Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the “Bayoud” disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S- (Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars (“Medjool”, “Ashrasi”, “Gish Rabi”, “Khineze”, and yellow- and red-“Kabkab”) have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.     

Molecular characterization of Mauritanian date palm cultivars using plasmid-like DNAs markers

Mauritanian date palm cultivars and progenies of two controlled crosses were analyzed according to the identity of mitochondrial plasmid-like DNAs. Starting from total genomic DNA and appropriate primers, polymerase chain reaction was designed to amplify either a 373-bp or a 265-bp fragments corresponding to the S and the R-plasmid respectively. Data proved that 5 cultivars out of 10 studied have exhibited the R-plasmid suggesting their resistance to the fusariosis. The existence of intra-cultivar variability has also been revealed in the cv. Ahmar. In addition, analysis throughout progenies of two controlled crosses suggested the strict maternal transmission of the date palms’ mitochondrial genome.     

A mitochondrial molecular marker of resistance to Bayoud disease in date palm

We have previously shown that two circular plasmid-like DNAs (the S and the R plasmids) can be found in the mitochondria of date palm (Phoenix dactylifera L.), a dioecious monocotyledonous tree. The two plasmids differ essentially by the absence, in the R plasmid, of a 109-bp DNA segment. Using 36 date palm varieties and employing a PCR-based approach, we show that the simultaneous presence of the R plasmid and absence of the S plasmid can be considered as a reliable molecular marker of resistance to a vascular wilt (Bayoud disease) caused by the fungus Fusarium oxysporum f. sp. albedinis. Conversely, the simultaneous presence of the S plasmid and absence of the R plasmid is correlated to Bayoud disease susceptibility The availibility of this diagnostic tool for plasmid characterization should subsequently allow simple, rapid and efficient selection of Bayoud-resistant individuals from the large number of date palms obtained by natural crosses which display good date quality. Received: 14 August 2000 / Accepted: 10 November 2000     

Distribution of mitochondrial plasmid-like DNA in cultivated rice (Oryza sativa L.) and its relationship with varietal groups

Summary Mitochondrial (mt) plasmid-like DNA was found in most of more than 100 rice cultivars (Oryza sativa L.) by the use of 0.7% agarose gel electrophoresis (AGE). The DNA varied in molecular weight and number. By electron microscopy, small circular DNAs of different sizes could be detected in addition to the DNAs of high molecular weight, even in cultivars in which mt plasmid-like DNA was not detected by AGE. The detection of the mt plasmid-like DNAs by AGE did not depend on their presence or absence, but on their high stoichiometry. The relationship between cytoplasms with mt plasmid-like DNAs and varietal (for example, Indica rice) groups was close. The geographical distribution of cytoplasms is discussed.     

Comparative electrophoretical analysis of plasmid-like mitochondrial DNAs in Vicia faba and in some other legumes

Minicircular DNAs found in mitochondria of 6-d-old etiolated seedlings of Vicia faba L. were also present in mitochondria isolated from cell suspension cultures and from green leaves of this plant. These results support the suggestion that the plasmid-like molecules found in mitochondria of V. faba are an essential component of the mitochondrial genome. The minicircular DNAs were, apparently, peculiar for the species V. faba since they were found in all three cultivars of this species which were studied. The distribution pattern of plasmid-like DNAs in Vicia villosa L. was completely different and mitochondria from Medicago sativa L. also contained specific minicircular DNAs. Thus, minicircular DNAs are typical for the mitochondrial genomes of several legumes and different plant species have their specific mitochondrial plasmid-like DNAs     

Identification of mitochondrial plasmid-like DNAs in Picea abies (L.) Karst.

The Norway spruce (Picea abies (L.) Karst.) mitochondrial DNA has been extracted from embryonal suspensor masses. In addition to a master chromosome, a family of plasmid-like DNAs were identified. These latter shared cross homologies but had no evident sequence homology with the master chromosome. The occurrence of mitochondrial plasmid-like DNAs was investigated in trees from different provenances. A vast majority of trees displayed extrachromosomal DNA elements of variable stoechiometry. For some trees, the sequences homologous to the extrachromosomal DNA elements were found associated with high molecular weight DNA. Received: 9 July 1997 / Revision received: 29 January 1998 / Accepted: 29 November 1998     

Linkage analysis of the nuclear homologues of mitochondrial plasmid-like DNAs in rice.

A genetical study on the nucleotide sequences of the nuclear DNAs which share homology with rice mitochondrial plasmid-like DNAs, B1, B2, B3 and B4 was carried out. Restriction fragments of the nuclear DNAs hybridized with these plasmid-like DNAs showed polymorphisms in their length between Indica and Japonica rice cultivars. The hybridized signals found specifically in Indica or Japonica cultivars segregated in the F2 population derived from a cross between these two subspecies. The observed ratio of the nuclear homologues in the F2 population demonstrated that they were transmitted according to the Mendelian inheritance. The co-segregation of homologues was examined and the linkage was detected between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica, and also between the nuclear homologues of B2 and B3 of Indica. The linkage between the B1-nuclear homologue of Japonica and the B4-nuclear homologue of Indica was conserved in the different rice cultivars.     

Genetic variance between some Egyptian Date Palm cultivars using PCR-based markers with emphasis on the prevalence of Al wijam disease

Date Palm is one of the most important crops in Egypt. At present, Egypt ranked second in world date production. The objectives of this study are the identification the genetic variance between some local varieties of Date Palm, in line with the establishment of a molecular diagnostic method to study the aetiology and the prevalence of Al Wijam disease. Date palm plantations have been suffering from number of pests and diseases infestation, mainly due to Dubas Bug, Red Palm Weevil, Lesser Date moth, and Al Wijam disease. Genomic DNAs were extracted from 18 Date Palm cultivars collected from Date Palm Research Institute, ARC using three different extraction methods. Nine common cultivars (Zaghlool, Hayani, Oraibi, Samani, Sultan, Bent-Aisha, Amrey, Aglani, and Barhee) and nine Siwi cultivars (Siwi, Fryhee, Taktakt, Quaipe, Karama Ghazally, Helw Ghanem, Gorm Agazal, and Oshbeigel,) were used in this study. In order to determine the genetic polymorphism and discriminate between each of these cultivars, RAPD and ISSR analysis were conducted on the isolated DNA samples from each cultivar. DNA fingerprints of the studied cultivars were conducted using 10 RAPD and eight ISSR primers. Dendrograms representing genetic relationships as well as genetic similarity matrices were performed for the studied cultivars using the UPGMA (Unweighted Pair Group Method with Arithmetic Average). Furthermore, 40 date palm trees representing the studied cultivars were collected from El-Marazik, El-Badrasheen, El-Wahat, and Demiate Governorates to study the prevalence of Al-Wijam disease using the DNA hybridisation technique. Symptoms including retardation in terminal bud growth, whole crown of leaves (rosetting symptoms), and yellow longitudinal lines on the midribs were observed. Non-radioactive Dig-labeled probes specific for 16s rDNA region of virus like agents were used to detect the infected trees.     

Characterization of ten date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia using AFLP and ISSR markers

Date palm is the most economically important plant in the Middle East due to its nutritionally valuable fruit. The development of accurate DNA fingerprints to characterize cultivars and the detection of genetic diversity are of great value for breeding programs. The present study explores the usefulness of ISSR and AFLP molecular markers to detect relationships among 10 date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia. Thirteen ISSR primers and six AFLP primer combinations were examined. The level of polymorphism among cultivars for ISSRs ranged from 20% to 100% with an average of 85%. Polymorphism levels for AFLPs ranged from 63% to 84% with an average of 76%. The total number of cultivar-specific markers was 241, 208 of which were generated from AFLP analysis. AJWA cultivar had the highest number of cultivar-specific ISSR markers, whereas DEK, PER, SUK-Q, SHA and MOS-H cultivars had the lowest. RAB and SHA cultivars had the most and least AFLP cultivar-specific markers, respectively. The highest pairwise similarity indices for ISSRs, AFLPs and combined markers were 84% between DEK (female) and PER (female), 81% between SUK-Q (male) and RAB (male), and 80% between SUK-Q (male) and RAB (male), respectively. The lowest similarity indices were 65% between TAB (female) and SUK-Q (male), 67% between SUK-A (female) and SUK-Q (male), and 67% between SUK-A (female) and SUK-Q (male). Cultivars of the same sex had higher pairwise similarities than those between cultivars of different sex. The Neighbor-Joining (NJ) tree generated from the ISSR dataset was not well resolved and bootstrap support for resolved nodes in the tree was low. AFLP and combined data generated completely resolved trees with high levels of bootstrap support. In conclusion, AFLP and ISSR approaches enabled discrimination among 10 date palm cultivars of from Saudi Arabia, which will provide valuable information for future improvement of this important crop.     

黄瓜线粒体类质粒pC1,pC4在品种间的分布及同源性研究

在黄瓜（津研四号）线粒体中存在4种线粒体类质粒（pCl,pC2,pC3,pC4),对14个黄瓜品种的线粒体类质分布情况进行了研究,发现在津春二号,津春五号,津新密刺,津绿四号和津研四吃5个品种中存在线粒体类质粒,其余9个品种无线粒体类质粒,类质粒的存在有一定随机性,不同品种中的同一种类质粒间具有同源性,pC4类质粒不仅与自身的核DNA同源,也与其他品种的核基因组有同源性,pC4同源序列在黄瓜核基因组中为多拷贝的,拷贝之间是不连续的,在丝瓜和西葫芦的核基因组中也有pC4的同源序列,因此,推测pC4可能在葫芦科分化的早期就已存在并整合于核中,某些品种缺乏类质粒是由于在进化过程中发生的类质粒的丢失。     

Plasmid-like DNAs associated with mitochondria of cytoplasmic male-sterile Sorghum

Summary Plasmid-like, linear DNAs were detected in preparations of mitochondrial DNA from cytoplasmic male-sterile sorghum. Designated N-1 and N-2, the DNAs exhibited molecular sizes of ca. 5,700 and 5,300 bp, respectively. The DNAs occurred in only the IS1112C entry among 24 entries examined. Electron microscopy of the DNAs indicated that the molecules were linear as isolated. Nick translation of N-1 and N-2 followed by membrane hybridization indicated substantial homology between the two DNAs, and some homology to the S-1 and S-2 maize plasmid-like DNAs. At least four additional DNA species, ranging from ca. 1,000–4,000 bp if linear, were also detected in sorghum mitochondrial DNA. The detection of plasmid-like DNAs in sorghum, with homology to the plasmid-like DNAs of maize, suggests an etiological relationship of the molecules to the inheritance and expression of cytoplasmic male sterility in sorghum, perhaps in a manner analogous to the postulated role of these elements in maize.Cooperative Investigations of Agricultural Research Service, U.S. Department of Agriculture, Institute of Food and Agricultural Sciences, University of Florida, Texas Agricultural Experiment Station, and Department of Genetics, North Carolina State University. Florida Experiment Stations Journal Series No. 3578     

Terminal protein association and sequence homology in linear mitochondrial plasmid-like DNAs of sorghum and maize

The linear extrachromosomal mitochondrial plasmid-like DNAs from the Ru cytoplasm of maize, and M35-1 and IS1112C cytoplasms of sorghum, possess 5 terminally-attached proteins. These molecules required proteinase K treatment for mobility in agarose gels and were susceptible to exonuclease III but not lambda exonuclease cleavage. Hybridizations, under stringent conditions, indicated that the sorghum plasmid-like DNAs, N1 and N2, did not possess DNA sequence homology to cloned central regions of S1 and S2, the linear mitochondrial plasmid-like DNAs present in S cytoplasm of maize. In addition, a novel 4.2kb, DNAase sensitive, RNAase insensitive band, exhibiting homology to internal sequences from maize S2, was observed in the sorghum IS1112C cytoplasm only.     

Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis

In the present study, two polymerase chain reaction (PCR)-based methods namely, randomly amplified polymophic DNA (RAPD) and amplification fragment length polymorphism (AFLP) were employed to assess genetic variations, which may appeared, in tissue culture-derived date palm (Phoenix dactylifera) offshoots. Analysis of RAPD banding patterns generated by PCR amplification using 37 random primers gave no evidences for somaclonal variations and the percentage of polymorphic bands in a total of 259 scored bands was zero. Meanwhile, analysis of AFLP banding patterns generated using 13 primer combinations pointed to minor genetic variations in the AFLP banding patterns. The percentage of genetic variations (polymorphism) in tissue culture-derived date palm offshoots belonging to cultivars Sakkoty, Gandila and Bertamoda was 2.6, 0.79 and 1 %, respectively, as revealed by AFLP analysis. The low percentage of genetic variations confirms the genetic stability of tissue culture-derived dry date palm cultivars.     

Date Palm DNA Mini-Preparation without Liquid Nitrogen

We have developed a new mini-procedure for isolation of total cellular DNA from date palm (Phoenix dactylifera L.). The procedure, which does not use liquid nitrogen, has proved useful due to temporary disruptions in supplies of liquid nitrogen that occur in countries where date palm trees are cultivated. DNA suitable for RFLP and PCR analyses is obtained.     

Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.)

Genetics of control mechanisms that underlies sex differentiation in date palm is not known. Sex of the plants becomes known only at the time of first flowering, which takes around 5 years. In comparison, molecular diagnosis (if available/feasible) promises quick and reliable identification of sex types very early when plantlets are growing in seedbeds. To develop such an assay, genomic DNA from 45 individual plants (25 female and 20 male) belonging to different varieties of date palm was subjected to PCR amplification using 100 random amplified polymorphic DNA (RAPD) and 104 intersimple sequence repeat (ISSR) primers. Initially, two bulk genomic DNA samples (each made by pooling DNA from ten male and female plants, separately) were used. A primer showing sex-specific band in bulked samples was further used for amplification of the genomic DNA of the individual samples of that bulk. Only one RAPD primer, OPA-02, amplified a fragment of ~1.0 kb in all the individual samples of male genotypes, whereas this fragment was absent in all the female genotypes. This male-specific fragment was cloned and sequenced (GenBank accession no. JN123357), and a sequence-characterized amplified region (SCAR) primer pair was designed that amplified a 406-bp fragment in both female and male genotypes and a unique fragment of 354 bp in only male genotypes. The SCAR marker was further validated using 25 female and ten male date palm plants belonging to different varieties collected from different locations.     

A randomized controlled trial of interventions to impede date palm sap contamination by bats to prevent nipah virus transmission in bangladesh

Background

Drinking raw date palm sap is a risk factor for human Nipah virus (NiV) infection. Fruit bats, the natural reservoir of NiV, commonly contaminate raw sap with saliva by licking date palm’s sap producing surface. We evaluated four types of physical barriers that may prevent bats from contacting sap.

Methods

During 2009, we used a crossover design and randomly selected 20 date palm sap producing trees and observed each tree for 2 nights: one night with a bamboo skirt intervention applied and one night without the intervention. During 2010, we selected 120 trees and randomly assigned four types of interventions to 15 trees each: bamboo, dhoincha (local plant), jute stick and polythene skirts covering the shaved part, sap stream, tap and collection pot. We enrolled the remaining 60 trees as controls. We used motion sensor activated infrared cameras to examine bat contact with sap.

Results

During 2009 bats contacted date palm sap in 85% of observation nights when no intervention was used compared with 35% of nights when the intervention was used [p<0.001]. Bats were able to contact the sap when the skirt did not entirely cover the sap producing surface. Therefore, in 2010 we requested the sap harvesters to use larger skirts. During 2010 bats contacted date palm sap [2% vs. 83%, p<0.001] less frequently in trees protected with skirts compared to control trees. No bats contacted sap in trees with bamboo (p<0.001 compared to control), dhoincha skirt (p<0.001) or polythene covering (p<0.001), but bats did contact sap during one night (7%) with the jute stick skirt (p<0.001).

Conclusion

Bamboo, dhoincha, jute stick and polythene skirts covering the sap producing areas of a tree effectively prevented bat-sap contact. Community interventions should promote applying these skirts to prevent occasional Nipah spillovers to human.     

De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera)

Date palm is one of the most economically important woody crops cultivated in the Middle East and North Africa and is a good candidate for improving agricultural yields in arid environments. Nonetheless, long generation times (5-8 years) and dioecy (separate male and female trees) have complicated its cultivation and genetic analysis. To address these issues, we assembled a draft genome for a Khalas variety female date palm, the first publicly available resource of its type for a member of the order Arecales. The ～380 Mb sequence, spanning mainly gene-rich regions, includes >25,000 gene models and is predicted to cover ～90% of genes and ～60% of the genome. Sequencing of eight other cultivars, including females of the Deglet Noor and Medjool varieties and their backcrossed males, identified >3.5 million polymorphic sites, including >10,000 genic copy number variations. A small subset of these polymorphisms can distinguish multiple varieties. We identified a region of the genome linked to gender and found evidence that date palm employs an XY system of gender inheritance.     

Molecular study of two distinct phytoplasma species associated with streak yellows of date palm in Iran

A disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.     

Use of Infrared Camera to Understand Bats’ Access to Date Palm Sap: Implications for Preventing Nipah Virus Transmission

Pteropus bats are commonly infected with Nipah virus, but show no signs of illness. Human Nipah outbreaks in Bangladesh coincide with the date palm sap harvesting season. In epidemiologic studies, drinking raw date palm sap is a risk factor for human Nipah infection. We conducted a study to evaluate bats’ access to date palm sap. We mounted infrared cameras that silently captured images upon detection of motion on date palm trees from 5:00 pm to 6:00 am. Additionally, we placed two locally used preventative techniques, bamboo skirts and lime (CaCO₃) smeared on date palm trees to assess their effectiveness in preventing bats access to sap. Out of 20 camera-nights of observations, 14 identified 132 visits of bats around the tree, 91 to the shaved surface of the tree where the sap flow originates, 4 at the stream of sap moving toward the collection pot, and no bats at the tap or on the collection pots; the remaining 6 camera-nights recorded no visits. Of the preventative techniques, the bamboo skirt placed for four camera-nights prevented bats access to sap. This study confirmed that bats commonly visited date palm trees and physically contacted the sap collected for human consumption. This is further evidence that date palm sap is an important link between Nipah virus in bats and Nipah virus in humans. Efforts that prevent bat access to the shaved surface and the sap stream of the tree could reduce Nipah spillovers to the human population.     

Molecular markers derived from RAPD,SCAR, and the conserved 18S rDNA sequences for classification and identification in<Emphasis Type="Italic"> Pyrus pyrifolia</Emphasis> and<Emphasis Type="Italic"> P</Emphasis>.<Emphasis Type="Italic"> communis</Emphasis>

We generated RAPD, SCAR, and conserved 18S rDNA markers for classifying and identifying cultivars of Pyrus pyrifolia (Japanese pear) and P. communis (European pear). PCR amplification with selected specific primers—LCH327UP and LCH327DOWN—was performed using DNA extracted from 25 P. pyrifolia and P. communis cultivars. The 1,380-bp fragment was amplified from P. communis cvs. Beurre Giffard, Cascade, Conference, Clapps Favorite, Packhams Triumph, and Winter Nelis. RAPD has only a dominant single band of 1,380-bp, however, SCAR has one or more band of the same size. Amplification involving sequence-specific primer pairs LCH346UP and LCH346DOWN resulted in a loss of polymorphism. The 1,190-bp fragment was amplified from all P. pyrifolia cultivars. The conserved sequences of the 18S rDNA fragment of 25 pear cultivars were amplified and analyzed with 42 restriction enzymes. Compared with P. pyrifolia cultivars, they lacked the restriction enzyme site of KpnI and had one less RsaI site. Cultivar Gamcheonbae had a specific PstI restriction site, while cvs. Mansoo and Conference pear digested with AluI showed a different presentation than other cultivars. For the Okusankichi and Shinil pears TaqI was best marker for identification in P. pyrifolia. These results can be adopted for identifying pear cultivars; to date there is no standard marker for identifying the cultivars of fruit trees in Korean fruit tree breeding programs.Communicated by J.S. Heslop-Harrison