Zinc finger knuckle genes are associated with tolerance to drought and dehydration in chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is a very important food legume and needs improved drought tolerance for higher seed production in dry environments. The aim of this study was to determine diversity and genetic polymorphism in zinc finger knuckle genes with CCHC domains and their functional analysis for practical improvement of chickpea breeding. Two CaZF-CCHC genes, Ca04468 and Ca07571, were identified as potentially important candidates associated with plant responses to drought and dehydration. To study these genes, various methods were used including Sanger sequencing, DArT (Diversity array technology) and molecular markers for plant genotyping, gene expression analysis using RT-qPCR, and associations with seed-related traits in chickpea plants grown in field trials. These genes were studied for genetic polymorphism among a set of chickpea accessions, and one SNP was selected for further study from four identified SNPs between the promoter regions of each of the two genes. Molecular markers were developed for the SNP and verified using the ASQ and CAPS methods. Genotyping of parents and selected breeding lines from two hybrid populations, and SNP positions on chromosomes with haplotype identification, were confirmed using DArT microarray analysis. Differential expression profiles were identified in the parents and the hybrid populations under gradual drought and rapid dehydration. The SNP-based genotypes were differentially associated with seed weight per plant but not with 100 seed weight. The two developed and verified SNP molecular markers for both genes, Ca04468 and Ca07571, respectively, could be used for marker-assisted selection in novel chickpea cultivars with improved tolerance to drought and dehydration.

SNP Genotyping with Amplifluor-Like Method.

For SNP genotyping, amplification of fluorescence (Amplifluor) is a popular and actively developing method in the plant sciences. The "Amplifluor-like" is a "home-made" modification of the original commercial Amplifluor method. Amplifluor-like genotyping requires two essential components: (1) two allele-specific forward primers targeting the SNP site with one common reverse primer; and (2) a universal part with two non-allele-specific molecular probes containing one of the two used fluorophores and a quencher. Allele discrimination is based on the fluorescence score, where the dominance of one dye over the other confirms the presence of each specific SNP allele. The Amplifluor-like method is similar to commercial KASP and original Amplifluor methods but is much cheaper because all components can be ordered as regular and modified oligos. The easily adaptable Amplifluor-like method can be modified by any researcher to make it suitable for available instruments, reagents and conditions in low-budget laboratories for SNP genotyping of any plant species with identified genetic polymorphism.

Height to first pod: A review of genetic and breeding approaches to improve combine harvesting in legume crops.

Height from soil at the base of plant to the first pod (HFP) is an important trait for mechanical harvesting of legume crops. To minimise the loss of pods, the HFP must be higher than that of the blades of most combine harvesters. Here, we review the genetic control, morphology, and variability of HFP in legumes and attempt to unravel the diverse terminology for this trait in the literature. HFP is directly related to node number and internode length but through different mechanisms. The phenotypic diversity and heritability of HFP and their correlations with plant height are very high among studied legumes. Only a few publications describe a QTL analysis where candidate genes for HFP with confirmed gene expression have been mapped. They include major QTLs with eight candidate genes for HFP, which are involved in auxin transport and signal transduction in soybean [Glycine max (L.) Merr.] as well as MADS box gene SOC1 in Medicago trancatula, and BEBT or WD40 genes located nearby in the mapped QTL in common bean (Phaseolus vulgaris L.). There is no information available about simple and efficient markers associated with HFP, which can be used for marker-assisted selection for this trait in practical breeding, which is still required in the nearest future. To our best knowledge, this is the first review to focus on this significant challenge in legume-based cropping systems.

Expression of Specific Alleles of Zinc-Finger Transcription Factors, HvSAP8 and HvSAP16, and Corresponding SNP Markers, Are Associated with Drought Tolerance in Barley Populations.

Two genes, HvSAP8 and HvSAP16, encoding Zinc-finger proteins, were identified earlier as active in barley plants. Based on bioinformatics and sequencing analysis, six SNPs were found in the promoter regions of HvSAP8 and one in HvSAP16, among parents of two barley segregating populations, Granal × Baisheshek and Natali × Auksiniai-2. ASQ and Amplifluor markers were developed for HvSAP8 and HvSAP16, one SNP in each gene, and in each of two populations, showing simple Mendelian segregation. Plants of F6 selected breeding lines and parents were evaluated in a soil-based drought screen, revealing differential expression of HvSAP8 and HvSAP16 corresponding with the stress. After almost doubling expression during the early stages of stress, HvSAP8 returned to pre-stress level or was strongly down-regulated in plants with Granal or Baisheshek genotypes, respectively. For HvSAP16 under drought conditions, a high expression level was followed by either a return to original levels or strong down-regulation in plants with Natali or Auksiniai-2 genotypes, respectively. Grain yield in the same breeding lines and parents grown under moderate drought was strongly associated with their HvSAP8 and HvSAP16 genotypes. Additionally, Granal and Natali genotypes with specific alleles at HvSAP8 and HvSAP16 were associated with improved performance under drought via higher 1000 grain weight and more shoots per plant, respectively.

Modified "Allele-Specific qPCR" Method for SNP Genotyping Based on FRET.

The proposed method is a modified and improved version of the existing "Allele-specific q-PCR" (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

Salt-induced expression of intracellular vesicle trafficking genes, CaRab-GTP, and their association with Na+ accumulation in leaves of chickpea (Cicer arietinum L.).

BACKGROUND: Chickpea is an important legume and is moderately tolerant to salinity stress during the growing season. However, the level and mechanisms for salinity tolerance can vary among accessions and cultivars. A large family of CaRab-GTP genes, previously identified in chickpea, is homologous to intracellular vesicle trafficking superfamily genes that play essential roles in response to salinity stress in plants. RESULTS: To determine which of the gene family members are involved in the chickpea salt response, plants from six selected chickpea accessions (Genesis 836, Hattrick, ICC12726, Rupali, Slasher and Yubileiny) were exposed to salinity stress and expression profiles resolved for the major CaRab-GTP gene clades after 5, 9 and 15 days of salt exposure. Gene clade expression profiles (using degenerate primers targeting all members of each clade) were tested for their relationship to salinity tolerance measures, namely plant biomass and Na+ accumulation. Transcripts representing 11 out of the 13 CaRab clades could be detected by RT-PCR, but only six (CaRabA2, -B, -C, -D, -E and -H) could be quantified using qRT-PCR due to low expression levels or poor amplification efficiency of the degenerate primers for clades containing several gene members. Expression profiles of three gene clades, CaRabB, -D and -E, were very similar across all six chickpea accessions, showing a strongly coordinated network. Salt-induced enhancement of CaRabA2 expression at 15 days showed a very strong positive correlation (R2 = 0.905) with Na+ accumulation in leaves. However, salinity tolerance estimated as relative plant biomass production compared to controls, did not correlate with Na+ accumulation in leaves, nor with expression profiles of any of the investigated CaRab-GTP genes. CONCLUSION: A coordinated network of CaRab-GTP genes, which are likely involved in intracellular trafficking, are important for the salinity stress response of chickpea plants.

Intracellular Vesicle Trafficking Genes, RabC-GTP, Are Highly Expressed Under Salinity and Rapid Dehydration but Down-Regulated by Drought in Leaves of Chickpea (Cicer arietinum L.).

Intracellular vesicle trafficking genes, Rab, encoding small GTP binding proteins, have been well studied in medical research, but there is little information concerning these proteins in plants. Some sub-families of the Rab genes have not yet been characterized in plants, such as RabC - otherwise known as Rab18 in yeast and animals. Our study aimed to identify all CaRab gene sequences in chickpea (Cicer arietinum L.) using bioinformatics approaches, with a particular focus on the CaRabC gene sub-family since it featured in an SNP database. Five isoforms of the CaRabC gene were identified and studied: CaRabC-1a, -1b, -1c, -2a and -2a∗ . Six accessions of both Desi and Kabuli ecotypes, selected from field trials, were tested for tolerance to abiotic stresses, including salinity, drought and rapid dehydration and compared to plant growth under control conditions. Expression analysis of total and individual CaRabC isoforms in leaves of control plants revealed a very high level of expression, with the greatest contribution made by CaRabC-1c. Salinity stress (150 mM NaCl, 12 days in soil) caused a 2-3-fold increased expression of total CaRabC compared to controls, with the highest expression in isoforms CaRabC-1c, -2a∗ and -1a. Significantly decreased expression of all five isoforms of CaRabC was observed under drought (12 days withheld water) compared to controls. In contrast, both total CaRabC and the CaRabC-1a isoform showed very high expression (up-to eight-fold) in detached leaves over 6 h of dehydration. The results suggest that the CaRabC gene is involved in plant growth and response to abiotic stresses. It was highly expressed in leaves of non-stressed plants and was down-regulated after drought, but salinity and rapid dehydration caused up-regulation to high and very high levels, respectively. The isoforms of CaRabC were differentially expressed, with the highest levels recorded for CaRabC-1c in controls and under salinity stress, and for CaRabC-1a - in rapidly dehydrated leaves. Genotypic variation in CaRabC-1a, comprising eleven SNPs, was found through sequencing of the local chickpea cultivar Yubileiny and germplasm ICC7255 in comparison to the two fully sequenced reference accessions, ICC4958 and Frontier. Amplifluor-like markers based on one of the identified SNPs in CaRabC-1a were designed and successfully used for genotyping chickpea germplasm.

Genes Encoding Transcription Factors TaDREB5 and TaNFYC-A7 Are Differentially Expressed in Leaves of Bread Wheat in Response to Drought, Dehydration and ABA.

Two groups of six spring bread wheat varieties with either high or low grain yield under the dry conditions of Central and Northern Kazakhstan were selected for analysis. Experiments were set up with the selected wheat varieties in controlled environments as follows: (1) slowly progressing drought imposed on plants in soil, (2) rapid dehydration of whole plants grown in hydroponics, (3) dehydration of detached leaves, and (4) ABA treatment of whole plants grown in hydroponics. Representatives of two different families of transcription factors (TFs), TaDREB5 and TaNFYC-A7, were found to be linked to yield-under-drought using polymorphic Amplifluor-like SNP marker assays. qRT-PCR revealed differing patterns of expression of these genes in the leaves of plants subjected to the above treatments. Under drought, TaDREB5 was significantly up-regulated in leaves of all high-yielding varieties tested and down-regulated in all low-yielding varieties, and the level of expression was independent of treatment type. In contrast, TaNFYC-A7 expression levels showed different responses in the high- and low-yield groups of wheat varieties. TaNFYC-A7 expression under dehydration (treatments 2 and 3) was higher than under drought (treatment 1) in all high-yielding varieties tested, while in all low-yielding varieties the opposite pattern was observed: the expression levels of this gene under drought were higher than under dehydration. Rapid dehydration of detached leaves and intact wheat plants grown in hydroponics produced similar changes in gene expression. ABA treatment of whole plants caused rapid stomatal closure and a rise in the transcript level of both genes during the first 30 min, which decreased 6 h after treatment. At this time-point, expression of TaNFYC-A7 was again significantly up-regulated compared to untreated controls, while TaDREB5 returned to its initial level of expression. These findings reveal significant differences in the transcriptional regulation of two drought-responsive and ABA-dependent TFs under slowly developing drought and rapid dehydration of wheat plants. The results obtained suggest that correlation between grain yield in dry conditions and TaNFYC-A7 expression levels in the examined wheat varieties is dependent on the length of drought development and/or strength of drought; while in the case of TaDREB5, no such dependence is observed.