Karyotype Analysis of elite Varieties of date Palm (Phoenix Dactylifera L.) Growing in Sindh, Pakistan

Abstract

Date palm (Phoenix dactylifera L.) is a dioecious, perennial, monocotyledonous fruit tree, belongs to Arecaceae family. The genus Phoenix comprised of 14 species. Date palm is cultivated in more than 39 countries of the world. Pakistan is one of the leading countries in terms of area and production of date palm. All four provinces of Pakistan are blessed with climatic conditions which favor date palm farming, therefore, date palm is cultivated throughout the country. In this era of modern molecular techniques, cytology is still a valuable tool for taxonomy, phylogeny and diversity studies. Karyotype analysis has been extensively used in plant phylogenetics and diversity studies. The information like chromosome number, size and morphology has been remained of considerable value in understanding interrelationships and delimitation of taxa. The karyotype shows chromosomal characteristics of a somatic cell of an individual. Date palm has uncertain diploidy levels because of scarcity of cytogenetic knowledge. Hence, the date palm cytogenetic studies were taken in hand to investigate the chromosome number and develop karyotype of commercial date palm varieties growing in Sindh province of Pakistan. These varieties include Aseel, Asul Khurmo, Dedhi, Kupro, Kashuwari, Karblain, Otakin Dhakki, Gulistan, Shamran, Zahidi, Begam Jangi, Hussaini, Pashna, a tissue culture derived Gulistan, three exotic varieties i.e. Safawi, Barhee & Rotana and four wild fruit bearing female date palms denoted with Wild01, Wild02, Wild03 and Wild04. The seeds of all varieties including tissue cultured date palm were collected from the desired sources and were sown in the pots for germination. The germinated roots of all date palm varieties were harvested for conducting karyotype analysis in the Date Palm Research Institute (DPRI), Shah Abdul Latif University, Khairpur, Pakistan and Plant Biology Lab, University of Illinois, USA. The karyotype of all 21 varieties was constructed on the basis of total chromosome number, chromosome type, total chromosome length, arm ratio (long/short arm), centromeric index value and relative length percentage. The results revealed that all varieties including three exotic used in this study have 2n=36 diploid chromosome number. The detailed karyotypes of all date palm varieties are presented for the first time. Particularly, Pakistani varieties remained virgin as nobody has ever conducted such cytological work. However, variations have been observed in physical appearance of chromosomes among all the varieties. The chromosome complement xii consists of metacentric, submetacentric, subtelocentric and telocentric chromosomes but interestingly no any two varieties have same number of chromosomes for all four categories. Total length of haploid set of chromosomes ranges from 26.6μm (Rotana) to 58.78μm (Kupro). The length of chromosomes varies from 0.71μm (Zahidi) to 6.46μm (Kashuwari) and centromeric index is highest in var. Begam Jangi having 37.03 value and lowest in var. Asul Khurmo having 19.64 value. The varieties showing symmetric karyotype includes Wild03 (11m & 4sm), var. Zahidi (11m & 3sm), var. Dhakki (10m & 3sm), var. Begam Jangi (10m & 3sm), var. Barhee (8m & 4sm) and var. Safawi (10m & 3sm). The karyotype of these six varieties is primitive in nature as its value is up to 2.00. Whereas, karyotype of rest of the varieties is between symmetric and asymmetric with value more than 2.00. The highest value is shown by var. Asul Khurmo (2.88), Wild01 (2.72), var. Karblain (2.55), and Wild02 (2.50), hence, these values show that these four varieties fall in category of advanced karyotype. Mother plant and tissue culture derived plants of variety Gulistan showed same number of diploid chromosomes 2n=36. Both kinds of plants have same number of meta to telocentric chromosomes and no significant variations in length of chromosomes. The highest mean length of chromosome number one in tissue culture plants was 3.97µm and 3.75µm in mother plant, while lowest mean length was 1.35µm and 1.38µm in tissue culture derived and mother plants respectively. The data obtained from the centromere length coefficient of 17 date plan varieties and 4 Wild plants was utilized for cluster analysis. The dendrogram obtained from UPGMA cluster analysis of the DIST coefficient classified into two clusters. The first cluster was further divided into two subgroups, the first one included vars. Kashuwari and Wild04 plant and second subgroups included var. Karblain and Wild03 plant. The second cluster has 15 varieties and rest of the 2 Wild plants. The second group was also further divided into two subgroups, where the first one has vars. Rotana, Barhee, Safawi and Wild02 plant and remaining 12 varieties and Wild01 plant in second subgroup. The varieties present in subgroups have close relationship. The sex determination in date palm at early stages of growth remained need of the day but it was only possible when plants reach to maturity and produce flowers. An accurate method has been developed and optimized for early sex determination in this most important cash crop of world which requires use of DNA markers and fluorescent in situ Hybridization (FISH). The SCAR primer produced a common 406 bp band in both male and female genotypes and a unique allele of 354 bp in male genotypes only in var. Medjool. The SCAR fluorescent signals xiii showed male and female specific complementary DNA on XX and XY chromosomes by FISH. The DP-2 marker amplified a unique fragment of 300 bp only in female plants while absent in male plants of var. Dedhi and showed physical location on XX chromosome only. The male specific PCR molecular marker SRY determined gender in var. Dedhi by showing only male DNA sequence of 400 bp and male allele on Y chromosome. The 45S rDNA showed two signals in female sample and three in male where one extra signal is most probably present on Y chromosome in var. Gulistan. The DNA content of 50 date palm varieties and four wild plants was estimated based on relative fluorescent density of each sample compared to corn plant because Zea maize L. was used as internal reference. The genome size of fifty commercially important date palm varieties and four wildly grown date palm plants ranged from 1.595 pg 2C-1 to 1.847 pg 2C-1 . Whereas, the overall mean of genome size of studied date palm varieties was found to be 1.726 pg & 843.912 Mbp. The number of events/nuclei varied significantly from cultivar to cultivar and was independent to the size of genome. Since, the var. Shakri (Balochistan, Pakistan) appeared with highest number of events/nuclei (6859) despite having a medium amount of genomic size (1.709 pg & 835.606 Mbp) as compared to var. Karblain (Sindh, Pakistan) having the highest genomic size (1.847 pg & 903.063 Mbp) with 3914 number of events/nuclei. The exotic date palm varieties i.e. Anber, Sugae, Medjool, Safawi and Ajwa showed great stability in genome size but no any significant difference was observed in all five varieties grown in Pakistan and their native cultivating regions.