The Application of Fluorescence In Situ Hybridization in Different Ploidy Levels Cross-Breeding of Lily

21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid ‘Freya’ had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true.     

Characterization of B chromosomes in Lilium hybrids through GISH and FISH

Supernumerary (B) chromosomes and small aberrant chromosomes were detected in Lilium hybrids and characterized through genomic in situ hybridization (GISH) and florescence in situ hybridization (FISH). Two small, supernumerary or B chromosomes were detected as extra chromosomes in a tetraploid plant derived from chromosome doubling of a hybrid (2n = 2x = 24) between a cultivar of the Longiflorum (L) and the Trumpet (T) group. When this tetraploid LLTT hybrid was crossed with a triploid LLO hybrid (O = Oriental), the B chromosome was transmitted to 73.4 % of the progenies. Based on GISH and FISH characterization, it was shown that the B chromosome consisted of two identical arms, with 5S rDNA hybridizing to the majority of it, which were flanked by normal telomeres, suggesting that this is an isochromosome. In another population, which is a backcross progeny between a F1 hybrid of Longiflorum × Asiatic (LA) and its Asiatic parent, the former produced functional 2n gametes which resulted in a triploid LAA progeny (2n = 3x = 36), in which three exceptional plants possessed 35 normal chromosomes and a small aberrant chromosome instead of the expected normal number of 36. In all three cases, the small aberrant chromosomes were isochromosomes which had obviously originated during the first backcross generation. These three chromosomes showed normal telomeres and mitosis. In addition, one of the new generated chromosomes possessed two 45S rDNA sites in the proximal positions. These new arisen isochromosomes were proposed to originate from centric breakage and fusion of two short arms of the missing chromosome in three genotypes, respectively, based on the comparison of arm lengths as well as rDNA loci. Their relevance to the origin of Bs is discussed.     

送春与多花兰种间杂交后代的ISSR分析

该文使用简单重复序列间( ISSR)分子标记,对送春与多花兰种间杂交后代进行了研究。结果表明：从80个ISSR引物中筛选出14个扩增效果稳定的ISSR引物,对两亲本和59个F1代个体进行了ISSR扩增,得到107个扩增位点,扩增的片段大小位于90~2100 bp之间,平均每个引物扩增7.64条条带,得到11种类型的带。 ISSR标记在送春×多花兰的F1代中表现出一定的多态性,分离频率为44.86％,分离位点有83.33％符合孟德尔1︰1或3︰1的分离规律,产生偏孟德尔分离的位点占12.50％,余下的4.17％属于特殊分离带型。可能导致后代变异的位点为偏孟德尔分离的6条带、缺失的8条带或新生成的2条带。聚类图中父本和母本与F1代个体间的遗传距离较远,59个杂交后代先聚集成一组,再同母本相聚为一组,最后才同父本聚在一起,59个杂种均偏母本型。送春与多花兰的杂交后代在植株形态、染色体、遗传物质方面都具备双亲特点,61个个体间的ISSR分子量标记结果和植株形态学特征都说明,59个F1代杂种包含送春和多花兰的遗传特性是真杂种;F1代杂种既有双亲的互补特征带,又有双亲的重组片断即产生新的特异带,这说明送春与多花兰的杂交后代具有遗传变异的特点。该研究结果可以有效地对杂交后代进行定向选择,为兰花的杂交育种提供了分子依据。     

Tracing B-genome chromatin in Brassica napus x B. juncea interspecific progeny.

We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demonstrate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus x B. juncea interspecific progeny. The B-genome--specific repeat sequence pBNBH35 was used to generate PCR products and FISH probes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspecific hybrid and the first backcross. B-genome--positive PCR assays were found in 34/51 fertile F2 progeny (67%), which was more than double the proportion found in fertile BC(1) progeny. Four B-genome--positive F(2)-derived families and 1 BC(1)-derived family were fixed or segregating for B. juncea morphology in the F(4) and BC(1)S(2), respectively, but in only 2 of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISH signals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F(4)-derived line that exhibited isolated and weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome--positive PCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomes tend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines containing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.     

Physical Mapping of rRNA Gene Loci and Inter-specific Relationships in Wild <Emphasis Type="Italic">Lilium</Emphasis> Distributed in Korea

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1–7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber’s classification of the genus Lilium.     

Assessment of the genetic diversity and genetic relationships of Lilium in China using ISSR markers

China is a center of natural distribution and diversity of genus Lilium around the world. In the study, the genetic diversity and genetic relationships of Lilium in China were analyzed by inter-simple sequence Repeat (ISSR) markers. The 6 highly polymorphic ISSR primers were selected to amplify the 20 Lilium species. The results showed that a total of 114 DNA bands were amplified, all of which were polymorphic loci (P = 100%), the effective number of alleles (Ne) was 1.2753, and the difference value between observed number of alleles (Na) and effective number of alleles (Ne) was 0.7247, and the Nei's genetic diversity (H) was 0.2048, and the Shannon's information index (I) was 0.3503. These results indicated that there is significant genetic difference among Lilium species in China. Taking the average genetic similarity coefficient (Gs) 0.5313 as the threshold, the 20 tested Lilium species were clustered into 5 groups, which was not entirely consistent with traditional clustering by morphological traits. The results obtained from this study can provide a reference for the molecular study of Lilium germplasm resources.     

Analysis of the coat protein gene and untranslated region of RNA3 of Cucumber Mosaic Virus isolates infecting various Lilium species and hybrids: association of the isolate infecting asiatic hybrid lily with subgroup II

The variability in the coat protein gene of Cucumber mosaic virus (CMV) isolates from various Lilium species and hybrids namely L. longiflorum, L. tigrinum, Asiatic hybrid and Oriental hybrid lilies was studied by sequence comparison of ～900 bp regions spanning the entire coat protein, intercistronic regions and 3′-UTR. CMV isolate characterised from Asiatic hybrid lily showed the highest homology with subgroup II isolates (94 – 97%), whereas 73 – 76% homology was observed with those belonging to subgroup I. Similarly, another three isolates showed 91 – 98% amino acid sequence homology with subgroup I and 74 – 76% sequence homology with subgroup II. Based on the criteria for classification of CMV isolates all the Indian isolates fall in subgroup I, except the one characterized from Asiatic Hybrid lily which falls into subgroup II. Other lily isolates from world were placed in subgroup II. This is the first case of Asiatic hybrid lily CMV isolate belonging to subgroup II.     

Inheritance of Cry1Ac resistance and associated biological traits in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

The analysis of reciprocal genetic crosses between resistant Helicoverpa armigera strain (BH-R) (227.9-fold) with susceptible Vadodara (VA-S) strain showed dominance (h) of 0.65-0.89 and degree of dominance (D) of 0.299-0.782 suggesting Cry1Ac resistance as a semi-dominant trait. The D and h values of F₁ hybrids of female resistant parent were higher than female susceptible parent, showing maternally enhanced dominance of Cry1Ac resistance. The progeny of F₂ crosses, backcrosses of F₁ hybrid with resistant BH-R parent did not differ significantly in respect of mortality response with resistant parent except for backcross with female BH-R and male of F₁ (BH-R × VA-S) cross, suggesting dominant inheritance of Cry1Ac resistance. Evaluation of some biological attributes showed that larval and pupal periods of progenies of reciprocal F₁ crosses, backcrosses and F₂ crosses were either at par with resistant parent or lower than susceptible parent on treated diet (0.01 μg/g). The susceptible strain performed better in terms of pupation and adult formation than the resistant strain on untreated diet. In many backcrosses and F₂ crosses, Cry1Ac resistance favored emergence of more females than males on untreated diet. The normal larval period and the body weight (normal larval growth) were the dominant traits associated with susceptible strain as contrast to longer larval period and the lower body weight (slow growth) associated with resistance trait. Further, inheritance of larval period in F₂ and backcross progeny suggested existence of a major resistant gene or a set of tightly linked loci associated with Cry1Ac sensitivity.     

Photoautotrophic culture conditions and photosynthetic photon flux influence growth of <Emphasis Type="Italic">Lilium</Emphasis> bulblets <Emphasis Type="Italic">in vitro</Emphasis>

Summary Lilium Asiatic hybrid ‘Mona’ bulblets were cultured in vitro for 100 d under photoautotrophic (CO₂-enriched conditions and without sucrose in the medium) and heterotrophic (non-enriched CO₂ conditions and sucrose-supplemented medium) methods and under various levels of photosynthetic photon flux (PPF). Bulblet growth and net photosynthetic rate (NPR) were analyzed. CO₂₋ and PPF-enriched conditions enhanced the overall growth of bulblets, scale leaves, and roots. Heterotrophic conditions enhanced bulblet growth but higher PPF levels were inhibitory to the development of scale leaves. These results indicate the CO₂₋ and PPF-enriched conditions (photoautotrophic conditions) are beneficial for the production of high-quality bulblets of Asiatic hybrid lilies in vitro     

Production and molecular characterization of somatic hybrids between Pleurotus florida and Lentinula edodes

Nine inter-generic somatic hybrids named as pfle were produced through PEG-mediated protoplast fusion between Pleurotus florida and Lentinula edodes using double selection method. Hybridity of the newly developed strains was established on the basis of colony morphology, mycelial growth, hyphal traits, fruit-body productivity and inter single sequence repeat (ISSR) marker profiling. Hybrid population was assessed with different phenotypic variables by one-way analysis of variance. Principal component matrices were analyzed for the six phenotypic variables in scatter plot showing maximum positive correlation between each variable for all strains examined. Six ISSR primers generated 66 reproducible fragments with 98.48 % polymorphism. The dendrogram thus created based on unweighted pair-group method with mathematic averages method of clustering and Euclidean distance which exhibited three major groups between the parents and pfle hybrids. Though P. florida parent remained in one group but it showed different degrees of genetic distance with all the hybrid lines belonging to the other two groups while L. edodes was most distantly related to all the hybrid lines. L. edodes specific sequence-rich ISSR amplicon was recorded in all the hybrid lines and in L. edodes but not in P. florida. All the fruit body generating pfle hybrid lines could produce basidiocarp on paddy straw in sub-tropical climate and showed phenotypic resemblance to the P. florida parent.     

Study on diversity of endophytic bacterial communities in seeds of hybrid maize and their parental lines

The seeds of plants are carriers of a variety of beneficial bacteria and pathogens. Using the non-culture methods of building 16S rDNA libraries, we investigated the endophytic bacterial communities of seeds of four hybrid maize offspring and their respective parents. The results of this study show that the hybrid offspring Yuyu 23, Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38 and 2 OTUs of bacteria, respectively. The parents Ye 478, Chang 7-2, Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12 and 2 OTUs, respectively. In the hybrid Yuyu 23, the dominant bacterium Pantoea (73.38?%) was detected in its female parent Ye 478, and the second dominant bacterium of Sphingomonas (26.62?%) was detected in both its female (Ye 478) and male (Chang 7-2) parent. In the hybrid Zhengdan 958, the first dominant bacterium Stenotrophomonas (41.67?%) was detected in both the female (Zheng 58) and male (Chang 7-2) parent. The second dominant bacterium Acinetobacter (9.26?%) was also the second dominant bacterium of its male parent. In the hybrid Jingdan 28, the second dominant bacterium Pseudomonas (12.78?%) was also the second dominant bacterium of its female parent, and its third dominant bacterium Sphingomonas (9.90?%) was the second dominant bacterium of its male parent and detected in its female parent. In the hybrid Jingyu 11, the first dominant bacterium Leclercia (73.85?%) was the third dominant bacterium of its male parent, and the second dominant bacterium Enterobacter (26.15?%) was detected in its male parent. As far as we know, this was the first research reported in China on the diversity of the endophytic bacterial communities of the seeds of various maize hybrids with different genotypes.     

Selection of generally applicable SSR markers for evaluation of genetic diversity and identity in Lilium

There are approximately 100 species and 10,000 cultivars of Lilium and in general their phylogeny is understood. Difficulties remain, however, in understanding the breeding relationships of cultivars and commercial hybrids. One solution to this problem is to identify a selection of validated and transferable SSR markers for use in genotyping. Although over 100 Lilium SSRs have been developed, they have not been validated for use with broad populations. Here, were-evaluated 112 SSRs with 69 lily accessions from different sources, and selected 70 SSRs as easy to score, transferable and polymorphic in all accessions tested. Based on the marker data from 70 SSRs, two main clusters were established for 69 accessions using TREECON, one includes Asiatic hybrids, Longiflorum × Asiatic hybrids and Asiatic local landraces (Lilium brownii, L. brownii var. giganteum, Lilium pumilum, Lilium davidii var. unicolor and Lilium lancifolium), the other is composed primarily of Oriental hybrids and Oriental × Trumpet hybrids, which is in agreement with previous studies and the breeding pedigree. The utility of the 70 SSR markers for establishing parentage and taxon identity of landraces was validated. Our study offers valuable information and validated markers for Lilium systematic classification and the establishment of identity.     

Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates

Self-fertilization (selfing) favours reproductive success when mate availability is low, but renders populations more vulnerable to environmental change by reducing genetic variability. A mixed-breeding strategy (alternating selfing and outcrossing) may allow species to balance these needs, but requires a system for regulating sexual identity. We explored the role of DNA methylation as a regulatory system for sex-ratio modulation in the mixed-mating fish Kryptolebias marmoratus. We found a significant interaction between sexual identity (male or hermaphrodite), temperature and methylation patterns when two selfing lines were exposed to different temperatures during development. We also identified several genes differentially methylated in males and hermaphrodites that represent candidates for the temperature-mediated sex regulation in K. marmoratus. We conclude that an epigenetic mechanism regulated by temperature modulates sexual identity in this selfing species, providing a potentially widespread mechanism by which environmental change may influence selfing rates. We also suggest that K. marmoratus, with naturally inbred populations, represents a good vertebrate model for epigenetic studies.     

Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification

In this study, apical meristems from adventitious buds of three lily (Lilium L.) cultivars were successfully cryopreserved by droplet-vitrification. The most effective techniques were as follows. Excised meristems from in vitro plantlets which had been sub-cultured for about 2 months were cold-hardened at 4 °C for 1 week, precultured on MS medium supplemented with 0.3 mol L⁻¹ sucrose for 2 days, osmoprotected in loading solution for 20-40 min at room temperature and then soaked in PVS2 solution for 90-120 min at 0 °C, frozen in microdroplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. The meristems were then rapidly rewarmed by dilution solution, transferred to regeneration medium and stored in the dark for two weeks at 20 °C, and then cultured under white fluorescent light at an intensity of 2000 lux, with a 16 h photoperiod at 20 °C. The highest post-thaw survival percentages of three cultivars ‘Siberia’ (Lilium × siberia), Lilium lancifolium Thunb. and ‘Snow Queen’ Lilium × longiflorum were 65.0%, 83.8% and 43.3%, and regeneration percentages were 62.0%, 67.6% and 35.0%, respectively. The study demonstrated that cryopreservation by droplet-vitrification increased survival and regeneration percentages of certain lily cultivars compared with vitrification. Thus to cryopreserve lily meristems, droplet-vitrification method is preferable to the vitrification method.     

Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines

Chromosomal rearrangements may complicate construction of Arabidopsis with multiple TDNA-insertion mutations. Here, crossing two lines homozygous for insertions in AtREV3 and AtPOLH (chromosomes I and V, respectively) and selfing F1 plants yielded non-Mendelian F2 genotype distributions: frequencies of +/++/+ and 1/1 2/2 progeny were only 0.42 and 0.25%. However, the normal development and fertility of double mutants showed AtPOLH-1 and AtREV3-2 gametes and 1/1 2/2 embryos to be fully viable. F2 distributions could be quantitatively predicted by assuming that F1 selfing produced inviable (1,2) and (+,+) gametophytes 86% of the time. Some defect intrinsic to the F1 selfing process itself thus appeared responsible. In selfing AtREV3 ^+/2 single mutants, imaging of ovules and pollen showed arrest or abortion, respectively, of half of gametophytes; however, gametogenesis was normal in AtREV3 ^2/2 homozygotes. These findings, taken together, suggested that T-DNA insertion at AtREV3 on chromosome I had caused a reciprocal I–V translocation. Spreads of meiosis I chromosomes in selfing AtREV3 ^+/2 heterozygotes revealed the predicted cruciform four-chromosome structures, which fluorescence in situ hybridization showed to invariably include both translocated and normal chromosomes I and V. Sequencing of the two junctions of T-DNA with AtREV3 DNA and the two with gene At5g59920 suggested translocation via homologous recombination between independent inverted-repeat T-DNA insertions. Thus, when crosses between TDNA-insertion mutants yield anomalous progeny distributions, TDNA-linked translocations should be considered. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Resource Allocation and the Evolution of Self-Fertilization in Plants

This article develops a simple evolutionarily stable strategy (ESS) model of resource allocation in partially selfing plants, which incorporates reproductive and sex allocation into a single framework. The analysis shows that, if female fitness gain increases linearly with resource investment, total reproductive allocation is not affected by sex allocation, defined as the fraction of reproductive resources allocated to male function. All else being equal, the ESS total reproductive allocation increases with increasing selfing rate if the fitness of selfed progeny is more than half that of outcrossed progeny, while the ESS sex allocation is always a decreasing function of the selfing rate. Self-fertilization is much more common in annual than in perennial plants, and this association has been commonly interpreted in terms of an effect of life history on mating system. The model in this article shows that self-fertilization can itself cause the evolution of the annual habit. Incorporating the effects of pollen discounting may not have any influence on total reproductive allocation if female fitness gain is a linear function of resource investment, although the evolutionarily stable sex allocation is altered. Evolution of the selfing rate is found to be independent of reproductive and sex allocation under the mass-action assumption that self- and outcross pollen are deposited simultaneously on receptive stigmas and compete for access to ovules.     

Transmission ratio distortion of molecular markers in a doubled haploid population originated from a natural hybrid between Osmunda japonica and O. lancea

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.