The availability of well-assembled genome sequences and reduced sequencing costs have enabled the resequencing of many additional accessions in several crops, thus facilitating the rapid discovery and development of simple sequence repeat (SSR) markers. Although the genome sequence of inbred spinach line Sp75 is available, previous efforts have resulted in a limited number of useful SSR markers. Identification of additional polymorphic SSR markers will support genetics and breeding research in spinach. This study aimed to use the available genomic resources to mine and catalog a large number of polymorphic SSR markers. A search for SSR loci on six chromosome sequences of spinach line Sp75 using GMATA identified a total of 42,155 loci with repeat motifs of two to six nucleotides in the Sp75 reference genome. Whole-genome sequences (30x) of additional 21 accessions were aligned against the chromosome sequences of the reference genome and in silico genotyped using the HipSTR program by comparing and counting repeat numbers variation across the SSR loci among the accessions. The HipSTR program generated SSR genotype data were filtered for monomorphic and high missing loci, and a final set of the 5986 polymorphic SSR loci were identified. The polymorphic SSR loci were present at a density of 12.9 SSRs/Mb and were physically mapped. Out of 36 randomly selected SSR loci for validation, two failed to amplify, while the remaining were all polymorphic in a set of 48 spinach accessions from 34 countries. Genetic diversity analysis performed using the SSRs allele score data on the 48 spinach accessions showed three main population groups. This strategy to mine and develop polymorphic SSR markers by a comparative analysis of the genome sequences of multiple accessions and computational genotyping of the candidate SSR loci eliminates the need for laborious experimental screening. Our approach increased the efficiency of discovering a large set of novel polymorphic SSR markers, as demonstrated in this report.
Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) has become a key pest in the sugarcane (Saccharum officinarum) fields of center-south Brazil. Although some control technologies have shown to be efficient, the damage promoted by this spittlebug species and its interaction with sugarcane are poorly characterized. At high infestation levels the symptoms are similar to those of severe water restriction. This work was conducted to determine whether the stress promoted by spittlebug infestation can be measured in terms of free proline accumulation. The water restriction tolerance of two sugarcane genotypes was also compared. Two experiments were set up in a greenhouse and arranged in a completely randomized design in a 2 × 2 × 4 factorial, with two cultivars (SP80-1816 and RB72454), two stress levels (control and ten nymphs per plant or 50% water restriction), and four sampling dates. The water deficit caused by spittlebug nymphs sucking xylem sap does not result in proline accumulation, illustrating that there are different mechanisms to sense when the water deficit is caused by insect feeding or water potential variation in root cells. The cultivar RB72454 accumulates more free-proline, and the dry mass accumulation and stalk growth are less affected in this cultivar under water restriction. The levels of compatible solutes probably cannot be used to measure spittlebug infestation stress in sugarcane and RB72454 is more tolerant to water shortage than SP80-1816. Key words: Mahanarva fimbriolata, Saccharum spp., proline accumulation, water deficit ACÚMULO DE PROLINA EM CANA-DE-AÇÚCAR SOB ESTRESSE HÍDRICO OU SUBMETIDA AO ATAQUE DE CIGARRINHA-DAS-RAÍZESRESUMO: A cigarrinha-das-raízes Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) tornou-se uma praga importante da cultura da cana-de-açúcar na região centro-sul do Brasil. Estratégias de controle têm sido desenvolvidas, mas os danos promovidos por essa praga e sua interação com a cana-de-açúcar ainda são pouco caracterizados. Em altos níveis de infestação da praga, os sintomas nas plantas são muito semelhantes à restrição hídrica severa. Este trabalho foi conduzido com o objetivo de determinar se o estresse promovido pela infestação de ninfas de cigarrinha-das-raízes resulta em acúmulo de prolina livre, além de comparar dois genótipos de cana-de-açúcar quanto à tolerância ao déficit hídrico. Dois experimentos foram conduzidos em delineamento inteiramente casualizado, em esquema fatorial 2 × 2 × 4 com duas cultivares (SP80-1816 e RB72454), dois níveis de estresse (controle e dez ninfas por planta ou 50% de restrição hídrica) e quatro épocas. O déficit hídrico promovido por M. fimbriolata não resultou em acúmulo de prolina, ilustrando a existência de mecanismos distintos para a percepção do estresse hídrico promovido pela praga e pela variação no potencial osmótico nas células das raízes. A cultivar RB72454 acumula maiores teores de prolina livre, e o acúmulo de massa seca e o crescimento dos colmos são menos afetados nessa cultivar sob restrição hídric...
This study was designed to characterize morphological, physiological and molecular responses of sugarcane genotypes to a simulated water deficit stress. Two genotypes (TSP05-4: Drought-tolerant; TCP02-4589: Drought-sensitive) were subjected to a 20-day water deficit treatment and an 8-day recovery period. Leaf photosynthesis (P n), transpiration rate (E), stomatal conductance (G s), leaf greenness index (SPAD) and variable-to-maximum chlorophyll a fluorescence ratio (F v /F m), were evaluated before, during and after water deficit. Root-to-shoot ratio (R/S), stalk height (SH), diameter (SD) and stalk weight (SW) were evaluated at the end of the experiment. Real-time RT-PCR confirmed seven differentially-expressed transcript-derived fragments (TDFs) identified by cDNA-AFLP. P n rates were similar between the genotypes under well-watered conditions. However, under water deficit, TSP05-4 had higher P n rates. SPAD, F v /F m and R/S were also generally higher in TSP05-4, regardless of soil moisture status. Water deficit-induced reductions in SH and SW were greater in TCP02-4589 than in TSP05-4. Three TDFs showing sequence similarities to genes encoding a putative expressed pentatricopeptide, a protein kinase CK2 regulatory subunit CK2β3, and a glucose-6phosphate/phosphate translocator 2 were identified in TCP02-4589. One TDF similar to a droughtinducible protein was identified in TSP05-4. Recovery of physiological processes and gene expression patterns to the water stress levels was fast.
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