Single nucleotide polymorphism in sugar pathway and disease resistance genes in sugarcane

Key message

Single nucleotide polymorphism in sugar pathway and disease resistance genes showing genetic association with sugar content and red rot resistance would be useful in marker-assisted genetic improvement of sugarcane.

Abstract

Validation and genotyping of potential sequence variants in candidate genes are necessary to understand their functional significance and trait association potential. We discovered, characterized, validated and genotyped SNPs and InDels in sugar pathway and disease resistance genes of Saccharum complex and sugarcane varieties using amplicon sequencing and CAPS assays. The SNPs were abundant in the non-coding 3′UTRs than 5′UTRs and coding sequences depicting a strong bias toward C to T transition substitutions than transversions. Sequencing of cloned amplicons validated 61.6 and 45.2 % SNPs detected in silico in 21 sugar pathway and 16 disease resistance genes, respectively. Sixteen SNPs in four sugar pathway genes and 10 SNPs in nine disease resistance genes were validated through cost-effective CAPS assay. Functional and adaptive significance of SNP and protein haplotypes identified in sugar pathway and disease resistance genes was assessed by correlating their allelic variation with missense amino acid substitutions in the functional domains, alteration in protein structure models and possible modulation of catalytic enzyme activity in contrasting high and low sugar and moderately red rot resistant and highly susceptible sugarcane genotypes. A strong genetic association of five SNPs in the sugar pathway and disease resistance genes, and an InDel marker in the promoter sequence of sucrose synthase-2 gene, with sugar content and red rot resistance, was evident. The functionally relevant SNPs and InDels, detected and validated in sugar pathway and disease resistance genes, and genic CAPS markers designed, would be of immense use in marker-assisted genetic improvement of sugarcane for sugar content and disease resistance.

     

Phenotypic polymorphism and allele differentiation of isozymes in fodder beet,multigerm sugar beet and monogerm sugar beet

Summary Thirteen enzymes (MDH, SDH, LAP, PGM, PX, IDH, GPI, 6PGD, APH, GOT, GDH, ME and SOD) of 3 cultivated beet (B. vulgaris L.) gene pools, comprising 12 accessions of fodder beet, 11 of old multigerm sugar beet and 10 of modern monogerm sugar beet, were investigated using horizontal starch gel electrophoresis. Eleven accessions of primitive or wild B. vulgaris were also included for the comparison of isozymes. Variation in isozyme phenotypes was investigated to detect diversity in the three cultivated forms of beet. Phenotypic variation was observed in all except ME and SOD, which were monomorphic. A high degree of phenotypic polymorphism (Pj) was found in GDH, PGM, IDH, APH and MDH. Differences in phenotypic polymorphism in MDH, GPI and PX were recognized between fodder beet and both sugar beet groups. Average polymorphism for 13 enzymes in both sugar beets was significantly higher than that in fodder beet. For 13 enzymes, the existence of high isozyme diversity in both sugar beet gene pools was revealed. Allele frequencies in 13 alleles of five enzyme-coding loci, Lap, Px-1, Aph-1, Got-2 and Gdh-2, were investigated. New alleles, Px-1 ¹ and Got-2 ¹, were found in fodder beet accessions. No significant differences of average allele frequencies of five loci between fodder beet and both sugar beets were recognized. Several unique alleles and different isozyme phenotypes were observed in the accessions of B. vulgaris ssp. macrocarpa and ssp. adanensis. Future utilization of cultivated beet gene pools for sugar beet breeding is discussed from the viewpoint of genetic resources.     

Photosynthesis in sugar beet depends on root growth

Summary Sugar-beet plants, germinated in growth cabinets at 20°C and transplanted into the field after 3 weeks, developed much larger roots than plants grown from seed drilled directly into the soil. At the end of the season, the roots of transplants were 39% greater than from drilled seed—an increase of 14 m tons per hectare. The increased yield was mainly due to a sustained increase in photosynthesis because of the larger sink for carbohydrates provided by plants from the growth cabinets.     

Sucrose uptake by sugar beet tap root tissue

Sucrose uptake by discs of mature sugar beet root tissue incubated in [¹⁴C]-sucrose exhibited nonsaturating kinetics over the concentration range of 1 to 500 millimolar. Uptake was inhibited by dinitrophenol, sodium cyanide, low O₂, and penetrating sulfhydryl inhibitors. ATPase inhibitors, sodium fluoride, and oligomycin reduced uptake by 20 and 40%, respectively. Uptake as asymmetrically labeled sucrose ([¹⁴C]glucose) occurred with approximately 80% retention of asymmetry, indicating a nonhydrolytic pathway. Uptake was against a concentration gradient and required metabolic energy.     

Adenylate patterns of autumn-sown sugar beet differ from spring-sown sugar beet. Implications for root quality

Sugar beet ( Beta vulgaris L) is generally cultivated using two different planting and harvest patterns. In northern zones, spring sugar beet is sown in spring and harvested in autumn, whereas in subtropical latitudes, autumn sugar beet is sown in autumn and harvested in summer. The industrial quality of the root is frequently higher in spring-sown sugar beet crops. In order to explore physiological changes associated with this fact, this study has been focused on the seasonal changes of adenosine 5'-triphosphate and adenosine 5'-diphosphate levels in the storage roots of sugar beet plants, as an index of its metabolic status. The results obtained correspond to a different metabolic status of spring and autumn sugar beet at the moment of harvest. The adenylate patterns of autumn beets suggested a functional and active respiratory system. On the contrary, the patterns shown by spring beets corresponded to those we would expect to see in plants becoming dormant. The proline and glucose contents, which decrease the industrial quality of the root, and the respiratory rate measured in autumn-sown sugar beets, were nearly twice those of spring-sown sugar beets. The combination of an active respiratory system, which allows the carbohydrate catabolism and the synthesis of stress molecules, with the environmental factors at the time of the harvest, could be the underlying physiological mechanism causing some of the differences between spring- and autumn-sown sugar beet crops.     

Comparative effectiveness of sugar beet microsatellite markers isolated from genomic libraries and GenBank ESTs to map the sugar beet genome

Sugar beet (Beta vulgaris) is an important root crop for sucrose production. A study was conducted to find a new abundant source of microsatellite (SSR) markers in order to develop marker assistance for breeding. Different sources of existing microsatellites were used and new ones were developed to compare their efficiency to reveal diversity in mapping population and mapping coverage. Forty-one microsatellite markers were isolated from a B. vulgaris ssp maritima genomic library and 201 SSRs were extracted from a B. vulgaris ssp vulgaris library. Data mining was applied on GenBank B. vulgaris expressed sequence tags (ESTs), 803 EST-SSRs were identified over 19,709 ESTs. Characteristics, polymorphism and cross-species transferability of these microsatellites were compared. Based on these markers, a high density genetic map was constructed using 92 F₂ individuals from a cross between a sugar and a table beet. The map contains 284 markers, spans over 555 cM and covers the nine chromosomes of the species with an average markers density of one marker every 2.2 cM. A set of markers for assignation to the nine chromosomes of sugar beet is provided.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Opportunities for regulation of sugar beet storage root growth

The percentage of sucrose in sugar beet storage root fresh and dry matter is closely related to root structure. It has been suggested that the sucrose content might be increased by using plant growth regulators to modify storage root structure through control of cambial development, cell division and cell expansion. During storage root development correlations were found between the changing phytohormone profiles and the formation of secondary cambia and their subsequent cell division and expansion. Sugar beet root derived cell suspension cultures were used for detailed studies of the roles of endogenous phytohormones. The gibberellin synthesis inhibitor paclobutrazol was tested in cell cultures and whole plants. The observations provide a basis for development of plant growth regulator regimes to optimise sucrose yield from sugar beet.     

Single nucleotide polymorphism discovery in common bean

Single nucleotide polymorphisms (SNPs) were discovered in common bean (Phaseolus vulgaris L.) via resequencing of sequence-tagged sites (STSs) developed by PCR primers previously designed to soybean shotgun and bacterial artificial chromosome (BAC) end sequences, and by primers designed to common bean genes and microsatellite flanking regions. DNA fragments harboring SNPs were identified in single amplicons from six contrasting P. vulgaris genotypes of the Andean (Jalo EEP 558, G 19833, and AND 277) and Mesoamerican (BAT 93, DOR 364, and Rudá) gene pools. These genotypes are the parents of three common bean recombinant inbred line mapping populations. From an initial set of 1,880 PCR primer pairs tested, 265 robust STSs were obtained, which could be sequenced in each one of the six common bean genotypes. In the resulting 131,120?bp of aligned sequence, a total of 677 SNPs were identified, including 555 single-base changes (295 transitions and 260 transversions) and 122 small nucleotide insertions/deletions (indels). The frequency of SNPs was 5.16 SNPs/kb and the mean nucleotide diversity, expressed as Halushka??s theta, was 0.00226. This work represents one of the first efforts aimed at detecting SNPs in P. vulgaris. The SNPs identified should be an important resource for common bean geneticists and breeders for quantitative trait locus discovery, marker-assisted selection, and map-based cloning. These SNPS will be also useful for diversity analysis and microsynteny studies among legume species.     

Effect of irrigation frequency on root water uptake in sugar beet

A 2-year trial was performed on autumn-sown sugar beet grown in pots in order to study the influence of irrigation frequency on the water used by plants along the soil profile. The outdoor pots, containing one plant each, were 1.3 m high and had circular openings, through which Time Domain Reflectometry (TDR) apparatus wave guides could be inserted. Three irrigation intervals were compared and plants were watered whenever the soil layer explored by roots had lost 30% (SWD₁), 50% (SWD₂) and 70% (SWD₃) of the total available water (TAW). During the irrigation season, the water extracted by the plants from each layer along the soil profile (RWU) was determined by monitoring volumetric soil moisture content (), by TDR. At harvest time, root length density (RLD) along the soil profile was assessed using the Tennant method. The applied irrigation frequencies significantly affected the RWU. With the SWD₃ protocol, irrigation was at longer irrigation intervals (9 days) and watering volumes were as high as 84 mm. In this treatment, the plants lost almost 60% of total water from the lower soil layer (0.6–1.0 m). In treatment SWD₁, the irrigation interval was very short (3 days), and water extraction from 0.0–0.6 m soil depth was 92.0%. In the intermediate treatment, the irrigation interval was 5.5 days and a more uniform water depletion was observed along the root zone, approximately equal between the 0–0.6 and 0.6–1.0 m soil layer. Water extraction of sugar beet plants at the deeper soil layers in response to long irrigation intervals was related to an increase in water uptake efficiency of the deeper younger roots and not to an increase in root length density, which, on the contrary, decreased. This morpho-physiological acclimatization to progressive soil water deficit was coupled with an increase of the root/shoot ratio.     

Radial and axial water transport in the sugar beet storage root

To evaluate the contribution of transcellular, apoplastic and symplastic pathways to water movements, horizontal (axial pathway) and vertical (radial pathway) sugar beet root (Beta vulgaris L.) slices were studied. Volume flows (Jv) were measured under hydrostatic and/or osmotic gradients, using a computer-based data-acquisition system. When tissues were tested under hydrostatic gradients (0.3 MPa m^-1) a much more important permeability was observed in the axial pathway, as compared with the radial one. Negative pressure gradients (tensions) were as effective as positive ones in inducing a net water movement. After the establishment of a concentration gradient in the radial pathway (obtained by adding 300 M m^-3 mannitol to the employed solution) an osmotic flux, sensitive to HgCI2, was observed. The inhibitory effect of mercurial compounds was reversed by -mercaptoethanol while [¹⁴C] mannitol unidirectional fluxes were not affected by mercurial agents. In the axial pathway, the presence of a mannitol gradient did not develop a sustained osmotic flux. After an initial Jv in the expected direction, the Jv reversed and moved in the opposite way. It is concluded that, in the sugar beet root, water channels play a significant role in water transfers in the radial pathway. On the other side, water and solutes are transported by a hydrostatic gradient in the xylem vessels. In general, these results extend and adapt to a storage root the 'composite transport model' first proposed by Steudle et al.     

Bird damage to sugar beet

Pest damage to sugar beet, including that by birds, has been recorded since 1957. During that time damage by rooks has decreased almost to nil, but some other bird damage has greatly increased, most probably as a result of changing agronomic practices, especially the extensive use of herbicides, the introduction of monogerm seed, and the increasing practice of ‘planting-to-a-stand’. The most severe bird damage in the spring is grazing by several species, and in early summer localized felling of plants by pheasants. Observations in the mid–1960's of causes of seedling and plant losses suggested that birds were then of minor importance; the British Sugar Corporation currently consider that birds are the most serious pest of sugar beet. The distribution of the reported damage does not seem to follow any national pattern. In small-plot field trials possible repellent materials such as anthraquinone, methiocarb or thiram, applied to seed or foliage, did not decrease the extent of grazing.     

黄土高原甜菜叶片生长特性及其对块根产量、含糖量的影响

通过对旱地甜菜叶片生长特性及摘除不同叶组对块根产量,含糖量,显微结构的影响研究,结果表明：甜菜第10-20片叶的叶龄最长,积温最高,是甜菜的主要功能叶;甜菜从第20片叶期起进入块根,糖份增长期,从第55叶期起进入糖份积累期;摘除不同叶组的叶片对甜菜块根产量,含糖量及显微结构均有不同程度降低作用,摘除前期叶组对甜菜块根产量,产糖量,根径减幅较大,摘除后期叶组对块根含糖量,维管束环数,维管束环密度减幅较大;摘除第1-30片叶对甜菜影响最大。     

Bacterial artificial chromosome-derived molecular markers for early bolting in sugar beet

Early bolting in sugar beet (Beta vulgaris L.) is controlled by the dominant gene B. From an incomplete physical map around the B gene, 18 bacterial artificial chromosomes (BACs) were selected for marker development. Three BACs were shotgun-sequenced, and 61 open reading frames (ORFs) were identified. Together with 104 BAC ends from 54 BACs, a total number of 55,464 nucleotides were sequenced. Of these, 37 BAC ends and 12 ORFs were selected for marker development. Thirty-one percent of the sequences were found to be single copy and 24%, low copy. From these sequences, 15 markers from ten different BACs were developed. Ten polymorphisms were determined by simple agarose gel electrophoresis of either restricted or non-restricted PCR products. Another five markers were determined by tetra-primer amplification refractory mutation system-PCR. In order to select candidate BACs for cloning the gene, genetic linkage between seven markers and the bolting gene was calculated using 1,617 plants from an F₂ population segregating for early bolting. The recombination values ranged between 0.0033 and 0.0201. In addition, a set of 41 wild and cultivated Beta accessions differing in their early bolting character was genotyped with seven markers. A common haplotype encompassing two marker loci and the b allele was found in all sugar beet varieties, indicating complete linkage disequilibrium between these loci. This suggests that the bolting gene is located in close vicinity to these markers, and the corresponding BACs can be used for cloning the gene.     

Search for actinomycetes--antagonists of fungi causing sugar beet root rot

Actinomycetes belonging to Streptomyces were isolated from the rhizosphere of sugar beet grown on an infected area after cultivation for many years. 44.1 per cent of them proved to be antagonists of phytopathogenic test fungi. The majority of the antagonists were detected among the Cinereus. The lowest number of the antagonists was detected among the Azureus, the maximum number of the antagonists was observed in the middle and at the end of the sugar beet vegetation period. During various periods of the plant development, the number of the antagonists belonging to different sections changed. The majority of the actinomycetes belonging to Streptomyces were active against Fusarium solani and Botrytis cinerea. The antagonists of the fungi were mainly detected among the representatives of the Cinereus and Helvolo-Flavus.     

Molecular characterization of UV-treated sugar beet somaclones using RFLP markers

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment length polymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants.     

Single nucleotide polymorphism discovery in barley using autoSNPdb

Molecular markers are used to provide the link between genotype and phenotype, for the production of molecular genetic maps and to assess genetic diversity within and between related species. Single nucleotide polymorphisms (SNPs) are the most abundant molecular genetic marker. SNPs can be identified in silico , but care must be taken to ensure that the identified SNPs reflect true genetic variation and are not a result of errors associated with DNA sequencing. The SNP detection method autoSNP has been developed to identify SNPs from sequence data for any species. Confidence in the predicted SNPs is based on sequence redundancy, and haplotype co-segregation scores are calculated for a further independent measure of confidence. We have extended the autoSNP method to produce autoSNPdb, which integrates SNP and gene annotation information with a graphical viewer. We have applied this software to public barley expressed sequences, and the resulting database is available over the Internet. SNPs can be viewed and searched by sequence, functional annotation or predicted synteny with a reference genome, in this case rice. The correlation between SNPs and barley cultivar, expressed tissue type and development stage has been collated for ease of exploration. An average of one SNP per 240 bp was identified, with SNPs more prevalent in the 5' regions and simple sequence repeat (SSR) flanking sequences. Overall, autoSNPdb can provide a wealth of genetic polymorphism information for any species for which sequence data are available.     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

Single nucleotide polymorphism for animal fibre identification

Animal fibres are highly valuable industrial products often adulterated during marketing. Currently, there is no precise method available to identify and differentiate the fibres. In this study, a PCR-RFLP technique was exploited to differentiate cashmere and wool fibres derived from goat and sheep, respectively. The presence of DNA in animal hair shafts has enabled the isolation of DNA from scoured cashmere and wool fibres. The mitochondrial cytochrome b sequences of both species were amplified by PCR using primers designed from conserved regions. The polymorphism observed between the two species was detected by restricting the amplified product by endonucleases viz., BamH1 and Ssp1. The RFLP profile clearly distinguishes the cashmere and wool fibres and this technique can also be exploited to test adulteration in animal fibres qualitatively.