast, and reliable system for the genetic diversity studies as reported by Liu et al (2011). Our results also suggest that the CT sequences may be very abundant in the genomes of wild ornamental species because the primers that were used here have been also useful for determining the genetic diversity of other ornamental wild species such as Lilium maculatum (Arzate-Fernández et al, 2005), Tigridia pavonia (Piña-Escutia et al, 2010a), and Sprekelia formosissima (Bautista-Puga et al, 2011).…”

“…The mean of total and the polymorphic fragments that were generated per primer were 6.4 and 4.2, respectively. ISSR markers have been successfully used to estimate the extent of genetic diversity in a wide range of crop and wild species, including lily (Arzate-Fernández et al, 2005), jacaranda (Escandón et al, 2005), citronella (Bhattacharya et al, 2010), and ginseng (Li et al, 2011). The ISSR marker efficiency has been attributed to motif sequences, as well as the sequence of its anchor.…”

“…The extraction procedure was the cetyltrimethylammonium bromide method that was reported by Arzate-Fernández et al (2005). The DNA samples were stored at -20°C prior to analysis.…”