Genome classification of banana cultivars from South India using IRAP markers

Summary The banana cultivars are originated from the intra- and inter-specific hybridization of two wild diploid species, Musa acuminata Colla and Musa balbisiana Colla, contributing the A and B genomes, respectively. They are classified into genomic groups by scoring morphological features. Molecular markers provide a quick and reliable system of genome characterization and manipulation in breeding lines. In the present study a PCR based molecular marker specific for B genomes is been reported. The IRAP primer, designed based on the LTR sequence of banana Ty3-gypsy-like retroelement (Musa acuminata Monkey retrotransposon, AF 143332), was used to identify the B genome in the banana cultivars. Further a primer pair designed from B specific bands of Musa balbisiana `Pisang Gala' was used to classify AAB and ABB cultivars in the collection. Among the 36 cultivars tested with this primer, the B specific band was absent in the AA and AAA cultivars (except in one AAA and AAB cultivar) but present in all other AB, AAB and ABB cultivars. Among the triploid AAB/ABB, the PCR products with B specific primers showed restriction pattern polymorphism with AluI. In ABB genomes the band intensity was high whereas low intensity band observed in AAB genomes. Four cultivars reported to have the ABB genome showed a pattern similar to AAB, and one cultivar reported to have AAA genome showed a pattern similar to ABB genome, suggesting missampling or misidentification. The primers used in this study are useful to identify the presence of B genome in banana cultivars, and band intensity may be a preliminary indicator of ploidy level of the B genome but needs further studies with competitive PCR for clarification. These authors contributed equally in this paper.     

香蕉ACC合成酶反义基因转化香蕉的研究

香蕉是重要的热带、亚热带地区作物,典型的呼吸跃变型果实,不耐贮藏.传统的保鲜方法存在诸多弊端,利用现代生物技术培育耐贮藏新品种,已经成为我们解决香蕉保鲜问题的重要课题.本研究利用果实特异性ACC合成酶基因反义植物表达载体,采用基因枪和农杆菌介导法转化海南主栽的巴西香蕉及红香蕉,并比较了不同转化方法及不同栽培品种的转化效果.结果表明农杆菌侵染法好于基因枪法,在农杆菌侵染方法中,对红香蕉的转化效果好于巴西香蕉.经PCR检测获得了9株转化植株,进一步采用Southern blot分析的方法,其中4株杂交信号较强,确认ACC合成酶反义基因已经整合进香蕉基因组中.本研究为香蕉的转基因研究和耐储藏新品种的培育奠定了一定的基础.     

A review on adaptation of banana (Musa spp.) to cold in subtropics

Global production of bananais adversely affected by abiotic stresses, namely, drought, salinity and low temperature which cause changes in morphology, anatomy, physiology and biochemical metabolism in plant. Different banana cultivars respond differently when grown in subtropical regions due to changes in temperature mainly at the time of flowering and fruiting. The subtropical regions persist cold in winter which become a major constraint for proper growth and development of banana. Only few cultivars are identified for cold tolerance. The cold tolerance is a complex phenomenon, involving numbers of interrelated metabolic pathways. With the onset of cold, growth decrease through alteration in the synthesis of metabolites which are regulated by expression of genes and their interactions. Recently, complete genome sequencing, mutation and transgenesis provided deep insight into the complex mechanism of cold tolerance in banana. In this review, efforts are made to compile the findings and to interpret the better application of technologies in understanding the cold tolerance which may assist in banana breeding.     

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of 'streamlined’ laboratory techniques and data analysis to Developing Countries are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nematode resistance in bananas: screening results on some wild and cultivated accessions of Musa spp.

Bananas cultivated for export all belong to Cavendish cultivars and are all recognized as very susceptible to nematodes, particularly to the burrowing nematode Radopholus similis and the lesion nematode Pratylenchus coffeae. Even if there have been many changes in the management of banana nematodes in large commercial banana plantations, chemical control still remains most often the last resort method to manage the nematodes, although the number of registered products is definitely declining. Therefore, nematode control though genetic improvement is gaining new interest worldwide. In this study, 55 banana accessions mostly diploids from the Musa acuminata genome group (AA) but including some triploid accessions (AAA), some diploids of the Musa balbisiana genome group (BB) and some interspecific hybrids (AAB, AB) were evaluated for resistance to four nematode species R. similis, P. coffeae, Meloidogyne incognita and M. arenaria. These experiments were conducted in a growth chamber under controlled conditions. All banana accessions were susceptible to nematode species, although many different levels of susceptibility were detected. This study confirmed the good resistance status to R. similis of some cultivars from the Pisang jari buaya and Pisang batuau subgroups and the partial resistance of 17 diploid accessions significantly different from the susceptible reference cv. Grande Naine. This study also showed that 12 diploid accessions exhibited a partial resistance to P. coffeae, including some usual or potential genitors belonging to the wild diploids subspecies burmannica (cvs. Long Tavoy 1 and 2) and burmannicoides (cv. Calcutta 4). No source of resistance to Meloidogyne spp. was found. These screening results, combining for the first time four nematode species, are discussed within the scope of banana breeding in order to produce parental diploid lines with single or combined nematode resistances and further develop triploids that can substitute existing susceptible commercial cultivars.     

香蕉枯萎病菌对不同香蕉品种防御酶系的影响

摘要：本文研究了不同香蕉品种在接种香蕉枯萎病菌后防御酶系的变化。结果表明,经病原菌接种后,SOD酶活性的大小与不同香蕉品种的耐、感病之间呈负相关;PO酶和内切几丁质酶活性的变化趋势与香蕉品种的抗病性之间不存在相关性;PAL酶、β-1, 3-葡聚糖酶和外切几丁质酶的活性变化趋势与香蕉品种抗、感病品种关系密切,可以作为香蕉品种抗枯萎病一个重要的生物化学指标。     

同品种香蕉外观品质和内在品质的比较研究

为解决如何评价不同品种香蕉品质优劣的问题,对广东省湛江市香蕉资源圃8个香蕉品种的果实外观品质进行调查评价,并分析测定果实的糖、有机酸、VC、单宁、淀粉等的含量。结果表明,香蕉不同品种之间的外观品质存在显著差异,‘威廉斯’、‘巴西诱变’、‘北大矮蕉2号’外观品质较优;所测定的香蕉不同品种间各内在品质指标有显著差异,淀粉、VC、单宁含量各品种间变化幅度较大,可溶性糖、有机酸含量不同品种间变化幅度较小。综合分析可知,‘威廉斯’、‘巴西诱变’、‘北大矮蕉2号’的品质较优。     

Agronomic and molecular characterization of gamma ray induced banana (<Emphasis Type="Italic">Musa</Emphasis> sp.) mutants using a multivariate statistical algorithm

Bananas are tropical fruits grown worldwide playing a key role in market trade and especially used as main food source for low income populations. In Brazil, bananas are mainly consumed in natura, occupying the second largest internal market. Nevertheless, this crop presents low availability of productive commercial varieties with good agronomic characteristics. A strategy undertaken to solve this problem is the development of new cultivars through conventional genetic breeding methods. However, this strategy presents some obstacles such as female sterility and low number of seeds. In order to overcome these shortcomings, use of mutation induction aiming the selection of mutants with desirable agronomic characteristics seems to have great potential for developing new cultivars. The objective of the present work was to evaluate the genetic variability in putative banana ‘Pacovan’ (AAB genome, subgroup Prata Type) mutants submitted to gamma ray irradiation, using a set of agronomical and molecular data (ISSR markers). The distance between the putative ‘Pacovan’ mutants varied from 0.26 to 0.64 with cophenetic correlation coefficient of 0.7669. Four mutants were selected based on best agronomical characteristics and height. This data also shows that there is variability that can be explored after the irradiation of ‘Pacovan’ banana mutants, which can be used in the genetic breeding program of banana aiming to develop short new varieties that also present good agronomic characteristics. This is the first attempt to use combined data in order to evaluate the genetic variability in putative banana mutants.     

The potential of high-resolution BAC-FISH in banana breeding

The genetic complexity in the genus Musa has been subject of study in many breeding programs worldwide. Parthenocarpy, female sterility, polyploidy in different cultivars and limited amount of genetic and genomic information make the production of new banana cultivars difficult and time consuming. In addition, it is known that part of the cultivars and related wild species in the genus contain numerous chromosomal rearrangements. In order to produce new cultivars more effectively breeders must better understand the genetic differences of the potential crossing parents for introgression hybridization, but extensive genetic information is lacking. As an alternative to achieve information on genetic collinearity we make use of modern chromosome map technology known as high-resolution fluorescent in situ hybridization (FISH). This article presents the technical aspects and applications of such a technology in Musa species. The technique deals with BAC clone positioning on pachytene chromosomes of Calcutta 4 (Musa acuminata ssp. burmanicoides, A genome group, section Eumusa) and M. velutina (section Rodochlamys). Pollen mother cells digestion with pectolytic enzymes and maceration with acetic acid were optimized for making cell spread preparations appropriate for FISH. As an example of this approach we chose BAC clones that contain markers to known resistance genes and hybridize them for establishing their relative positions on the two species. Technical challenges for adapting existing protocols to the banana cells are presented. We also discuss how this technique can be instrumental for validating collinearity between potential crossing parents and how the method can be helpful in future mapping initiatives, and how this method allows identification of chromosomal rearrangements between related Musa species and cultivars.     

香蕉遗传转化方法研究进展

香蕉是热带亚热带发展中国家重要的粮食作物和碳水化合物来源。但近年来,香蕉生产受到严重的病虫危害。大多数香蕉栽培品种是三倍体,生长周期长,而且不孕。由于没有种子,给繁殖和育种带来一定的困难。遗传转化技术的发展为香蕉品种的改良提供了一种有效的手段。香蕉的遗传转化方法有电激法、基因枪法、农杆菌介导法等。农杆菌介导法的应用是香蕉品种改良的一个重大突破。香蕉遗传转化的外植体也发展到多种,有原生质体,胚性细胞悬浮系,分生组织,以及横切薄片等。近几年,随着分子生物学的发展,出现了转化效率更高,重复性更好的香蕉遗传转化技术。如农杆菌和基因枪结合法,离心辅助农杆菌介导法、真空渗透技术等。这些新技术新方法的出现,必将推动香蕉产业高速发展。     

Identification of discriminant factors after treatment of resistant and susceptible banana leaves with Fusarium oxysporum f. sp. cubense culture filtrates

Among the most important crops in developing countries are banana and plantain. However, the production is threatened by increasingly virulent forms of Fusarium wilt, and therefore, intensive breeding programmes are being carried out worldwide. As conventional field studies of banana resistance to this disease are time‐consuming and destructive, an easy‐to‐do procedure was previously developed to differentiate field‐grown resistant and susceptible banana cultivars at leaf level. Such a procedure involved the in vitro treatment of fungal culture filtrates on to field‐grown adult leaves and the measurement of lesion areas 48 h later. The present report includes measurements of other indicators such as biochemical compounds. The cultivar ‘Gross Michel’ (susceptible) and cv. ‘FHIA‐01’ (resistant) leaves were treated with Fusarium oxysporum f. sp. cubense race 1 culture filtrates. Evaluations were performed 48 h after leaf treatment. Compared with culture medium‐treated leaves (control treatment), fungal metabolites produced leaf lesions, decreased freephenolic contents and increased protein levels in both cultivars. In ‘FHIA‐01’, the culture filtrate increased contents of cell wall‐linked phenolics and the pool of aldehydes (except malondialdehyde). Fungal metabolites did not cause variations in peroxidase activity, chlorophyll pigment contents or malondialdehyde level in any cultivar. The use of Fisher's linear discriminant analysis to differentiate resistant and susceptible banana cultivars in breeding programmes is also a novel aspect of this report. Such an estimation was performed from a data matrix that included the effects of the fungal metabolites (leaf lesion area and levels of free and cell wall‐linked phenolics, aldehydes, except malondialdehyde, and proteins) on banana leaves of seven cultivars (four susceptible and three resistant).     

DNA typing of hops (Humulus lupulus) through application of RAPD and microsatellite marker sequences converted to sequence tagged sites (STS)

Summary Both random amplified polymorphic DNA and microsatellite repeat sequences were investigated as DNA markers for distinguishing hop cultivars. Microsatellite sequences converted to STS markers proved to be most successful. The relative abundance of microsatellite repeat sequences in the hop genome varied depending on the sequence class. Of the repeat types investigated the dinucleotide repeats (GA)_n and (GT)_n are the most highly represented in the hop genome. Microsatellite repeat sequences in hops have been shown to be highly polymorphic and are very informatives as STS molecular markers. A DNA typing system using sequence-tagged microsatellite site markers has been developed which will not only be useful for hop cultivar identification but also marker assisted breeding and quality control purposes.     

Development of cultivar-specific DNA markers based on retrotransposon-based insertional polymorphism in Japanese pear

We developed retrotransposon-based insertional polymorphism (RBIP) markers based on the long terminal repeat (LTR) sequences of copia-like retrotransposon Ppcrt4 and flanking genome sequences, which were derived from 454 sequencing data from Japanese pear (Pyrus pyrifolia) ‘Hosui’. Out of 40 sequences including both LTR and flanking genome regions, we developed 22 RBIP markers and used them for DNA profiling of 80 pear cultivars: 64 Japanese, 10 Chinese (Pyrus ussuriensis) and 6 European (Pyrus communis). Three RBIP markers were enough to differentiate ‘Hosui’ from the other Japanese pear cultivars. The 22 RBIP markers could also distinguish 61 of the 64 Japanese pear cultivars. European pears showed almost no amplification of the 22 RBIP markers, which might suggest that retrotransposons had transposed during Asian pear evolution or reflect the genetic relationship between Asian and European pears. Sixteen of the RBIP markers could be positioned on a genetic linkage map of ‘Hosui’. The RBIP loci were distributed in 10 linkage groups, and some loci were very closely located within the same linkage group. The information obtained will be applicable to developing cultivar-specific RBIP marker sets in plants.     

Breeding banana (Musa spp.) for drought tolerance: A review

Drought is a major abiotic stress affecting banana production worldwide, leading to yield losses of up to 65%. Consequently, numerous efforts to understand and mitigate drought effects that include developing tolerant crop varieties are ongoing in several banana breeding programmes. The breeding efforts, however, have been greatly slowed down by inherent banana problems (polyploidy and male or female sterility) and complexity of drought tolerance (reportedly controlled by several genes). This review summarizes the pertinent research findings on water requirements of banana for its proper growth and productivity, symptoms of drought-sensitive varieties and field management strategies to cope with drought stress. The coping strategies deployed by resistant cultivars include high assimilation rates and water retention capacity as well as minor losses in leaf area and gaseous exchange. Reduced bunch weight, leaf chlorosis, wilting and strangled birth are underlined to be directly associated with drought susceptibility. Integration of conventional, molecular breeding and biotechnological tools as well as exploitation of the existing banana genetic diversity presents a huge opportunity for successful banana improvement.     

Application of degenerate oligonucleotide primed PCR (DOP-PCR) for SNP discovery in soybean

As the majority of methods for single nucleotide polymorphism (SNP) identification are highly cost-prohibitive, it is necessary to develop new strategies that are more suitable for small and medium-scale laboratories. In this paper, we investigate the potential of degenerate oligonucleotide primed PCR (DOP-PCR) for SNP discovery in soybean. PCR fragments were amplified from two soybean cultivars, ‘Pureunkong’ and ‘Jinpumkong 2,’ shotgun cloned and sequenced. The sequences of both cultivars were then assembled and examined for occurrence of SNPs. The effectiveness of SNP discovery was much lower than expected. Over 1,300 analyzed sequences were grouped in 144 contigs, but only 51 putative SNP sites were found in 18 of these contigs. About 50% of the contigs contained identical sequences and in more than one-third (35.4%), putative paralogous fragments were assembled. Subsequent validation of SNPs allowed the confirmation of only eight SNP sites. The failure to validate the remaining SNPs was mostly due to amplification of duplicated or multiplicated genomic regions. A relatively high proportion of chloroplast and mitochondrial DNA sequences was another limitation of effective SNP detection. Although the DOP-PCR technique was not efficient enough for SNP discovery because of the degree of soybean genome complication, the relatively large number of paralogous sequences in our data collection can be used for further detailed analysis of the genome structure of this species.     

栽培稻种内rDNA基因IGS序列分析及系统学意义

以普通野生稻为对照, 将rDNA基因间区(IGS)序列用于栽培稻种内不同亚种以及亚种内不同品种的亲缘关系分析。结果表明, 栽培稻种内IGS序列长度为2 130~2 145 bp, G+C含量为74.59%~75.29%, 变异位点229个, 占10.70%, 信息位点76个, 占3.51%。IGS序列中籼亚种和粳亚种之间有38个亚种标志性碱基差异, 主要分布在IGS 5′ 端近400 bp的序列中。用IGS序列构建的系统树能将栽培稻的籼亚种和粳亚种分为两大类, 亚种内不同亲缘关系的品种也能区分开。本研究结果支持爪哇稻为栽培稻中一个独立亚种的观点。     

Low temperature induced changes in activity and protein levels of the enzymes associated to conversion of starch to sucrose in banana fruit

Storage at low temperature is the most frequently used method to extend the shelf life of banana fruit, and is fundamental for extended storage and transport over long distances. However, storage and transport conditions must be carefully controlled because of the high susceptibility of many commercial cultivars to chilling injury. The physiological behavior of bananas at low temperatures has been studied to identify possible mechanisms of resistance to chilling injury. The aim of this work was to evaluate differences in the starch-to-sucrose metabolism of a less tolerant and susceptible (Musa acuminata, AAA cv. Nanicão) and a more tolerant (M. acuminata × Musa balbusiana, AAB, cv. Prata) banana cultivar to chilling injury. Fruits of these cultivars were stored in chambers at 13 °C for 15 d, at which point they were transferred to 19 °C, where they were left until complete ripening. The low temperature induced significant changes in the metabolism of starch and sucrose in comparison to fruit ripened only at 19 °C. The sucrose accumulation was slightly higher in cv. Prata, and different patterns of starch degradation, sucrose synthesis, activity and protein levels of the α- and β-amylases, starch phosphorylase, sucrose synthase and sucrose phosphate synthase were detected between the cultivars. Our results suggest that starch-to-sucrose metabolism is likely part of the mechanism for cold acclimation in banana fruit, and the cultivar-dependent differences contribute to their ability to tolerate cold temperatures.     

矮生香蕉品种特性及栽培技术研究

矮生香蕉品种可在南北方发展温室栽培,是优良的经济树种与观赏树种,研究矮生香蕉的品种特性与配套栽培技术,可为发展矮生香蕉的观赏与鲜食应用提供基础和指南。试验选取了矮生香蕉品种‘中蕉12 号’为材料,评估其枯萎病抗性,开展地培及盆栽试验研究其栽培特性,总结其温室栽培技术要点。试验结果表明,矮生香蕉‘中蕉12 号’株高仅为85.7~103.3 cm,树形矮小,树姿优美,生长期较短约为10 个月,与易感品种‘巴西香蕉’相比具较强枯萎病抗病性,产量适中（6.0~7.8 kg/株）,品质优良,果实品质媲美常规鲜食品种‘巴西香蕉’。矮生香蕉品种‘中蕉12 号’可鲜食与观赏两用,用于休闲观光以及生态农业建设,具有良好的应用前景,符合现代都市农业的发展需求。     

Use of culture-derived Fusarium oxysporum f. sp. cubense, race 1 filtrates for rapid and non-destructive in vitro differentiation between resistant and susceptible clones of field-grown banana

Banana and plantain are among the most important food crops in developing countries but production is threatened by increasing virulent forms of Fusarium oxysporum f. sp. cubense. Chemical control is not economically effective and,therefore, breeding programs are necessary. Traditional field studies of new genotype resistance to this disease are time-consuming and destructive. Therefore,we developed a rapid and non-destructive procedure to differentiate field-grown banana resistant from susceptible clones. This procedure implicates application of culture filtrates of Fusarium oxysporum f. sp. cubense race 1 onto banana leaves. The relationship between duration of the fungal in vitro incubation, and the fungal culture fresh mass, the culture filtrate absorbency, and the Gross Michel (susceptible cultivar)leaf lesion area (after application of the culture filtrate) were similar and at 24day-incubation the highest values of the recorded indicators were observed. A comparison between Gross Michel and FHIA-01(resistant) was also performed. The most relevant differences between cultivars were observed at 48 hours after application of the culture filtrate, and in the middle-aged leaves. The position of the culture filtrate application in the leaf limb (distal, middle, proximal) was not determinant. A wider comparison among banana cultivars confirmed previous results informed by other researchers using different systems to study this plant-fungus interaction. Such a confirmation validates the effectiveness of the procedure described here to select rapid and non-destructively banana resistance to this disease at field level. This revised version was published online in August 2006 with corrections to the Cover Date.