Genetic Improvement of Adapted Ethiopian Chickpea (Cicer Arietinum L.) Cultivar for Drought Tolerance Through Conventional and Marker-Assisted Backcross Breeding Methods
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2017-04-05
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Addis Ababa University
Abstract
Chickpea (Cicer arietinum L.) is the world's third most important food legume both in area and production next to common bean (Phaseolus vulgaris L.) and field pea (Pisum sativum L.), grown mainly by small-scale farmers in the semi-arid tropics, West Asia and North Africa, and sub-Saharan regions, including Ethiopia. It an affordable cheap source of protein, one of the export commodities for cash and plays important role in soil amelioration. However, currently its average national yield is 1.91 t/ha when its potential could reach 5.5 t/ha. The low productivity is attributed to biotic and abiotic stresses, of which drought is accounting for 40-50% globally. Genetic manipulation of the crops through breeding for drought tolerance is among the ways to combat this problem. In recent years, tremendous progresses have been made in the development of novel genetic tools such as DNA molecular markers, dense genetic maps, and whole-genome transcription profiling techniques to identify genomic regions and genes underlying plant stress responses. The root traits such as root length-density have been also proposed as the main drought avoidance traits that contribute to yield under terminal drought environments in chickpea. Major genomic region containing QTL for drought related traits contributing up to 36% phenotypic variation have been identified by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). However, these markers have not yet been validated and been deployed into the Ethiopian chickpea breeding program. Even the already released Ethiopian chickpea cultivars for optimum environment have never been systematically evaluated for their response to drought tolerance. Ethiopia has vast area coverge under drought-prone zones and smallholder farmers are growing chichpea under this sub-optimal environment, but not specific breeding program in place for drougt tolerance. In this study, therefore, I have conducted two set of experiments: (1) introgression of drought tolerance genes from a drought tolerant donor parent (ICC 4958 obtained from ICRISAT, India) to the adapted high yielder Ethiopian chickpea cultivar, Ejere and (2) screening the improved cultivars for drought tolrance.
A totao of 208 BC3F4 isogenic lines (ILs) were developed through MABC from by crossing ICC 4958 and Ejere, these ILs were tested in multi-location field trial under full-water (FW) and under water-stress (WS) conditions to select ILs tolerant to moisture stress with good agronomic traits. Data were collected on number of days to flowering (DTF), maturity (DTM) A Doctoral Dissertation Mussa Jarso Addis Ababa University April 2017 Page xxix
and grain filling (DTGF), number of pods per plant (PPP) and seeds per pod (SPP), plant height (PHT), hundred seed weight (HSW), biomass (BMY) and grain yields (GYLD) and harvest index (HI), and subjected to statistical analyses. The result of the ANOVA indicated that there were significant differences among the ILs for the traits at each location and combined over locations under both FW and WS conditions. Significant difference was also observed beteen the locations and genotype by location interaction for almost all the traits. There relative reduction (RR) in traitsā means due to WS for almost all the traits except DTF and SPP ranged from 0.2% for DTF to 41% for GYLD. The RR of GYLD for different genotypes ranged from 19.0 to 41% with average of 37.5%. The highest RR in GYLD was recorded for the recurrent parent (41%) followed by the ILs without markers (26.1%), while the least (19%) was for best ILs with markers for drought tolerance. Of the yield components, PPP had the highest RR ranging from 23%- 41%. Moderate to high RR were recorded for the rest of the traits too. Under FW condition, ILs 5-9, 7-19, 37-3, and 37-4 with markers and ILs lines 5-10, 18-3, 18-5, and 19-4 without markers were among top 10% selected for high GYLD. Under WS condition, the ILs 9-7, 10-10, 29-6, and 38-2 with markers and ILs 7-14, 27-2, 30-5, and 34-2 without markers were among selected top 10%. The analysis of drought tolerance index (DSI) revealed that HI, GYLD, DTF and PPP had high (above unity) DSI while SPP had the least (below unity) under FW; but under WS, DTF, HI and HSW had high DSI while GYLD, SPP, BMY, and PPP had low DSI. The selected ILs under WS tolerated the stress by low susceptibility of GYLD, BMY, SPP and DTGF.
The cluster analysis has grouped the 208 ILs and the parents into 12 distinct clusters under FW and 10 clusters under WS. There was high genetic divergence for the genotypes under FW than under WS as revealed by the distances among the clusters. The principal component analysis has also revealed that PPP, BMY, HI and GYLDs accounted for larger proportion of the variations in the genotypes under both FW and WS while DTGF under WS only. The variance component analysis has showed the existence of high genetic variations among the tested ILs for studied traits. High genetic variations were recorded for GYLD, BMY and PPP along with high genetic coefficient of variation (GCV) under both FW and WS. Broad sense heritability (H2b) of the traits ranged from 69% for DTF to 96% for PHT under FW and 55% for SPP to 85% for PPP under WS. The GCV and H2b were higher under FW than under WS condition. The expected genetic gain (GA) from selection and the GA as a A Doctoral Dissertation Mussa Jarso Addis Ababa University April 2017 Page xxx
percentage of the mean (GAM) under FW condition were high for PPP (10 pods and 36%), GYLD (14.3g and 29.6%), PHT (7.7cm and 24.2%), HI (6.6% and 21.4%) and HSW (5.3g and 18.4%), respectively. Under WS condition, GA and GAM were high for GYLD (10.7g and 29.9%), PPP (7.2 pods and 25.7%), BMY (27.9g and 24.2%), respectively. Traits with high GCV and H2b values have resulted in high GA and GAM as compared to traits with low GCV and / or H2b. The genetic correlations (rG) among traits ranged from -0.41 to 0.98 under FW and -0.67 to 0.96 under WS conditions. Grain yield had strong positive correlations with PPP, BMY, and HI (rG=0.67 to 0.99) under both FW and WS; it had strong positive correlation with DTM (rG =0.40) and DTGF (rG=0.53) whereas strong negative correlation with DTF (rG=-0.44) only under WS. GYLD had medium positive or negative genetic correlation with SPP, PHT and HSW under FW and WS conditions. In the sequence analysis of the selected two ILs (5-12 and 38-2) and the two parents, 293.3 million paired end (PE) raw reads were generated from control and stress root tissue samples. A total of 272 (92.8%) million PE reads were aligned of which 88.8% were concordantly aligned and 11.2 % were non-concordantly aligned. A total of 48,698 transcripts were assembled using Cufflinks and subjected to differential gene expression analysis. Gene expression was identified up- and down- regulated and differentially expressed genes. A total of 1,088 differentially expressed unique transcripts were functionally categorized based on gene ontology (GO) into three principal categories: molecular function (401), biological process (436) and cellular component (251). In total, 9,393,545 SNPs identified among different combinations across the eight chickpea chromosomes that could be further exploited for molecular breeding of chickpea.
The ANOVA for the released cultivars tested for drought tolerance showed significant differences between the cultivars for most of the agronomic traits under both FW and WS conditions. There was significant difference between the soils for GYLD, RWC, LAPP, LAI and WUEGY under both FW and WS while for EV and HI under WS condition only. Soil by cultivar interaction was significant for EV, DTF, HI, GYLD and WUEGY under both FW and WS, while for DSI under WS condition only. There was also significant differences between the soils, the crop stages and the cultivars for BMY, leaf, stem, root and shoot dry weights (LDWt, StDWt, RDWt, and ShDWt, respectively), shoot-root ratio (SRR), transpiration (TP) and WUEBM. Almost all the interactions were significant for all the traits. Under WS condition, there was significant difference between the soils, destruction levels and cultivars for all the traits and all the interaction, except for A Doctoral Dissertation Mussa Jarso Addis Ababa University April 2017 Page xxxi
WUEBM. Mean GYLD of the cultivars under FW ranged from 6.65 to 28.45g per five plants with mean of 15.93g and 3.54 to 27.42g under WS condition. Cultivars Shasho, Marye, Natoli, Ejere, and ICC 4958 had high GYLD while DZ-10-4 and Dz-10-11 had low GYLD under FW; cultivars Arerti, Natoli, ICC 4958 and Minjar had high GYLD while Acos Dubie, Akaki, Akuri and Dz-10-11 had low GYLD under WS. The high GYLD of the cultivars was due to high BMY, PPP and HI under both FW and WS. In addition, these cultivars had high TP, WUEBM and WUEGY under both FW and WS, and high ShDWt, SRR and LAI under FW while high RDWt, LAPP, RWC under WS. For agronomic traits, the highest overall mean reduction (MR) due to WS was scored for GYLD (28%) followed by BMY (24%). The overall MR in DTM, DTGF, SPP, and HSW was negligible. There was clear difference between the cultivars for MR; Mean GYLD reduction for the cultivars ranged from 12 to 70%. For physiological traits, the MR for LDWt (28%), ShDWt (23%), LAPP (23%), LAI (21%), StDWt (20%), WUEGY (16%), RDWt (12%), and SRR (11%). The WUE of the cultivars for GYLD ranged from 2.7 to 9.4% with overall mean of 5.4% under FW and 5.7 to 19.4% with overall mean of 12.2% under WS condition. Cultivar Shasho was the most efficient (9.4%) while Dz-10-4 and Dz-10-11 were the least (2.7% each) for GYLD under FW while Arerti was the most efficient (9.4%) followed by ICC 4958 (8.3%) and the least for Dz-10-11 (1.9%) under WS condition. Water-use efficiency of the cultivars for BMY had similar trend with that of GYLD and it ranged from 5.7 to 19.4% with overall mean of 12.2% under FW and from 4.5 to 19.6% with the overall mean of 10.1% under WS. This result clearly revealed that a cultivar that is water use efficient under FW condition may not necessary be efficient under WS and vice-versa. Cluster analysis grouped the 20 cultivars into five distinct clusters under both FW and WS conditions and are recommended based on their efficiency to the two environments. The pair wise generalized square distances among the clusters were significant for most of the cluster pairs indicating genetic divergence between cultivars in the different clusters. Principal component analysis confirmed that trait like GYLD, StDW, ShDWt, WUEBM, WUEGY have accounted for the highest proportion of the genetic variability in the tested cultivars under both stress levels.
In conclusion, the introgression of drought tolerance QTLs into the adapted high yielding Ethiopian cultivar has resulted in ILs with high yield and water-stress tolerance; screening of the already released cultivars for optimum environment has also enabled identification of some cultivars that are relatively better tolerant to water-stress condition with reasonable grain yields. A Doctoral Dissertation Mussa Jarso Addis Ababa University April 2017
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Keywords
Genetic Improvement, Adapted Ethiopian, Marker-Assisted, Breeding Methods