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Title: Studies on metaxenial effect and use of molecular approaches for detection of hybrids, genetic diversity and sex in date palm (Phoenix dactylifera L.)
Authors: Maryam
Keywords: Applied Sciences
Agriculture & related technologies
Issue Date: 2015
Abstract: Date palm (Phoenix dactylifera L.) has been an important crop in the desert regions of Asia and North Africa for centuries know adopted in many countries. Alexander the great and Arab conquerors introduced date fruit in Indo-Pak subcontinent after that date extended as a food item. Date palm is among the oldest cultivated plants in history of mankind, being used as food for 6000 years. Date is a world agricultural industry with total production of 7.5 million metric tonnes (MT) of fruit and Pakistan contribution is 10.34 metric tonnes. Date palm (2n=36) is dioecious monocotyledonous belonging to Arecaceae family. Fertilization and fruit set are consequences of pollination as there is an interesting but uncommon effect known as metaxenia, the direct influence of pollen on maternal tissues of date palm. Pollen not only control the fruit set, size of fruits and seeds but also effect the time of ripening. As pollen grains caused the great diversity and variations in size, quality and ripening time so 9 different pollen sources were selected to study the differential behaviour of pollen parents on fruit characteristics of two female cultivars i.e Hillawi and Khadrawy. Fruit was harvested at Khalal stage from controlled pollinated treatments and pysicochemical analysis was carried out to study the metaxenial effect of different pollen. Physical characters including ripening time, bunch weight, fruit length, fruit width, fruit area, pulp weight, seed weight and biochemical characters, total sugars, DPPH, ascorbic acid, total phenolic contents, total soluble solids, total proteins, sucrose, glucose, fructose, and enzymatic activities (peroxidase, catalaze, superoxidase ans protease) were highly variables. Data regarding all physicochemical characters was analyzed statistically using the two factor factorial in Randomized Complete Block Design (RCBD) that showed the significant differences in all variables. Duncan multiple range test was used to discrete the means (using the statistica 9.1) revealed that different pollen sources respond variably. Among 9 pollen sources M8 ( 32) was superior because it was positively associated with most of the variables. Fruits developed from M8 pollination were prominent with high bunch weight more sweetly in taste (high total sugars), good fruit area, enriched with protein, reduced seed weight, high antioxidant and enzymatic activity and reduced ripening time. M7 (collected from Uni campus) retained highest capability for ascorbic acid, reduced ripening time, good fruit length and width, reduced seed weight. For more positive correlation of pollen parents M8 was followed by M7, M3, M5 and M4. Strong positive correlation was found among most of 16 the traits. All the new combinations developed from pollinating the 2 date palm cultivars with 9 pollen sources were categorized by principle component analysis. The morphological and biochemical dendrogram generated from Wards method can distinguished these combinations into 3 and 2 clusters for cultivar Hillawi and Khadrawy respectively. Our results showed that physicochemical traits (using XLSTAT software) were very appropriate for studying the metaxenail effect that can be used for further breeding targets as economically very important regarding yield and quality. For hybrid identification 12 Simple Sequence Repeats were used to identify the true hybrid of date palm developed from the seeds, resulted from the controlled pollination from their respective parents. Main objective of this study was to identify the true hybrids respective to their parents to carry out the further breeding programmes. Out of 12, only 6 SSR primers clearly produced the SSR amplification with the size ranging from 125 to 500 bp. These six microsatellite primers resulted in 66 microsatellite loci with a mean of 11 loci per primer. Total 6 primers identified the true to type as well as ambiguous hybrids. Primer mpdCIR-10 identified the maximum no of true hybrids 13 (81.25%) out of 16 while 3 ambiguous hybrids (18.75%). In comparison, DP159 detected only 10 (62.5%) true hybrids and maximum ambiguous hybrids i.e 6 (37.5%). 75% true hybrids were determined with primer mpdCIR16 and mpdCIR25 followed by the mpdCIR15 and mpdCIR93 (68.75%). 12 microsatellite primers were used with 30 date palm samples which showed that microsatellites were highly polymorphic, having a great number of alleles. Total 124 alleles were scored with a mean of 9.08 alleles per locus while allelic range varied from 5 to 16 using primers mpdCIR15 to mpdCIR57, respectively. With primer mpdCIR10 highest microsatellite loci were amplified while with mpdCIR15 only 4 different microsatellite loci were scored. Our objective was to identify microsatellite markers that could be used to differentiate between male and female date palm (Phoenix dactylifera) in saplings to cultivate the large number of productive female trees. These primers produced 15 polymorphic loci specifically in male date palm samples and the seedlings harboring the unique fragments were further characterized as male plants. Increasingly 38.46% of these loci were scored as homozygous alleles while 61.53% heterozygous allelic loci were determined. Primer mpdCIR48 produced a specific locus (250/250) in all male samples where as the same locus was absent in female samples. Similarly a locus of 300/310 bp 17 reoccurred in 6 date palm male samples which indicated that this is a promising candidate marker to detect the sex in date palm. The data resulted from combination of 12 primers enabled the seedling samples of date palm cultivars to divide into two groups i.e., male and female regarding their sex expression comparative to the parents (male + female) using the principle coordinate analysis (PCoA). Genetic diversity in date palm breeding parents and its population was investigated using 19 loci of microsatellite (SSR) primers. 6 female accessions, 8 male parent and 16 new combinations resulted from controlled crossing were included as reference material. Our objective was to determine the genetic distance and relatedness among breeding parents and its population for the improvement of the existing date palm germplasm. Microsatellites were highly polymorphic with wide range of alleles and reproducible fragments with the scorable size of 60 to 500 bp. Total 135 alleles with the mean of 7.50 per locus were scored. Average major allele frequency was 0.65 ranged from 0.50 in marker mpdCIR-15 to 0.95 in primer pair DP-175. The pylogenetic tree separated all the parent from the progeny but 31% and 69% variations were observed among and within the populations respectively using the analysis of molecular variance. High level of heterozygosity (0.66) was found among the parents and its population. The results depicted that male and females were distinct from each other but progeny developed from male and female combinations fall between these two parents. These co-dominant markers will be a starting point for researchers making use of the markers for genetic mapping and diversity analysis of date palm. Results showed that physicochemical traits (using XLSTAT software) were appropriate for studying the metaxenial effect that can be used for further breeding targets as economically very important regarding yield and quality. These new hybrids may facilitate to exploit the new plant material in future. These preliminary results emphasized the importance of breeding of more diversified parents, progeny development and sex determination to expand the germplasm base and add more commercial cultivars in Pakistan. 18 CHAPTER 1 INTRODUCTION Date palm (Phoenix dactyliferaL.) has been an important crop in the desert regions of Asia and North Africa for centuries but now adopted in many countries. It is believed to be native from Arabian Gulf and North Africa. Alexander the great and Arab conquerors introduced date fruit in Indo-Pak subcontinent after that date extended as a food item. Date is a world agricultural industry with total production of 7.5 million metric tonnes (MT) of fruit and Pakistan contribution is 10.34 metric tonnes (FAO, 2010). In Pakistan, it is the 3rd major fruit crop after citrus and mango, and about 325 varieties of date palm are reported (Botes and Zaid, 2002). In Pakistan most significant areas regarding dates production are; Punjab (Jhang, Muzafar Garh, Bahawalpur, Dera Ghazi Khan), Sindh (Khairpur), Khyber Pakhtunkhwa (Dera Ismail Khan) and Balochistan (Turbat and Khuzdar) (Markhand et al., 2010). From the worldwide 100 million date trees distribution, 62 million are found in Arab world and has formed the basis of fortitude for many ancient nomade (Mohammad et al., 1983). Date palm (2n=36) is dioecious monocotyledonous belonging to Arecaceae family. The Arecaceae family consists of 200 genera and 500 species (Dowson, 1982). Phoenix is an important genus in the family which is most captivating (Munier, 1973) as they exert great variation in their reproductive morphology where single sex flowers are present on more than 85% palms (Dransfield et al., 2008). The date palm fruit contains high sugar (mainly reducing) contents and it provides 314 kcal of energy from 100 g of the date flesh (Al Farsi and Lee, 2008). The date palm fruits are rich source of nutrition especially in the arid regions where very few plants can grow due to the harsh environmental conditions. In formal language dates are known as Sugar Palm (Al-Shahib and Marshall, 2003). Due to unique characteristics and its role in survival of people living in hot and arid environments it can be truly called as “tree of life”. Without the presence of date palm, survival and development of human race in hot and barren areas of world was not feasible. As well as date is prominent in its cultural and religious values throughout the world. Its very important in the religious ceremonies of Islam, Hinduism, Judaism and Christianity.
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