Development and characterization of non-coding RNA-derived simple sequence repeat markers in coconut (Cocos nucifera L.).

Non-coding RNA (ncRNA)-based SSR markers are highly useful in molecular breeding as ncRNAs play a significant role in gene regulation. In the present study, for the first time in coconut, we have identified 597 ncRNA-derived SSR markers, including 509 long non-coding RNASSRs (lncRNASSRs) and 88 micro RNASSRs (miRNASSRs). Of these, 20 primers (10 each from lncRNA-SSR and miRNA-SSR) were selected, screened on 6 coconut accessions, and 50% produced polymorphic fragments. These 10 polymorphic primers were used for genotyping 96 palms of 16 coconut accessions, comprising eight tall and dwarf accessions each. The number of alleles ranged from 2 to 9 per SSR marker, with an average of 4.6 alleles per locus. The average heterozygosity and Shannon index were 0.5 and 1.1, respectively, suggesting that ncRNA-SSRs show high polymorphism level. Distance-based cluster analyses revealed that all the tall and dwarf accessions were differentiated and grouped in different clusters. The study demonstrates the usefulness of ncRNA-based SSR markers for assessing genetic diversity and genetic improvement in coconut.

Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.).

Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient characterization of genetic diversity. Microsatellites or simple sequence repeats (SSRs), mined from expressed sequence tags (ESTs), constitute an important resource for analysis of genetic diversity as they are abundant, polymorphic and represent function regions of the genome. We have identified a total of 318,528 putative EST-SSRs from 130,942 unigenes utilizing a leaf transcriptome dataset of coconut. Among the EST-SSRs, dinucleotide repeats were abundant (219,912; 69.04%) followed by trinucleotide (70,722; 22.2%) and tetra-nucleotide repeats (6281; 1.9%). Among the dinucleotide repeat motifs, the dominant repeat was AG/CT (35.87%), followed by AT/AT (18.59%), while the dominant trinucleotide repeat was AAG/CTT (4.59%). One hundred and twenty EST-SSR primer pairs were designed and utilized to amplify six DNA samples of coconut accessions. Fifty primers (41.7%) produced reproducible polymorphic fragments of expected sizes, from which a total of 10 primers were selected for the diversity assessment in 186 palms of 50 coconut accessions, comprising of 25 each of tall and dwarf accessions. A total of 137 alleles were detected with an average of 13.7 alleles per SSR locus. The number of alleles observed at each locus in the data set ranged from 7 to 22. All the loci showed 100% polymorphism with respect to the samples screened. The average observed heterozygosity was 0.46. The PIC values ranged from 0.79 (CnKGDEST129 and CnKGDEST100) to 0.91 (CnKGDEST117 and CnKGDEST122) with a mean value of 0.85, indicating the capacity of the EST-SSR markers to detect high levels of polymorphism. The cluster analysis revealed that accessions were generally clustered based on their relative similarity and irrespective of their geographic origins. The present study demonstrates the usefulness of transcriptome sequencing as a rapid and cost-effective methodology for the development of molecular markers. The EST-SSR markers generated through this study constitute useful and reliable tools for assessment of genetic diversity and marker-assisted selection in coconut.

Coconut (Cocos nucifera l.) pollen cryopreservation.

BACKGROUND: Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. OBJECTIVE: The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. MATERIALS AND METHODS: Pollen of two coconut varieties (West Coast Tall WWCTW and Chowghat Orange Dwarf CODC) was collected in March-May over three successive years, desiccated to 7.5 % moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. RESULTS: Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32 % in desiccated pollen whereas it was between 26 and 29 % in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31 % in desiccated pollen, while it was between 28 and 32 % in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 nm and 226-396 mm for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1 % after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31 % and 25-49 % germination, respectively. CONCLUSION: These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

Understanding Physiology and Impacts of High Temperature Stress on the Progamic Phase of Coconut (Cocos nucifera L.).

The reproductive phase of coconut is extremely sensitive to high temperature, manifesting as button (female flower) shedding and poor nut set. The progamic phase, which elapses from pollination to fertilization, is one of the most critical phases during the sexual reproduction processes in annuals and fruit trees and is extremely vulnerable to high temperature. Hence, we investigated the progamic phase of the tall coconut cultivar West Coast Tall (WCT) and the effect of high temperature on the phase under both in vivo and in vitro conditions. Coconut has a long pistil and its length was found to be 18.2 ± 4.9 mm in WCT. Pollen germination on stigma occurred one day after pollination and the pollen tube traversed through the pistil and reached micropyle of ovule four days after pollination at 29 °C. However, high temperature (Tmax > 33 °C), both under in vivo and in vitro conditions, significantly reduced pollen tube growth through the pistil, suggesting its inability to reach the ovule on time to effect fertilization. High temperature also advanced nectar secretion and stigma receptivity and the receptive stigma was dry without nectar, rendering it unappealing to insect pollinators. Thus, both poor pollination and the inability of pollen tube to reach the ovule on time to effect fertilization could be the cause of poor nut set in the coconut variety WCT under high temperature. However, it was encouraging to note that the pollen tube growth was less vulnerable to elevated temperature under high humidity, suggesting that climate change effect on coconut in coastal regions with high humidity might be less severe.