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.     

Levels of host plant resistance to banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), in African Musa germplasm

Forty-five Musa clones, including endemic and introduced cultivars plus hybrids, were evaluated for resistance against the banana weevil, Cosmopolites sordidus, in a field trial in Uganda. The predominant groups of staple crops, East African highland bananas (Musa spp. AAA) and plantains (Musaspp. AAB), as well as plantain-derived hybrids (AAB × AA), showed the highest levels of susceptibility to this pest. These were followed by dessert bananas (Musa spp. AAA), exotic bananas (Musa spp. ABB) and finally diploids of M. acuminata (AA). Hybrids of banana origin were highly resistant. Some East African highland cultivars, especially brewing types (e.g., Kabula, Bagandeseza, Ediirira), showed intermediate levels of resistance. Among the non-highland bananas, high levels of resistance were observed in Yangambi-Km5 (AAA), Cavendish (AAA), Gros Michel (AAA), Kayinja (ABB, Pisang Awak subgroup), Ndiizi (AB, Ney Poovan subgroup)and Kisubi (Ney Poovan subgroup). The highest resistance was observed in banana hybrids TMB2×7197-2, TMB2×8075-7 and the wild banana Calcutta-4 (AA). These were considered the best sources of resistance for a weevil resistance-breeding programme with the two hybrids commonly used as improved male parents. 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.     

Genetic relationships among cultivated bananas and plantains from Asia and the Pacific

Summary Isozyme variation was studied to determine genetic relationships among 563 accessions of Musa, including diploid (AA and BB), triploid (AAA, AAB, and ABB), and a few tetraploid (ABBB) clones from Asia and the Pacific. Several open-pollinated seedling progenies of wild, diploid M. acuminata and M. balbisiana were also studied. Cryogenic preservation of leaf tissue in liquid nitrogen allowed sampling of a wide array of germplasm from Papua New Guinea and several Pacific Islands without transporting propagules which are subjected to quarantine regulations. Electrophoretic variation was recorded in three enzyme systems, MDH, PGI and PGM. In total, 52 distinct electromorphs were identified among 192 different isozyme phenotypes (zymotypes). Multivariate analyses of the data clearly differentiated the major genome groups and revealed patterns of association within groups. The isozyme data suggest that the genes contributed by the M. acuminata genome to the triploid Pacific plantain AAB subgroup are similar to those of the acuminata/banksii complex of Papua New Guinea. It is likely that the Pacific plantain subgroup, including the Hawaiian Maoli, Pp'ulu and Iholena cultivars, originated in Papua New Guinea/Melanesia, rather than in Asia or the Malay Archipelago.Journal Series no. 3783 of the Hawaii Institute of Tropical Agriculture and Human Resources.     

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.     

Inter-Alu PCR like genomic profiling in banana

Alu sequences are a major repetitive element of the primate genome. To date Alu sequences have seldom been reported in plant genomes. We report here an inter-Alu PCR like genomic profiling in banana using a single primer designed from a human Alu sequence. The different Musa species involved in the complex genome of banana cultivars can thus be discriminated. The use of this technique for monitoring germplasm or genotyping cultivars is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation of bunch orientation in plantain and banana hybrids

Bunch orientation is an economically important trait in plantain (Musa spp. AAB group) and banana (Musa spp. AAA or ABB groups). Pendulous bunches are more symmetrical than subhorizontal, horizontal, or erect bunches and are, therefore, better adapted for transportation. Erect, horizontal, subhorizontal, and pendulous bunches were observed in segregating populations derived from crosses between plantains and bananas and among bananas. A proposed genetic model controlling this trait was tested in four populations. The true breeding diploid banana line, 'Calcutta 4' shows a pendulous bunch and has recessive alleles at three bunch orientation loci. The diploid banana cultivar 'Pisang lilin', which is heterozygous for the three loci, has a subhorizontal bunch. The triploid AAB plantain cultivar 'Bobby Tannap' has two simplex and one duplex loci, which also results in subhorizontal bunch orientation. The other plantain cultivar, 'Obino l'Ewai', which is simplex for two loci and nulliplex for the third, has a pendulous bunch due to dosage effects at the triploid level of the recessive alleles at simplex loci. Two tetraploid hybrids (TMPx 582-4 and 1187-8) have a subhorizontal bunch because of its duplex genotype for two loci and simplex genotype for the other locus. Bunch orientation might be an oligogenic trait regulated by the epistatic effects of at least three dominant loci. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Detecting genetic diversity in diploid bananas using PCR and primers from a highly repetitive DNA sequence

Summary The polymerase chain reaction (PCR) was used to detect polymorphisms among 29 diploid clones of Musa acuminata Colla. from Papua New Guinea. Primer sequences were derived from a 520 bp highly repetitive DNA sequence isolated from M. acuminata ssp. malaccensis. Primers, used individually, detected a total of 48 polymorphisms that were scored as unit characters and used to generate a Jaccard's similarity index. Principal coordinate analysis (PCO) was used to cluster clones and the unweighted paired-group method of analysis (UPGMA) was used to compute genetic distance among the materials examined. The abundance of diversity within the PNG diploids examined reflects the extreme genetic variability within the M. acuminata gene pool. PCR, utilizing primers from a highly repetitive sequence, is a rapid means of detecting genetic diversity in M. acuminata.     

Physiological and biochemical changes during banana ripening and finger drop

Fruit drop of banana is due to breaking at the junction of the pedicel and pulp, and we found no true abscission zone. The breakage seems therefore due to weakening of the peel. We investigated pectin hydrolysis and some properties at the rupture zone, using ‘Hom Thong’ (Musa acuminata, AAA Group) and ‘Namwa’ (Musa x paradisiaca, ABB Group) fruit, which show massive drop and no drop, respectively. During the period of finger drop, the water content of the peel in ‘Namwa’ was similar to that of ‘Hom Thong’ and thus water content does not account for the high breakage in ‘Hom Thong’. The peel thickness at the rupture area in the two cultivars was not significantly different. During the period of finger drop, the level of water-soluble pectin in the peel at the rupture area of ‘Hom Thong’ was higher than that of ‘Namwa’, indicating pectin degradation. CDTA soluble pectin and insoluble pectin was lower in ‘Hom Thong’, also indicating more pectin breakdown in this cultivar. Polygalacturonase activity in the peel at the rupture area of ‘Hom Thong’ bananas rapidly increased, but not clearly more than in ‘Namwa’ bananas. Pectinesterase activity in the peel at the rupture area of ‘Hom Thong’ was much lower than that of ‘Namwa’ bananas, and thus does not account for the breakage. Pectate lyase in the peel of ‘Hom Thong’ was considerably higher than in that of ‘Namwa’. The present data indicate that the much higher pectate lyase activity in ‘Hom Thong’ might be responsible, at least partially, for the finger drop.     

Leaf stomatal conductance and stomatal morphology of Musa germplasm

Drought tolerance combined with resistance to black sigatoka (BS) disease are two desirable traits for plantains in some parts of the tropics. Field evaluation of leaf stomatal conductance, single leaf transpiration rate, photochemical efficiency, and role of stomata in BS resistance, was done for 18 genotypes of Musa spp., which included diploid and triploid banana and plantains and tetraploid plantain hybrids, at a sub-humid and a humid site. Conductances were significantly (P ≤ 0.01) higher for leaf abaxial than adaxial surfaces (S), while clonal (C) differences were significant (P ≤ 0.05). Interactions between sampling period (T: morning vs afternoon) and location (L), and clone and location (C × L) were significant for both conductance and transpiration; (C × T × L) interaction was significant for conductance. Significant interactions for (C × L) and (C × T) suggested that conductance and transpiration of a specific clone need to be considered for a given environment. Total variation in conductance was explained by the following variables: S, C, (C × L), (C × T), and (C × L × T). In general, ABB cooking banana had higher conductances than other Musa taxonomic groups (AAB, AA, AAA, and AAAB). Evaluation of morphological and physiological traits of leaf stomata indicated that the resistance to BS disease is due to non-stomatal mechanisms. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Race 1 fusarium wilt tolerance on banana plants selected by fusaric acid

Summary The selection of tolerant variants to race 1 fusarium wilt of banana was carried out through the effects of fusaric acid onin vitro banana multiple bud clumps (MBCs). The MBCs of Maçã cultivar (Musa sp., AAB, Silk), which is susceptible to the race 1 fusarium wilt, were used. And, Nanicão cultivar (AAA, Cavendish subgroup) was used in the tolerant tests as control of a disease tolerant variety. Firstly, to aim at determining an appropriate concentration of the toxin for the tolerant selection, the MBCs were cultured on the Modified Murashige & Skoog medium supplemented with 0.05 to 1.6 mM fusaric acid. The growth on both cultivars was completely inhibited on the medium containing 0.1 mM fusaric acid.Tolerant variants of Maçã were, then, selected with 0.1 mM fusaric acid after chemical mutagen treatment. The results showed an increased tolerance of the selected Maçã plants to the race 1 fungus in greenhouse tests. In vitro selection by fusaric acid is a very useful method for obtaining fusarium disease tolerance, although the tolerance mechanism of the selected plants may be different from that of existing tolerant cultivars.     

Nematode resistance in bananas: screening results on some new Mycosphaerella resistant banana hybrids

Banana hybrids with resistance to Yellow Sigatoka and Black Leaf Streak disease were evaluated for resistance to the burrowing nematode Radopholus similis and to the lesion nematode Pratylenchus coffeae in a growth chamber at 24–28°. Plants produced by tissue culture were acclimatised for 6 weeks prior to inoculation. Forty-five days after inoculation with nematodes, the root systems were processed and nematode numbers assessed. Two cultivars of Grande Naine (Musa AAA, Cavendish subgroup, ITC1256 and cv902) and one cultivar of Yangambi Km5 (Musa AAA, Ibota subgroup, ITC1123) were used respectively as susceptible and resistant controls. Results based on multiplication rates and root infestations showed that three of these hybrids (FB918, FB919 and FB924) were not significantly different from the resistant control Yangambi Km5 with a lower multiplication of R. similis. Similarly four of these hybrids (FB918, FB919, FB920, FB924) showed a lower multiplication of P. coffeae, not significantly different from the same resistant control. This is the first study that shows a partial resistance to both nematode species, R. similis and P. coffeae within synthetic hybrids of M. acuminata, adding an important extra value to these dessert banana hybrids formerly bred to resist to Mycosphaerella leaf spot diseases.     

Genome-specific markers of tetraploid wheats and their putative diploid progenitor species

The objective of this study was to isolate genome‐specific markers from the genomes of tetraploid wheats and the putative donor diploid species on the basis of random amplified polymorphic DNA analysis followed by cross‐hybridization. Twenty different Triticum and Aegilops species and accessions were analysed by polymerase chain reaction (PCR) using 30 random primers. The polymorphic PCR fragments were then isolated, labelled and used in cross‐hybridization screenings. The hybridization results established that one marker was specific to the Ae. speltoides S genome, two to the A genome, one to the B genome and five to the G genomes of polyploid species (and to the genomes of the corresponding progenitor species). Four markers were identified that were specific to both the B and G genomes. Analysis of the Triticum and Aegilops species and accessions supported the notion that Ae. speltoides is more closely related to the B and G genomes of polyploid wheat species than were other members of the Sitopsis section. The data also indicated that the B and G genomes had originated from different accessions of Ae. speltoides.     

利用SNP分型技术鉴别油菜FLC1与FLC3基因的A、C组基因型

甘蓝犁油菜是由白菜型油菜(A组基因型)与甘蓝(C组基因型)杂交后自然加倍的异源四倍体芸薹属植物.其中含有A组与C组两种基因型的染色体,而FLC1与FLC3是A、C组中共同拥有的FLC基因.通过序列比对,分别寻找出A、C组基因中FLC1与FLC3基因的特异性SNP位点,并设计了3对SNP特异性引物对.采用特异性引物延伸法,分别对来自白菜型油菜、甘蓝以及甘蓝型油菜的不同材料进行检测.利用这3对特异性SNP引物对,成功地对油菜中A、C组的FLC1与FLC3基因进行了有效的分型,为研究甘蓝型油菜中A、C组来源的FLC1与FLC3表达奠定了基础.     

Discovery of single nucleotide polymorphism in <Emphasis Type="Italic">Capsicum</Emphasis> and SNP markers for cultivar identification

Molecular markers based on single nucleotide polymorphisms (SNPs) are abundant and evenly distributed in a whole genome enough to distinguish individuals in a population. In recent years, sets of SNP markers have been designed and applied for cultivar identification in various crop species. This paper is the first to report the development of a panel of SNP markers for variety identification in peppers. We used conserved ortholog set II (COSII) markers developed from conserved unigenes between tomato and Arabidopsis to identify SNPs in peppers. We tested 438 COSII primer sets amplified as single PCR products out of a total 600 COSII primer sets. Among the 438 COSII primers, 170 primer sets (38.8%) showed polymorphisms between Capsicum annuum ‘RNaky (RN)’and C. chinense ‘PI 159234 (234)’. In contrast, only 48 primer sets (11.0%) out of 438 primers sets were polymorphic between C. annuum ‘Perennial (PER), and ‘Dempsey (DEMP)’. The average frequency of SNPs plus InDels between C. annuum and C. chinense was 1/189 bp and between C. annuum spp. was 1/948 bp. Primer sets showing SNP between C. annuum PER and DEMP were re-designed to Allele Specific PCR (AS-PCR) primers and we finally selected a total of 40 SNP markers for cultivar identification. As the result, we were able to discriminate 97.5% of the 81 commercial hot cultivars and 100% of the 17 sweet pepper cultivars. We conclude the paper by discussing the use of the SNP marker set for cultivar identification and other applications.     

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.     

Duplication of the Bx7 high-molecular-weight glutenin subunit gene in bread wheat (Triticum aestivum L.) cultivar'Red River 68'

SDS-PAGE analysis of seed proteins of the cultivar‘Red River 68’showed a considerably higher staining intensity of the band corresponding to HMW-GS Bx7 relative to the equivalent band in the cultivars‘Chinese Spring’and‘Cheyenne’. Southern blots of restriction enzyme fragments from DNA of these three cultivars were analyzed densitometrically to reveal that the band corresponding to the Bx7 gene of‘Red River 68’had a double staining intensity compared to the equivalent bands from the other two cultivars, which indicates that in‘Red River 68’a duplication of the Bx7 gene has occurred. Although the possibility of the gene copy being a pseudogene was not ruled out, the greater amount of protein corresponding to Bx7 in‘Red River 68’most likely is in accord with an increase in active gene number. SDSPAGE analysis of the proteins showed also that the mobility of Bx7 in‘Cheyenne’was slightly different from the mobilities of the Bx7 subunits of‘Red River 68’and‘Chinese Spring’. The same difference was observed at the gene level by PCR amplification of the genes encoding these subunits.