Interspecific hybridization in vanilla and molecular characterization of hybrids and selfed progenies using RAPD and AFLP markers

Vanilla, Vanilla planifolia Andrews, is native to Mexico and Central America, but is now cultivated in other parts of the tropics. Continuous clonal propagation has resulted in very little variability for crop improvement programmes in vanilla. In this study, an attempt has been made to increase the spectrum of variation by interspecific hybridization with Vanilla aphylla, an Indian species which is tolerant to Fusarium. Interspecific hybrids were successfully produced and morphological characters and molecular profiles revealed the true hybridity of the progenies. Ten seedling progenies of V. planifolia, and four interspecific hybrids obtained from crosses between V. planifolia (female) and V. aphylla (male) using a number of different loci as markers were evaluated and 319 amplified fragment length polymorphisms (AFLPs) and 83 random amplified polymorphic DNAs (RAPDs) loci were marked. The profiles indicate similarity between the parents, selfed progenies and interspecific hybrids and that all the progenies tested were variable when compared to each other, which can be exploited for crop improvement in vanilla. This is the first report in vanilla, indicating that RAPD and AFLP profiles coupled with morphological characters can be utilized to assess the variability and hybrid nature of genotypes and of successful interspecific hybridization and production of hybrids between V. planifolia and V. aphylla.     

Interspecific hybridization between three closely related ornamental Eucalyptus species: E. macrocarpa,E. youngiana and E. pyriformis

Summary

Interspecific hybridization between thre Eucalyptus species with horticultural merit, E. macrocarpa, E. pyriformis and E. youngiana, was undertaken to investigate the likelihood of success of such crosses. All combinations produced fertile seed, with interspecific crosses as successful as intraspecific. There were no differences between male species in fertility but there were differences between female trees within a species. Discriminant analysis of each cross indicated a high degree of interspecific hybridization for hybrid 166 seedlings, with all except two seedlings clustering separately from a mixture of selfed and outcrossed seedlings of either parent, when measured for a range of leaf and stem characters at three different nodes. The success of this programme in producing both large numbers of seed and large numbers of hybrids indicated that it is possible that these species could hybridize naturally if they were growing in the same location. It also demonstrated the potential of controlled pollination, between closely related species of similar floral morphology, and seedling-based hybrid recognition as a method to produce eucalypt hybrids for further evaluation for commercial horticulture.     

Reciprocal difference of interspecific hybridization between three different colours of Iris dichotoma and I. domestica

Previously, we found that reciprocal F₁ hybrids of purple-flowered Iris dichotoma and I. domestica were different. To further characterize this reciprocal difference, we carried out reciprocal crosses between three different colours of I. dichotoma and I. domestica. Morphological and cytological characters of F₁ hybrids of six interspecific crosses between three different colours of I. dichotoma and I. domestica (three direct and their three reciprocals) were evaluated. The results showed that the fruit-setting rates of three different colours of I. dichotoma as female and I. domestica as male were lower than reciprocal cross combinations, but their seed germination rates were significantly higher than reciprocal ones. Reciprocal differences of F₁ hybrids were observed for most morphological characters. The F₁ hybrids of three different colours of I. dichotoma as female were totally different from their parents; however, the F₁ hybrids of I. domestica as female were extremely similar to the mother. The six interspecific hybrids had the same chromosome number (2n = 32) as their parents. Reciprocal differences were detected for karyotype. Parents’ and F₁ hybrids’ karyotypes focused on 1A to 2B. In these cases, the direction of the cross must be kept in mind in order to improve the efficacy of future breeding programmes.     

Identification of parental species of the Alstroemeria cv. ‘Jubilee’ using AFLP marker technique

Twelve Alstroemeria species, two hybrids, one cv. ‘Jubilee’, an anther-cultured plant from cultivar ‘Jubilee,’ and Bomarea salsilla and Leontochir ovallei (the latter two were chosen as outgroup) were evaluated using the AFLP marker technique in order to identify putative parental genotypes of the Alstroemeria cv. ‘Jubilee’ and of known interspecific hybrids, and to estimate their genetic relationships within the genus Alstroemeria. A total of 297 AFLP markers were scored by using the primer combination (E + ACCA/M + CTAG). In order to discriminate all Alstroemeria genotypes, cluster analysis (UPGMA) and principal coordinates analysis were performed. The Alstroemeria cv. ‘Jubilee’, of which the parents are unknown, had genetic distance (GD) 0.54 from Alstroemeria exserens, GD 0.57 from Alstroemeria garaventae, GD 0.62 from Alstroemeria gayana, and GD 0.66 from Alstroemeria hookeri cumminghiana. Thus, these four species are considered as putative parental genotypes. An interspecific hybrid (Alstroemeria aurea × Alstroemeria inodora), showed the smallest genetic distance from A. aurea (GD 0.56) and A. inodora (GD 0.45). The Alstroemeria ligtu group was distantly allocated from other Chilean species. We conclude that the AFLP marker technique appears to be a satisfactory tool for identifying the parental genotypes of interspecific hybrids in Alstroemeria.     

A PCR-based assessment of genetic diversity,and parentage analysis among commercial mango cultivars and hybrids

Summary

Three different PCR methods [Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR), and Directed Amplification of Minisatellite DNA (DAMD)] were used to analyse genetic diversity and parentage among 20 mango cultivars, including 18 landraces and two hybrids (‘Amrapali’ and ‘Mallika’). These hybrids together with a third hybrid (‘Ratna’), and an out-group species (Mangifera sylvatica) were also analysed for parentage. Fifteen, seven and four primers were used to amplify a total of 158, 69 and 59 distinct DNA fragments by RAPD, ISSR and DAMD, respectively. Of these, approx. 85%, 64% and 90% were polymorphic, respectively. Genetic distances between pairs of mango cultivars were measured separately by each method and depicted graphically as a Neighbor Joining (NJ) tree. The three methods revealed different groupings of cultivars and hybrids. A NJ tree based on the cumulative data from all methods correlated well with the parentage of the mango hybrids, and the grouping of cultivars on a regional basis. Genetic markers likely to be associated with important agronomic traits were identified by further analysing the hybrids, with their respective parents, using all three methods. On the basis of the highest number of polymorphic bands observed (90%), DAMD was judged to be the best method with which to analyse mango germplasm.     

Morphological and molecular analyses of Rosa damascena × R. bourboniana interspecific hybrids

Rosa damascena Mill is the most important scented rose species cultivated for rose oil production. Rosa bourboniana L. (Edward rose), a related species, is popular on account of its longer blooming period and ease of propagation. With an aim to combine the oil quality of R. damascena and recurrent flowering habit of R. bourboniana, two cultivars (Jwala and Himroz) of R. damascena were crossed with R. bourboniana. The F1 hybrids obtained were evaluated using morphological, random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers. Twenty-two selected RAPD and three SSR primer pairs were utilized for hybrid identification. According to presence or absence of bands RAPD and SSR markers were classified into seven types of markers. The bands specific for the pollen parent and occurring in the hybrids were good markers to confirm the hybridity. The non-parental bands expressing uniquely in hybrids were effective in distinguishing the hybrids from each other. Cluster analysis, based on Jaccard's similarity coefficient using unweighted pair group method based on arithmetic mean (UPGMA), reliably discriminated the hybrids into two main clusters. These results indicate the practical usefulness of RAPD and SSR markers in hybrid identification in scented roses. The approach is advantageous for its rapidity and simplicity, for identification of hybrids at the juvenile stage. One of the studied morphological traits – prickle density, can also complement in the identification of interspecific hybrids between R. damscena (♀) and R. bourboniana (♂).     

In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum × Vaccinium ashei) with colchicine treatment

Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent.     

Flowering ability of commercial Calibrachoa cultivars as compared to that of natural species

To evaluate and describe the current status of Calibrachoa cultivars as bedding plants, the seasonal flowering of 91 commercial cultivars were compared to those of natural species using principal components analysis. The predominant flowering of the cultivars, that is, floriferous in late spring to early summer with a tendency to reduce flower production toward winter, seems to have remained unchanged from that of natural species. However, properties desired in bedding plants, such as early flowering and continuous flowering under short-day and/or low temperature conditions, appear to have been improved in a considerable number of cultivars, although there is still room for improvement. Seasonal and total floral counts were higher in many cultivars compared to natural species. Several natural species were selected for their unique flowering ability and should be evaluated as gene resources for improving the flowering of Calibrachoa cultivars.     

Hybridization among Epimedium (Berberidaceae) species native to China

The hybridization of fourteen plant populations belonging to seven Epimedium species native to China was studied by self-cross, infra-population cross, inter-population cross within species, and interspecific cross. Self-pollination studies on nine populations indicated high incompatibility; capsule-set rates were higher than zero in only three (4.61–6.76%). Interspecific cross-pollinations demonstrated high crossabilities in most cross combination (15.38–92.44% capsule-set rate), and the F₁ seeds possessed high germination rates (>20%). The F₁ hybrids of three interspecific cross combinations were raised to maturity. The morphology, karyomorphology of somatic cells, and pollen mother cell (PMC) meiosis of these F₁ plants revealed that they were all highly fertile (>76.10% in pollen viability), that there were few structural differences in chromosomes among species, and that most PMCs had 6 bivalents at MI. Abnormal chromosomal behaviors occurred in a minority, including chromosome bridges, unequal segregation of chromosome number, lagging chromosomes, and micronuclei. A series of experimental crosses provided strong evidence for an outbreeding system and a weak internal barrier to hybridizations in the taxa studied.     

Aneuploidy in the progenies of inter-specific hybrid (AAAB genome) tetraploid potatoes

Summary

Cultivated potatoes (Solanum tuberosum L.) are autotetraploid (AAAA). Wild allotetraploid (AABB) relatives that share the A genome with cultivated potatoes are important for potato breeding; however, it is difficult to perform direct crossings between these two groups due to a post-zygotic crossing barrier called the endosperm balance number (EBN). In this study, the progenies of three exceptional sesquiploid (AAAB) hybrids (stbr₁, srbr₂ and stbr₃) from S. stoloniferum (sto) × S. tuberosum (tbr) were analysed. Five progenies, including selfed stbr, stbr₁ × sto, stbr₁ × tbr, stbr₂ × tbr, and stbr₃ × tbr were investigated in detail in terms of their somatic chromosome numbers. As expected, based on the genomic formula of hybrids, the incidence of aneuploidy was high in the progenies. However, most were aneuploid, with only one or two extra or deleted chromosomes. The results are discussed based on EBN theory.     

Studies on genetic variation in cherry germplasm using RAPD analysis

Random amplified polymorphic DNA (RAPD) variation among eight cherry species and two interspecific progenies were analyzed. Out of 130, 48 arbitrary oligonucleotide primers were screened for PCR amplification to generate polymorphisms. The phylogenetic analysis was carried out using two distance-matrix methods and a dendrogram was generated to show the relationships among species and cultivars. The results showed that there were 840 amplified loci in total; 23 sweet cherry and four sour cherry cultivars were clustered together with 569 and 247 polymorphic loci respectively which accounted for 67.74% and 29.40% of the total variation. Prunus tomentosa T., Prunus fruticosa var. aucta P. and Prunus humilis B. formed a monophyletic group. A relationship between Prunus pseudocerasus L. and Colt, which formed another closely related group, was observed while Prunus avium L., Prunus cerasus L. and other cherry species were more divergent. The range of genetic distance was from 0.0623 to 0.2719 among the Prunus species, which were genetically distinct. The topology of the tree was generally in agreement with taxonomic classification. The results indicated that with the exception of the sweet cherry variety “Hongdeng”, there were one or more cultivar-specific RAPD markers in cherry species and cultivars. Using these specific markers, cherry species and varieties could be identified and there is therefore the potential to select for good characteristics of hybrids at an early stage.     

Genetics of resistant to tomato yellow leaf curl virus in tomato

The genetics of resistance to tomato yellow leaf curl virus (TYLCV) were studied in TYLCV-resistant lines developed by crossing wild species of Lycopersicon pimpinellifolium, L. hirsutum and L. peruvianum resistant to TYLCV with susceptible L. esculentum cv. Special Back. Crosses between TYLCV-resistant lines derived from the same wild species produced progenies similar to their parents in their level of resistance. However, progenies from interspecific crosses showed greater resistance than either parent suggesting that the genes for TYLCV resistance contributed by different wild species are probably not the same (non-allelic). The gene action for TYLCV resistance also varied with the source of resistance. Analysis of F₁, F₂ and BC populations for lines derived from L. pimpinellifolium showed that resistance to TYLCV in these lines is a quantitative trait with some dominance.     

Genetic characterization of asparagus doubled haploids collection and wild relatives

The genus Asparagus is very large consisting of around 150 species found as herbaceous perennials, tender woody shrubs and vines. The cultivated species (Asparagus officinalis L., diploid) is a highly prized vegetable, grown in different environments ranging from cool temperate zones to deserts, Mediterranean climates and tropical areas. As a consequence, Asparagus breeders have developed different cultivars that differ for their morpho-agronomic traits, habit and ploidic status (few triploid and tetraploid cultivars are used). Several breeding methods are used for developing cultivars, among which a well developed in vitro anther culture technique produces homozygous clones useful for F₁ hybrids constitution. A fluorescent based AFLP (amplified fragment length polymorphism) technique were applied with the aim to assess genetic diversity among a collection of 173 doubled haploid (DH) androgenetic clones, five Asparagus wild species and interspecific hybrids obtained among the cultivated species and two wild relatives. The average number of AFLP fragments generated per primer set was 105, varying in size from 50 to 550 bp. A total of 1054 AFLP fragments were detected, 20% of which were polymorphic. Genetic similarity based on DNA polymorphisms, showed that a few number of AFLP primer combinations are able to distinguish the cultivated DH clones from the wild species. Indeed, from one DH clone for each anther donors and the wild species were used to construct a dendrogram using Dice's coefficient and the unweighted pair group method with the arithmetic mean (UPGMA). Genetic distances among all DH clones were calculated using the C.S. Chord distance; and a neighbour-joining (NJ) consensus tree was constructed in order to support the breeder for parental genotype choice for asparagus hybrid constitution.     

毛百合×有斑百合种间杂种的育成

以毛百合(Lilium dauricum)为母本、有斑百合(L.concolor var.prlchellum)为父本进行常规杂交,通过胚培养成功获得了种间远缘杂交种,对种问杂交亲和性、胚培养和杂种的形态特征进行了调查研究.结果表明:毛百合×有斑百合蒴果膨大率达46.1%,种子有胚率为1.30%,胚培养萌发率为15.2%;4个杂交后代生长旺盛,株高表现出较明显的超亲现象,其他性状呈趋中趋势,杂种植株的花色更偏向橙色.4个杂交后代虽然在花色上与亲本有一定差异,但其它性状均具有双亲的特征,从形态上能证明是真杂种.     

东北草莓×凤梨草莓种间杂种一代的细胞遗传学观察与RAPD分析

以利用我国原产草莓二倍体野生种东北草莓与八倍体栽培种凤梨草莓栽培品种‘圣星’杂交, 通过胚拯救获得的种间杂种为试材, 经体细胞鉴定种间杂交后代为五倍体, 育性差。叶柄茸毛和花梗茸毛与野生种母本东北草莓相近; 花序着生高度部分与母本一致, 部分与父本一致, 各占一半; 植株高度多数介于双亲之间, 趋向于栽培品种。杂交F1代的花粉母细胞减数分裂观察表明, 染色体构型为6.1Ⅰ +10.6Ⅱ + 2.1Ⅲ + 0.35Ⅳ, 后期出现少量畸形多分孢子。RAPD分析表明, 杂种后代具有亲本扩增片段, 后代个体间存在遗传异质性。     

黄瓜栽培种、近缘野生种、种间杂种及其回交后代的RAPD 分析

 黄瓜栽培种、黄瓜野生变种、近缘野生种、种间杂交种及其与黄瓜回交自交的后代以及甜瓜为试材, 采用RAPD 方法探讨其亲缘关系。对23 份试材进行扩增, 筛选出31 条随机引物的扩增产物进行结果分析, 共产生375 个位点, 具多态性位点占90. 0 % , 平均每条扩增出6. 3 条。通过UPGMA 聚类分析,以遗传距离0. 37 为阀值, 可将23 份试材归为黄瓜、近缘野生种、种间杂交种及甜瓜亚属种4 类。     

Current status of commercial Calibrachoa cultivars as assessed by morphology and other traits

To study the morphological diversity of the popular ornamental crop Calibrachoa, traded since the 1990s, floral and vegetative characters of 91 commercial cultivars were compared to those of natural species. Floral colours were classified according to the ISCC-NBS system and the CIE LAB system. The numerical data obtained by measuring 19 floral and vegetative characters was tested with principal component (PC) analysis. Floral colour classes of commercial cultivars were divided into 37 groups according the ISCC-NBS system, whereas those of natural species were divided into 9 groups. These tendencies were readily observable in the scatter diagrams of the a* vs. b* values and the C* vs. L* values. As the result of PC analysis, six PCs were obtained. For PC4, which negatively correlated with the degree of nocturnal corolla limb closure, the majority of natural species showed negative scores whereas the majority of cultivars had positive PC4 scores. In terms of PC2 (flower size), PC3 (growth form), PC5 (floral shape) and PC6 (leaf shape), natural species were dispersed in a range wider than the cultivars. These results suggest that Calibrachoa cultivars possess more highly variable floral colour than natural species, and breeding programmes to diversify the floral colour seem to have progressed rapidly. The tendency of natural species to close the corolla limb toward evening has been removed almost completely from the cultivars. The floral size, growth form, floral shape and leaf shape of the cultivars were rather uniform compared to those of natural species.     

LA杂交系百合特征花香释放规律及关键基因研究

使用顶空固相微萃取（HS–SPME）-气相色谱-质谱（GC–MS）联用技术和实时荧光定量技术,分析LA杂交系百合（Lilium longiflorum × Asiatic hybrids）花香释放代谢途径的生物化学规律和遗传特征。对比LA系百合、麝香百合（Longiflorum hybrids）、亚洲百合（Asiatic hybrids）、OT系百合（Oriential hybrids × Trumpet hybrids）4个杂交系的5个品种挥发物释放差异;利用同源克隆从LA百合中克隆得到3个甲羟戊酸合成途径（MVA）中的关键基因。结果表明：5个百合品种花香成分释放存在显著差异,萜烯类化合物的释放规律与香气程度基本一致。LA百合‘爱神’在初开期花香释放达到顶峰,随后逐渐降低;花瓣的香味释放量最大。不同杂交系百合MVA途径合成酶基因（HMGR、MVD、FPS、TPS）的表达模式不同,关键基因的表达量影响最终产物的生成。在LA百合‘爱神’中克隆得到LlaTPS,在开花进程中,LlaTPS表达趋势与萜烯类化合物释放规律相似,表明其是控制萜烯类化合物挥发量的关键基因。LlaTPS cDNA全长1 799 bp,具开放阅读框1 764 bp,共编码587个氨基酸。系统进化树分析得出LlaTPS与同属百合‘Siberia’亲缘关系最近,属于TPSb亚家族。克隆得到合成倍半萜前提物质FPP的关键基因--法尼基焦磷酸合酶基因LlaFPPS,cDNA全长1 072 bp,开放阅读框1 056 bp,共编码351个氨基酸;其编码蛋白与单子叶植物FPPS蛋白亲缘关系较近。克隆得到焦磷酸甲羟戊酸脱羧酶（MVD）基因LlaMVD（KX034060）,cDNA全长1 439 bp,开放阅读框1 275 bp,共编码424个氨基酸;百合MVD与油棕（Elaeis guineensis）、短花药野生稻（Oryza brachyantha）等单子叶植物亲缘关系较近。     

Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (Brassica oleracea var. acephala) to produce hybrids with the aid of ovule culture

The objective of this study was to produce interspecific hybrids between an Ogura-cytoplasmic male sterile (CMS) line of zicaitai (Brassica campestris var. purupurea, 2n = 20) and cultivars of ornamental kale (Brassica oleracea var. acephala, 2n = 18) to develop a CMS system for hybrid seed production. Pollination with pollen grains of ornamental kales irradiated at a power output of 9.0 mW with a He–Ne laser for 3 min could overcome the cross-incompatibility between the species concerned. Intact hybrids could be efficiently produced from ovules cultured on Murashige and Skoog media supplemented by 0.2 mg l⁻¹ 6-benzyladenine. Chromosome number of hybrids was confirmed to be 2n = 19. Hybrids resembled ornamental kales in leaf morphology and in vernalization response. Pollens of hybrids had a sterile appearance. Moreover the hybridity of the putative hybrids was confirmed by RAPD data on a DNA fragment of 820 bp.