METHODS

1. Gender Markers in Nepenthes Michael Taylor and Dr. Cheryld Emmons Division of Biology, Alfred University, Alfred, NY USA ACKNOWLEDGEMENTS ABSTRACT METHODS RESULTS The carnivorous plant Nepenthes, endemic to Southeast-Asia, is dioecious (male and female flowers on separate plants). Commercially grown nepenthes are difficult to reproduce due to the poor success of tissue culture methods and identification of the sex of plants when they are young. Currently the only method to identify gender in the plants is by the flower structures. Recent studies have indicated that there may be a genetic method for identifying sex in Nepenthes. Specific DNA PCR primers have been tested in this study to determine the possibility of identifying sex using molecular genetic techniques. The primers used are Ms45 a gene derived from maize that when mutated produces male sterility. Salix a gene derived from Willows that is involved in partial sterility. And OP15 a primer used in a previous study that determined sex in Nepenthes. However the primers tested showed some inconclusive results. Plant tissue samples were obtained from Bruce Lee Bednar of Lee's Botanical Garden in Florida, William McLaughlin US Botanical Garden in Washington DC, and a private collection. They were designated N1-N8 (N1-N5, from Lee’s Botanical, N8 from US Botanical, and N6 &N7 from private collection). N5 and N8 were previously identified as female. DNA samples were extracted and isolated using MoBio UltraClean Plant Isolation Kit. The DNA was then amplified using Polymerase Chain Reaction (PCR) in a PCR master mix from Promega (Taq DNA polymerase (50units/mL), dNTP (400uM each) and MgCl2 (3mM)). The Primers used Salix, Ms45, OPP08, and OPA15 were all previously successful in identifying sex-linked genes (OPA15 (Mokkamul et.al. 2007).The thermocycler was run the same as Mokkamul et. al., incubated at 94C for 3 min. for 35 cycles, denaturing 1 min. at 94C, annealing 2 min. at 36C, 2 min. at 72C and 7min final extension at 72C. PCR products were then run on 4 gels,each for the four primers used. The 0.8% agarose gel was run at 120 volts and stained using SYBRSafe. Gels were viewed using a UV imaging system. Special thanks to: Dr. Alfred Butner of the Brenda Berstein Butner Fund for providing funding for this study. Bruce Lee Bednar of Lee’s Botanical Garden and William McLaughlin from the US Botanical Garden for donating tissue samples. Primers OPP08, Ms45, Salix had confirmed bands show up while OPA15 had no visible DNA fragments. Only N5 and N6 had visible DNA fragment bands. OPP08 and Ms45 both had visible bands on N5 and N6 (Fig. 1 A and B). Salix only had confirmed bands under N6 (Fig. 1 C). Figure 1. Gel Electrophoresis on 0.8% agarose gel for primers Ms45, OPP08, and Salix. (A) shows DNA amplified by Ms45 main fragment roughly 700bp long on both N5 and N6. (B) shows DNA amplified using OPP08, fragments are between 500-1kbp long. ( C) amplified using Salix shows only fragments for sample N6 between 500 and 1kbp long. OPA15 not shown , had no fragments. INTRODUCTION REFERENCES Nepenthes is a dioescious plant from southeast asia. The genetic mechanisms of how the sex is determined is still unknown. Currently the only means to identify the sex is by floral structures. Only quite recently has there been a confirmed genetic marker that was specific to a males in the Nepenthes (Mokkamul et. al. 2007). This indicates that there is a genetic difference between males and females. Amateur cultivators of Nepenthes have yet to see a Nepenthes change from one sex to another. However a confirmed case of a hermaphroditic plant was discovered (Bednar 1985). Different primers have been studied previously that gave some insight on different aspects of dioecious strategies. The primers tested in this study were Ms45, OPP08, OPA15, and Salix. Ms45 a sequence that when mutated produce male sterile plants in maize (Zea mays) (Cigan et. al. 2001). OPP 08 a primer used to identify males in Silene (Mulcahy et. al. 1992). OPA15 was the only successful primer used in the Mokkamul study. Salix named after the willow (Salix) has been observed to be involved in partial sterility and has been used to identify females in willows (Alstrom-Rapaport et. al. 1998). CONCLUSION & DISCUSSION Bednar, Bruce (1985) An Unusual mirabilis Plant. Carniv. Pl. Newslett. 14(4):91 Mulcahy, D. L., Weeden, N. F., Kesseli, R., Carroll, S. B.“DNA probes for the Y-chromosome of Silene latifolia, a dioecious angiosperm”. Sexual Plant Reproduction. 5. 1 (1992). 86-88 Dellaporta, Stephen L. and Calderon-Urrea, Alejandro. “Sex Determination in Flowering Plants”. The Plant Cell. 5 (1993): 1241-1251. Alstrom-Rapaport, C., Lascoux, M., Wang, Y. C., Roberts, G., Tuskan, G. A.“Identification of a RAPD Marker Linked to Sex Determination in the Basket Willow (Salix viminalis L.)”. Journal of Heredity. 48. 1(1998). 44-49. Cigan, A. M., Unger, E., Xu, R.-j., Kendall, Fox, T.W.“Phenotypic complementation of ms45 maize requires tapetal expression of MS45”. Sexual Plant Reproduction. 14. 3(2001): 135-142. Mokkamul, Piya, Arunrat Chaveerach, Runglawan Sudmoon, and Tawatchai Tanee. “Species Identification and Sex Determination of the Genus Nepenthes (Nepenthaceae)”. Pakistan Journal of Biological Sciences. 10. 4 (2007): 561-567.  The visible bands of Ms45 indicates that the same sequence in maize is present in Nepenthes. Since Ms45 has been seen to be involved in male sterility in maize (Cigan et. al. 2001) it may give insight that the primer can be used to identify females. As a result N6 may be female. However this is hard to confirm until the plant flowers. The same result came with the OPP 08 primer. Which is quite fascinating due to previous study’s findings that the primer was used as a male identification marker (Mulcahy et. al. 1992). Salix showed some interesting results the only visible bands were of N6 however not on N5. This supported the Ms45 results that N6 could be female since Salix also was used to identify females in willows genus, Salix (Alstrom-Rapaport et. al. 1998). The failure of OPA15 is somewhat surprising because it is the only one of these that has been confirmed to be a sex specific marker for Nepenthes (Mokkamul 2007). With the poor success of OPA15 and the lack of visible bands on any N8 and Salix N5 the results seem to be very inconclusive as to the identification of sex in Nepenthes.