Discovery of the human homolog of sex-determining region (SRY) gene in dioecious plants

Sex determination in the early developmental stages of dioecious crops is economically-beneficial. During this study, a human homology of SRY gene was successfully identified in dioecious crops. SRY gene sequences of date palm and jojoba were submitted to GenBank under the accession numbers KC577225 and MK991776, respectively. This is the first report regarding the novel sex-determination methodology of four dioecious plants (jojoba, date palm, papaya, and pistachios). SRY sex gene was found in all the tested dioecious plant and human samples. This novel approach is simple and of significant importance for breeders. It facilitates the unambiguous selection of jojoba and date palm female plants at an early age and reduces the plantation cost of cultivating non-productive male plants. This is a rapid sex-determination technique for dioecious plants and mammals at an early stage. This technique specifically targets the SRY sequence that has been comprehensively investigated in humans. The kit development for the SRY-based sex determination of various crops is in progress.     

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.     

Cloning and Sequencing of Phytoplasma Ribosomal DNA (rONA) Associated with Kerala Wilt Disease of Coconut Palms

Using the polymerase chain reaction (PCR) the 165 rRNA gene of phytoplasma associated with Kerala wilt disease of coconut palm (Cocos nucifera L) was amplified from infected leaf samples. Within the three universal primer pairs P1/P6, P1/P7and P41 P7, the primer pair P4/P7 only showed an amplification of 650 bp DNA fragment. 5ince P4/P7 amplifies the 16S-23S intergenic spacer region of 165 rRNA gene, the PCR product 650 bp of Kerala wilt disease palm indicates the phytoplasma DNA. The amplified fragment was sequenced and deposited in Genbank data library (Accession No. AY158660). The absence of restriction sites for Bcll and Rsa/l in 650 bp indicates phytoplasmic nature of DNA and its strain difference. A comparison of the 650 bp sequence with other phytoplasmas and its restriction profile indicates Kerala wilt disease phytoplasma as a separate 165 rRNA group in the classification of phytoplasmas. To our knowledge, this report records the first finding of the phytoplasma DNA using universal primers and its sequence analysis in coconut palms of Kerala, south India.     

Molecular characterization and in silico analysis of RNA polymerase alpha subunit gene (rpoA) in Date Palm (Phoenix dactylifera L.) cv. Deglet Nour

The rpoA gene coding for the ??-subunit of DNA-dependent RNA polymerase located in the chloroplastic genome of date palm has been characterized from the elite cultivar Deglet Nour by cloning and sequencing of the PCR amplification product. The full length of rpoA-Pd (Phoenix dactylifera) gene was 1014 bp. The comparison in Genbank showed that the rpoA gene has a 100% homology with the Khalas cultivar of date palm and a strong homology with Oil Palm (99%). The deduced protein full length is 337 amino acid corresponding to 38,692 Da polypeptide. It contained an Alpha N-terminal domain (alpha-NTD) between 1 to 233 (aa) and Alpha C-terminal domain (alpha-CTD) between 266 to 337 (aa). Initially, we have compared the sequences of the full-length DNA rpoA gene from Date Palm and Oil palm, 15 mutations have been detected, 4 do not affect amino acid sequences. A multiple alignment of protein sequences of Date Palm and other plants shows 6 mutations specific for palms family and one is specific to monocots species. A multiple alignment of 35 nucleotide sequences from different plant species shows 3 SNPs specific to Date Palm, 6 SNPs specific to Palms family and 6 other to monocot species. The phylogenetic analysis performed in this work shows a strong similarity between Pd-rpoA and rpoA genes from other plant species, but it shows a great divergence with the rpoA of E. coli. To explain whether the separation of the two clades was due to selection pressure. We calculate the ratio Ka/Ks for different species. A synteny analyses of rpoA genes was effected, a high genomic synteny is observed for the ropA in all the species included in this study.     

Sex-Biased Temporal Gene Expression in Male and Female Floral Buds of Seabuckthorn (Hippophae rhamnoides)

Seabuckthorn is an economically important dioecious plant in which mechanism of sex determination is unknown. The study was conducted to identify seabuckthorn homologous genes involved in floral development which may have role in sex determination. Forty four putative Genes involved in sex determination (GISD) reported in model plants were shortlisted from literature survey, and twenty nine seabuckthorn homologous sequences were identified from available seabuckthorn genomic resources. Of these, 21 genes were found to differentially express in either male or female flower bud stages. HrCRY2 was significantly expressed in female flower buds only while HrCO had significant expression in male flowers only. Among the three male and female floral development stages (FDS), male stage II had significant expression of most of the GISD. Information on these sex-specific expressed genes will help in elucidating sex determination mechanism in seabuckthorn.     

Occurrence of 16SrIV Subgroup A Phytoplasmas in Roystonea regia and Acrocomia mexicana Palms with Lethal Yellowing‐like Syndromes in Yucatán,Mexico

The lethal yellowing (LY) disease and LY‐type syndromes affecting several palm species are associated with 16SrIV phytoplasmas in the Americas. In Mexico, palms of the species Roystonea regia and the native Acrocomia mexicana were found to exhibit LY‐type symptoms, including leaf decay, starting with mature leaves, necrosis and atrophy of inflorescences. DNA extracts obtained from these palms could be amplified by nested‐PCR using phytoplasma‐universal primer pair P1/P7 followed by LY‐group‐specific primer pair LY16Sr/LY16Sf. Blast analysis of the sequences obtained revealed an identity of 100% for R. regia and 99.27% for A. mexicana with 16SrIV‐A strain associated with LY in Florida, USA (Acc. AF498309 ). Computer‐simulated RFLP analysis showed that the patterns for the phytoplasma DNA of the two palm species were highly similar to that for 16SrIV subgroup A strain. A neighbour‐joining tree was constructed, and the sequences of the two palm species clustered in the same clade of group 16SrIV subgroup A. The results therefore support that LY‐type syndromes observed in palms of R. regia and A. mexicana in the Yucatan region of Mexico are associated with 16SrIV subgroup A phytoplasmas.     

Design and testing of a plant-specific PCR primer for ecological and evolutionary studies

A polymerase chain reaction (PCR) primer, 28KJ (5-GGCGGTAAATTCCGTCC-3), was developed to specifically amplify plant DNA. This primer is located approximately 250 bases downstream of the 5′ end of the 28S ribosomal RNA gene, and it was used in combination with the universal primer 28C. The specificity of this primer combination was tested against 31 angiosperms, 9 conifers, 1 alga and 30 fungi (21 basidiomycetes and 9 ascomycetes). Both herbarium specimens and fresh samples were tested. The 28KJ/28C primer combination successfully amplified all angiosperm and conifer DNAs, but no fungal or algal DNAs. Plant DNA was amplified from plant/fungal symbioses (ectomycorrhizae of conifers and ericoid mycorrhizae of Ericaceous plants), and the plants involved in these symbioses were identified by comparing DNA sequences or restriction enzyme digest patterns of the mycorrhizal DNAs to those of known plant samples. These methods allow rapid and accurate identification of plant associates in complex plant/ fungal systems when the identity of the roots is unclear.     

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.     

Preimplantation embryo sexing by polymerase chain reaction amplification of the sry gene on single mouse blastomeres

Accurate and rapid sex determination of preimplantation embryos has great potential both in animal breeding and in human pathology. In the past, sex determination has been accomplished by cytogenetic or immunologic means and by polymerase chain reaction amplification of Y-chromosome-specific repetitive sequences. More recently, amplification of the Y-specific single-copy ZFY gene has been used in humans for sex determination of preimplantation embryos. The experiments reported here indicate that another Y-chromosome-specific single-copy gene, the sex-determining region gene (sry) can be successfully amplified from single mouse blastomeres. Blastocysts positive for sry amplification were reimplanted to foster mothers, and six of six newborns were male. We conclude that sry gene amplification can represent a good marker for embryo sex determination.     

Rapidly evolving sex-specific sequences in <Emphasis Type="Italic">Calamus travancoricus</Emphasis> Bedd. ex. Becc. and <Emphasis Type="Italic">Calamus nagbettai</Emphasis> R.R.Fernald &amp; Dey

Gene sequences mediating sexual reproduction are more divergent within and between closely related species. Microsatellite or simple sequence repeat (SSR) markers are valuable molecular tools for analysis of genetic variability, phylogeny, and also for identifying sex at seedling stage in dioecious plant species. Calamus travancoricus Bedd. ex. Becc. and Calamus nagbettai R.R.Fernald & Dey are economically important rattan species. The dioecious nature of the Calamus spp. limits its breeding and cultivation. The sex of rattans can only be identified after attaining reproductive maturity which ranges from 5 to 15 years. A study was carried out in this background and 9 putative sex-specific PCR products were identified as sex markers for C. travancoricus and C. nagbettai and sequenced by Sanger method. The sequence homology search revealed occurrence of identical sequences in many plant species across different families indicating the conserved nature of the sequences. However, these sequences were not present in opposite sex in the studied species, indicating divergent evolution favoring sex determination. Annotation of these sequences revealed that most of these are mediating sexual reproduction by and large. An adequate sex ratio is to be maintained for these dioecious palms in natural habitat for producing offsprings having equal gene complements for continual evolution and sustainable utilization. Developing scientific management strategies and improved utilization of canes could help to generate employment locally and thus contribute to the socioeconomics sustainably.     

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.     

Male-specific DNA in the dioecious species Atriplex garrettii (Chenopodiaceae)

The mechanism of sex determination in dioecious species of the genus Atriplex (Chenopodiaceae) has not been determined. This paper reports the discovery of a male-specific DNA fragment in the diploid dioecious species A. garrettii. DNA samples extracted individually from ten male and ten female plants were bulked by sex. Random amplified polymorphic DNA (RAPD) fragments were generated in the two bulks in order to identify markers that were polymorphic between male and female plants. A total of 158 decamer primers were tested. A 2075 base-pair (bp) male-specific DNA fragment generated with the OPAF-14 primer was identified. The fragment was cloned and partially sequenced and 24-mer primers that exclusively amplified this fragment were constructed. When 124 male plants, 126 female plants, and one hermaphroditic plant were tested individually, the male-specific 2075-bp DNA fragment was present in the hermaphrodite and all but one of the male plants, and was absent in all female plants. A smaller DNA fragment (~1800 bp) that was homologous to the 2075-bp fragment was amplified from the single male plant that lacked the 2075-bp fragment. Cytogenetic analysis revealed no apparent heteromorphic sex chromosomes. These observations suggest that sex determination in A. garrettii is genetic, with no evidence of heteromorphic sex chromosomes.     

Lethal yellowing-type diseases of palms associated with phytoplasmas newly identified in Florida, USA

A nested polymerase chain reaction (PCR) assay was used to detect phytoplasmas in stem tissues from declining silver date ( Phoenix sylvestris ), Canary Island date ( Phoenix canariensis ), edible date ( Phoenix dactylifera ), Queen ( Syagrus romanozoffiana ) and Mexican fan ( Washingtonia robusta ) palms, all displaying symptoms similar to lethal yellowing (LY) disease, in a tri-county region of west central Florida. Restriction fragment length polymorphism analysis or sequencing of PCR-amplified rDNA products (1.6 kb) identified three distinct group 16SrIV phytoplasma strains among 38 infected palms. Most palms (86.8%) contained Texas Phoenix decline (TPD) phytoplasma, a subgroup 16SrIV-D strain, while two P. canariensis harboured palm LY phytoplasma, a subgroup 16SrIV-A strain. A novel phytoplasma, initially detected in W. robusta and tentatively classified as a subgroup 16SrIV-F strain, also occurred with LY phytoplasma as mixed infections in two P. dactylifera palms. Collectively, these findings extend the known geographic range of TPD in the USA to include Florida and the listing of TPD-susceptible palm species to include P. dactylifera , P. sylvestris and S. romanozoffiana . Moreover, discovery of a novel subgroup 16SrIV-F strain in W. robusta adds to the list of phytoplasma host palm species and complexity of phytoplasma-vector-palm pathosystems newly identified in the west central region of the state. The emergence of new lethal disease of palms beyond southern subtropical region of the state is unprecedented but follows similar developments in other regions where LY is known to occur.     

Fot 1 Insertions in the Fusarium oxysporum f. sp. albedinis Genome Provide Diagnostic PCR Targets for Detection of the Date Palm Pathogen

Populations of Fusarium oxysporum f. sp. albedinis, the causal agent of Bayoud disease of date palm, are derivatives of a single clonal lineage and exhibit very similar Fot 1 hybridization patterns. In order to develop a sensitive diagnostic tool for F. oxysporum f. sp. albedinis detection, we isolated several DNA clones containing a copy of the transposable element Fot 1 from a genomic library of the date palm pathogen. Regions flanking the insertion sites were sequenced, and these sequences were used to design PCR primers that amplify the DNA regions at several Fot 1 insertion sites. When tested on a large sample of Fusarium isolates, including 286 F. oxysporum f. sp. albedinis isolates, 17 other special forms, nonpathogenic F. oxysporum isolates from palm grove soils, and 8 other Fusarium species, the primer pair TL3-FOA28 allowed amplification of a 400-bp fragment found only in F. oxysporum f. sp. albedinis. Sequence analysis showed that one of the Fot 1 copies was truncated, lacking 182 bp at its 3′ terminus. The primer pair BI03-FOA1 amplified a 204-bp fragment which overlapped the Fot 1 truncated copy and its 3′ site of insertion in the F. oxysporum f. sp. albedinis genome and identified 95% of the isolates. The primer pairs BIO3-FOA1 and TL3-FOA28 used in PCR assays thus provide a useful diagnostic tool for F. oxysporum f. sp. albedinis isolates.     

Sequence length polymorphisms within primate amelogenin and amelogenin-like genes: usefulness in sex determination

Sequence length polymorphisms between the amelogenin (AMELX) and the amelogenin-like (AMELY) genes both within and between several mammalian species have been identified and utilized for sex determination, species identification, and to elucidate evolutionary relationships. Sex determination via polymerase chain reaction (PCR) assays of the AMELX and AMELY genes has been successful in greater apes, prosimians, and two species of old world monkeys. To date, no sex determination PCR assay using AMELX and AMELY has been developed for new world monkeys. In this study, we present partial AMELX and AMELY sequences for five old world monkey species (Mandrillus sphinx, Macaca nemestrina, Macaca fuscata, Macaca mulatta, and Macaca fascicularis) along with primer sets that can be used for sex determination of these five species. In addition, we compare the sequences we generated with other primate AMELX and AMELY sequences available on GenBank and discuss sequence length polymorphisms and their usefulness in sex determination within primates. The mandrill and four species of macaque all share two similar deletion regions with each other, the human, and the chimpanzee in the region sequenced. These two deletion regions are 176-181 and 8 nucleotides in length. In analyzing existing primate sequences on GenBank, we also discovered that a separate six-nucleotide polymorphism located approximately 300 nucleotides upstream of the 177 nucleotide polymorphism in sequences of humans and chimps was also present in two species of new world monkeys (Saimiri boliviensis and Saimiri sciureus). We designed primers that incorporate this polymorphism, creating the first AMELX and AMELY PCR primer set that has been used successfully to generate two bands in a new world monkey species.     

Phylogenetic analyses of peanut resistance gene candidates and screening of different genotypes for polymorphic markers

The nucleotide-binding-site-leucine-rich-repeat (NBS–LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. Here, we describe a collection of peanut NBS–LRR resistance gene candidates (RGCs) isolated from peanut (Arachis) species by mining Gene Bank data base. NBS–LRR sequences assembled into TIR-NBS-LRR (75.4%) and non-TIR-NBS-LRR (24.6%) subfamilies. Total of 20 distinct clades were identified and showed a high level of sequence divergence within TIR-NBS and non-TIR-NBS subfamilies. Thirty-four primer pairs were designed from these RGC sequences and used for screening different genotypes belonging to wild and cultivated peanuts. Therefore, peanut RGC identified in this study will provide useful tools for developing DNA markers and cloning the genes for resistance to different pathogens in peanut.     

Genetic structure of the date palm (Phoenix dactylifera) in the Old World reveals a strong differentiation between eastern and western populations

Background and Aims Date palms (Phoenix dactylifera, Arecaceae) are of great economic and ecological value to the oasis agriculture of arid and semi-arid areas. However, despite the availability of a large date palm germplasm spreading from the Atlantic shores to Southern Asia, improvement of the species is being hampered by a lack of information on global genetic diversity and population structure. In order to contribute to the varietal improvement of date palms and to provide new insights on the influence of geographic origins and human activity on the genetic structure of the date palm, this study analysed the diversity of the species.Methods Genetic diversity levels and population genetic structure were investigated through the genotyping of a collection of 295 date palm accessions ranging from Mauritania to Pakistan using a set of 18 simple sequence repeat (SSR) markers and a plastid minisatellite.Key Results Using a Bayesian clustering approach, the date palm genotypes can be structured into two different gene pools: the first, termed the Eastern pool, consists of accessions from Asia and Djibouti, whilst the second, termed the Western pool, consists of accessions from Africa. These results confirm the existence of two ancient gene pools that have contributed to the current date palm diversity. The presence of admixed genotypes is also noted, which points at gene flows between eastern and western origins, mostly from east to west, following a human-mediated diffusion of the species.Conclusions This study assesses the distribution and level of genetic diversity of accessible date palm resources, provides new insights on the geographic origins and genetic history of the cultivated component of this species, and confirms the existence of at least two domestication origins. Furthermore, the strong genetic structure clearly established here is a prerequisite for any breeding programme exploiting the effective polymorphism related to each gene pool.     

Development of PCR‐Based Markers to Determine the Sex of Kelps

Sex discriminating genetic markers are commonly used to facilitate breeding programs in economically and ecologically important animal and plant species. However, despite their considerable economic and ecological value, the development of sex markers for kelp species has been very limited. In this study, we used the recently described sequence of the sex determining region (SDR) of the brown algal model Ectocarpus to develop novel DNA-based sex-markers for three commercially relevant kelps: Laminaria digitata, Undaria pinnatifida and Macrocystis pyrifera. Markers were designed within nine protein coding genes of Ectocarpus male and female (U/V) sex chromosomes and tested on gametophytes of the three kelp species. Seven primer pairs corresponding to three loci in the Ectocarpus SDR amplified sex-specific bands in the three kelp species, yielding at least one male and one female marker for each species. Our work has generated the first male sex-specific markers for L. digitata and U. pinnatifida, as well as the first sex markers developed for the genus Macrocystis. The markers and methodology presented here will not only facilitate seaweed breeding programs but also represent useful tools for population and demography studies and provide a means to investigate the evolution of sex determination across this largely understudied eukaryotic group.     

The Chloroplast Genome Sequence of Date Palm (Phoenix dactylifera L. cv. ‘Aseel’)

Date palm (Phoenix dactylifera L.) is an economically important and widely cultivated palm of the family Arecaceae. We sequenced the complete date palm chloroplast genome (cpDNA) from Pakistani cv. ??Aseel??, using a combination of Sanger-based and next-generation sequencing technologies. Being very similar to a sequence from a Saudi Arabian date palm cultivar ??Khalas?? published recently, the size of the genome was 158,458?bp with a pair of inverted repeat (IR) regions of 27,276?bp that were separated by a large single-copy (LSC) region of 86,195?bp and a small single-copy (SSC) region of 17,711?bp. Genome annotation demonstrated a total of 138 genes, of which 89 were protein coding, 39 were tRNA, and eight were rRNA genes. Comparison of cpDNA sequences of cultivars ??Aseel?? and ??Khalas?? showed following intervarietal variations in the LSC region; (a) two SNPs in intergenic spacers and one SNP in the rpoc1 gene, (b) polymorphism in two mono-nucleotide simple sequence repeats (SSR), and (c) a 4-bp indel in the accD-psaI intergenic spacer. The SSC region has a polymorphic site in the mono-nucleotide SSR located at position 120,710. We also compared cv. ??Aseel?? cpDNA sequence with partial P. dactylifera cpDNA sequence entries deposited in Genbank and identified a number of potentially useful polymorphisms in this species. Analysis of date palm cpDNA sequences revealed a close relationship with Typha latifolia. Occurrence of small numbers of forward and inverted repeats in date palm cpDNA indicated conserved genome arrangement.