果实红色与黄色番茄品种植株茎部内生细菌群落结构及代谢功能特征

[目的]比较果实颜色分别为红色和黄色的番茄植株茎部内生细菌群落组成及代谢功能特征,旨在探究番茄果色形成与植株内生细菌的关联,为构建番茄育种新型的评价体系,以及开发利用有益微生物功能提供理论依据和技术支撑.[方法]基于MiSeq高通量测序技术,分析果实颜色分别为红色和黄色番茄品种植株茎部内生细菌群落组成及相关代谢功能生物...     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

Nonfunctional alleles of long‐day suppressor genes independently regulate flowering time

Due to the remarkable adaptability to various environments, rice varieties with diverse flowering times have been domesticated or improved from Oryza rufipogon. Detailed knowledge of the genetic factors controlling flowering time will facilitate understanding the adaptation mechanism in cultivated rice and enable breeders to design appropriate genotypes for distinct preferences. In this study, four genes (Hd1, DTH8, Ghd7 and OsPRR37) in a rice long‐day suppression pathway were collected and sequenced in 154, 74, 69 and 62 varieties of cultivated rice (Oryza sativa) respectively. Under long‐day conditions, varieties with nonfunctional alleles flowered significantly earlier than those with functional alleles. However, the four genes have different genetic effects in the regulation of flowering time: Hd1 and OsPRR37 are major genes that generally regulate rice flowering time for all varieties, while DTH8 and Ghd7 only regulate regional rice varieties. Geographic analysis and network studies suggested that the nonfunctional alleles of these suppression loci with regional adaptability were derived recently and independently. Alleles with regional adaptability should be taken into consideration for genetic improvement. The rich genetic variations in these four genes, which adapt rice to different environments, provide the flexibility needed for breeding rice varieties with diverse flowering times.     

Advanced backcross QTL analysis in tomato. I. Identification of QTLs for traits of agronomic importance from Lycopersicon hirsutum

 Advanced backcross QTL (AB-QTL) analysis is a new strategy for studying the effect of unadapted alleles on the agronomic performance of elite cultivated lines. In this paper we report results from the application of the AB-QTL strategy to cultivated tomato using the wild species Lycopersicon hirsutum LA1777 as the donor parent. RFLP genomic fingerprints were determined for 315 BC₂ plants and phenotypic data were collected for 19 agronomic traits from approximately 200 derived BC₃ lines which were grown in replicated field trials in three locations worldwide. Between 1 and 12 significant QTLs were identified for each of the 19 traits evaluated, with a total of 121 QTLs identified for all traits. For 25 of the QTLs (20%) corresponding to 12 traits (60%), the L. hirsutum allele was associated with an improvement of the trait from a horticultural perspective, despite the fact that L. hirsutum is overall phenotypically inferior to the elite parent. For example, L. hirsutum has fruit that remains green when ripe (lack of red pigment) yet alleles were found in this species that significantly increase red color when transferred into cultivated tomatoes. Wild alleles were also associated with increases in total yield and soluble solids (up to 15%) and brix×red yield (up to 41%). These results support the idea that one cannot predict the genetic potential of exotic germplasm based on phenotype alone and that marker-based methods, such as the AB-QTL strategy, should be applied to fully exploit exotic germplasm. Received: 27 October 1997 / Accepted: 25 November 1997     

Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags

Single nucleotide polymorphisms (SNPs) are useful for characterizing allelic variation, for genome-wide mapping, and as a tool for marker-assisted selection. Discovery of SNPs through de novo sequencing is inefficient within cultivated tomato (Lycopersicon esculentum Mill.) because the polymorphism rate is more than ten-fold lower than the sequencing error rate. The availability of expressed sequence tag (EST) data has made it feasible to discover putative SNPs in silico prior to experimental verification. By exploiting redundancy among EST data available for different varieties among 148,373 tomato ESTs, we have identified candidate SNPs for use within cultivated germplasm pools. 1,245 contigs having three EST sequences of Rio Grande and three EST sequences of TA496 were used for SNP discovery. We detected 1 SNP for every 8,500 bases analyzed, with 101 candidate SNPs in 44 genes identified. Sixty-six SNPs could be recognized by restriction enzymes, and subsequent experimental verification using restriction digestion or CEL I digestion confirmed 83% of the putative polymorphisms tested. SNPs between TA496 and Rio Grande have a high probability (53%) of detecting polymorphisms between other L. esculentum varieties. Twenty-six SNPs in 18 unigenes were mapped to specific chromosomes. Two SNPs, LEOH23 and LEOH37, were shown to be linked to quantitative trait loci contributing to fruit color within elite breeding populations. These results suggest that the growing databases of DNA sequence will yield information that facilitates improvement within the germplasm pools that have contributed to productive modern varieties.     

Soybean seed lipoxygenase genes: molecular characterization and development of molecular marker assays

Soybean seeds contain three lipoxygenase (Lox) enzymes that are controlled by separate genes, Lox1, Lox2 and Lox3. Lipoxygenases play a role in the development of unpleasant flavors in foods containing soybean by oxidation of polyunsaturated fatty acids. Null alleles for all three enzymes have been identified, lox1, lox2 and lox3, and are known to be inherited as simple recessive alleles. Previous studies determined that a missense mutation rendered Lox2 inactive; however, the genetic cause of either lox1 or lox3 mutation was not known. The objectives of this study were the molecular characterization of both lox1 and lox3 mutant alleles and the development of molecular markers to accelerate breeding for Lox-free soybean varieties. We identified two independent mutant alleles as the genetic causes of the lack of Lox1 in seeds of two lox1 mutant soybean lines. Similarly, a mutant allele that truncates Lox3 in a lox3 mutant soybean line was identified. Molecular markers were designed and confirmed to distinguish mutant, wild type, and heterozygous individuals for Lox1, Lox2 and Lox3 genes. Genotype and Lox phenotype analysis showed a perfect association between the inheritance of homozygous lox mutant alleles and the lack of Lox activity. Molecular characterization of a seed-lipoxygenase-free soybean line led to the discovery that an induced recombination event within the Lox1 gene was responsible for breaking the tight linkage in repulsion phase between mutant alleles at the Lox1 and Lox2 loci. The molecular resources developed in this work should accelerate the inclusion of the lipoxygenase-free trait in soybean varieties.     

Using Heading date 1 preponderant alleles from indica cultivars to breed high‐yield,high‐quality japonica rice varieties for cultivation in south China

Heading date 1 (Hd1) is an important gene for the regulation of flowering in rice, but its variation in major cultivated rice varieties, and the effect of this variation on yield and quality, remains unknown. In this study, we selected 123 major rice varieties cultivated in China from 1936 to 2009 to analyse the relationship between the Hd1 alleles and yield‐related traits. Among these varieties, 19 haplotypes were detected in Hd1, including two major haplotypes (H8 and H13) in the japonica group and three major haplotypes (H14, H15 and H16) in the indica group. Analysis of allele frequencies showed that the secondary branch number was the major aimed for Chinese indica breeding. In the five major haplotypes, SNP₃₁₆(C‐T) was the only difference between the two major japonica haplotypes, and SNP₄₉₅(C‐G) and SNP₆₁₄(G‐A) are the major SNPs in the three indica haplotypes. Association analysis showed that H16 is the most preponderant allele in modern cultivated Chinese indica varieties. Backcrossing this allele into the japonica variety Chunjiang06 improved yield without decreasing grain quality. Therefore, our analysis offers a new strategy for utilizing these preponderant alleles to improve yield and quality of japonica varieties for cultivation in the southern areas of China.     

Physiology of the tomato mutant alcobaca

Alcobaca is commonly regarded as an abnormally ripening mutant of the tomato (Lycopersicon esculentum Mill.). Alcobaca fruits were found to be similar to cv. Rutgers fruits in the following characteristics: time between full anthesis and the onset of ripening, response to ethephon, flavor, pH and concentrations of titratable acids, total soluble solids and reducing sugars. The pattern of CO₂ and ethylene climacteric are similar in the two plant types, but the peak levels were lower and occurred later in alcobaca than in ‘Rutgers’. The mutant fruits differed from fruits of normal varieties in their greatly prolonged shelf life, their relatively low activity of polygalacturonase (PG) and polymethylgalacturonase (PMG), and their low level of endogenous ethylene. Fruits of the mutant harvested before the onset of ripening failed to reach normal pigmentation and remained yellow. Fruits harvested at the onset of ripening reached an orange color, while fruits ripened while attached to the plant reached almost normal pigmentation. These results suggest that alcobaca is a slow ripening mutant and does not belong to the category of non-ripening mutants.     

The tomato calcium-permeable channel 4.1 (SlOSCA4.1) is a susceptibility factor for pepino mosaic virus

The hyperosmolality-gated calcium permeable channel 4.1 (OSCA4.1) belongs to an evolutionarily conserved small family of mechano-sensitive channels. OSCA members may represent key players in plant resistance to drought and to pathogen infection but are scarcely studied. After screening for resistance to pepino mosaic virus (PepMV) a collection of 1000 mutagenized tomato families, we identified a mutant showing no symptoms and reduced virus accumulation. Resistance was mapped to chromosome 2 between positions 46 309 531 to 47 044 163, where a missense mutation caused the putative truncation of the OSCA4.1 protein. A CRISPR/Cas9 slosca4.1 mutant was resistant to PepMV, but not to tobacco mosaic virus or potato virus X. Inoculation of mutant and wild type tomato protoplasts showed that resistance was expressed in single cells, suggesting a role for SlOSCA4.1 in early viral function(s); congruently, SlOSCA4.1 re-localized to structures reminiscent of viral replication complexes. We propose that SlOSCA4.1 contributes to the correct regulation of the Ca²⁺ homeostasis necessary for optimal PepMV infection. PepMV is a pandemic virus that causes significant losses in tomato crops worldwide. In spite of its importance, no tomato-resistant varieties have been deployed yet; the mutant identified here has great potential to breed tomato varieties resistant to PepMV.     

Stock-Scion Interactions of Normal and Fruit Ripening Mutants rin and nor in Tomato

Scions of the non-ripening rin and nor tomato strains (Lycopersicum esculentum Mill.) were grafted on normal understock plants (cv. Rutgers) in an effort to study the influence of roots and vegetative tissue on the ripening behavior of the tomato fruit. Receiprocal grafts of ‘Rutgers’ scions on rin and nor understocks as well as grafted and ungrafted controls were also established. No alteration in the ethylene, and CO₂ evolution and color development of either mutant fruits on normal understock or of normal fruits on mutant understock occurred. We suggest that the inability of rin and nor mutant fruits to ripen normally stems either from the presence in mutant fruit of a non-translocatable ripening inhibitor, or from the absence of a non-translocatable ripening factor.     

A TILLING approach to generate broad‐spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy

Genetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLING‐induced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh–eIF4E1^PI24–pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re‐establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss‐of‐function alleles to generate resistance may be counter‐productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.     

Evidences of introgression from cultivated rice to Oryza rufipogon (Poaceae) populations based on SSR fingerprinting: implications for wild rice differentiation and conservation

Crop-to-wild introgression may play an important role in evolution of wild species. Asian cultivated rice (Oryza sativa L.) is of a particular concern because of its cross-compatibility with the wild ancestor, O. rufipogon Griff. The distribution of cultivated rice and O. rufipogon populations is extensively sympatric, particularly in Asia where many wild populations are surrounded by rice fields. Consequently, gene flow from cultivated rice may have a potential to alter genetic composition of wild rice populations in close proximity. In this study, we estimated introgression of cultivated rice with O. rufipogon based on analyses of 139 rice varieties (86 indica and 53 japonica ecotypes) and 336 wild individuals from 11 O. rufipogon populations in China. DNA fingerprinting based on 17 selected rice simple sequence repeat (SSR) primer pairs was adopted to measure allelic frequencies in rice varieties and O. rufipogon samples, and to estimate genetic associations between wild and cultivated rice through cluster analysis. We detected consanguinity of cultivated rice in O. rufipogon populations according to the admixture model of the STRUCTURE program. The analyses showedz that four wild rice populations, DX-P1, DX-P2, GZ-P2, and HL-P, contained some rare alleles that were commonly found in the rice varieties examined. In addition, the four wild rice populations that scattered among the rice varieties in the cluster analysis showed a closer affinity to the cultivars than the other wild populations. This finding supports the contention of substantial gene flow from crop to wild species when these species occur close to each other. The introgressive populations had slightly higher genetic diversity than those that were isolated from rice. Crop-to-wild introgression may have accumulative impacts on genetic variations in wild populations, leading to significant differentiation in wild species. Therefore, effective measure should be taken to avoid considerable introgression from cultivated rice, which may influence the effective in-situ conservation of wild rice species.     

Detection of major loci associated with the variation of 18 important agronomic traits between Solanum pimpinellifolium and cultivated tomatoes

Wild species can be used to improve various agronomic traits in cultivars; however, a limited understanding of the genetic basis underlying the morphological differences between wild and cultivated species hinders the integration of beneficial traits from wild species. In the present study, we generated and sequenced recombinant inbred lines (RILs, 201 F₁₀ lines) derived from a cross between Solanum pimpinellifolium and Solanum lycopersicum tomatoes. Based on a high‐resolution recombination bin map to uncover major loci determining the phenotypic variance between wild and cultivated tomatoes, 104 significantly associated loci were identified for 18 agronomic traits. On average, these loci explained ~39% of the phenotypic variance of the RILs. We further generated near‐isogenic lines (NILs) for four identified loci, and the lines exhibited significant differences for the associated traits. We found that two loci could improve the flower number and inflorescence architecture in the cultivar following introgression of the wild‐species alleles. These findings allowed us to construct a trait–locus network to help explain the correlations among different traits based on the pleiotropic or linked loci. Our results provide insights into the morphological changes between wild and cultivated tomatoes, and will help to identify key genes governing important agronomic traits for the molecular selection of elite tomato varieties.     

Self-incompatibility alleles in Polish wild pear (Pyrus pyraster (L.) Burgsd.): a preliminary analysis

Wild pear (Pyrus pyraster, syn.P. communis var.pyraster) is thought to be one of the species that gave rise to all other members of the genusPyrus, although intraspecific hybridizations with cultivated varieties could cause the disappearance of original species characteristics. S-RNase alleles from 7 different wild pear individuals, collected from various regions of Poland, were cloned on the basis of the PCR method and nucleotide sequence analyses. The hypervariable (HV) region is responsible for allele-specific S-RNase activity in the self-incompatibility mechanism. The high level of polymorphism of its sequences may constitute a source of valuable phylogenetic information. From all individuals, 14 sequences were obtained successfully, and 9 of them were novel alleles. Phylogenetic analysis of these alleles was based on the amino acid sequence interpretation of coding regions and intron nucleotide sequences. The research conducted on a limited pool of availableP. pyraster alleles gives only an initial insight into possible S-RNase allele polymorphisms in wild populations. At this stage, the results do not confirm a strong influence of cultivated pear species on the wild pear.     

Characterization and mapping of a gene controlling shoot regeneration in tomato

The superior regeneration capacity of Lycopersicon peruvianum was introduced into the cultivated tomato Lycopersicon esculentum by backcrossing hybrid material with the tomato genotype VF11. In segregating material derived from these backcrosses, the ability to regenerate shoots on root explants cultured on a zeatin-containing medium, was highly correlated with the ability to regenerate shoots on established callus cultures. The efficient shoot-regenerating root explant system permitted us to study the genetics of this trait and to locate the genes involved, using a set of morphological markers defining all 12 tomato chromosomes. Depending on the tomato genotype, mono, -di- or trigenic ratios were observed. It is concluded that a dominant L. peruvianum allele at a locus (Rg-1) near the middle of chromosome 3 determines efficient shoot regeneration on root explants in tomato in combination with dominant alleles at one or two other loci of either L. peruvianum or L. esculentum origin. The map location of the Rg-1 locus was refined further using a number of chromosome-3-specific RFLPs. The addition of new classical and RFLP linkage data to existing literature data and subsequent processing resulted in a revised and integrated map of tomato chromosome 3. From a morphological and physiological analysis of genotypes differing in Rg phenotype, it is concluded that the genetic component associated with regeneration determines the maintenance of morphogenetic competence and not the sensitivity to hormones.     

Comparative analysis of polymorphism and chromosomal location of tomato microsatellite markers isolated from different sources

Previously isolated tomato (Lycopersicon esculentum) microsatellite markers were mainly clustered in the centromeric heterochromatin and not located in euchromatic regions. To achieve a more-uniform distribution of microsatellite markers for genome mapping purposes, a set of tomato microsatellite markers containing dinucleotide simple sequence repeats were developed by screening genomic libraries enriched for single-copy sequences, and screening the tomato EST database. The tomato microsatellites isolated in these ways were characterized by combinations of different types of repeated motifs and they were polymorphic in a set of L. esculentum varieties detecting up to four alleles. A total of 20 markers were placed on the genetic map of tomato. Interestingly, all markers isolated from genomic libraries enriched for single-copy sequences by PstI-pre-digestion mapped into the centromeric regions. The majority of markers derived from EST sequences contained predominantly AT microsatellites and were located in euchromatic regions. Received: 22 December 2000 / Accepted: 4 May 2001     

Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum)

Xanthophylls, the pigments responsible for yellow to red coloration, are naturally occurring carotenoid compounds in many colored tissues of plants. These pigments are esterified within the chromoplast; however, little is known about the mechanisms underlying their accumulation in flower organs. In this study, we characterized two allelic tomato (Solanum lycopersicum L.) mutants, pale yellow petal (pyp) 1‐1 and pyp1‐2, that have reduced yellow color intensity in the petals and anthers due to loss‐of‐function mutations. Carotenoid analyses showed that the yellow flower organs of wild‐type tomato contained high levels of xanthophylls that largely consisted of neoxanthin and violaxanthin esterified with myristic and/or palmitic acids. Functional disruption of PYP1 resulted in loss of xanthophyll esters, which was associated with a reduction in the total carotenoid content and disruption of normal chromoplast development. These findings suggest that xanthophyll esterification promotes the sequestration of carotenoids in the chromoplast and that accumulation of these esters is important for normal chromoplast development. Next‐generation sequencing coupled with map‐based positional cloning identified the mutant alleles responsible for the pyp1 phenotype. PYP1 most likely encodes a carotenoid modifying protein that plays a vital role in the production of xanthophyll esters in tomato anthers and petals. Our results provide insight into the molecular mechanism underlying the production of xanthophyll esters in higher plants, thereby shedding light on a longstanding mystery.     

Molecular Characterization of the pun Allele of the Mouse Pink-Eyed Dilution Locus

The mouse pink eyed dilution locus, p, located on chromosome 7, mediates coat and eye color. The human correlate of this gene may underlie some forms of tyrosinase-positive oculocutaneous albinism. Mutations at the p locus result in a reduction in pigmentation of the eyes and coat. Although most mutant p alleles (including all spontaneous mutations) affect only pigmentation, several mutant alleles (all radiation induced) are also associated with a variety of other phenotypes. We have focused our attention on the p^un mutant allele, a spontaneous mutation, exhibiting one of the highest reversion frequencies reported for a mammalian mutation. Using a new technique, genome scanning, we have cloned fragments of genomic DNA from the p locus that are associated with a DNA duplication in p^un DNA. These fragments can now be used to locate the p gene-encoding sequences and aid in the molecular characterization of complex mutant p alleles.     

Molecular diversity at the self-incompatibility locus is a salient feature in natural populations of wild tomato (Lycopersicon peruvianum)

A cDNA encoding a stylar protein was cloned from flowers of self-incompatible wild tomato (Lycopersicon peruvianum). The corresponding gene was mapped to the S locus, which is responsible for self-incompatibility. The nucleotide sequence was determined for this allele, and compared to other S-related sequences in the Solanaceae. The S allele was used to probe DNA from 92 plants comprising 10 natural populations of Lycopersicon peruvianum. Hybridization was conducted under moderate and permissive stringencies in order to detect homologous sequences. Few alleles were detected, even under permissive conditions, underscoring the great sequence diversity at this locus. Those alleles that were detected are highly homologous. Sequences could not be detected in self-incompatible Nicotiana alata, self-compatible L. esculentum (cultivated tomato) or self-compatible L. hirsutum. However, hybridization to an individual of self-incompatible L. hirsutum revealed a closely related sequence that maps to the S locus in this reproductively isolated species. This supports the finding that S locus polymorphism predates speciation. The extraordinarily high degree of sequence diversity present in the gametophytic self-incompatibility system is discussed in the context of other highly divergent systems representing several kingdoms.