Developing dwarf specific scar markers from WCT inbreds of coconut (Cocos nucifera L.)

Abstract

Coconut is an important subsistence and cash crop in the humid tropical regions of the world. The coconut palm is often called “the tree of life” because of its numerous uses as food, drink, fuel, building materials and so on. Coconut cultivars are mainly classified into two: tall and dwarf. West Coast Tall (WCT) is the most common and superior tall cultivar extensively cultivated all over the west coast region, especially in Kerala. One of the main methods of crop improvement in coconut is hybridization between tall and dwarf types. However the heterozygous nature of tall types is a hurdle in selection of mother palms and seedlings. Efforts to develop homozygous inbreds of WCT were started as early as 1924. Chethana (2016) studied the extent of inbreeding depression in second generation (S2) inbred palms and recorded morphological observations of one year old third generation (S3) inbred seedlings. Based on height, she classified the S3 seedlings into Low, Medium and High, before planting in the field. She also carried out the characterization of selected third generation (S3) inbreds using RAPD marker system along with tall (WCT) and dwarf palms (COD, CGD). The results revealed two dwarf specific RAPD bands in two of the S3 seedlings belonging to IAS3-1 and IBS3-1 family, when amplified with primers OPAU03 and OPAW15. However, as RAPD markers have low specificity and sometimes lack repeatability, converting them into Sequence Characterized Amplified Region (SCAR) markers is more advantageous. A tall specific SCAR marker was reported in coconut using primer OPA 09 (Rajesh et al., 2013). The present investigation was carried out in the departments of Plant Biotechnology, College of Agriculture, Padannakkad and College of Horticulture, Vellanikkara during the period 2016-2018 was undertaken with an objective of developing SCAR markers from dwarf specific RAPD marker for screening superior inbreds with dwarfing nature. DNA samples were extracted from total 54 genotypes, 22 seedlings belonging to the inbred families IAS3-1and IBS3-1, 10 seedlings from other three families viz, IIS3, IIIS3 and VS3, 14 dwarf seedlings (including COD, CGD and MYD) and 8 tall seedlings (WCT). These were subjected to RAPD analysis using the 3 reported primers viz, OPAU 03, OPAW 15 and OPA 09. Though OPAU03 showed polymorphism between tall and dwarfs, the bands lacked repeatability and hence this was not included in further analysis. OPA 09, which was reported to produce a tall specific band (Rajesh et al., 2013), confirmed the result in the present study also. Primer OPAW15 was successful in amplifying a polymorphic band of size around 320 base pair specifically in dwarf seedlings and it was not present in the tall cultivar (WCT). The polymorphic dwarf specific band produced by primer OPAW 15 was eluted and cloned in pTZ57R/T vector and transformed into E. coli DH5 alpha cells. Cloned cells were subjected to blue white screening and transformed ones were selected, the plasmid was isolated and sequenced. The sequence after vector screening was subjected to homology search using BLASTn. The DNA sequence of polymorphic band showed similarity with chromosome 2 of Solanum lycopersicum. Based on the sequence, one pair of SCAR primer was designed and tested for dwarf specific band. Dwarf specific amplicon was found in all the dwarfs but absent in the tall WCT seedlings. Hence, this was used to screen the S3 inbred seedlings which were classified as Low, Medium, and High based on their height at the time of planting, to confirm the effectiveness of this classification based on morphology. The result showed that the dwarf specific marker is present in S3 inbreds belonging to Low and Medium types but absent in all those seedlings classified as ‘High’ based on height. This shows that the classification based on height in the one year old seedlings is effective to some extent. However, some of the seedlings in ‘Low’ and ‘Medium’ category lack the marker, which indicate that a classification based on morphological and molecular marker is more effective than classification based on morphology alone. The dwarf specific SCAR marker developed in the present study after validation using other tall and dwarf cultivars, along with the earlier reported tall specific SCAR marker can be successfully employed in breeding experiments for marker assisted selection. The SCAR markers which distinguish tall and dwarf coconut cultivars can be used in screening seedlings at an early stage which would be of immense importance to nurseries and growers while establishing coconut plantations.