Molecular characterization and testing hybridity of interspecific crosses in black pepper (Piper Nigrum L.)
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Date
2012
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College of Horticulture, Vellanikkara
Abstract
Although India is one of the leading producer of the economically important spice crop black pepper (Piper nigrum L.), the productivity of the crop is low due to various factors among which infestation by Phytophthora capsici has been identified as a major factor. Wild relatives of crops are valuable sources of desirable characteristics for genetic improvement of crops. Attempts were made in the past to transfer genes for disease resistance through interspecific hybridization in black pepper also using the related species like P. attenuatum (Sasikumar et al., 1999), P. colubrinum (Vanaja et al., 2008) etc. However, the major problem was cross incompatibility and hybrid sterility caused by the difference in chromosome number between the different species. As it is a perennial crop, a reliable method for identification of hybrids at the early stage of development, preferably at seedling stage itself, is essential. Hence the study entitled “Molecular characterization and testing hybridity of interspecific crosses in black pepper (Piper nigrum L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2010-2012. The objectives of the study were to characterize the partially fertile interspecific hybrid (Culture P5PC-1) from the cross P. nigrum x P. colubrinum tolerant to Phytophthora foot rot and to test the hybridity of putative F1 hybrids developed at Pepper Research Station (PRS), Panniyur, using Randomly Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSR) markers. Six interspecific hybrids produced by crossing two cultivars (Uthirankotta, Karimunda) and four high yielding varieties (Panniyur 1, Panniyur 2, Panniyur 3 and Panniyur 5) of P. nigrum as female parents and P. colubrinum as male parent were used for the study. Morphological observations were taken from the field grown vines of parents and putative hybrids maintained at PRS, Panniyur. The hybrids resembled the respective female parents for the most of the leaf, stem and spike characters recorded as per the descriptor for Piper sp. except for berry setting percentage which was found significantly low in hybrids compared to respective female parents. For SSR and RAPD assay, genomic DNA was extracted from the first leaf from tip of the stem of all the plants using the CTAB procedure reported by Rogers and Bendich (1994) with slight modification which yielded good quality DNA for further analysis. Thirty RAPD primers and fifty four SSR primer pairs were screened with DNA of black pepper var. Panniyur 5 for amplification and those which gave reliable distinct banding pattern were selected for further analysis. Genomic DNA of six interspecific hybrids and their parents were amplified with 10 selected decamer primers and 11 SSR primer pairs. The presence or absence of the markers were scored and entered into a binary data matrix and was used for calculating the similarity coefficient using Dice coefficient (Nei and Li, 1979) using software DARwin (Version 5.0.158) and Jaccard’s coefficient (Jaccard, 1908) using software NTSYS pc version 2.02i (Rohlf, 1993). Cluster analysis was done using the UPGMA method and dendrograms were constructed by neighbor joining. The marker data were analyzed separately as well as in combination for the two marker systems. In the dendrogram with NTSYS pc and DARwin, all the interspecific hybrids showed highest similarity with their respective female parents and values of similarity ranges from 96 per cent (in Panniyur 1, Panniyur 2, Panniyur 3 and Panniyur 5) to 100 per cent (in Uthirankotta, Karimunda). All interspecific hybrids showed greater diversity from the male parent P. colubrinum (52-72%). The markers were efficient in revealing the varietal difference of the various genotypes of P. nigrum and grouped each of the putative hybrid along with the respective genotype used as the female parent. However, with respect to RAPD assay, one band each present in female parent was absent in the hybrids P5PC and P2PC. The Polymorphic Information Content (PIC) worked out for the different primers ranged between 0.72 to 0.87 in RAPD and 0.23 to 0.57 in SSR analysis; indicating the capacity of the primers selected to distinguish hybrids. Based on PIC, five SSR and two RAPD primers selected for further charaterization of open pollinated progeny of hybrid culture P5PC-1. The seeds produced by the partially fertile hybrid (Culture P5PC-1) (Vanaja et al., 2008) were subjected to germination test and the seedlings obtained from viable seeds were used for molecular characterization using RAPD and SSR primers selected based on PIC, along with the reported hybrid P5PC-1 and grandparents Panniyur 5 and P. colubrinum. Most of seedlings showed SSR banding pattern similar to Panniyur 5 and female parent. In RAPD, primer OPA 30 showed a polymorphic band which was present in Panniyur 5 and the Hybrid P5PC-1 but absent in three seedlings. The present study using 87 RAPD and 33 SSR markers and the morphological characters revealed significantly high similarity of interspecific hybrids to respective female parents. However the low berry setting percentage as well as the presence of one or two polymorphic bands in two hybrids needs further investigation. Also the number of black pepper specific SSR markers reported is insufficient to cover the large genome of black pepper. Hence more SSR markers as and when they are available as well as efficient markers like AFLP and SNP may be included for genome wide coverage during screening of these hybrids to develop hybrid specific markers.
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