Haplotypes of ATP-Binding Cassette CaABCC6 in Chickpea from Kazakhstan Are Associated with Salinity Tolerance and Leaf Necrosis via Oxidative Stress.

Salinity tolerance was studied in chickpea accessions from a germplasm collection and in cultivars from Kazakhstan. After NaCl treatment, significant differences were found between genotypes, which could be arranged into three groups. Those that performed poorest were found in group 1, comprising five ICC accessions with the lowest chlorophyll content, the highest leaf necrosis (LN), Na+ accumulation, malondialdehyde (MDA) content, and a low glutathione ratio GSH/GSSG. Two cultivars, Privo-1 and Tassay, representing group 2, were moderate in these traits, while the best performance was for group 3, containing two other cultivars, Krasnokutsky-123 and Looch, which were found to have mostly green plants and an exact opposite pattern of traits. Marker-trait association (MTA) between 6K DArT markers and four traits (LN, Na+, MDA, and GSH/GSSG) revealed the presence of four possible candidate genes in the chickpea genome that may be associated with the three groups. One gene, ATP-binding cassette, CaABCC6, was selected, and three haplotypes, A, D1, and D2, were identified in plants from the three groups. Two of the most salt-tolerant cultivars from group 3 were found to have haplotype D2 with a novel identified SNP. RT-qPCR analysis confirmed that this gene was strongly expressed after NaCl treatment in the parental- and breeding-line plants of haplotype D2. Mass spectrometry of seed proteins showed a higher accumulation of glutathione reductase and S-transferase, but not peroxidase, in the D2 haplotype. In conclusion, the CaABCC6 gene was hypothesized to be associated with a better response to oxidative stress via glutathione metabolism, while other candidate genes are likely involved in the control of chlorophyll content and Na+ accumulation.

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.

SSR Genotyping and Marker-Trait Association with Yield Components in a Kazakh Germplasm Collection of Chickpea (Cicer arietinum L.).

Genetic diversity and marker-trait association with yield-related components were assessed in 39 chickpea accessions from a germplasm collection with either spring or autumn-sown seeds in South-Eastern Kazakhstan. Chickpea accessions originated from Azerbaijan, Germany, Kazakhstan, Moldova, Russia, Türkiye, Ukraine, Syria, and the International Center for Agricultural Research in the Dry Areas (ICARDA). Eleven SSR markers were used for molecular genotyping. Yield and yield components were evaluated in nine traits in experiments with spring and autumn seed sowing. The number of alleles of polymorphic markers varied from 2 to 11. The greatest polymorphism was found in the studied chickpea genotypes using SSR marker TA22 (11 alleles), while NCPGR6 and NCPGR12 markers were monomorphic. In the studied chickpea accessions, unique alleles of the SSR loci TA14, TA46, TA76s, and TA142 were found that were not previously described by other authors. An analysis of correlation relationships between yield-related traits in chickpea revealed the dependence of yield on plant height, branching, and the setting of a large number of beans. These traits showed maximal values in experiments with chickpea plants from autumn seed sowing. An analysis of the relationship between the SSR markers applied and morphological yield-related traits revealed several informative markers associated with important traits, such as plant height, height to first pod, number of branches, number of productive nodes, number of pods per plant, hundred seed weight, seed weight per plant, and seed yield.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Marker-Assisted Selection for Early Maturing E Loci in Soybean Yielded Prospective Breeding Lines for High Latitudes of Northern Kazakhstan.

The photoperiodic sensitivity of soybean (Glycine max L.) is one of the limiting factors affecting plant growth and yield. At higher latitudes, early flowering and maturity with neutral reaction to photoperiods are required for adaptation of soybean plants to long-day conditions. Currently, the production and distribution of new varieties of soybeans adapted to widespread agricultural regions in northern Kazakhstan is in strong demand. Eleven soybean hybrid populations were obtained from crosses between 17 parents with four maturity groups, MG 000, 00, 0, and I. Marker-assisted selection (MAS) was assessed for suitable SSR markers and successfully applied for genes E1, E3, E4, and E7, targeting homozygous genotypes with recessive alleles. The identified and selected genotypes were propagated and tested in the conditions of 53° N latitude in the Kostanay region of northern Kazakhstan. Finally, 20 early maturing F4 breeding lines were identified and developed with genotypes e1 e3 E4 e7, e1 E3 E4 e7, and e1 E3 e4 e7, all completing their growth period within 92-102 days. These breeding lines were developed by MAS and should provide very prospective superior varieties of soybean for northern Kazakhstan through a strategy that may be very helpful to other countries with high latitudes.

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.

Modified Allele-Specific qPCR (ASQ) Genotyping.

The allele-specific qPCR (ASQ) method for SNP (single nucleotide polymorphism) detection is based on the FRET (fluorescence resonance energy transfer) system, a system using position-dependent fluorescent dyes and quenches. The modified ASQ method requires two separate components: (1) the allele-specific part, two AS primers targeting the SNP with identity in the penultimate positions at the 3'-end and specific tags in the 5'-end, and (2) the universal part, two universal probes (UPs) with corresponding tags and different fluorescent dyes in the 5'-end and a single common universal probe with a quencher in the 3'-ends (Uni-Q), complementary to all UP tags. There are two major variations of the ASQ method, with either short 4-bp tags (variant A) or longer 6-bp tags (variant B), both of which have been successfully used for SNP genotyping in plants. The modified ASQ method is much cheaper compared to other similar FRET-based methods because the most expensive parts, the universal probes, have a short and linear structure, where fluorophores and quenchers are located in the ends but not incorporated inside of the sequences.

Molecular-Genetic Basis of Plant Breeding.

Traditional plant breeding can be improved significantly through the application of molecular and genetic approaches [...].

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.

Organ-specific expression of genes involved in iron homeostasis in wheat mutant lines with increased grain iron and zinc content.

Background: Iron deficiency is a well-known nutritional disorder, and the imbalance of trace-elements, specifically iron, is the most common nutrient deficiency of foods across the world, including in Kazakhstan. Wheat has significant nutritional relevance, especially in the provision of iron, however many bread wheat varieties have low iron despite the need for human nourishment. In this study, the expression profiles of wheat homologous genes related to iron homeostasis were investigated. The work resulted in the development of two new M5 mutant lines of spring bread wheat through gamma-irradiation (200 Gy) with higher grain iron and zinc content, lower phytic acid content, and enhanced iron bioavailability compared to the parent variety. Mutant lines were also characterized by higher means of yield associated traits such as grain number per main spike, grain weight per main spike, grain weight per plant, and thousand-grain weight. Methods: The homologous genes of bread wheat from several groups were selected for gene expression studies exploring the tight control of iron uptake, translocation rate and accumulation in leaves and roots, and comprised the following: (1) S-adenosylmethionine synthase (SAMS), nicotianamine synthase (NAS1), nicotianamine aminotransferase (NAAT), deoxymugineic acid synthetase (DMAS), involved in the synthesis and release of phytosiderophores; (2) transcription factor basic helix-loop-helix (bHLH); (3) transporters of mugineic acid (TOM), involved in long-distance iron transport; (4) yellow stripe-like (YSlA), and the vacuolar transporter (VIT2), involved in intracellular iron transport and storage; and lastly (5) natural resistance-associated macrophage protein (NRAMP) and ferritin (Fer1A). Results: The wheat homologous genes TaSAMS, TaNAS1, and TaDMAS, were significantly up-regulated in the roots of both mutant lines by 2.1-4.7-fold compared to the parent variety. The combined over-expression of TaYSlA and TaVIT2 was also revealed in the roots of mutant lines by 1.3-2.7-fold. In one of the mutant lines, genes encoding intracellular iron transport and storage genes TaNRAMP and TaFer1A-D showed significant up-regulation in roots and leaves (by 1.4- and 3.5-fold, respectively). The highest expression was recorded in the transcription factor TabHLH, which was expressed 13.1- and 30.2-fold in the roots of mutant lines. Our research revealed that genotype-dependent and organ-specific gene expression profiles can provide new insights into iron uptake, translocation rate, storage, and regulation in wheat which aid the prioritization of gene targets for iron biofortification and bioavailability.

Developments and prospects for doubled haploid wheat.

Doubled haploid production is a valuable biotechnology that can accelerate the breeding of new wheat varieties by several years through the one-step creation of 100% homozygous plants. The technology also plays important role in studying the genetic control of traits in wheat, in marker-assisted selection, in genomics and in genetic engineering. In this paper, recent advances in androgenesis and gynogenesis techniques, emphasizing predominantly the in vitro culture phase, as well as the emerging innovative approaches in researching and producing wheat doubled haploids are reviewed. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing, that allows targeted mutagenesis and gene targeting, is being tested extensively as a powerful and precise tool to induce doubled haploids in wheat. The review provides the reader with recent examples of gene modifications in wheat to induce haploidy.

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.

TaDrAp1 and TaDrAp2, Partner Genes of a Transcription Repressor, Coordinate Plant Development and Drought Tolerance in Spelt and Bread Wheat.

Down-regulator associated protein, DrAp1, acts as a negative cofactor (NC2α) in a transcription repressor complex together with another subunit, down-regulator Dr1 (NC2ß). In binding to promotors and regulating the initiation of transcription of various genes, DrAp1 plays a key role in plant transition to flowering and ultimately in seed production. TaDrAp1 and TaDrAp2 genes were identified, and their expression and genetic polymorphism were studied using bioinformatics, qPCR analyses, a 40K Single nucleotide polymorphism (SNP) microarray, and Amplifluor-like SNP genotyping in cultivars of bread wheat (Triticum aestivum L.) and breeding lines developed from a cross between spelt (T. spelta L.) and bread wheat. TaDrAp1 was highly expressed under non-stressed conditions, and at flowering, TaDrAp1 expression was negatively correlated with yield capacity. TaDrAp2 showed a consistently low level of mRNA production. Drought caused changes in the expression of both TaDrAp1 and TaDrAp2 genes in opposite directions, effectively increasing expression in lower yielding cultivars. The microarray 40K SNP assay and Amplifluor-like SNP marker, revealed clear scores and allele discriminations for TaDrAp1 and TaDrAp2 and TaRht-B1 genes. Alleles of two particular homeologs, TaDrAp1-B4 and TaDrAp2-B1, co-segregated with grain yield in nine selected breeding lines. This indicated an important regulatory role for both TaDrAp1 and TaDrAp2 genes in plant growth, ontogenesis, and drought tolerance in bread and spelt wheat.

Identification, gene expression and genetic polymorphism of zinc finger A20/AN1 stress-associated genes, HvSAP, in salt stressed barley from Kazakhstan.

BACKGROUND: A family of genes designated as the Zinc finger A20/AN1 Transcription factors encoding stress-associated proteins (SAP) are well described in Arabidopsis and rice, and include 14 AtSAP and 18 OsSAP genes that are associated with variable tolerances to multiple abiotic stresses. The SAP gene family displays a great diversity in its structure and across different plant species. The aim of this study was to identify all HvSAP genes in barley (Hordeum vulgare L.), to analyse the expression of selected genes in response to salinity in barley leaves and develop SNP marker for HvSAP12 to evaluate the association between genotypes of barley plants and their grain yield in field trials. RESULTS: In our study, 17 HvSAP genes were identified in barley, which were strongly homologous to rice genes. Five genes, HvSAP5, HvSAP6, HvSAP11, HvSAP12 and HvSAP15, were found to be highly expressed in leaves of barley plants in response to salt stress in hydroponics compared to controls, using both semi-quantitative RT-PCR and qPCR analyses. The Amplifluor-like SNP marker KATU-B30 was developed and used for HvSAP12 genotyping. A strong association (R2 = 0.85) was found between KATU-B30 and grain yield production per plant of 50 F3 breeding lines originating from the cross Granal × Baisheshek in field trials with drought and low to moderate salinity in Northern and Central Kazakhstan. CONCLUSIONS: A group of HvSAP genes, and HvSAP12 in particular, play an important role in the tolerance of barley plants to salinity and drought, and is associated with higher grain yield in field trials. Marker-assisted selection with SNP marker KATU-B30 can be applied in barley breeding to improve grain yield production under conditions of abiotic stress.

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.