Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.     

Genetic Diversity of the Wild Banana Musa acuminata Colla in Malaysia as Evidenced by AFLP

Musa acuminata Colla (Musaceae), the wild progenitor of thecultivated banana, is highly variable in Malaysia and presentsseveral unresolved nomenclatural problems. AFLP was employedto distinguish among three subspecies of Musa acuminata(subsp.truncata and subsp. malaccensis from peninsular Malaysia andsubsp. microcarpa from Borneo) and to examine whether subsp.truncata is a distinct taxon. Eight primer combinations revealedmolecular markers specific for each of the three taxa. UPGMAcluster analysis showed the three taxa were distinct. Subspeciesmalaccensis which is endemic in peninsular Malaysia and subsp.microcarpa which is endemic in Borneo were found to be moresimilar to each other in their DNA patterns than they are tosubsp. truncata, which is endemic to peninsular Malaysia. Sincesubsp. truncata is genetically separate from subsp. malaccensisand subsp.microcarpa , it cannot be regarded as synonymous witheither of these subspecies. This paper sheds light on the nomenclatureof the three subspecies of Musa acuminata. Copyright 2001 Annalsof Botany Company Musa acuminata Colla, truncata, malaccensis, microcarpa, Musaceae, wild banana, genetic diversity, AFLP, DNA fingerprinting     

Identification and evaluation of two diagnostic markers linked to Fusarium wilt resistance (race 4) in banana (Musa spp.)

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (FOC4) results in vascular tissue damage and ultimately death of banana (Musa spp.) plants. Somaclonal variants of in vitro micropropagated banana can hamper success in propagation of genotypes resistant to FOC4. Early identification of FOC4 resistance in micropropagated banana plantlets is difficult, however. In this study, we identified sequence-characterized amplified region (SCAR) markers of banana associated with resistance to FOC4. Using pooled DNA from resistant or susceptible genotypes and 500 arbitrary 10-mer oligonucleotide primers, 24 random amplified polymorphic DNA (RAPD) products were identified. Two of these RAPD markers were successfully converted to SCAR markers, called ScaU1001 (GenBank accession number HQ613949) and ScaS0901 (GenBank accession number HQ613950). ScaS0901 and ScaU1001 could be amplified in FOC4-resistant banana genotypes (“Williams 8818-1” and Goldfinger), but not in five tested banana cultivars susceptible to FOC4. The two SCAR markers were then used to identify a somaclonal variant of the genotype “Williams 8818-1”, which lost resistance to FOC4. Hence, the identified SCAR markers can be applied for a rapid quality control of FOC4-resistant banana plantlets immediately after the in vitro micropropagation stage. Furthermore, ScaU1001 and ScaS0901 will facilitate marker-assisted selection of new banana cultivars resistant to FOC4.     

SAMPL: A technique for somaclonal variation fingerprinting inMusa

SAMPL primers designed for genomic profiling in chickpea (Cicer arietinum L.) were tested for their applicability to fingerprinting of DNA of banana cultivars and soma-clonal variants. Most of the chickpea primers allowed amplification of genomic DNA of banana and detection of sequence polymorphisms within theMusa acuminata genome (A). Our results demonstrate that the highly resolving SAMPL technique is useful in banana genomics, especially for the distinction and characterization of commercially important cultivars and promising somaclonal variants containing the A genome.     

Somatic embryogenesis and plant regeneration through cell suspensions inMusa acuminata

Summary Cell suspensions ofMusa acuminata sspburmannicoides andMusa acuminata sspmalaccensis were obtained by culturing embryogenic callus initiated from immature zygotic embryos in liquid medium. Plant regeneration was then achieved through somatic embryogenesis. Germination of these embryos occurred in a modified MS medium containing auxin and cytokinin. Plant recovery frequencies were 20 to 36%. This method may allow a better utilization of biotechnologies in genetic improvement of theMusa diploid species, essential for banana and plantain breeding.     

RFLP-based phylogeny of Musa species in Papua New Guinea

Summary Random genomic probes were used to detect restriction fragment length polymorphisms (RFLPs) in 26 accessions of Musa representing eight species from Papua New Guinea (PNG), M. textilis, M. jackeyi and one accession of Ensete. Ninety-eight phylogenetically informative characters were scored and analyzed cladistically and phenetically. Results generally agreed with previous morphology-based phylogenetic analyses. However, the closest wild relative of the edible M. fehi (fe'i banana) appears to be M. lolodensis. Musa angustigemma is sister species with M. boman and M. jackeyi and is distinct from M. peekelii, with which it is often united. Musa boman is unambiguously placed in section Australimusa. The diploid parthenocarpic landraces of section Musa unique to PNG are closely related to, but apparently distinct from, M. acuminata ssp. banksii. The evolution of the fe'i bananas and the M. acuminata-derived diploid landraces of PNG are discussed.     

Genetic Diversity in Musa acuminata Colla and Musa balbisiana Colla and some of their natural hybrids using AFLP Markers

Genetic diversity and relationships were assessed in 28 accessions of Musa acuminata (AA) Colla and Musa balbisiana (BB) Colla, and some of their natural hybrids, using the amplified fragment length polymorphisms (AFLP) technique. Fifteen AFLP +3 primer pairs produced 527 polymorphic bands among the accessions. Neighbor-joining and principal co-ordinate (PCO) analyses using Jaccard's similarity coefficient produced four major clusters that closely corresponded with the genome composition of the accessions (AA, BB, AAB and ABB). The AFLP data distinguished between the wild diploid accessions and suggested new subspecies relationships in the M. acuminata complex that are different from those based on morphological data. The data suggested that there are three subspecies within the M. acuminata complex (ssp. burmannica Simmonds, malaccensis Simmonds, and microcarpa Simmonds). 'Tjau Lagada' (ssp. microcarpa), 'Truncata' [ssp truncata (Ridl.) Shepherd] and 'SF247' [ssp. banksii (F.Muell) Simmonds] clustered very closely with 'Gros Michel' and 'Km 5', indicating that more than one M. acuminata subspecies may be involved in the origin of triploid AAA bananas. 'Calcutta 4' (ssp. burmannicoides De Langhe &; Devreux) and 'Long Tavoy' (ssp. burmannica) were closely related and could be together in the same subspecies. This study also showed that there is much more genetic diversity within M. balbisiana that was split into two groups: (1) 'I-63' and 'HND' and (2) 'Los Banos', 'MPL' (Montpellier), '10852', 'Singapuri', 'Etikehel', and 'Butohan 1' as the other.     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

Assessment of a species-specific element (Brep 1) in banana

 The nuclear genome of wild-type banana accessions was investigated for repetitive elements. We report here the occurrence, in the banana genome, of a sequence family of species-specific repetitive elements: Brep 1. This sequence family is distributed throughout the Musaceae with various copy numbers. The two species Musa acuminata and M. schizocarpa carry the highest copy numbers in contrast to M. balbisiana and tested representatives of different other sections. PCR primers were defined in the core consensus sequence for specific amplifications, which allow representatives of this sequence family to be easily detected in wild and cultivated banana clones. Sequence data were analysed and hypotheses on the evolution of banana cultivars from the wild-type banana clones are discussed. Received: 17 January 1997 / Accepted : 7 March 1997     

Genetic diversity and species-specific PCR-based markers from AFLP analyses of Thai bananas

A large amount of banana genetic resource has been found in Thailand which is believed to be one of the centers of its origins. To assess genetic diversity and determine genetic relationships of edible bananas in Thailand, 110 accessions of banana species and cultivars collected from villages and natural locations were investigated. UPGMA clustering of numerical data from Amplified Fragment Length Polymorphism (AFLP) patterns showed two large groups which corresponded to genome designations of Musa acuminata (AA) and Musa balbisiana (BB), the known ancestors of most edible cultivars. The AFLP data suggested that among Thai bananas, AA and AAA cultivars were closely related to M. acuminata subsp. malaccensis, while some of ‘B’ genome contained ones closely related to wild M. balbisiana in Thailand and some may have been imported. Eight species-specific PCR-based primer pairs, generated from the AFLP results clearly identify ‘A’ and ‘B’ genomes within cultivars and hybrids. The analyses were useful to readily and easily infer progenitors of these cultivars and pronounce wide genetic diversity of the bananas in Thailand.     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

Using flow cytometry and cytological analyses to assess the genetic stability of somatic embryo-derived plantlets from embryogenic Musa acuminata Colla (AA) ssp. malaccensis cell suspension cultures

Flow cytometry and chromosome counts were used to analyze the genetic stability of plants regenerated via Musa acuminata Colla (AA) ssp. malaccensis embryogenic cell suspension (ECS) cultures. These cultures were initiated from immature zygotic embryos (IZE) on Murashige and Skoog medium using nine different plant growth regulator (PGR) treatments. Highest percentage of embryogenic calli (EC) formation occurred on media with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D, 97 %), and 8.2 μM of picloram (Pi, 80 %) followed by 2.2 μM 2,4-D (75 %). Embryonic development was synchronized in liquid medium by filtration, and somatic embryo development was achieved with ECS aliquots overlaid on PGR-free medium. The EC medium composition and elapsed time of both short-term (~5 months old) and long-term (~2 years old) ECS cultures influenced plant regeneration, resulting in 65–99 % embryo germination and 50 to 100 % plant conversion. The mean 2C DNA content (1.23 ± 0.002 pg) and chromosome number (2n = 2x = 22) of M. acuminata ssp. malaccensis IZEs, seedlings and sucker plantlets were similar to the reported values. No significant differences were detected among IZEs before culturing, and none were found among the IZEs and leaves of control plants and the plants regenerated from short-term ECS lines when initiated with 2,4-D or Pi vis-à-vis the accession from which they originated. However, plants regenerated from the long-term ECS-L3 culture remained diploid, had the highest DNA content (2C = 1.283 ± 0.01 pg) and were clearly separate from the other regenerated and control plants.     

Detection of RAPD polymorphisms inGladiolus cultivars with differing sensitivities toFusarium oxysporum f. sp.gladioli

Fusarium oxysporum Schlecht. Fr. F. sp.gladioli (FOG) is the most important gladiolus pathogen. One of the most environmentally friendly methods to control its spread is to use cultivars that are minimally sensitive to the pathogen. Infected corm tissues in more resistant varieties have been shown to produce suberin layers that inhibit fungal iphae growth. RAPD analysis of genomes from 9 selected gladiolus cvs, chosen to be the most resistant and sensitive to FOG, were performed to verify DNA polymorphism levels. Total nucleic acid extraction was carried out with a chloroform-phenol method from tissues of plants in 3 growth stages. RAPD experiments were performed using 14 primers with varyingTaq polymerases and primer concentrations. Five of the primers tested gave no polymorphic profiles. Five primers produced polymorphic bands, allowing us to obtain RAPD profiles typical for one or more of the more resistant cvs. All the tested growth stages provided repeatable results, indicating the reliability of detected polymorphisms. Cloning the more interesting polymorphic DNA fragments in the future will verify the presence of specific genes related to FOG resistance mechanisms in gladiolus.     

中国西南地区特有水生植物海菜花微卫星引物开发

水鳖科(Hydrocharitaceae)海菜花(Ottelia acuminata)是中国西南地区特有的水生单子叶植物。基于AFLP技术的磁珠富集快速分离技术(Fast Isolation by AFLP of Sequences Containing Repeats,FIASCO),共筛选出9对多态性引物并对3个居群45个个体进行分析。结果表明:三个居群的等位基因数目为1~3个,观测杂合度从0.000~0.933,期望杂合度从0.000~0.605。这些筛选出的微卫星引物将用于海菜花后续的谱系地理学和生态遗传学研究。     

香蕉肉桂醇脱氢酶基因的克隆及表达分析

肉桂醇脱氢酶(CAD)在木质素合成过程中起关键作用。通过RACE(rapid-amplification of cDNA ends)方法从香蕉根系cDNA均一化全长文库中获得一个肉桂醇脱氢酶基因,命名为MaCAD1(GenBank登录号为KF582533)。MaCAD1是香蕉MYB基因编码框全长cDNA,包含一个1 077bp的最大开放阅读框(ORF),编码358个氨基酸。蛋白质序列同源比对发现,其含有完整的醇脱氧酶的典型保守结构域,属于典型的CAD蛋白。系统进化树比对分析表明,MaCAD1与水稻OsCAD6(CAD39907)的亲缘关系较近。组织特异性研究表明MaCAD1基因组成型表达于香蕉各个组织。在耐病和感病品种中,MaCAD1均上调表达,但在耐病品种中MaCAD1在所有时间点相对于对照增加的倍数均高于感病品种,表明MaCAD1基因在香蕉的抗病性中起着重要作用,MaCAD1可以作为一个新的响应枯萎病侵染的标记基因。     

Analysis of genetic diversity and relationships in East African banana germplasm

The genetic diversity and phylogenetic relationships of 29 East African highland banana (Musa spp.) cultivars and two outgroup taxa, M. acuminata Calcutta 4 and Agbagba were surveyed by RAPD analysis. A genetic similarity matrix was established based on the presence or absence of polymorphic amplified fragments. Phylogenetic relationships were determined by UPGMA cluster analysis. RAPDs showed that the highland bananas are closely related with a narrow genetic base. Nevertheless, there were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram was divisible into a major cluster composed of all the AAA highland banana cultivars and Agbagba (AAB) and a minor cluster consisting of Kisubi (AB), Kamaramasenge (AB) and Calcutta 4 (AA). Several subgroups are recognized within the major cluster. RAPD data did not separate beer and cooking banana cultivars. Our study showed that RAPD markers can readily dissect genetic differences between the closely related highland bananas and provide a basis for the selection of parents for improvement of this germplasm. Received: 28 June 2000 / Accepted: 1 August 2000