Long Non-Coding RNAs: New Players in Plants.

During the process of growth and development, plants are prone to various biotic and abiotic stresses. They have evolved a variety of strategies to resist the adverse effects of these stresses. lncRNAs (long non-coding RNAs) are a type of less conserved RNA molecules of more than 200 nt (nucleotides) in length. lncRNAs do not code for any protein, but interact with DNA, RNA, and protein to affect transcriptional, posttranscriptional, and epigenetic modulation events. As a new regulatory element, lncRNAs play a critical role in coping with environmental pressure during plant growth and development. This article presents a comprehensive review on the types of plant lncRNAs, the role and mechanism of lncRNAs at different molecular levels, the coordination between lncRNA and miRNA (microRNA) in plant immune responses, the latest research progress of lncRNAs in plant growth and development, and their response to biotic and abiotic stresses. We conclude with a discussion on future direction for the elaboration of the function and mechanism of lncRNAs.

WGCNA Identifies a Comprehensive and Dynamic Gene Co-Expression Network That Associates with Smut Resistance in Sugarcane.

Sugarcane smut is a major fungal disease caused by Sporisorium scitamineum, which seriously reduces the yield and quality of sugarcane. In this study, 36 transcriptome data were collected from two sugarcane genotypes, YT93-159 (resistant) and ROC22 (susceptible) upon S. scitamineum infection. Data analysis revealed 20,273 (12,659 up-regulated and 7614 down-regulated) and 11,897 (7806 up-regulated and 4091 down-regulated) differentially expressed genes (DEGs) in YT93-159 and ROC22, respectively. A co-expression network was then constructed by weighted gene co-expression network analysis (WGCNA), which identified 5010 DEGs in 15 co-expressed gene modules. Four of the 15 modules, namely, Skyblue, Salmon, Darkorange, and Grey60, were significantly associated with smut resistance. The GO and KEGG enrichment analyses indicated that the DEGs involving in these four modules could be enriched in stress-related metabolic pathways, such as MAPK and hormone signal transduction, plant-pathogen interaction, amino acid metabolism, glutathione metabolism, and flavonoid, and phenylpropanoid biosynthesis. In total, 38 hub genes, including six from the Skyblue module, four from the Salmon module, 12 from the Darkorange module, and 16 from the Grey60 module, were screened as candidate hub genes by calculating gene connectivity in the corresponding network. Only 30 hub genes were amplifiable with RT-qPCR, of which 27 were up-regulated upon S. scitamineum infection. The results were consistent with the trend of gene expression in RNA-Seq, suggesting their positive roles in smut resistance. Interestingly, the expression levels of AOX, Cyb5, and LAC were higher in ROC22 than in YT93-159, indicating these three genes may act as negative regulators in response to S. scitamineum infection. This study revealed the transcriptome dynamics in sugarcane challenged by S. scitamineum infection and provided gene targets for smut resistance breeding in sugarcane.

Allelic divergence and cultivar-specific SSR alleles revealed by capillary electrophoresis using fluorescence-labeled SSR markers in sugarcane.

Though sugarcane cultivars (Saccharum spp. hybrids) are complex aneupolyploid hybrids, genetic evaluation and tracking of clone- or cultivar-specific alleles become possible through capillary electrophoresis (CE) using fluorescence-labeled SSR markers. Twenty-four sugarcane cultivars, 12 each from India and the USA, were genetically assessed using 21 fluorescence-labeled polymorphic SSR markers. These markers primed the amplification of 213 alleles. Of these alleles, 161 were common to both Indian and US cultivars, 25 were specific to the Indian cultivars, and 27 were observed only in the US cultivars. Only 10 alleles were monomorphic. A high level of heterozygosity was observed in both Indian (82.4%) and US (91.1%) cultivars resulting in average polymorphism information content (PIC) values of 0.66 and 0.77 and marker index (MI) values of 5.07 and 5.58, respectively. Pearson correlation between PIC and MI was significant in both sets of cultivars (r = 0.58 and 0.69). UPGMA clustering separated cultivars into three distinct clusters at 59% homology level. These results propose the potential utility of six Indian cultivar-specific SSR alleles (mSSCIR3_182, SMC486CG_229, SMC36BUQ_125, mSSCIR74_216, SMC334BS_154, and mSSCIR43_238) in sugarcane breeding, vis a vis transporting CE-based evaluation in clone or variety identity testing, cross fidelity assessments, and genetic relatedness among species of the genus Saccharum and related genera.

Cultivar evaluation and essential test locations identification for sugarcane breeding in China.

The discrepancies across test sites and years, along with the interaction between cultivar and environment, make it difficult to accurately evaluate the differences of the sugarcane cultivars. Using a genotype main effect plus genotype-environment interaction (GGE) Biplot software, the yield performance data of seven sugarcane cultivars in the 8th Chinese National Sugarcane Regional Tests were analyzed to identify cultivars recommended for commercial release. Fn38 produced a high and stable sugar yield. Gn02-70 had the lowest cane yield with high stability. Yz06-407 was a high cane yield cultivar with poor stability in sugar yield. Yz05-51 and Lc03-1137 had an unstable cane yield but relatively high sugar yield. Fn39 produced stable high sugar yield with low and unstable cane production. Significantly different sugar and cane yields were observed across seasons due to strong cultivar-environment interactions. Three areas, Guangxi Chongzuo, Guangxi Baise, and Guangxi Hechi, showed better representativeness of cane yield and sugar content than the other four areas. On the other hand, the areas Guangxi Chongzuo, Yunnan Lincang, and Yunnan Baoshan showed strong discrimination ability, while the areas Guangxi Hechi and Guangxi Liuzhou showed poor discrimination ability. This study provides a reference for cultivar evaluation and essential test locations identification for sugarcane breeding in China.

Photosynthetic and canopy characteristics of different varieties at the early elongation stage and their relationships with the cane yield in sugarcane.

During sugarcane growth, the Early Elongation stage is critical to cane yield formation. In this study, parameters of 17 sugarcane varieties were determined at the Early Elongation stage using CI-301 photosynthesis measuring system and CI-100 digital plant canopy imager. The data analysis showed highly significant differences in leaf area index (LAI), mean foliage inclination angle (MFIA), transmission coefficient for diffused light penetration (TD), transmission coefficient for solar beam radiation penetration (TR), leaf distribution (LD), net photosynthetic rate (PN), transpiration rate (E), and stomatal conductance (GS) among sugarcane varieties. Based on the photosynthetic or canopy parameters, the 17 sugarcane varieties were classified into four categories. Through the factor analysis, nine parameters were represented by three principal factors, of which the cumulative rate of variance contributions reached 85.77%. A regression for sugarcane yield, with relative error of yield fitting less than 0.05, was successfully established: sugarcane yield = -27.19 - 1.69 × PN + 0.17 × E + 90.43 × LAI - 408.81 × LD + 0.0015 × NSH + 101.38 × D (R(2) = 0.928**). This study helps provide a theoretical basis and technical guidance for the screening of new sugarcane varieties with high net photosynthetic rate and ideal canopy structure.

A Genome-Wide Association Study and Genomic Prediction for Fiber and Sucrose Contents in a Mapping Population of LCP 85-384 Sugarcane.

Sugarcane (Saccharum spp. hybrids) is an economically important crop for both sugar and biofuel industries. Fiber and sucrose contents are the two most critical quantitative traits in sugarcane breeding that require multiple-year and multiple-location evaluations. Marker-assisted selection (MAS) could significantly reduce the time and cost of developing new sugarcane varieties. The objectives of this study were to conduct a genome-wide association study (GWAS) to identify DNA markers associated with fiber and sucrose contents and to perform genomic prediction (GP) for the two traits. Fiber and sucrose data were collected from 237 self-pollinated progenies of LCP 85-384, the most popular Louisiana sugarcane cultivar from 1999 to 2007. The GWAS was performed using 1310 polymorphic DNA marker alleles with three models of TASSEL 5, single marker regression (SMR), general linear model (GLM) and mixed linear model (MLM), and the fixed and random model circulating probability unification (FarmCPU) of R package. The results showed that 13 and 9 markers were associated with fiber and sucrose contents, respectively. The GP was performed by cross-prediction with five models, ridge regression best linear unbiased prediction (rrBLUP), Bayesian ridge regression (BRR), Bayesian A (BA), Bayesian B (BB) and Bayesian least absolute shrinkage and selection operator (BL). The accuracy of GP varied from 55.8% to 58.9% for fiber content and 54.6% to 57.2% for sucrose content. Upon validation, these markers can be applied in MAS and genomic selection (GS) to select superior sugarcane with good fiber and high sucrose contents.

A genome-wide association study and genomic prediction for Phakopsora pachyrhizi resistance in soybean.

Soybean brown rust (SBR), caused by Phakopsora pachyrhizi, is a devastating fungal disease that threatens global soybean production. This study conducted a genome-wide association study (GWAS) with seven models on a panel of 3,082 soybean accessions to identify the markers associated with SBR resistance by 30,314 high quality single nucleotide polymorphism (SNPs). Then five genomic selection (GS) models, including Ridge regression best linear unbiased predictor (rrBLUP), Genomic best linear unbiased predictor (gBLUP), Bayesian least absolute shrinkage and selection operator (Bayesian LASSO), Random Forest (RF), and Support vector machines (SVM), were used to predict breeding values of SBR resistance using whole genome SNP sets and GWAS-based marker sets. Four SNPs, namely Gm18_57,223,391 (LOD = 2.69), Gm16_29,491,946 (LOD = 3.86), Gm06_45,035,185 (LOD = 4.74), and Gm18_51,994,200 (LOD = 3.60), were located near the reported P. pachyrhizi R genes, Rpp1, Rpp2, Rpp3, and Rpp4, respectively. Other significant SNPs, including Gm02_7,235,181 (LOD = 7.91), Gm02_7234594 (LOD = 7.61), Gm03_38,913,029 (LOD = 6.85), Gm04_46,003,059 (LOD = 6.03), Gm09_1,951,644 (LOD = 10.07), Gm10_39,142,024 (LOD = 7.12), Gm12_28,136,735 (LOD = 7.03), Gm13_16,350,701(LOD = 5.63), Gm14_6,185,611 (LOD = 5.51), and Gm19_44,734,953 (LOD = 6.02), were associated with abundant disease resistance genes, such as Glyma.02G084100, Glyma.03G175300, Glyma.04g189500, Glyma.09G023800, Glyma.12G160400, Glyma.13G064500, Glyma.14g073300, and Glyma.19G190200. The annotations of these genes included but not limited to: LRR class gene, cytochrome 450, cell wall structure, RCC1, NAC, ABC transporter, F-box domain, etc. The GWAS based markers showed more accuracies in genomic prediction than the whole genome SNPs, and Bayesian LASSO model was the ideal model in SBR resistance prediction with 44.5% ~ 60.4% accuracies. This study aids breeders in predicting selection accuracy of complex traits such as disease resistance and can shorten the soybean breeding cycle by the identified markers.

Identification of QTLs and critical genes related to sugarcane mosaic disease resistance.

Mosaic viral diseases affect sugarcane productivity worldwide. Mining disease resistance-associated molecular markers or genes is a key component of disease resistance breeding programs. In the present study, 285 F1 progeny were produced from a cross between Yuetang 93-159, a moderately resistant variety, and ROC22, a highly susceptible variety. The mosaic disease symptoms of these progenies, with ROC22 as the control, were surveyed by natural infection under 11 different environmental conditions in the field and by artificial infections with a mixed sugarcane mosaic virus (SCMV) and sorghum mosaic virus (SrMV) inoculum. Analysis of consolidated survey data enabled the identification of 29 immune, 55 highly resistant, 70 moderately resistant, 62 susceptible, and 40 highly susceptible progenies. The disease response data and a high-quality SNP genetic map were used in quantitative trait locus (QTL) mapping. The results showed that the correlation coefficients (0.26~0.91) between mosaic disease resistance and test environments were significant (p< 0.001), and that mosaic disease resistance was a highly heritable quantitative trait (H2 = 0.85). Seven mosaic resistance QTLs were located to the SNP genetic map, each QTL accounted for 3.57% ~ 17.10% of the phenotypic variation explained (PVE). Furthermore, 110 pathogen response genes and 69 transcription factors were identified in the QTLs interval. The expression levels of nine genes (Soffic.07G0015370-1P, Soffic.09G0015410-2T, Soffic.09G0016460-1T, Soffic.09G0016460-1P, Soffic.09G0017080-3C, Soffic.09G0018730-3P, Soffic.09G0018730-3C, Soffic.09G0019920-3C and Soffic.03G0019710-2C) were significantly different between resistant and susceptible progenies, indicating their key roles in sugarcane resistance to SCMV and SrMV infection. The seven QTLs and nine genes can provide a certain scientific reference to help sugarcane breeders develop varieties resistant to mosaic diseases.

Genome-wide characterization of sugarcane catalase gene family identifies a ScCAT1 gene associated disease resistance.

In plants, catalase (CAT) mainly scavenges H2O2 from reactive oxygen species (ROS) and regulates the growth and development. So far, genome-wide identification of CAT gene family in Saccharum has not yet been reported. Here, 16 SsCAT genes were identified based on a Saccharum spontaneum genome. They were clustered into three subfamilies, with closer genes sharing similar structures. Most SsCAT proteins contained three conserved amino acids, one active catalytic site, one heme-ligand signature, and three peroxisomal targeting signal 1 (PTS1) sequences. The cis-regulatory element prediction revealed that SsCAT genes were involved in growth and development, and in response to various hormones and stresses. RNA-Seq databases showed that SsCAT genes were differentially expressed in Saccharum tissues and under cold, drought, and Sporisorium scitamineum stresses. The ScCAT1 gene transcript (GenBank accession number KF664183) and relevant CAT activity were up-regulated under S. scitamineum stress. Overexpression of ScCAT1 gene in Nicotiana benthamiana could enhance its resistance to pathogen infection through scavenging of excessive toxic ROS and up-regulated expressions of genes related to hypersensitive response (HR), ROS and salicylic acid (SA) pathways. This study provides comprehensive information for the CAT gene family and sets up a basis for its function identification in sugarcane.

Molecular characterization and phylogenetic analysis of Sugarcane yellow leaf virus isolates from China.

Sugarcane yellow leaf virus (SCYLV) (genus Polerovirus, family Luteoviridae), the causal agent of sugarcane yellow leaf disease (YLD), was first detected in China in 2006. To assess the distribution of SCYLV in the major sugarcane-growing Chinese provinces, leaf samples from 22 sugarcane clones (Saccharum spp. hybrid) showing YLD symptoms were collected and analyzed for infection by the virus using reverse transcription PCR (RT-PCR), quantitative RT-PCR, and immunological assays. A complete genomic sequence (5,879 nt) of the Chinese SCYLV isolate CHN-FJ1 and partial genomic sequences (2,915 nt) of 13 other Chinese SCYLV isolates from this study were amplified, cloned, and sequenced. The genomic sequence of the CHN-FJ1 isolate was found to share a high identity (98.4-99.1 %) with those of the Brazilian (BRA) genotype isolates and a low identity (86.5-86.9 %) with those of the CHN1 and Cuban (CUB) genotype isolates. The genetic diversity of these 14 Chinese SCYLV isolates was assessed along with that of 29 SCYLV isolates of worldwide origin reported in the GenBank database, based on the full or partial genomic sequence. Phylogenetic analysis demonstrated that all the 14 Chinese SCYLV isolates clustered into one large group with the BRA genotype and 12 other reported SCYLV isolates. In addition, five reported Chinese SCYLV isolates were grouped with the Peruvian (PER), CHN1 and CUB genotypes. We therefore speculated that at least four SCYLV genotypes, BRA, PER, CHN1, and CUB, are associated with YLD in China. Interestingly, a 39-nt deletion was detected in the sequence of the CHN-GD3 isolate, in the middle of the ORF1 region adjacent to the overlap between ORF1 and ORF2. This location is known to be one of the recombination breakpoints in the Luteoviridae family.

Genetic identification of SNP markers and candidate genes associated with sugarcane smut resistance using BSR-Seq.

Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases worldwide. In this study, a cross was made between a smut-resistant variety YT93-159 and a smut-susceptible variety ROC22, and 312 progenies were obtained. Two bulks of progenies were then constructed, one consisted of 27 highly smut resistant progenies and the other 24 smut susceptible progenies. Total RNAs of the progenies of each bulk, were pooled and subject to bulked segregant RNA-sequence analysis (BSR-Seq). A total of 164.44 Gb clean data containing 2,341,449 SNPs and 64,999 genes were obtained, 7,295 of which were differentially expressed genes (DEGs). These DEGs were mainly enriched in stress-related metabolic pathways, including carbon metabolism, phenylalanine metabolism, plant hormone signal transduction, glutathione metabolism, and plant-pathogen interactions. Besides, 45,946 high-quality, credible SNPs, a 1.27 Mb region at Saccharum spontaneum chromosome Chr5B (68,904,827 to 70,172,982), and 129 candidate genes were identified to be associated with smut resistance. Among them, twenty-four genes, either encoding key enzymes involved in signaling pathways or being transcription factors, were found to be very closely associated with stress resistance. RT-qPCR analysis demonstrated that they played a positive role in smut resistance. Finally, a potential molecular mechanism of sugarcane and S. scitamineum interaction is depicted that activations of MAPK cascade signaling, ROS signaling, Ca2+ signaling, and PAL metabolic pathway and initiation of the glyoxalase system jointly promote the resistance to S. scitamineum in sugarcane. This study provides potential SNP markers and candidate gene resources for smut resistance breeding in sugarcane.

Genetic analysis of the sugarcane (Saccharum spp.) cultivar 'LCP 85-384'. I. Linkage mapping using AFLP, SSR, and TRAP markers.

Sugarcane hybrids are complex aneu-polyploids (2n = 100-130) derived from inter-specific hybridization between ancestral polyploid species, namely S. officinarum L. and S. spontaneum L. Efforts to understand the sugarcane genome have recently been enhanced through the use of new molecular marker technologies. A framework genetic linkage map of Louisiana's popular cultivar LCP 85-384 was constructed using the selfed progeny and based on polymorphism derived from 64 AFLP, 19 SSR and 12 TRAP primer pairs. Of 1,111 polymorphic markers detected, 773 simplex (segregated in 3:1 ratio) and 182 duplex (segregate in 77:4 ratio) markers were used to construct the map using a LOD value of ≥ 4.0 and recombination threshold of 0.44. The genetic distances between pairs of markers linked in the coupling phase was computed using the Kosambi mapping function. Of the 955 markers, 718 simplex and 66 duplex markers were assigned to 108 co-segregation groups (CGs) with a cumulative map length of 5,617 cM and a density of 7.16 cM per marker. Fifty-five simplex and 116 duplex markers remained unlinked. With an estimated genome size of 12,313 cM for LCP 85-384, the map covered approximately 45.6% of the genome. Forty-four of the 108 CGs were assigned into 9 homo(eo)logous groups (HGs) based on information from locus-specific SSR and duplex markers, and repulsion phase linkages detected between CGs. Meiotic behavior of chromosomes in cytogenetic studies and repulsion phase linkage analysis between CGs in this study inferred the existence of strong preferential chromosome pairing behavior in LCP 85-384. This framework map marks an important beginning for future mapping of QTLs associated with important agronomic traits in the Louisiana sugarcane breeding programs.

Sugarcane Mosaic Disease: Characteristics, Identification and Control.

Mosaic is one of the most important sugarcane diseases, caused by single or compound infection of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and/or Sugarcane streak mosaic virus (SCSMV). The compound infection of mosaic has become increasingly serious in the last few years. The disease directly affects the photosynthesis and growth of sugarcane, leading to a significant decrease in cane yield and sucrose content, and thus serious economic losses. This review covers four aspects of sugarcane mosaic disease management: first, the current situation of sugarcane mosaic disease and its epidemic characteristics; second, the pathogenicity and genetic diversity of the three viruses; third, the identification methods of mosaic and its pathogen species; and fourth, the prevention and control measures for sugarcane mosaic disease and potential future research focus. The review is expected to provide scientific literature and guidance for the effective prevention and control of mosaic through resistance breeding in sugarcane.

Utilization of a Sugarcane100K Single Nucleotide Polymorphisms Microarray-Derived High-Density Genetic Map in Quantitative Trait Loci Mapping and Function Role Prediction of Genes Related to Chlorophyll Content in Sugarcane.

Chlorophyll is the most important pigment for plant photosynthesis that plays an important role in crop growth and production. In this study, the chlorophyll content trait was explored to improve sugarcane yield. Two hundred and eighty-five F1 progenies from the cross YT93-159 × ROC22 with significantly different chlorophyll contents were included as test materials. The chlorophyll content of the +1 leaves during elongation phase was measured using a SPAD-502 meter through a three-crop cycle (plant cane, first ratoon, and second ratoon). Linkage analysis was conducted on a high-density genetic map constructed based on the sugarcane 100K SNP chip. In addition, Fv/Fm, plant height, stalk diameter, brix data were collected on plant cane during the elongation and maturation phases. The results showed that the +1 leaf SPAD values, which can be used as an important reference to evaluate the growth potential of sugarcane, were significantly and positively correlated with the Fv/Fm during elongation phase, as well as with plant height, stalk diameter, and brix during maturity phase (P < 0.01). The broad sense heritability (H 2) of the chlorophyll content trait was 0.66 for plant cane crop, 0.67 for first ratoon crop, and 0.73 for second ratoon crop, respectively, indicating that this trait was mainly controlled by genetic factors. Thirty-one quantitative trait loci (QTL) were detected by QTL mapping. Among them, a major QTL, qCC-R1, could account for 12.95% of phenotypic variation explained (PVE), and the other 30 minor QTLs explained 2.37-7.99% PVE. Twenty candidate genes related to chlorophyll content were identified in the QTLs plus a 200-Kb extension region within either sides, of which four were homologous genes involved in the chlorophyll synthesis process and the remaining 16 played a certain role in chlorophyll catabolic pathway, chloroplast organization, or photosynthesis. These results provide a theoretical reference for analyzing the genetic mechanism of chlorophyll synthesis and subsequent improvement of photosynthetic characteristics in sugarcane.

[In-situ Phosphorus Removal Activity and Impact of the Organic Matter Concentration on Denitrifying Phosphorus Removal in Sludge Aggregates].

In this work, the redox potential, dissolved oxygen, and phosphate microelectrodes were used to quantitatively study the in-situ activity of dephosphorization bacteria and the impact of the organic matter concentration on denitrifying phosphorus removal in sludge aggregates in a sequencing batch reactor. The results showed that the maximum net volume release rate of phosphorus was 3.29 mg·(cm3·h)-1 in the initial anaerobic sludge aggregates, which was approximately 3 times the maximum net volume uptake rate of phosphorus at the initial anoxic stage. The release rate of phosphorus clearly decreased at the final anaerobic stage, and the maximum net volume release rate of phosphorus was only half of that at the initial anaerobic stage. At the final anoxic stage, the maximum net volume uptake rate of phosphorus decreased to 0.14 mg·(cm3·h)-1, and the phenomenon of secondary phosphorus release occurred in the deep area below 1800 µm. When the concentration of COD decreased from 350 mg·L-1 to 250 mg·L-1 and 150 mg·L-1, the maximum net volume release rate of phosphorus of dephosphorization bacteria decreased from 3.27 mg·(cm3·h)-1 to 2.44 mg·(cm3·h)-1 and 2.01 mg·(cm3·h)-1, respectively, and the rapid uptake area of phosphorus narrowed to the surface of the sludge aggregates.

[Effect of Ozone Dosage on Sludge Settleability and Biological Nutrient Removal in SBR System].

The effect of ozone dosage on sludge settleability and biological nutrient removal performance in a sequencing batch reactor was investigated by inoculating the bulking sludge with the SVI of 280 mL·g-1 from a wastewater treatment plant in winter. The filamentous mycelium was interrupted, and the SVI was decreased to 125 mL·g-1 after ozone dosage with a low concentration of 0.085 g·g-1(O3/MLSS) for 20 days, which indicated the disappearance of the sludge bulking. The performance of nitrification and phosphorus removal efficiency was not affected obviously. However, the sludge settleability deteriorated with a high dosage of ozone, and the phosphorus removal efficiency was decreased to around 60%. Further study showed that PS/PN had a positive correlation with SVI with the correlation coefficient of 0.9381, which can be used to characterize sludge settleability. A low ozone dosage not only interrupted the filamentous mycelium, but it also affected the content and composition of the EPS, which led to improved settleability.

Genetic diversity and population structure analysis of Saccharum and Erianthus genera using microsatellite (SSR) markers.

In order to understand the genetic diversity and structure within and between the genera of Saccharum and Erianthus, 79 accessions from five species (S. officinarum, S. spontaneum, S. robustum, S. barberi, S. sinense), six accessions of E. arundinaceus, and 30 Saccharum spp. hybrids were analyzed using 21 pairs of fluorescence-labeled highly poloymorphic SSR primers and a capillary electrophoresis (CE) detection system. A total of 167 polymorphic SSR alleles were identified by CE with a mean value of polymorphic information content (PIC) of 0.92. Genetic diversity parameters among these 115 accessions revealed that Saccharum spp. hybrids were more diverse than those of Saccharum and Erianthus species. Based on the SSR data, the 115 accessions were classified into seven main phylogenetic groups, which corresponded to the Saccharum and Erianthus genera through phylogenetic analysis and principle component analysis (PCA). We propose that seven core SSR primer pairs, namely, SMC31CUQ, SMC336BS, SMC597CS, SMC703BS, SMC24DUQ, mSSCIR3, and mSSCIR43, may have a wide appicability in genotype identification of Saccharum species and Saccharum spp. hybrids. Thus, the information from this study contibites to manage sugarcane genetic resources.

High-throughput procedure for single pollen grain collection and polymerase chain reaction in plants.

Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.

Heterologous expression of a Glyoxalase I gene from sugarcane confers tolerance to several environmental stresses in bacteria.

Glyoxalase I belongs to the glyoxalase system that detoxifies methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates. The concentration of MG increases rapidly under stress conditions. In this study, a novel glyoxalase I gene, designated as SoGloI was identified from sugarcane. SoGloI had a size of 1,091 bp with one open reading frame (ORF) of 885 bp encoding a protein of 294 amino acids. SoGloI was predicted as a Ni2+-dependent GLOI protein with two typical glyoxalase domains at positions 28-149 and 159-283, respectively. SoGloI was cloned into an expression plasmid vector, and the Trx-His-S-tag SoGloI protein produced in Escherichia coli was about 51 kDa. The recombinant E. coli cells expressing SoGloI compared to the control grew faster and tolerated higher concentrations of NaCl, CuCl2, CdCl2, or ZnSO4. SoGloI ubiquitously expressed in various sugarcane tissues. The expression was up-regulated under the treatments of NaCl, CuCl2, CdCl2, ZnSO4 and abscisic acid (ABA), or under simulated biotic stress conditions upon exposure to salicylic acid (SA) and methyl jasmonate (MeJA). SoGloI activity steadily increased when sugarcane was subjected to NaCl, CuCl2, CdCl2, or ZnSO4 treatments. Sub-cellular observations indicated that the SoGloI protein was located in both cytosol and nucleus. These results suggest that the SoGloI gene may play an important role in sugarcane's response to various biotic and abiotic stresses.

A Comparative Study of Three Detection Techniques for Leifsonia xyli Subsp. xyli, the Causal Pathogen of Sugarcane Ratoon Stunting Disease.

The ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most economically devastating diseases impacting sugarcane. RSD causes significant yield losses and variety degradation. Diagnosis of RSD is challenging because it does not exhibit any discernible internal and external symptoms. Moreover, the Lxx bacteria are very small and difficult to isolate, cultivate, and detect. In this study, conventional polymerase chain reaction (PCR), real-time quantitative PCR (RT-qPCR), and Lxx-loop-mediated isothermal amplification (Lxx-LAMP) were utilized to specifically detect the presence of Lxx pathogens in the juice from Lxx-infected sugarcane stalks and an Lxx-pMD18-T recombinant plasmid. The results showed that Lxx was a highly specific causal pathogen for RSD. All three techniques provided great reproducibility, while Lxx-LAMP had the highest sensitivity. When the DNA extract from Lxx-infected sugarcane juice was used as a template, Lxx-LAMP was 10 and 100 times more sensitive than RT-qPCR and conventional PCR, respectively. When the Lxx-pMD18-T recombinant plasmid was used as a template, Lxx-LAMP was as sensitive as RT-qPCR but was 10 times more sensitive than conventional PCR. Based on the Lxx-LAMP detection system established, adding 0.4 µM loop primers (LF/LP) can accelerate the reaction and reduce the total time required. In addition, the optimal amount of Bst DNA polymerase for Lxx-LAMP reactions was determined to be 6.0 U. The results provide technical support for the detection of RSD Lxx pathogen that will help manage sugarcane RSD.