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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Genetic diversity and phylogenetic analysis of <italic>Citrus</italic> (L) from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA</title><author><name sortKey="Hynniewta, Marlykynti" sort="Hynniewta, Marlykynti" uniqKey="Hynniewta M" first="Marlykynti" last="Hynniewta">Marlykynti Hynniewta</name></author><author><name sortKey="Malik, Surendra Kumar" sort="Malik, Surendra Kumar" uniqKey="Malik S" first="Surendra Kumar" last="Malik">Surendra Kumar Malik</name></author><author><name sortKey="Rao, Satyawada Rama" sort="Rao, Satyawada Rama" uniqKey="Rao S" first="Satyawada Rama" last="Rao">Satyawada Rama Rao</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25606407</idno><idno type="pmc">4287869</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287869</idno><idno type="RBID">PMC:4287869</idno><idno type="doi">10.1016/j.mgene.2014.01.008</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">001065</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Genetic diversity and phylogenetic analysis of <italic>Citrus</italic> (L) from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA</title><author><name sortKey="Hynniewta, Marlykynti" sort="Hynniewta, Marlykynti" uniqKey="Hynniewta M" first="Marlykynti" last="Hynniewta">Marlykynti Hynniewta</name></author><author><name sortKey="Malik, Surendra Kumar" sort="Malik, Surendra Kumar" uniqKey="Malik S" first="Surendra Kumar" last="Malik">Surendra Kumar Malik</name></author><author><name sortKey="Rao, Satyawada Rama" sort="Rao, Satyawada Rama" uniqKey="Rao S" first="Satyawada Rama" last="Rao">Satyawada Rama Rao</name></author></analytic><series><title level="j">Meta Gene</title><idno type="eISSN">2214-5400</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The north-eastern region of India is reported to be the center of origin and rich in diversity of <italic>Citrus</italic> (L.) species, where some wild and endangered species namely <italic>Citrus indica</italic>, <italic>Citrus macroptera</italic>, <italic>Citrus latipes</italic>, <italic>Citrus ichagensis</italic> and <italic>Citrus assamensis</italic> exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various <italic>Citrus</italic> species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs) analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of <italic>Citrus</italic> species confirming our cytogenetical interpretations. The three true basic species i.e. <italic>Citrus medica</italic>, <italic>Citrus maxima</italic> and <italic>Citrus reticulata</italic> with their unique status have been resolved into distinct clades with molecular approaches as well. <italic>C. indica</italic> which occupies a unique position in the phylogenetic ladder of the genus <italic>Citrus</italic> has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in <italic>C. indica</italic> also echo and support its unique position. From our study it is amply clear that <italic>C. reticulata</italic> also has close relation to <italic>C. ichagensis</italic>, as these species have clustered together, denoting their close genetic relationship. On the other hand, our studies did not demonstrate a clear differentiation between subgenera <italic>Citrus</italic> and <italic>Papeda</italic> at the rDNA level. The combined approach of cytogenetical and molecular analysis did complement our early karyological findings and helped in resolving many a taxonomic riddles.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Abkenar, A A" uniqKey="Abkenar A">A.A. Abkenar</name></author><author><name sortKey="Isshiki, S" uniqKey="Isshiki S">S. Isshiki</name></author><author><name sortKey="Tashino, Y" uniqKey="Tashino Y">Y. Tashino</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Agarwal, P K" uniqKey="Agarwal P">P.K. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Meta Gene</journal-id><journal-id journal-id-type="iso-abbrev">Meta Gene</journal-id><journal-title-group><journal-title>Meta Gene</journal-title></journal-title-group><issn pub-type="epub">2214-5400</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25606407</article-id><article-id pub-id-type="pmc">4287869</article-id><article-id pub-id-type="publisher-id">S2214-5400(14)00010-3</article-id><article-id pub-id-type="doi">10.1016/j.mgene.2014.01.008</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Genetic diversity and phylogenetic analysis of <italic>Citrus</italic> (L) from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Hynniewta</surname><given-names>Marlykynti</given-names></name></contrib><contrib contrib-type="author"><name><surname>Malik</surname><given-names>Surendra Kumar</given-names></name></contrib><contrib contrib-type="author"><name><surname>Rao</surname><given-names>Satyawada Rama</given-names></name><email>srrao22@yahoo.com</email><xref rid="cr0005" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="af0005">Plant Biotechnology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong, 793022 Meghalaya, India</aff><aff id="af0010">Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, New Delhi, India</aff><author-notes><corresp id="cr0005"><label>⁎</label>Corresponding author at: Plant Biotechnology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong, 793022 Meghalaya, India.Tel.: + 91 364 2722404; fax: + 91 364 2550076. <email>srrao22@yahoo.com</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>28</day><month>3</month><year>2014</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="collection"><month>12</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>28</day><month>3</month><year>2014</year></pub-date><volume>2</volume><fpage>237</fpage><lpage>251</lpage><history><date date-type="received"><day>6</day><month>11</month><year>2013</year></date><date date-type="rev-recd"><day>25</day><month>1</month><year>2014</year></date><date date-type="accepted"><day>28</day><month>1</month><year>2014</year></date></history><permissions><copyright-statement>© 2014 The Authors</copyright-statement><copyright-year>2014</copyright-year><license license-type="CC BY-NC-ND" xlink:href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><license-p>This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).</license-p></license></permissions><abstract><p>The north-eastern region of India is reported to be the center of origin and rich in diversity of <italic>Citrus</italic> (L.) species, where some wild and endangered species namely <italic>Citrus indica</italic>, <italic>Citrus macroptera</italic>, <italic>Citrus latipes</italic>, <italic>Citrus ichagensis</italic> and <italic>Citrus assamensis</italic> exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various <italic>Citrus</italic> species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs) analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of <italic>Citrus</italic> species confirming our cytogenetical interpretations. The three true basic species i.e. <italic>Citrus medica</italic>, <italic>Citrus maxima</italic> and <italic>Citrus reticulata</italic> with their unique status have been resolved into distinct clades with molecular approaches as well. <italic>C. indica</italic> which occupies a unique position in the phylogenetic ladder of the genus <italic>Citrus</italic> has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in <italic>C. indica</italic> also echo and support its unique position. From our study it is amply clear that <italic>C. reticulata</italic> also has close relation to <italic>C. ichagensis</italic>, as these species have clustered together, denoting their close genetic relationship. On the other hand, our studies did not demonstrate a clear differentiation between subgenera <italic>Citrus</italic> and <italic>Papeda</italic> at the rDNA level. The combined approach of cytogenetical and molecular analysis did complement our early karyological findings and helped in resolving many a taxonomic riddles.</p></abstract><kwd-group><title>Keywords</title><kwd>North-east India</kwd><kwd>Univalents</kwd><kwd>Quadrivalents</kwd><kwd>Bayesian inference</kwd><kwd>Maximum parsimony</kwd><kwd>Species diversity</kwd><kwd><italic>Citrus</italic></kwd></kwd-group></article-meta></front><body><sec id="s0005"><title>Introduction</title><p>The genus <italic>Citrus</italic> L., the sole source of the Citrus fruits of commerce, belongs to the orange subfamily Aurantioideae of the family Rutaceae and is grown in tropical and subtropical areas of the world (<xref rid="bb0305" ref-type="bibr">Webber, 1967</xref>). The genus includes some of the most commercially important fruits viz. mandarin (<italic>Citrus reticulata</italic> Blanco), sweet orange (<italic>Citrus sinensis</italic> (L.) Osbeck), grapefruit (<italic>Citrus paradisi</italic> Macf.), lemon (<italic>Citrus limon</italic> (L.) Burm. f.) and lime (<italic>Citrus aurantiifolia</italic> (Christm.) Swingle). India enjoys a remarkable position in the “<italic>Citrus</italic> belt of the world” due to her rich wealth of <italic>Citrus</italic> genetic resources, both wild and cultivated (<xref rid="bb0170" ref-type="bibr">Malik et al., 2013</xref>, <xref rid="bb0200" ref-type="bibr">Nair and Nayar, 1997</xref>). The north-eastern region of India is a rich treasure of various <italic>Citrus</italic> species. Natural and undisturbed populations of <italic>Citrus</italic> genepool observed during collection trips from time to time confirms the assumption that this area might be the center of origin of several <italic>Citrus</italic> species. As many as 17 <italic>Citrus</italic> species, their 52 cultivars and 7 probable natural hybrids are reported to have originated in the North-eastern region of India (<xref rid="bb0060" ref-type="bibr">Bhattacharya and Dutta, 1956</xref>). <italic>Citrus</italic> plants growing in deep forests undisturbed by abiotic factors have also been reported from the region, thus bestowing this area with a special status of “treasure house” of <italic>Citrus</italic> germplasm (<xref rid="bb0250" ref-type="bibr">Sharma et al. 2004</xref>). <italic>Citrus</italic> is the third most important fruit crop of India with an estimated production of 9441 MT from an area of 1039 ha (Annual <xref rid="bb0030" ref-type="bibr">reports, 2012</xref>). Annual production of <italic>Citrus</italic> species in North-east is 506.9 tons from 98.3 ha (<xref rid="bb0025" ref-type="bibr">Annual report, 2010</xref>).</p><p><italic>Citrus</italic> taxonomy and phylogeny are very complicated, controversial and ambiguous (<xref rid="bb0205" ref-type="bibr">Nicolosi et al., 2000</xref>) due to sexual compatibility among species, long history of cultivation, apomixis (adventives nucellar polyembryony), somatic bud mutation etc. Sexual compatibility even between <italic>Citrus</italic> and related genera like <italic>Fortunella</italic>, <italic>Poncirus</italic> etc. (<xref rid="bb0380" ref-type="bibr">Frost and Soost, 1968</xref>, <xref rid="bb0170" ref-type="bibr">Malik et al., 2013</xref>) has contributed to the taxonomic confusion. <italic>Citrus</italic> taxonomy was based mainly on morphological and geographical data and many classification systems have been formulated from time to time. Two of these systems suggested by <xref rid="bb0270" ref-type="bibr">Swingle and Reece (1967)</xref> and <xref rid="bb0295" ref-type="bibr">Tanaka (1977)</xref> have been the most widely accepted ones. The discrepancy between them is shown by the fact that Swingle's system recognizes just 16 species while Tanaka's system recognizes 162 species in the genus <italic>Citrus.</italic><xref rid="bb0240" ref-type="bibr">Scora (1975)</xref> and <xref rid="bb0050" ref-type="bibr">Barrett and Rhodes (1976)</xref> suggested that there are only three ‘basic’ true species of <italic>Citrus</italic> within the subgenus <italic>Citrus</italic> as follows: citron (<italic>Citrus medica</italic> L.), mandarin <italic>(C. reticulata</italic> Blanco), and pummelo (<italic>Citrus maxima</italic> (Burm) Merrill), other species within this subgenus are hybrids derived from these true species, species of subgenus <italic>Papeda</italic> or closely related genera. Taxonomic characterization leading to unambiguous identification of <italic>Citrus</italic> species and their genetic resources are essential requisites for <italic>Citrus</italic> breeding, Citriculture and <italic>Citrus</italic> industry. In a systematic account on Indian <italic>Citrus</italic>, <xref rid="bb0200" ref-type="bibr">Nair and Nayar (1997)</xref> followed primarily the scheme of <xref rid="bb0270" ref-type="bibr">Swingle and Reece (1967)</xref> and partly that of <xref rid="bb0295" ref-type="bibr">Tanaka (1977)</xref> including 18 taxa, comprising of eight species under subgenus <italic>Citrus</italic>, three under subgenus <italic>Papeda</italic>, and seven other indigenous <italic>Citrus</italic> varieties with a suspected hybrid origin and uncertain taxonomic affinities.</p><p>Most species of the genus <italic>Citrus</italic> are characterized by polyembryony, which consists of the production of 1 to 40 adventive embryos by the nucellus (<xref rid="bb0105" ref-type="bibr">Fusurato, 1957</xref>), so that two or more embryos develop in a single seed. The trait of adventive nucellar embryony in <italic>Citrus</italic> has long been a subject of interest to taxonomists. Among the three true species in sub-genus <italic>Citrus</italic> i.e. <italic>C. medica</italic> (citron), <italic>C. maxima</italic> (pummelo) and <italic>C. reticulata</italic> (mandarin), first two species are strictly monoembryonic with only sexual offspring whereas <italic>C. reticulata</italic> is polyembryonic.</p><p>Meiotic events in <italic>Citrus</italic> and its inter-specific and inter-generic hybrids are quite interesting. However, the meiotic behavior in <italic>Citrus</italic> is mainly regular; irregularities are infrequent (<xref rid="bb0020" ref-type="bibr">Agarwal, 1989</xref>, <xref rid="bb0120" ref-type="bibr">Iwamasa, 1966</xref>, <xref rid="bb0230" ref-type="bibr">Raghuvanshi, 1962</xref>). Analysis of meiotic chromosome pairing in hybrids not only is a classical and authentic approach to understand species relationships but it also helps in genetic stability of polyploids (<xref rid="bb0315" ref-type="bibr">Yan et al., 1997</xref>). Cultivated <italic>Citrus</italic> species have been hybridized with some wild relatives such as <italic>Murraya</italic>, <italic>Severinia</italic>, <italic>Atalantia</italic>, and <italic>Swinglea</italic>, in order to introduce desirable traits, mainly resistance to pests and pathogens (<xref rid="bb0055" ref-type="bibr">Barrett, 1977</xref>, <xref rid="bb0180" ref-type="bibr">Motomura et al., 1995</xref>). The identification of chromosomes of different genomes could be a simple method of identifying <italic>Citrus</italic> hybrids and is thus important for future work (<xref rid="bb0070" ref-type="bibr">Cameron and Frost, 1968</xref>). Therefore, the present study was taken up to resolve the cryptic hybridity of <italic>Citrus</italic> species present in their natural habitat through cytogenetical tools.</p><p>Since morphological characters are only of limited use and cytogenetical parameters are time consuming, alternate approaches, including application of appropriate molecular markers, have now been increasingly adopted to address the problems in <italic>Citrus</italic> taxonomy (<xref rid="bb0145" ref-type="bibr">Kumar et al., 2012</xref>). The perplexing condition of the <italic>Citrus</italic> phylogeny has drawn many workers to try and resolve the ambiguities using molecular markers such as isozymes (<xref rid="bb0110" ref-type="bibr">Herrero et al., 1996</xref>), RAPD and PCR–RFLP (<xref rid="bb0005" ref-type="bibr">Abkenar et al., 2004</xref>, <xref rid="bb0095" ref-type="bibr">Federici et al., 1998</xref>, <xref rid="bb0125" ref-type="bibr">Jena et al., 2009</xref>), RAPD and SCAR (<xref rid="bb0205" ref-type="bibr">Nicolosi et al., 2000</xref>), AFLP (<xref rid="bb0155" ref-type="bibr">Liang et al., 2007</xref>, <xref rid="bb0210" ref-type="bibr">Pang et al., 2007</xref>), SSR (<xref rid="bb0045" ref-type="bibr">Barkley et al., 2006</xref>), ISSR (<xref rid="bb0090" ref-type="bibr">Fang et al., 1998</xref>, <xref rid="bb0245" ref-type="bibr">Shahsavar et al., 2007</xref>) and sequence data analysis of ITS region of nrDNA (<xref rid="bb0150" ref-type="bibr">Kyndt et al., 2010</xref>, <xref rid="bb0225" ref-type="bibr">Pessina et al., 2011</xref>, <xref rid="bb0320" ref-type="bibr">Xu et al., 2006</xref>) and non-coding chloroplast DNA (cpDNA) regions (<xref rid="bb0035" ref-type="bibr">Araujo et al., 2003</xref>, <xref rid="bb0075" ref-type="bibr">Chase et al., 1999</xref>, <xref rid="bb0160" ref-type="bibr">Lu et al., 2011</xref>, <xref rid="bb0175" ref-type="bibr">Morton et al., 2003</xref>). The molecular phylogeny of Indian <italic>Citrus</italic> using PCR–RFLP of the <italic>trnD-trnT</italic> and <italic>rbcL</italic>-ORF 106 regions as well as sequence data analysis of the <italic>trnL-trnF</italic> intergenic spacer region of cpDNA was carried out by <xref rid="bb0125" ref-type="bibr">Jena et al. (2009)</xref> where they supported the recognition of <italic>C. maxima</italic>, <italic>C. medica</italic> and <italic>C</italic>. <italic>reticulata</italic> as the basal species of cultivated <italic>Citrus</italic>. Therefore, in order to have comprehensive and substantial information about the extent of genetic variability and occurrence of cryptic genomic hybridity among <italic>Citrus</italic> species, a combined approach involving morphological, cytogenetical and molecular phylogenetical approaches has been adopted in the present study. Thus our investigations constitute as a first multi-pronged approach to resolve several issues of <italic>Citrus</italic> taxonomy, which is beleaguered.</p></sec><sec id="s0010"><title>Material and methods</title><sec id="s0025"><title>Plant materials</title><p>Extensive surveys and exploration trips were conducted in different states of North-East India to collect wild, semi-wild and cultivated species of <italic>Citrus</italic>. Location, state and name of species are provided in <xref rid="t0005" ref-type="table">Table 1</xref>/<xref rid="f0005" ref-type="fig">Fig. 1</xref>. Germplasm was collected in the form of fruits, seeds and flower buds. The samples used in the present study are authenticated and are being maintained at the National Bureau of Plant Genetic Resource, New Delhi. The trees were marked and appropriately labeled before flowers and leaves were collected from them, which formed the basic material for detailed male meiotic studies and phylogenetic analysis.</p></sec><sec id="s0030"><title>Meiotic studies</title><p>Flower buds of appropriate size (1–2 cm in diameter) were harvested from mature trees of <italic>Citrus</italic> species and fixed on the spot in freshly prepared 1:3 glacial acetic acid: 95% ethanol mixture for a minimum of 24 h at room temperature and later stored in 70% ethanol at 10 °C. Anthers were squashed in 1% acetocarmine solution with ferric chloride solution as mordant. On average 25–30 PMCs were analyzed at diplotene/diakinesis/metaphase I to estimate the range of chromosome associations and recombination frequencies through chiasma analysis. On average 15–20 cells were analyzed at anaphase I/II to study the distributional pattern of chromosomes and chromatids. For percentage pollen stainability the pollen grains were stained in 1:1 glycerine:acetocarmine mixture and on average ten slides were scored for stainable pollen. Photomicrographs of cytological preparations were taken from temporary slides with <italic>Jenoptik</italic> CCD camera (Germany) attached to <italic>Labomed</italic> LX 400 brightfield microscope. The illustrations in the present investigation were magnified at 1000 × to the original dimensions of the image, with no further increase in the magnification during processing stage.</p></sec><sec id="s0035"><title>DNA extraction, amplification reaction and sequencing for nrDNA ITS</title><p>Genomic DNA of <italic>Citrus</italic> species was extracted following <xref rid="bb0195" ref-type="bibr">Murray and Thompson (1980)</xref>. The PCR primers ITS 4 and ITS 5 of <xref rid="bb0310" ref-type="bibr">White et al. (1990)</xref> were used to amplify the ITS region (ITS 1, 5.8S, and ITS 2) utilizing same primers for sequencing. The amplification program consisted of one cycle of initial denaturation at 94 °C for 4 min followed by 30 cycles of 94 °C for 1 min, 55 °C for 3 min and 72 °C for 1 min with final extension of 72 °C for 7 min. DNA amplification was performed in a thermal cycler system (Gene Amp® 2700 Applied Biosystems). Amplified PCR products were purified using QIAquick gel extraction kit (QIAGEN, Germany) and sequenced at Xcelris Scientific Pvt. Ltd., India.</p></sec><sec id="s0040"><title>Sequence alignment and indel coding</title><p>The boundaries of the ITS region for all 12 species of <italic>Citrus</italic> were determined by comparing published sequences and on the basis of the angiosperm consensus motif determined by <xref rid="bb0130" ref-type="bibr">Jobes and Thien (1997)</xref>. The putative start and end points of 5.8S regions in the aligned sequences were identified. <italic>Atalantia ceylanica</italic> was selected as out group and sequences obtained were subjected to multiple sequence alignment using Clustal X program (<xref rid="bb0300" ref-type="bibr">Thompson et al., 1997</xref>) with default settings. Clustal X generated alignments were further re-aligned manually. Gaps were included into analysis and coded automatically in a binary matrix using SeqState v.1.21 (<xref rid="bb0190" ref-type="bibr">Müller, 2005</xref>) applying the simple indel coding strategy (<xref rid="bb0255" ref-type="bibr">Simmons and Ochoterena, 2000</xref>).</p></sec><sec id="s0045"><title>Phylogenetic analysis</title><p>The sequence characteristics of the ITS region were calculated using MEGA version 4 (<xref rid="bb0280" ref-type="bibr">Tamura et al., 2007</xref>). Maximum parsimony (MP) method was used to analyze the aligned sequence data matrix. The tree was constructed using Phylip (<xref rid="bb0100" ref-type="bibr">Felsenstein, 2004</xref>). Bootstrap analysis was carried out with 999 random seed and 1000 replicates to examine the relative level of support for individual clades on the cladograms of each search. The Bayesian inference (BI) of phylogeny was also conducted using MRBAYES v.3.1.2 (<xref rid="bb0235" ref-type="bibr">Ronquist and Huelsenbeck, 2003</xref>). BI analysis was performed for 1,000,000 generations applying the default settings (MCMC, two runs with four chains each, heating temperature 0.2, saving one tree every 100 generations).</p></sec></sec><sec id="s0015"><title>Result</title><sec id="s0050"><title>Meiotic studies</title><p>The meiotic divisions in the wild and cultivated species of <italic>Citrus</italic> were studied. The details regarding the ten <italic>Citrus</italic> species analyzed in the present investigation, total number of PMCs analyzed and their association at diplotene, diakinesis and metaphase 1 are summarized in <xref rid="t0005" ref-type="table">Table 1</xref>. The data on total and mean number of chiasmata and its range along with number of terminalized chiasma, terminalisation co-efficient and its percentage pollen stainability is summarized in <xref rid="t0010" ref-type="table">Table 2</xref>. The distribution pattern of chromosome at anaphase I has been detailed in <xref rid="t0015" ref-type="table">Table 3</xref>. Most of these observations are illustrated in <xref rid="f0010" ref-type="fig">Fig. 2</xref>. From the data summarized in <xref rid="t0005" ref-type="table">Table 1</xref>, <xref rid="t0010" ref-type="table">Table 2</xref>, it is amply clear that all the species presently investigated were characteristic in showing nine bivalents at diplotene/diakinesis/metaphase I in all PMCs analyzed. The present study carried out in 10 species of <italic>Citrus</italic> revealed a gametic number of n = 9, without any variation (<xref rid="f0010" ref-type="fig">Fig. 2</xref>). The meiotic chromosome behavior in the ten <italic>Citrus</italic> species studied was regular where bivalent associations outnumbered other types of associations in pollen mother cells (PMCs) studied at diplotene, diakinesis and metaphase I. The mean value for ring bivalents ranged between 5.88 (<italic>C. medica</italic>) and 4.32 (<italic>Citrus limetta</italic>) while that of rod bivalents ranged between 4.24 (<italic>C. limetta</italic>) and 2.96 (<italic>C. medica</italic>). We also observed meiotic irregularities including univalents, quadrivalents, laggards, bridges and chromosome stickiness, though at low frequency in the PMC. Univalents were frequently encountered in most of the species namely <italic>Citrus indica</italic>, <italic>C. limetta</italic>, <italic>C. sinensis</italic>, <italic>Citrus jambhiri</italic>, <italic>C. medica</italic>, <italic>C. maxima</italic>, <italic>Citrus limon</italic>, <italic>Citrus latipes</italic> and <italic>Citrus macroptera</italic>, where a maximum of 4 univalents per PMC was found in <italic>C. indica</italic> and <italic>C. limetta</italic>. <italic>C. reticulata</italic> was unique in having no univalents. 1–3 bivalents were observed to be associated with the nucleolus per PMC. An occasional multivalent association in the form of quadrivalents was detected in <italic>C. indica</italic>. Ring bivalents per PMC ranged from 3 to 9 while 1–7 rod bivalents were also recorded per PMC. From the data presented in <xref rid="t0010" ref-type="table">Table 2</xref> it is apparent that the mean number of chiasmata per cell ranged from 22.6 (<italic>C. limetta</italic>) to 26.5 (<italic>C. medica</italic>). <italic>C. limetta</italic> (13.32) was observed to have minimum terminalized chiasmata whereas <italic>C. indica</italic> recorded the highest value of 15.68. Terminalization co-efficient ranged from 0.39 to 0.42 in all the ten species studied. Five species viz. <italic>C. latipes</italic>, <italic>C. macroptera</italic>, <italic>C. indica</italic>, <italic>C. jambhiri</italic> and <italic>C. limetta</italic> recorded similar value of terminalisation co-efficient of 0.41. The chiasma frequency in PMCs of the ten species studied ranged from 22.48 to 26.92. The highest value was recorded in <italic>C. indica</italic> (26.92) and the lowest in <italic>C. jambhiri</italic> (22.48). Chromosome distribution at anaphases I and II was observed to be regular, but a few laggards were detected in some of the PMC of a few species. Micronuclei or secondary associations of the chromosomes were also observed in 1–2 species. Pollen stainability in the ten species studied ranged from 44.4 to 90%. <italic>C. medica</italic>, <italic>C. jambhiri</italic> and <italic>C. limon</italic> exhibited low percentage of pollen stainability with 44.4%, 59.4% and 63% respectively.</p></sec><sec id="s0055"><title>ITS sequences data</title><p>The phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 loci using maximum parsimony method and Bayesian inference has thrown light on species inter-relationships and evolution of <italic>Citrus</italic> species. Sequence length in the 12 <italic>Citrus</italic> accessions ranged from 601 to 694 bp (ITS 1 and ITS 2 partial and 5.8S complete sequence) as compared to 649 bp in <italic>A. ceylanica</italic>. The ITS 1 and ITS 2 regions of twelve species of <italic>Citrus</italic> presently investigated showed variable sequence lengths and G + C content (%). The sequence lengths of ITS 1 for all the 12 species ranged from 210 to 310 bp while ITS 2 sequence lengths ranged from 207 to 270 bp (<xref rid="t0020" ref-type="table">Table 4</xref>). All the twelve <italic>Citrus</italic> species revealed a sequence length of 160 bp for 5.8S region. The ITS sequences were very rich in G + C content ranging from 59.8% (<italic>C. medica</italic>) to 64.4% (<italic>Citrus ichagensis</italic>) with an average of 64.2%. The G + C content (%) of ITS 1 was found to be slightly higher as compared to ITS 2 region and average G + C content of 64.3% and 63.2% were recorded for ITS 1 and ITS 2, respectively. The final aligned data matrix of the combination of ITS 1, 5.8S and ITS 2 yielded 732 characters including 385 conserved, 316 variable and 130 parsimony informative sites. For determining sequence statistics among <italic>Citrus</italic> species, 245 and 268 characters were aligned for ITS 1 and ITS 2 respectively. The addition of <italic>A. ceylanica</italic> (selected as out-group) resulted in an aligned length of 324 and 296 characters for ITS 1 and ITS 2 respectively. The 5.8S region has been found to be more conserved as evidenced from the number of conserved sites (153 out of 155, 98.71%), followed by ITS 1 (86.12%) and ITS 2 (69.40%). On the contrary, a higher sequence divergence though marginally was recorded for ITS 2 (<xref rid="t0025" ref-type="table">Table 5</xref>). Percentage of sequence divergence based on substitution plus indels was 34.8% for ITS 1; 28 for ITS 2 and 7.05% for 5.8S region respectively. ITS 1 recorded highest percentage (30.5%) of parsimony informative sites. The numbers of indels for ITS 1 and ITS 2 were 14 and 9 respectively. In ITS sequence (ITS 1 and ITS 2 partial and 5.8S complete sequence), the nucleotide frequencies were found as 0.208 (A), 0.310 (C), 0.300 (G), and 0.181 (T). Transition/transversion bias (R) was 0.83.</p><p>The Maximum Parsimony (MP) and Bayesian Inference (BI) methods were used to assess the phylogenetic relationship of the genus <italic>Citrus</italic> based on the combined nucleotide sequence data of ITS 1, 5.8S and ITS 2 (<xref rid="f0015" ref-type="fig">Fig. 3</xref>, <xref rid="f0020" ref-type="fig">Fig. 4</xref>). A clear relationship among subgenera is observed in all the trees generated through two phylogenetic methods. ITS sequence (ITS 1 and ITS 2 partial and 5.8S complete sequence), analysis showed moderate rate of nucleotide divergence within and among the <italic>Citrus</italic> taxa and <italic>A. ceylanica</italic>, genetic divergence within <italic>Citrus</italic> group ranged from 0 to 26.5%. The phylogenetic trees based on both maximum parsimony and Bayesian analyses show a clear separation between the three ‘basic’ species as proposed by <xref rid="bb0240" ref-type="bibr">Scora (1975)</xref> and <xref rid="bb0050" ref-type="bibr">Barrett and Rhodes (1976)</xref>. A clear relationship among subgenera in the maximum parsimony analysis is observed and the phylogenetic tree (<xref rid="f0015" ref-type="fig">Fig. 3</xref>) has been resolved into three major clusters. Major cluster I is further resolved into two sub-clusters viz. Sub-cluster Ia which consisted of <italic>C. medica</italic>, <italic>C. maxima</italic> and <italic>C. limon</italic> and sub-cluster Ib which had <italic>C. limetta</italic>, <italic>C. jambhiri</italic> and <italic>C. macroptera</italic>. Major cluster II consisted of <italic>C. latipes</italic> and <italic>C. assamensis</italic> while major cluster III is further resolved into two sub-clusters i.e. IIIa and IIIb. Sub-cluster IIIa comprised of <italic>C. reticulata</italic>, <italic>C. ichagensis</italic> and <italic>C. sinensis</italic> while sub-cluster IIIb had only <italic>C. indica</italic>. <italic>A. ceylanica</italic> was separately attached at the base of tree as the diverging <italic>Citrus</italic> relative's lineage.</p><p>Bayesian Inference (BI) method was used to assess the phylogenetic relationship of the genus <italic>Citrus</italic> based nrDNA sequences. The phylogenetic tree based on BI method (<xref rid="f0020" ref-type="fig">Fig. 4</xref>) has resolved into four major clusters. Major cluster I had only <italic>C. indica</italic> while major cluster II is further resolved into two sub-clusters. Sub-cluster IIa had <italic>C. medica</italic>, <italic>C. maxima</italic> and <italic>C. limon</italic> while sub-cluster IIb consisted of <italic>C. jambhiri</italic>, <italic>C. limetta</italic> and <italic>C. macroptera</italic>. Major cluster III comprised of <italic>C. sinensis</italic>, <italic>C. ichagensis</italic> and <italic>C. reticulata</italic> and lastly major cluster IV consisted of <italic>C. assamensis</italic> and <italic>C. latipes</italic>. <italic>A. ceylanica</italic> was separately attached at the base of tree as the diverging <italic>Citrus</italic> relative's lineage.</p></sec></sec><sec id="s0020"><title>Discussion</title><p><italic>Citrus</italic> classification is ambiguous and highly controversial. Various taxonomists have recognized 16 to 162 species in the genus <italic>Citrus</italic> (<xref rid="bb0275" ref-type="bibr">Swingle, 1943</xref> and <xref rid="bb0290" ref-type="bibr">Tanaka, 1954</xref>). Most of the confusion is due to free hybridization of different species and occurrences of intermediate forms. This study was an attempt to distinguish the intermediate forms through analysis of chromosomal associations and their behavior during meiosis. Interestingly it was found, by and large, normal for all the <italic>Citrus</italic> species investigated where bivalent associations outnumbered other types of associations in pollen mother cells (PMCs) studied at diplotene, diakinesis and metaphase I, the presence of regular bivalent formation in these species indicates that the genome of the species is homologous and does not have large structural differences. Univalents were frequently encountered in most of the species studied. <xref rid="bb0230" ref-type="bibr">Raghuvanshi (1962)</xref> found univalents in 17 out of 25 <italic>Citrus</italic> species analyzed. The presence of univalents in some of the species studied indicates certain degree of structural heterogeneity in the genetic makeup of the bivalents. <italic>C. limetta</italic>, <italic>C. indica</italic> and <italic>C. sinensis</italic> which recorded highest number of univalents per PMC confirm the heterogeneity within their genomes, which could possibly be of an intermediate nature. Up to 18 univalents were detected in intergeneric hybrids between <italic>Citrus</italic> and <italic>Poncirus</italic>, suggesting a lack of homology of different chromosomes (<xref rid="bb0120" ref-type="bibr">Iwamasa, 1966</xref>), though <xref rid="bb0230" ref-type="bibr">Raghuvanshi (1962)</xref> said this could be due to precocious separation of bivalents. Early separation of synapsed homologues is generally the reason for regular occurrence of univalents in many of the tree species (<xref rid="bb0325" ref-type="bibr">Kumar et al., 2002</xref>, <xref rid="bb0260" ref-type="bibr">Singh, 1993</xref>). This explanation is also given for the presence of univalents in other genera, for examples, in wild <italic>Saccharum</italic> species (<xref rid="bb0065" ref-type="bibr">Burner, 1991</xref>). Univalents may lead to unequal distribution at anaphase and consequently a decrease in fertility (<xref rid="bb0140" ref-type="bibr">Khazanehdari and Jones, 1997</xref>). The distribution of chromosomes at anaphases I and II in all the species studied was normal indicating that the pollen sterility in these species is genic controlled (<xref rid="bb0015" ref-type="bibr">Agarwal, 1987</xref>).</p><p>The association of some bivalents (<xref rid="bb0005" ref-type="bibr">Abkenar et al., 2004</xref>, <xref rid="bb0010" ref-type="bibr">Agarwal, 1984</xref>, <xref rid="bb0015" ref-type="bibr">Agarwal, 1987</xref>, <xref rid="bb0020" ref-type="bibr">Agarwal, 1989</xref>) with nucleolus in majority of the PMCs analyzed at diplotene/diakinesis might be indicative of nucleolar nature of representative chromosomes. Secondary association of chromosomes has been considered as an evidence of remote affinity between the chromosomes (<xref rid="bb0260" ref-type="bibr">Singh, 1993</xref>). Further the maximum grouping of these bivalents in groups of three indicates the basic chromosome number for <italic>Citrus</italic> as three, as has been reported earlier by <xref rid="bb0040" ref-type="bibr">Banerjee (1954)</xref> and <xref rid="bb0010" ref-type="bibr">Agarwal (1984)</xref>. The sporadic occurrence of quadrivalents may be attributed to plausible partial homology between otherwise non homologous chromosomes arising out of structural rearrangements (<xref rid="bb0265" ref-type="bibr">Stebbins, 1971</xref>). <xref rid="bb0015" ref-type="bibr">Agarwal (1987)</xref> suggested that the presence of tetravalents in four hybrid <italic>Citrus</italic> taxa indicated homology (or homoeology) among different genomes as well as the absence of large chromosomal differences. Chromosome structural/numerical changes apparently did not play any role in <italic>Citrus</italic> speciation and evolution. Most probably variations at gene level might have influenced speciation in <italic>Citrus</italic> as evident from its morphological diversity. <xref rid="bb0115" ref-type="bibr">Hore and Barua (2004)</xref> reported presence of several intermediate types, hinting at natural hybridization. Meiotic behavior of somatic hybrids provides valuable information for their practical utilization in <italic>Citrus</italic> breeding programs (<xref rid="bb0135" ref-type="bibr">Khan, 2007</xref>). Meiotic abnormalities such as chromosome bridges and chromosomes orientated away from the equatorial plate are frequently observed in hybrids resulting in different sizes of pollen grain and generally abnormal tetrad formation and irregular chromosome behavior with univalents or multivalent pairing which occur in somatic hybrid plants (<xref rid="bb0080" ref-type="bibr">Chen et al., 2004</xref>)<italic>.</italic> However, from our studies none of the species presently investigated from cytogenetical point of view could be regarded as hybrid origin due to lack of stabilized polyploidy events in the species. The relationship between the genomes of the parental species has great influence on the determination of the process of chromosome pairing and recombination and thus the extent of meiotic irregularities and viability of the gametes (<xref rid="bb0085" ref-type="bibr">De Jong et al., 1993</xref>). The taxonomic relationships between the genomes of these intergeneric species need to be re-evaluated by observing meiotic behavior and also molecular cytogenetics studies like FISH/GISH can play a main role in ascertaining the true hybrid nature of the intermediate types in more authentic manner.</p><p>The phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 using maximum parsimony method and Bayesian inference has thrown light on species inter-relationship and evolution of <italic>Citrus</italic> species. Among all the analyses BI method used for analysis of ITS 1 + 5.8 + ITS 2 sequenced data has given more convincing information which had the critical support of both morphological and cytogenetical analyses. The three true basic species i.e. <italic>C. medica</italic>, <italic>C. maxima</italic> and <italic>C. reticulata</italic> have resolved into distinct clades. <italic>C. indica</italic> occupies a unique position in the phylogenetic ladder of the genus <italic>Citrus</italic>. <italic>C. medica</italic>, <italic>C. limon</italic> and <italic>C. maxima</italic> have resolved into a distinct group, which is on expected lines and receives support from published literature (<xref rid="bb0125" ref-type="bibr">Jena et al., 2009</xref>). <italic>C. jambhiri</italic>, <italic>C. limetta</italic> and <italic>C. marcoptera</italic> have resolved into a distinct but separate cluster. It is quite interesting and intriguing to note that <italic>C. macroptera</italic> considered to be a member of sub-genus <italic>Papeda</italic> which shows similarity with the members (<italic>C. jambhiri</italic> and <italic>C. limetta</italic>) of sub genus <italic>Citrus.</italic> The grouping of <italic>C. reticulata</italic> with <italic>C. sinensis</italic> and <italic>C. ichagenesis</italic> is duly approved as the latter species are probable derivatives of <italic>C. reticulata</italic> as reported in literature (<xref rid="bb0215" ref-type="bibr">Penjor et al., 2010</xref>, <xref rid="bb0220" ref-type="bibr">Penjor et al., 2013</xref>). <italic>C. assamensis</italic> which belongs to sub genus <italic>Citrus</italic> and <italic>C. latipes</italic> which belongs to sub-genus <italic>Papeda</italic> are also grouped together. However, both the species exhibit distinct morphological and cytogenetical diversity from each other.</p><p><italic>Citrus indica</italic> is a true wild species endemic to the Garo Hills in Meghalaya. <xref rid="bb0285" ref-type="bibr">Tanaka (1928)</xref> was the first to describe it as a new species. He placed <italic>C. indica</italic> in section Acrumen of the Subgenus MetaCitrus. <italic>C. indica</italic> clustered distinctly from all the other species in the phylogenetic ladder of both MP and BI trees which is also reflected in the meiotic data with the presence of univalents indicating, probably a heterogeneous genome. The presences of quadrivalents in <italic>C. indica</italic> also echo its unique position. Therefore, elucidating its special taxonomic position as a true species or progenitor species of cultivated <italic>Citrus</italic> taxa. <italic>C. medica</italic> (citron), <italic>C. reticulata</italic> (mandarin) and <italic>C. maxima</italic> (pummelo) are defined as basic true species by <xref rid="bb0270" ref-type="bibr">Swingle and Reece (1967)</xref> a phylogenetic truth which was later supported by a number of workers (<xref rid="bb0050" ref-type="bibr">Barrett and Rhodes, 1976</xref>, <xref rid="bb0125" ref-type="bibr">Jena et al., 2009</xref>, <xref rid="bb0145" ref-type="bibr">Kumar et al., 2012</xref>, <xref rid="bb0150" ref-type="bibr">Kyndt et al., 2010</xref>, <xref rid="bb0240" ref-type="bibr">Scora, 1975</xref>). This undisputed taxonomical phenomenon also gains support from our investigation, though partially where <italic>C. reticulata</italic>, <italic>C. medica</italic> and <italic>C. maxima</italic> were resolved into separate clusters while <italic>C. maxima</italic> clustered itself closer to <italic>C. medica.</italic> The presence of maximum number of ring bivalents indicates the homology and stability of their genome. Univalents were recorded in both <italic>C. medica</italic> and <italic>C. maxima</italic> but are low in numbers and there was a total absence of univalents in <italic>C. reticulata</italic>, all these findings support the position of the three basic species. The chiasma frequency recorded in the <italic>C. medica</italic>, <italic>C. reticulata</italic> and <italic>C. maxima</italic> which indicates cryptic heterozygosity at molecular level which is confirmed by clustering patterns in the ITS analysis (<xref rid="f0015" ref-type="fig">Fig. 3</xref>). The species belonging to sub genus <italic>Papeda</italic> i.e. <italic>C. ichangensis</italic>, <italic>C. macroptera</italic> and <italic>C. latipes</italic> have clustered separately and show similarity with the members of sub genus <italic>Citrus.</italic><xref rid="bb0145" ref-type="bibr">Kumar et al. (2012)</xref> also could not find any clear cut differentiation between subgenera <italic>Citrus</italic> and <italic>Papeda</italic> as per Swingle's system. This supports the earlier findings of <xref rid="bb0205" ref-type="bibr">Nicolosi et al. (2000)</xref> and <xref rid="bb0210" ref-type="bibr">Pang et al. (2007)</xref>. Cytogenetically <italic>C. macroptera</italic> and <italic>C. latipes</italic> are alike having maximum number of bivalents which are resolved into ring (5.4 and 5.32) and rod bivalents (3.25 and 3.5). The genome size of <italic>C. ichagensis</italic> is considerably larger as reflected in chromosome size (unpublished data) as compared to <italic>C. latipes</italic> and <italic>C. macroptera</italic>. Thus <italic>C. ichangensis</italic> differs from the two papedian taxa significantly and also gets support from the molecular data of ITS analysis.</p><p><italic>C. reticulata</italic> which is considered as a basal species and possible progenitor of <italic>C. sinensis</italic> (<xref rid="bb0145" ref-type="bibr">Kumar et al., 2012</xref>, <xref rid="bb0165" ref-type="bibr">Mabberley, 2004</xref>, <xref rid="bb0185" ref-type="bibr">Moore, 2001</xref>) and <italic>C. ichangensis</italic> (<xref rid="bb0220" ref-type="bibr">Penjor et al., 2013</xref>) is characteristic of having extensive homogeneity in their genome by showing 100% bivalent association of which more number of ring bivalents and fewer rod bivalents followed by complete absence of univalents. On the other hand, <italic>C. sinensis</italic> which is supposedly a derivative of <italic>C. reticulata</italic> does exhibit cryptic structural hybridity in the form of fewer bivalents, with ring and rod in equal proportion followed by the presence of significant number of univalents which is also reflected in its low pollen stainability. <xref rid="bb0125" ref-type="bibr">Jena et al. (2009)</xref> based on their cpDNA data, also elucidated the involvement of <italic>C. reticulata</italic> as a maternal parent in the origin of sweet orange (<italic>C. sinensis</italic>) although few workers suggest that <italic>C. maxima</italic> may be the maternal parent of sweet orange (<xref rid="bb0035" ref-type="bibr">Araujo et al., 2003</xref>, <xref rid="bb0205" ref-type="bibr">Nicolosi et al., 2000</xref>, <xref rid="bb0220" ref-type="bibr">Penjor et al., 2013</xref>). The molecular data suggest that <italic>C. limon</italic> and <italic>C. maxima</italic> are closely related while <xref rid="bb0205" ref-type="bibr">Nicolosi et al<italic>.</italic> (2000)</xref> hypothesized, from chloroplastic CAPS and nuclear genome analysis, that <italic>C. limon</italic> was derived from hybridization between <italic>C. aurantium</italic> (♀) and <italic>C. medica</italic> (♂). Cytogenetical studies of both the species do not show significant difference either in chromosomes association of chiasma frequency, therefore it is difficult to support the above theory of <italic>C. maxima</italic> as parental species. 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Supplementary information with regards to plant materials and primers used in the present study.</p></caption><media xlink:href="mmc1.docx"></media></supplementary-material></p></sec><fn-group><fn id="s0065" fn-type="supplementary-material"><label>Appendix A</label><p>Supplementary data to this article can be found online at <ext-link ext-link-type="doi" xlink:href="10.1016/j.mgene.2014.01.008" id="ir0005">http://dx.doi.org/10.1016/j.mgene.2014.01.008</ext-link>.</p></fn></fn-group></back><floats-group><fig id="f0005"><label>Fig. 1</label><caption><p>Collection sites of wild and semi-wild <italic>Citrus</italic> species from North-east India.</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="f0010"><label>Fig. 2</label><caption><p>Different male meiotic stages in 10 <italic>Citrus</italic> species. 1–3: diplotene, diakinesis & metaphase I in <italic>C. limetta</italic>; 4–5: diakinesis & metaphase I in <italic>C. medica;</italic> 6–8: diplotene, diakinesis & metaphase I in <italic>C. latipes</italic>; 9–10: dilpotene & diakinesis in <italic>C. limon</italic>; 11–12: diplotene & metaphase I in <italic>C. sinensis</italic>; 13–14: diplotene & metaphase I in <italic>C. reticulata</italic>; 15–16: diplotene & diakinesis in <italic>C. macroptera</italic>; 17–19: diplotene, diplotene (I) & diplotene (IV) in <italic>C. indica</italic>; 20: metaphase I in <italic>C. jambhiri</italic>; 21: metaphase I in <italic>C. maxima</italic>; 22: anaphase I in <italic>C. reticulata</italic> (laggard); 23: anaphase II in <italic>C. limetta</italic>; 24: anaphase II in <italic>C. sinensis</italic> (laggards). Scale bar, 5 μm (applies to all images).</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="f0015"><label>Fig. 3</label><caption><p>50% majority-rule consensus phylogenetic trees for illustrating the relationship among Indian representatives of the genus <italic>Citrus</italic> based on ITS of the rDNA sequence data using maximum parsimony method. Bootstrap values given at the nodes. The tree is rooted with the <italic>Atalantia ceylanica</italic>. The meiotic data of chromosome association, chiasma frequency per cell, terminalization co-efficient and pollen stainability followed by number of ring, rod bivalents and univalents is also given.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="f0020"><label>Fig. 4</label><caption><p>Bayesian inference phylogenetic tree of 12 <italic>Citrus</italic> species based on ITS of the rDNA sequence data. The posterior probability is given on each node. The tree is rooted with the <italic>Atalantia ceylanica</italic>. The scale bar represents branch length (number of substitutions/site). The meiotic data of chromosome association, chiasma frequency per cell, terminalization co-efficient and pollen stainability followed by number of ring, rod bivalents and univalents is also given.</p></caption><graphic xlink:href="gr4"></graphic></fig><table-wrap id="t0005" position="float"><label>Table 1</label><caption><p>Mean number and range of chromosome associations at diplotene/diakinesis/metaphase I in 10 species of <italic>Citrus</italic> accessions.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Species</th><th align="left">Accession No.</th><th align="left">No. of cells analyzed</th><th align="left">2n</th><th colspan="13" align="left">Chromosomes associations<hr></hr></th></tr><tr><th align="left">Quadrivalents</th><th colspan="9" align="left">Bivalents<hr></hr></th><th colspan="3" align="left">Univalents<hr></hr></th></tr><tr><th colspan="3" align="left">Total</th><th colspan="3" align="left">Rings</th><th colspan="3" align="left">Rods<hr></hr></th><th align="left"><hr></hr></th><th align="left"><hr></hr></th><th align="left"><hr></hr></th></tr><tr><th align="left">No</th><th align="left">Mean</th><th align="left">Range</th><th align="left">No</th><th align="left">Mean</th><th align="left">Range</th><th align="left">No</th><th align="left">Mean</th><th align="left">Range</th><th align="left">No</th><th align="left">Mean</th><th align="left">Range</th></tr></thead><tbody><tr><td align="left"><italic>C. medica</italic></td><td align="left">IC 583259</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">222</td><td align="char">8.8 ± 0.3</td><td align="char">8–9</td><td align="left">147</td><td align="char">5.88 ± 1.56</td><td align="char">3–8</td><td align="char">74</td><td align="char">2.96 ± 1.54</td><td align="char">1–6</td><td align="char">6</td><td align="left">0.24 ± 0.66</td><td align="char">0–2</td></tr><tr><td align="left"><italic>C. latipes</italic></td><td align="left">IC 583263</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">222</td><td align="char">8.8 ± 0.33</td><td align="char">8–9</td><td align="left">133</td><td align="char">5.32 ± 1.31</td><td align="char">3–8</td><td align="char">89</td><td align="char">3.56 ± 1.41</td><td align="char">1–6</td><td align="char">6</td><td align="left">0.244 ± 0.66</td><td align="char">0–2</td></tr><tr><td align="left"><italic>C. macroptera</italic></td><td align="left">IC 558161</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">223</td><td align="char">8.92 ± 0.27</td><td align="char">8–9</td><td align="left">142</td><td align="char">5.4 ± 1.24</td><td align="char">3–8</td><td align="char">81</td><td align="char">3.25 ± 1.26</td><td align="char">1–6</td><td align="char">4</td><td align="left">0.16 ± 0.55</td><td align="char">0–2</td></tr><tr><td align="left"><italic>C. indica</italic></td><td align="left">IC 558179</td><td align="left">25</td><td align="left">18</td><td align="left">1</td><td align="left">215</td><td align="char">8.6 ± 0.76</td><td align="char">7–9</td><td align="left">141</td><td align="char">5.64 ± 1.65</td><td align="char">2–8</td><td align="char">73</td><td align="char">3 ± 1.5</td><td align="char">1–7</td><td align="char">16</td><td align="left">0.64 ± 1.38</td><td align="char">0–4</td></tr><tr><td align="left"><italic>C. maxima</italic></td><td align="left">IC 583271</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">223</td><td align="char">8.92 ± 0.27</td><td align="char">8–9</td><td align="left">125</td><td align="char">5 ± 1.35</td><td align="char">2–7</td><td align="char">98</td><td align="char">3.92 ± 1.41</td><td align="char">2–6</td><td align="char">4</td><td align="left">0.16 ± 0.55</td><td align="char">0–2</td></tr><tr><td align="left"><italic>C. sinensis</italic></td><td align="left">IC 558164</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">217</td><td align="char">8.68 ± 0.55</td><td align="char">7–9</td><td align="left">119</td><td align="char">4.76 ± 1.71</td><td align="char">2–8</td><td align="char">98</td><td align="char">3.92 ± 1.68</td><td align="char">1–6</td><td align="char">16</td><td align="left">0.64 ± 1.1</td><td align="char">0–4</td></tr><tr><td align="left"><italic>C. jambhiri</italic></td><td align="left">IC 278011</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">212</td><td align="char">8.83 ± 0.37</td><td align="char">8–9</td><td align="left">117</td><td align="char">4.87 ± 1.16</td><td align="char">3–7</td><td align="char">90</td><td align="char">3.9 ± 1.17</td><td align="char">1–6</td><td align="char">8</td><td align="left">0.34 ± 0.74</td><td align="char">0–2</td></tr><tr><td align="left"><italic>C. reticulata</italic></td><td align="left">IC 583264</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">225</td><td align="char">9 ± 0</td><td align="char">9</td><td align="left">128</td><td align="char">5.12 ± 1.36</td><td align="char">3–9</td><td align="char">97</td><td align="char">3.88 ± 1.36</td><td align="char">2–6</td><td></td><td align="left">–</td><td></td></tr><tr><td align="left"><italic>C. limon</italic></td><td align="left">IC 278013</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">224</td><td align="char">8.95 ± 0.2</td><td align="char">8–9</td><td align="left">122</td><td align="char">4.88 ± 1.66</td><td align="char">2–6</td><td align="char">102</td><td align="char">4.08 ± 1.7</td><td align="char">1–7</td><td align="char">2</td><td align="left">0.08 ± 0.4</td><td align="char">0–2</td></tr><tr><td align="left"><italic>C. limetta</italic></td><td align="left">IC 583244</td><td align="left">25</td><td align="left">18</td><td align="left"><bold>–</bold></td><td align="left">214</td><td align="char">8.56 ± 0.58</td><td align="char">7–9</td><td align="left">108</td><td align="char">4.32 ± 1.21</td><td align="char">2–7</td><td align="char">106</td><td align="char">4.24 ± 1.2</td><td align="char">1–7</td><td align="char">22</td><td align="left">0.88 ± 1.16</td><td align="char">0–4</td></tr></tbody></table></table-wrap><table-wrap id="t0010" position="float"><label>Table 2</label><caption><p>Mean number, range of chiasmata and terminalization coefficient in 10 species of <italic>Citrus</italic> accessions.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Species</th><th align="left">Accession number</th><th align="left">No. of cells analyzed</th><th align="left">n</th><th colspan="3" align="left">Total chiasma<hr></hr></th><th colspan="3" align="left">Terminalised chiasma<hr></hr></th><th colspan="3" align="left">Unterminalised chiasma<hr></hr></th><th align="left">Terminilization co efficient</th><th align="left">Chiasma frequency</th><th align="left">Pollen stainability studies (%)</th></tr><tr><th align="left">No</th><th align="left">Mean</th><th align="left">Range</th><th align="left">No</th><th align="left">Mean</th><th align="left">range</th><th align="left">No</th><th align="left">Mean</th><th align="left">Range</th></tr></thead><tbody><tr><td align="left"><italic>C. medica</italic></td><td align="left">IC 583259</td><td align="left">25</td><td align="left">9</td><td align="left">669</td><td align="char">26.5 ± 4.18</td><td align="char">20–33</td><td align="left">284</td><td align="char">11.45 ± 2.1</td><td align="char">9–15</td><td align="left">385</td><td align="char">15.4 ± 1.9</td><td align="char">12–18</td><td align="char">0.42</td><td align="char">26.76</td><td align="char">44.4</td></tr><tr><td align="left"><italic>C. latipes</italic></td><td align="left">IC 583263</td><td align="left">25</td><td align="left">9</td><td align="left">628</td><td align="char">25.12 ± 4</td><td align="char">19–31</td><td align="left">263</td><td align="char">10.52 ± 2.6</td><td align="char">6–14</td><td align="left">365</td><td align="char">14.6 ± 1.7</td><td align="char">12–19</td><td align="char">0.41</td><td align="char">25.12</td><td align="char">87</td></tr><tr><td align="left"><italic>C. macroptera</italic></td><td align="left">IC 558161</td><td align="left">25</td><td align="left">9</td><td align="left">661</td><td align="char">26.44 ± 3.7</td><td align="char">19–33</td><td align="left">272</td><td align="char">10.88 ± 2.29</td><td align="char">6–16</td><td align="left">389</td><td align="char">15.56 ± 1.7</td><td align="char">13–19</td><td align="char">0.41</td><td align="char">26.44</td><td align="char">80</td></tr><tr><td align="left"><italic>C. indica</italic></td><td align="left">IC 558179</td><td align="left">25</td><td align="left">9</td><td align="left">673</td><td align="char">26.92 ± 5.67</td><td align="char">18–37</td><td align="left">281</td><td align="char">11.24 ± 2.63</td><td align="char">7–14</td><td align="left">392</td><td align="char">15.68 ± 3.22</td><td align="char">11–21</td><td align="char">0.41</td><td align="char">26.92</td><td align="char">90</td></tr><tr><td align="left"><italic>C. maxima</italic></td><td align="left">IC 583271</td><td align="left">25</td><td align="left">9</td><td align="left">610</td><td align="char">24.4 ± 2.81</td><td align="char">17–28</td><td align="left">240</td><td align="char">9.6 ± 1.52</td><td align="char">7–13</td><td align="left">370</td><td align="char">14.8 ± 1.6</td><td align="char">7–13</td><td align="char">0.39</td><td align="char">24.4</td><td align="char">89</td></tr><tr><td align="left"><italic>C. sinensis</italic></td><td align="left">IC 558164</td><td align="left">25</td><td align="left">9</td><td align="left">572</td><td align="char">22.88 ± 5</td><td align="char">17–33</td><td align="left">224</td><td align="char">8.96 ± 2.68</td><td align="char">5–13</td><td align="left">348</td><td align="char">13.92 ± 2.64</td><td align="char">9–20</td><td align="char">0.39</td><td align="char">22.88</td><td align="char">78.9</td></tr><tr><td align="left"><italic>C. jambhiri</italic></td><td align="left">IC 278011</td><td align="left">25</td><td align="left">9</td><td align="left">562</td><td align="char">24.28 ± 3.27</td><td align="char">18–30</td><td align="left">231</td><td align="char">9.62 ± 1.68</td><td align="char">7–11</td><td align="left">354</td><td align="char">14.75 ± 1.77</td><td align="char">11–18</td><td align="char">0.41</td><td align="char">22.48</td><td align="char">59.4</td></tr><tr><td align="left"><italic>C. reticulata</italic></td><td align="left">IC 583264</td><td align="left">25</td><td align="left">9</td><td align="left">634</td><td align="char">25.36 ± 3.81</td><td align="char">17–30</td><td align="left">269</td><td align="char">10.76 ± 2.52</td><td align="char">5–14</td><td align="left">365</td><td align="char">14.6 ± 1.5</td><td align="char">12–16</td><td align="char">0.42</td><td align="char">25.36</td><td align="char">82</td></tr><tr><td align="left"><italic>C. limon</italic></td><td align="left">IC 278013</td><td align="left">25</td><td align="left">9</td><td align="left">604</td><td align="char">24.16 ± 3.92</td><td align="char">16–33</td><td align="left">241</td><td align="char">9.64 ± 2.21</td><td align="char">5–14</td><td align="left">365</td><td align="char">14.52 ± 1.93</td><td align="char">11–19</td><td align="char">0.40</td><td align="char">24.16</td><td align="char">63</td></tr><tr><td align="left"><italic>C. limetta</italic></td><td align="left">IC 583244</td><td align="left">25</td><td align="left">9</td><td align="left">565</td><td align="char">22.6 ± 3.52</td><td align="char">17–28</td><td align="left">236</td><td align="char">9.44 ± 1.95</td><td align="char">6–13</td><td align="left">322</td><td align="char">13.32 ± 1.65</td><td align="char">10–16</td><td align="char">0.41</td><td align="char">22.6</td><td align="char">80</td></tr></tbody></table></table-wrap><table-wrap id="t0015" position="float"><label>Table 3</label><caption><p>Anaphase I distribution in <italic>Citrus</italic> species.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Species</th><th align="left">Accession no.</th><th align="left">No of cell analyzed</th><th align="left">Chromosome distribution</th><th align="left">No. of cells</th><th align="left">Percentage</th></tr></thead><tbody><tr><td align="left"><italic>C. medica</italic></td><td align="left">IC 583259</td><td align="char">15</td><td align="left">9:9</td><td align="char">15</td><td align="char">100</td></tr><tr><td align="left"><italic>C. latipes</italic></td><td align="left">IC 583263</td><td align="char">10</td><td align="left">9:9</td><td align="char">10</td><td align="char">100</td></tr><tr><td align="left"><italic>C. macroptera</italic></td><td align="left">IC 558161</td><td align="char">20</td><td align="left">9:9, 9:1:8</td><td align="char">19</td><td align="char">95</td></tr><tr><td align="left"><italic>C. indica</italic></td><td align="left">IC 558179</td><td align="char">20</td><td align="left">9:9</td><td align="char">20</td><td align="char">100</td></tr><tr><td align="left"><italic>C. maxima</italic></td><td align="left">IC 583271</td><td align="char">15</td><td align="left">9:9</td><td align="char">15</td><td align="char">100</td></tr><tr><td align="left"><italic>C. sinensis</italic></td><td align="left">IC 558164</td><td align="char">9</td><td align="left">9:9</td><td align="char">9</td><td align="char">100</td></tr><tr><td align="left"><italic>C. jambhiri</italic></td><td align="left">IC 278011</td><td align="char">10</td><td align="left">9:9</td><td align="char">10</td><td align="char">100</td></tr><tr><td align="left"><italic>C. reticulata</italic></td><td align="left">IC 583264</td><td align="char">15</td><td align="left">9:9, 9:1:8</td><td align="char">14</td><td align="char">93</td></tr><tr><td align="left"><italic>C. limon</italic></td><td align="left">IC 278013</td><td align="char">15</td><td align="left">9:9</td><td align="char">15</td><td align="char">100</td></tr><tr><td align="left"><italic>C. limetta</italic></td><td align="left">IC 583244</td><td align="char">15</td><td align="left">9:9</td><td align="char">15</td><td align="char">100</td></tr></tbody></table></table-wrap><table-wrap id="t0020" position="float"><label>Table 4</label><caption><p>Summary of nrDNA sequences of 12 species of <italic>Citrus</italic> and the out-group <italic>Atalantia ceylanica.</italic></p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Sl. no.</th><th align="left">Taxon</th><th align="left">Accessions number</th><th align="left">Status</th><th align="left">ITS 1</th><th align="left">5.8S</th><th align="left">ITS 2</th><th align="left">Total</th><th align="left">G + C (%)</th></tr></thead><tbody><tr><td align="left">1</td><td align="left"><italic>C. assamensis</italic></td><td align="left">IC 285355</td><td align="left">Wild</td><td align="left">254</td><td align="left">160</td><td align="left">248</td><td align="left">662</td><td align="char">63.3</td></tr><tr><td align="left">2</td><td align="left"><italic>C. ichagensis</italic></td><td align="left">IC 591460</td><td align="left">wild</td><td align="left">258</td><td align="left">160</td><td align="left">240</td><td align="left">658</td><td align="char">64.4</td></tr><tr><td align="left">3</td><td align="left"><italic>C. indica</italic></td><td align="left">IC 558179</td><td align="left">Wild</td><td align="left">224</td><td align="left">160</td><td align="left">237</td><td align="left">621</td><td align="char">63</td></tr><tr><td align="left">4</td><td align="left"><italic>C. jambhiri</italic></td><td align="left">IC 278011</td><td align="left">Cultivated</td><td align="left">221</td><td align="left">160</td><td align="left">220</td><td align="left">601</td><td align="char">63</td></tr><tr><td align="left">5</td><td align="left"><italic>C. latipes</italic></td><td align="left">IC 583263</td><td align="left">Wild</td><td align="left">253</td><td align="left">160</td><td align="left">233</td><td align="left">646</td><td align="char">62.8</td></tr><tr><td align="left">6</td><td align="left"><italic>C. limon</italic></td><td align="left">IC 278013</td><td align="left">Cultivated</td><td align="left">255</td><td align="left">160</td><td align="left">261</td><td align="left">676</td><td align="char">61.2</td></tr><tr><td align="left">7</td><td align="left"><italic>C. limettioides</italic></td><td align="left">IC 583244</td><td align="left">Cultivated</td><td align="left">210</td><td align="left">160</td><td align="left">270</td><td align="left">640</td><td align="char">63</td></tr><tr><td align="left">8</td><td align="left"><italic>C. macroptera</italic></td><td align="left">IC 558161</td><td align="left">Wild</td><td align="left">255</td><td align="left">160</td><td align="left">220</td><td align="left">605</td><td align="char">63</td></tr><tr><td align="left">9</td><td align="left"><italic>C. maxima</italic></td><td align="left">IC 583271</td><td align="left">Cultivated</td><td align="left">255</td><td align="left">160</td><td align="left">237</td><td align="left">652</td><td align="char">62.1</td></tr><tr><td align="left">10</td><td align="left"><italic>C. medica</italic></td><td align="left">IC 583259</td><td align="left">Cultivated</td><td align="left">310</td><td align="left">160</td><td align="left">224</td><td align="left">694</td><td align="char">59.8</td></tr><tr><td align="left">11</td><td align="left"><italic>C. reticulata</italic></td><td align="left">IC 583264</td><td align="left">Cultivated</td><td align="left">264</td><td align="left">160</td><td align="left">237</td><td align="left">661</td><td align="char">61.3</td></tr><tr><td align="left">12</td><td align="left"><italic>C. sinensis</italic></td><td align="left">IC 558164</td><td align="left">Cultivated</td><td align="left">221</td><td align="left">160</td><td align="left">238</td><td align="left">619</td><td align="char">60</td></tr><tr><td align="left">13</td><td align="left"><italic>Atalantia ceylanica</italic></td><td align="left">–</td><td align="left">–</td><td align="left">256</td><td align="left">155</td><td align="left">238</td><td align="left">649</td><td align="char">64.4</td></tr></tbody></table></table-wrap><table-wrap id="t0025" position="float"><label>Table 5</label><caption><p>Sequence characteristics of ITS region of rDNA in <italic>Citrus</italic> species.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Sl. no.</th><th align="left">Parameters</th><th align="left">ITS</th><th align="left">ITS 1</th><th align="left">ITS 2</th><th align="left">5.8S</th></tr></thead><tbody><tr><td align="left">1</td><td align="left">Length range (in-group) (bp)</td><td align="left">601–694</td><td align="left">210–310</td><td align="left">207–270</td><td align="left">160</td></tr><tr><td align="left">2</td><td align="left">Length (out-group) (bp)</td><td align="left">649</td><td align="left">256</td><td align="left">238</td><td align="left">155</td></tr><tr><td align="left">3</td><td align="left">Aligned length (bp) including missing data<break></break>No. of conserved sites (%)<break></break>No. of variable sites (%)<break></break>No. of informative sites (%)</td><td align="left">732<break></break>385(52.5)<break></break>316(43.1)<break></break>130(25.4)</td><td align="left">324<break></break>181(55.6)<break></break>113(34)<break></break>99(30.5)</td><td align="left">296<break></break>183(61.8)<break></break>83(28)<break></break>81(27.3)</td><td align="left">170<break></break>155(91.1)<break></break>12(7)<break></break>3(1.76)</td></tr><tr><td align="left">4</td><td align="left">Indels</td><td align="left">20</td><td align="left">14</td><td align="left">9</td><td align="left">6</td></tr><tr><td align="left">5</td><td align="left">G + C content range (%)</td><td align="left">59.8–64.4</td><td align="left">57–69</td><td align="left">56.6–69.6</td><td align="left">52.4–55.8</td></tr><tr><td align="left">6</td><td align="left">G + C content mean (%)</td><td align="left">62.24</td><td align="left">64.3</td><td align="left">63.2</td><td align="left">54.2</td></tr><tr><td align="left">7</td><td align="left">Sequence divergence (%)</td><td align="left">26.5</td><td align="left">34.8</td><td align="left">28</td><td align="left">7.05</td></tr><tr><td align="left">8</td><td align="left">Nucleotide frequencies of<break></break>Adenine<break></break>Thymine<break></break>Cytosine<break></break>Guanine</td><td align="left">0.208<break></break>0.181<break></break>0.310<break></break>0.300</td><td align="left">0.185<break></break>0.136<break></break>0.346<break></break>0.333</td><td align="left">0.185<break></break>0.174<break></break>0.333<break></break>0.308</td><td align="left">0.25<break></break>0.217<break></break>0.263<break></break>0.27</td></tr><tr><td align="left">9</td><td align="left">Transition/transversion bias (R)</td><td align="left">0.83</td><td align="left">6.023</td><td align="left">1.048</td><td align="left">1.095</td></tr></tbody></table></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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