Agronomic Approach to Increase Seed Zinc Content and Productivity of Chickpea (Cicer Arietinum L.) Varieties on Zinc Deficient Soils of Southern Ethiopia

Low dietary intake of Zn is the major reason for the prevalence of Zn deficiency in the majority of the population in the Southern Ethiopia. Fertilizer application is one of the agronomic approaches that enhance nutrition quality of grains in addition to its role in raising productivity. Field experiment was conducted in three locations with zinc deficient soils of Southern Ethiopia during 2012 and 2013 cropping seasons to determine zinc fertilizer rate which improve seed zinc content and productivity of chickpea varieties. A factorial combination of three chickpea varieties (Habru, Mastewal and Local) and seven zinc fertilizer rates (0, 5, 10, 15, 20, 25, and 30 kg ZnSO4.7H2O ha-1) were laid in Randomized Complete Block design within three replications. Results revealed that Habru (the improved Kabuli type) was taller (9%) than Mastewal (the improved desi type) and the local landrace. Landrace produced 7% more pods per plant than Habru. Inversely, Habru had 60% heavier seed weight than the landrace. The significant interaction effect of variety by location on grain yield, seed zinc yield and straw zinc content indicated that Mastewal was superior in grain yield at Jolle andegna and Huletegna Choroko, while landrace performed better at Taba. The landrace and Habru were superior in seed zinc yield and straw zinc content across locations, in that order. There was no significant effect of zinc fertilization on agronomic performance of chickpeas. Seed zinc content and seed zinc yield significantly varied among zinc rates.  25 kg ZnSO4.7H2O ha-1 resulted in 7, 8, and 10% more seed zinc and straw zinc content and seed zinc yield over the control, respectively. Therefore, application of 25 kg ZnSO4.7H2O with either of the varieties can be recommended for chickpeas zinc enrichment under zinc deficient soil condition of southern Ethiopia. Keywords: seed zinc, zinc content, zinc deficient, enrichment, micronutrient, agronomic approach

Effect of Zinc Fertilizer Rates on Grain and Straw Zn Content, and Grain Yield of Chickpea Varieties in Southern Ethiopia

አህፅሮት  የዚንክ ንጥረ-ይዘት ለሚያጥርባቸው የእርሻ መሬት የዚንክ ማዳበሪያ መጨመር በምርትም ሆነ በምርት የዚንክ ንጥረ-ይዘት ላይ አወንታዊ እመርታ እንደሚያሰገኝ ይታመናል፡፡ ሆኖም የሚጨመረውን የማዳበሪያ መጠን የሚወስነዉ የአፈር ውስጥ የዚንክ ንጥረ ይዘት እጥረት መጠን፣ የአፈሩ ዓይነት፣ የስብል ዓይነትና ዝርያ እንዲሁም የአጨማመር ዘዴ ነዉ፡፡ የዚህ ጥናት ዓላማ የሽምብራን ምርትና የምርት ዚንክ ንጥረ-ይዘት መጠንን የሚያሻሽል የዚንክ ማዳበሪያ መጠንን ለዚንክ ንጥረ-ይዘት መጠን አነስተኛ ለሆነ መሬት ለመወሰን ነው፡፡ የአፈር ዚንክ መጠን ዝቅተኛ ከሆነበት የእርሻ መሬት እና ከዚሁ ማሳ በተሰበሰበ አፈር ላይ ሶስት የሽምብራ ዝርያዎች በሰባት የዚንክ ማዳበሪያ መጠን  በ2004 እና በ2005 የምርት ዘመን ተጠንቷል፡፡ በማሳ ላይ የተደረገው ጥናት ውጤት የሚያመለክተው ሁለተኛ ጮሮቆ ከጣባም ሆነ ከጆሌ አንደኛ አካባቢዎች በ46.67 በመቶ የተሸለ የምርት ዚንክ ንጥረ-ይዘት የተገኘበት ሲሆን  በልዩ መደብም ሆነ በማሳ ላይ  የተደረገው ጥናት ውጤት የሚያመለክተው ከዝርያዎቹ ማስተዋል የተባለ የሽምብራ ዝርያ ከሌሎቹ የተሸለ ምርት ሲገኝበት፣ ሀብሩ ደግሞ በገለባ የዚንክ ንጥረ-ይዘት ተሸሎ ተገኝቷል፡፡ የአካባቢው ዝርያ በምርት የዚንክ ንጥረ-ይዘት ከፍተኛ ሲሆን የዝርያና የማዳበሪያ ዉህደት በሽምብራ ምርትና በምርት ንጥረ-ይዘት ላይ እመርታዊ ጭማሪ ያስገኘ ሲሆን 25 ኪሎ ግራም ዚንክ ማዳበሪያ በሄክታር በሁሉም ዝርያዎች ከፍተኛ መሆኑን ያሳያል፡፡ ከዚህ በላይ የማዳበሪያ መጠኑን መጨመር የተለየ ዉጤት አላሳየም፡፡ ስለዚህ ይህ ጥናት የሚያረጋግጠው ሽምብራን በዚንክ ንጥረ-ይዘት ለማበልፀግ ዝርያና ማዳበሪያ አጠቃቀምን በመመጠን እንደሚቻል ነዉ፡፡   Abstract  Application of Zn had a significantly positive effect on grain Zn concentrations and also on grain yield especially under Zn deficient conditions. The amount of Zn required to alleviate Zn deficiency varied with severity of deficiency, soil types, nature of crops and cultivars. The response of chickpea varieties to Zn nutrition was studied in pots and on fields using zinc deficient soils during 2012 and 2013 cropping seasons to determine zinc fertilizer rate which improve zinc content and productivity of the crop. A factorial combination of three chickpea varieties and seven zinc fertilizer rates were laid in Randomized Complete Block design with three replications for both pot and field experiments. The result of pot experiment revealed that, variety Mastewal produced the highest grain yield (5.9 g pot-1) and Habru produced highest (35.99mg kg-1) straw zinc content. Conversely, local chickpea provided the highest (36.1mg kg-1) grain Zn. Chickpea varieties and zinc fertilizer rates interaction on grain yield was significant where 25kg ha-1 produced highest regardless of the varieties. Similarly, location had significant (p<0.01) effect of grain zinc content where Choroko produced 46.6 % more grain zinc content than both Taba and Jolle. Highest straw zinc (24.96 mg kg-1) obtained from variety Habru, while highest grain zinc obtained from the application of 25 kg ZnSO4 .7H2O ha-1 with either of the varieties which was at par with the highest Zinc rate (30kg ha-1). Significant interaction effect of variety by location on grain yield and straw zinc content was observed. The variety Mastewal was superior in grain yield at Jolle and Choroko, while landrace performed better at Taba. The landrace and Habru were higher in straw zinc content across locations. Moreover, 25 kg ZnSO4 .7H2O ha-1 resulted in 7 and 8% more grain and straw zinc content over the control, in that order. Thus, the current research inveterate a possibility of agronomic intervention for zinc fortification of chickpea through zinc fertilizer management. &nbsp

Genetic Analysis of NBS-LRR Gene Family in Chickpea and Their Expression Profiles in Response to Ascochyta Blight Infection

Ascochyta blight is one of the major diseases of chickpea worldwide. The genetic resistance to ascochyta blight in chickpea is complex and governed by multiple QTLs. However, the molecular mechanism of quantitative disease resistance to ascochyta blight and the genes underlying these QTLs are still unknown. Most often disease resistance is determined by resistance (R) genes. The most predominant R-genes contain nucleotide binding site and leucine rich repeat (NBS-LRR) domains. A total of 121 NBS-LRR genes were identified in the chickpea genome. Ninety-eight of these genes contained all essential conserved domains while 23 genes were truncated. The NBS-LRR genes were grouped into eight distinct classes based on their domain architecture. Phylogenetic analysis grouped these genes into two major clusters based on their structural variation, the first cluster with toll or interleukin-1 like receptor (TIR) domain and the second cluster either with or without a coiled-coil domain. The NBS-LRR genes are distributed unevenly across the eight chickpea chromosomes and nearly 50% of the genes are present in clusters. Thirty of the NBS-LRR genes were co-localized with nine of the previously reported ascochyta blight QTLs and were tested as potential candidate genes for ascochyta blight resistance. Expression pattern of these genes was studied in two resistant (CDC Corinne and CDC Luna) and one susceptible (ICCV 96029) genotypes at different time points after ascochyta blight infection using real-time quantitative PCR. Twenty-seven NBS-LRR genes showed differential expression in response to ascochyta blight infection in at least one genotype at one time point. Among these 27 genes, the majority of the NBS-LRR genes showed differential expression after inoculation in both resistant and susceptible genotypes which indicates the involvement of these genes in response to ascochyta blight infection. Five NBS-LRR genes showed genotype specific expression. Our study provides a new insight of NBS-LRR gene family in chickpea and the potential involvement of NBS-LRR genes in response to ascochyta blight infection

Table_5_Freezing stress response of wild and cultivated chickpeas.xlsx

Chickpea is an economically and nutritionally important grain legume globally, however, cold stress has adverse effects on its growth. In cold countries, like Canada where the growing season is short, having cold stress-tolerant varieties is crucial. Crop wild relatives of chickpea, especially Cicer reticulatum, can survive in suboptimal environments and are an important resource for crop improvement. In this study, we explored the performance of eleven C. reticulatum wild accessions and two chickpea cultivars, CDC Leader and CDC Consul, together with a cold sensitive check ILC533 under freezing stress. Freezing tolerance was scored based on a 1-9 scale. The wild relatives, particularly Kesen_075 and CudiA_152, had higher frost tolerance compared to the cultivars, which all died after frost treatment. We completed transcriptome analysis via mRNA sequencing to assess changes in gene expression in response to freezing stress and identified 6,184 differentially expressed genes (DEGs) in CDC Consul, and 7,842 DEGs in Kesen_075. GO (gene ontology) analysis of the DEGs revealed that those related to stress responses, endogenous and external stimuli responses, secondary metabolite processes, and photosynthesis were significantly over-represented in CDC Consul, while genes related to endogenous stimulus responses and photosynthesis were significantly over-represented in Kesen_075. These results are consistent with Kesen_075 being more tolerant to freezing stress than CDC Consul. Moreover, our data revealed that the expression of CBF pathway-related genes was impacted during freezing conditions in Kesen_075, and expression of these genes is believed to alleviate the damage caused by freezing stress. We identified genomic regions associated with tolerance to freezing stress in an F2 population derived from a cross between CDC Consul and Kesen_075 using QTL-seq analysis. Eight QTLs (P<0.05) on chromosomes Ca3, Ca4, Ca6, Ca7, Ca8, and two QTLs (P<0.01) on chromosomes Ca4 and Ca8, were associated with tolerance to freezing stress. Interestingly, 58 DEGs co-located within these QTLs. To our knowledge, this is the first study to explore the transcriptome and QTLs associated with freezing tolerance in wild relatives of chickpea under controlled conditions. Altogether, these findings provide comprehensive information that aids in understanding the molecular mechanism of chickpea adaptation to freezing stress and further provides functional candidate genes that can assist in breeding of freezing-stress tolerant varieties.</p

Towards Zinc Biofortification in Chickpea: Performance of Chickpea Cultivars in Response to Soil Zinc Application

A field experiment was conducted at three locations in the southern region of Ethiopia during the 2012 and 2013 cropping seasons to evaluate chickpea cultivars for their response to soil zinc application, including agronomic performance, grain yield, grain zinc concentration, zinc and agronomic efficiency. Fifteen chickpea cultivars were evaluated in a randomized complete block design with three replications at each location and year. The highest number of pods (237) plant−1 was obtained from Butajira local landrace. The cultivar Naatolii produced the highest grain yield (2895 kg·ha−1), while the breeding line FLIP03-53C had the lowest yield (1700 kg·ha−1). The highest zinc concentrations of 47.5, 47.4, and 46.4 mg·kg−1 grain were obtained from the cultivar Arerti, and the two breeding lines FLIP07-27C and FLIP08-60C, respectively. The highest zinc efficiency (88%) was obtained from the Wolayita local landrace, whereas the highest agronomic efficiency of 68.4 kg yield increase kg−1 zinc application was obtained from the cultivar Naatolii. The current research identified chickpea cultivars with high grain zinc concentration, zinc efficiency, agronomic efficiency, and grain yield. The identification of cultivars with high grain zinc concentration allows the use of chickpea as a potential alternative to help to correct zinc deficiency, which is highly prevalent in the population of the region

Table_4_Freezing stress response of wild and cultivated chickpeas.xlsx

Chickpea is an economically and nutritionally important grain legume globally, however, cold stress has adverse effects on its growth. In cold countries, like Canada where the growing season is short, having cold stress-tolerant varieties is crucial. Crop wild relatives of chickpea, especially Cicer reticulatum, can survive in suboptimal environments and are an important resource for crop improvement. In this study, we explored the performance of eleven C. reticulatum wild accessions and two chickpea cultivars, CDC Leader and CDC Consul, together with a cold sensitive check ILC533 under freezing stress. Freezing tolerance was scored based on a 1-9 scale. The wild relatives, particularly Kesen_075 and CudiA_152, had higher frost tolerance compared to the cultivars, which all died after frost treatment. We completed transcriptome analysis via mRNA sequencing to assess changes in gene expression in response to freezing stress and identified 6,184 differentially expressed genes (DEGs) in CDC Consul, and 7,842 DEGs in Kesen_075. GO (gene ontology) analysis of the DEGs revealed that those related to stress responses, endogenous and external stimuli responses, secondary metabolite processes, and photosynthesis were significantly over-represented in CDC Consul, while genes related to endogenous stimulus responses and photosynthesis were significantly over-represented in Kesen_075. These results are consistent with Kesen_075 being more tolerant to freezing stress than CDC Consul. Moreover, our data revealed that the expression of CBF pathway-related genes was impacted during freezing conditions in Kesen_075, and expression of these genes is believed to alleviate the damage caused by freezing stress. We identified genomic regions associated with tolerance to freezing stress in an F2 population derived from a cross between CDC Consul and Kesen_075 using QTL-seq analysis. Eight QTLs (P<0.05) on chromosomes Ca3, Ca4, Ca6, Ca7, Ca8, and two QTLs (P<0.01) on chromosomes Ca4 and Ca8, were associated with tolerance to freezing stress. Interestingly, 58 DEGs co-located within these QTLs. To our knowledge, this is the first study to explore the transcriptome and QTLs associated with freezing tolerance in wild relatives of chickpea under controlled conditions. Altogether, these findings provide comprehensive information that aids in understanding the molecular mechanism of chickpea adaptation to freezing stress and further provides functional candidate genes that can assist in breeding of freezing-stress tolerant varieties.</p

Fine Mapping of QTLs for Ascochyta Blight Resistance in Pea Using Heterogeneous Inbred Families

Ascochyta blight (AB) is an important disease of pea which can cause severe grain yield loss under wet conditions. In our previous study, we identified two quantitative trait loci (QTLs) abIII-1 and abI-IV-2 for AB resistance and these QTLs were consistent across locations and/or years in an inter-specific pea population (PR-19) developed from a cross between Alfetta (Pisum sativum) and P651 (P. fulvum). The objectives of this study were to fine map the abIII-1 and abI-IV-2 QTLs using a high density single nucleotide polymorphism (SNP)-based genetic linkage map and analyze identified markers in heterogeneous inbred family (HIF) populations. Selective genotyping of 51 PR-19 recombinant inbred lines was performed using genotyping-by-sequencing (GBS) and the resulting high density genetic linkage map was used to identify eight new SNP markers within the abI-IV-2 QTL, whereas no additional SNPs were identified within the abIII-1 QTL. Two HIF populations HIF-224 (143 lines) and HIF-173 (126 lines) were developed from F6 RILs PR-19-224 and PR-19-173, respectively. The HIF populations evaluated under field conditions in 2015 and 2016 showed a wide range of variation for reaction to AB resistance. Lodging score had significant positive (P &lt; 0.001) correlation with AB scores. HIFs were genotyped using SNP markers within targeted QTLs. The genotypic and phenotypic data of the HIFs were used to identify two new QTLs, abI-IV-2.1 and abI-IV-2.2 for AB resistance within the abI-IV-2 QTL. These QTLs individually explained 5.5 to 14% of the total phenotypic variation. Resistance to lodging was also associated with these two QTLs. Identified SNP markers will be useful in marker assisted selection for development of pea cultivars with improved AB resistance

Identification of Quantitative Trait Loci Associated with Seed Protein Concentration in a Pea Recombinant Inbred Line Population

This research aimed to identify quantitative trait loci (QTLs) associated with seed protein concentration in a recombinant inbred line (RIL) population of pea and aimed to validate the identified QTLs using chromosome segment-introgressed lines developed by recurrent backcrossing. PR-25, an RIL population consisting of 108 F7 bulked lines derived from a cross between CDC Amarillo (yellow cotyledon) and CDC Limerick (green cotyledon), was used in this research. The RIL population was genotyped using an Axiom 90K SNP array. A total of 10,553 polymorphic markers were used for linkage map construction, after filtering for segregation distortion and missing values. The linkage map represents 901 unique loci on 11 linkage groups which covered a map distance of 855.3 Centimorgans. Protein concentration was assessed using near-infrared (NIR) spectroscopy of seeds harvested from field trials in seven station-years in Saskatchewan, Canada, during the 2019–2021 field seasons. Three QTLs located on chromosomes 2, 3 and 5 were identified to be associated with seed protein concentration. These QTLs explained 22%, 11% and 17% of the variation for protein concentration, respectively. The identified QTLs were validated by introgression lines, developed by marker-assisted selection of backcross lines for introgression of corresponding chromosome segments (~1/4 chromosome) harboring the QTL regions. Introgression line PR-28-7, not carrying any protein-related QTLs identified in this study, was 4.7% lower in protein concentration than CDC Amarillo, the lower protein parent of PR-25 which carried one identified protein-related QTL. The SNP markers located at the peak of the three identified QTLs will be converted into breeder-friendly KASP assays, which will be used for the selection of high-protein lines from segregating populations