Molecular marker based linkage mapping in Black pepper (Piper nigrum l)
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Date
2009
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College of Horticulture, Vellanikkara
Abstract
The study on the Development of molecular marker based linkage mapping in black pepper (Piper nigrum L.) was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture and Pepper Research Station (PRS), Panniyur during the period of 2006-2008. The research work included mainly three aspects; the development of mapping population, generation of molecular marker data for linkage mapping and the analysis of RAPD data for linkage map construction. A molecular linkage map covering a large region of the genome with informative DNA markers is very useful for effective and efficient plant selection in breeding programs. It is also essential for identifying and isolating the genes responsible for various quantitative traits. Genetic mapping is based on the principle that genes (markers or loci) segregate via chromosome recombination during meiosis, thus allowing their analysis in the progeny. It requires two genetically divergent parents and segregating plant populations. The recombination frequency of the identified polymorphic markers is an indication of the relative position of the markers in a linkage group. Developing a linkage map in black pepper (2n=4x=52) genome would be very useful because of its specific disadvantages in breeding programmes such as long juvenile period, inherent heterozygosity, perennial nature etc. Two genetically diverse and heterozygous cultivars viz., Cheriyakaniakkadan and Uthirenkotta were selected as the male and female parents of black pepper for the present investigation. The progenies developed by selfing of Panniyur-1(the F1 progeny of these parents) was taken as the F2 population; the progenies developed by back crossing Panniyur-1 with one of the parents gave the BC1 generation and a fresh hybridization of the parental cultivars produced the F1 generation. The seedlings were germinated through in vitro and ex vitro methods. The seed set of the F1 population and germination percentage of in vitro raised seedlings were not satisfactory hence only ex vitro raised F2 population (67 selfed seedlings) was used for linkage analysis. To have an assessment about the general variability in the mapping population morphological observations viz. number of leaves, height, length and breadth of leaf and the pigmentation at the growing tip were recorded and the statistical parameters such as Mean, Coefficient of variation (C.V) and Standard deviation (S.D) were worked out. The result showed that maximum variation was observed in the height of seedlings with the value of Coefficient of variation (C.V) 65.32 per cent. Random Amplified Polymorphic DNA (RAPD) marker was used to identify the polymorphic loci across the male and female parents. Twenty four loci were found polymorphic between the parents using 10 random primers. The identified polymorphic loci were used to genotype the entire mapping population. Linkage analysis was done using the software MAPMAKER/EXP Version 3.0. The results showed that the markers were mainly grouped into four linkage groups. First group consisted of 12 markers with a total genetic distance of 84.8 cM, second group consisted of four markers of 56.6 cM and the last two groups consisted of only two markers each with a total genetic distance of 6.3 cM and 3.3 cM respectively This is a preliminary study carried out with and objective to standardize the method for linkage analysis in black pepper so as to develop a frame work map using molecular markers. Further analysis with larger mapping population and varied markers will provide enough polymorphic markers to supplement this map. Once the seedlings have attained full growth, the morphological and yield related characters could also be subjected to linkage analysis in order to produce a more comprehensive genetic map of all the 13 linkage groups in black pepper.
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