key: cord-0835938-8bimta6w authors: Eker, Tuba; Sari, Duygu; Sari, Hatice; Tosun, Hilal Sule; Toker, Cengiz title: A kabuli chickpea ideotype date: 2022-01-31 journal: Sci Rep DOI: 10.1038/s41598-022-05559-3 sha: ab5761c9d1b42e61d3f2b2f1c254b42c7cc06e2b doc_id: 835938 cord_uid: 8bimta6w The concept of ‘crop ideotype’ is coined as a desirable plant model expected to better perform for seed yield, oils and other useful characteristics when developed as a cultivar, and it consists of two major approaches, namely, (i) ‘defect elimination’, that is, integration of disease resistance to a susceptible genotype from a resistant genotype and (ii) ‘selection for yield’ by improving yield after crosses between desirable parents. For consideration of these approaches, here we introduced an ideotype in kabuli chickpea (Cicer arietinum L.) which is high-yielding, extra-large-seeded, and double- or multi-podded, has high plant height and imparipinnate-leafed traits, and is heat tolerant and resistant to ascochyta blight [Ascochyta rabiei (Pass.) Labr.], which causes considerable yield losses, via marker-assisted selection. F(3) and F(4) lines were evaluated for agro-morphological traits divided into six classes, namely, (i) imparipinnate-leafed and single-podded progeny, (ii) imparipinnate-leafed and double-podded progeny, (iii) imparipinnate-leafed and multi-podded progeny, (iv) unifoliolate-leafed and single-podded progeny, (v) unifoliolate-leafed and double-podded progeny, (vi) unifoliolate-leafed and multi-podded progeny. F(3:4) lines having 100-seed weight ≥ 45 g and double- or multi-podded traits were additionally assessed for resistance to ascochyta blight using molecular markers including SCY17(590) and CaETR-1. Superior lines having higher values than their best parents were determined for all studied traits indicating that economic and important traits including yield and seed size in chickpea could be improved by crossing suitable parents. Imparipinnate-leafed and multi-podded plants had not only the highest number of pods and seeds per plant but also the highest yield. On the other hand, imparipinnate-leafed and single podded progeny had the largest seed size, followed by imparipinnate-leafed and double-podded progeny. Multi-podded plants produced 23% more seed yield than that of single-podded plants, while multi-podded plants attained 7.6% more seed yield than that of double-podded plants. SCY17(590) and CaETR-1 markers located on LG4 related to QTL(AR2) and QTL(AR1) were found in 14 lines among 152 F(3:4) lines. Six superior lines were selected for being double- or multi-podded, imparipinnate-leafed, suitable for combine harvest, heat-tolerant, and resistant to ascochyta blight, and having both of two resistance markers and extra-large seeds as high as 50–60 g per 100-seed weight. Resistance alleles from two different backgrounds for resistance to ascochyta blight were integrated with double- or multi-podded kabuli chickpea lines having high yield, extra-large seeds, high plant height, imparipinnate-leaves and high heat tolerance, playing a crucial role for future demands of population and food security. These approaches seem to be applicable in ideotype breeding for other important crop plants. ing to the data analyses on descriptive statistics of the F 3 population, transgressive segregations were determined for all agro-morphological traits including 100-seed weight (Table 1) . Minimum and maximum values of days to first flowering of F 3 population were found to be 37 and 76 days, respectively, whereas days to 50% flowering in F 3 population ranged from 39 to 73 days, respectively. Days to first flowering and days to 50% flowering of Sierra and CA 2969 were 48.3-50.3 days and 50-52.3 days, respectively. Plant height of the genotypes in F 3 population varied from 19 to 68 cm, while the plant height of Sierra and CA 2969 was measured as 52.3 and 42.7 cm, respectively. The average first pod height in F 3 population was 30.9 cm, while it was 31.3 cm for Sierra and 33.3 cm for CA 2969 (Table 1 , Fig. 1 ). The number of main stems per plant in F 3 population was 1-6, whereas it was 2-3 in the Sierra and CA 2969. The canopy width in F 3 population varied between 10 and 96 cm, while it was 49 cm in the Sierra and 58.3 cm in the CA 2969. The number of seeds per plant in F 3 population was 1-267 and the average of this trait was recorded as 32.7 and 50.7 in the Sierra and CA 2969, respectively (Table 1, Fig. 2 ). Seed yield per plant ranged from 1 to 79 g in F 3 population, while mean seed yield per plant was 15.2 and 13.9 g in the Sierra and CA 2969, respectively. The 100-seed weight ranged from 7 to 64 g in F 3 population, while the 100-seed weight was recorded as 46.9 g and 27.4 g in the Sierra and CA 2969, respectively. Biological yield in F 3 population was determined as 4-147 g, while harvest index was 0.4-62.4%. Biological yield and harvest index in Sierra were determined as 39 g and 38.4%, while in CA 2969 they were calculated as 29.5 g and 47.5%, respectively ( Table 1, Fig. 3 ). Minimum and maximum values of days to first flowering in F 4 were found to be 55 Fig. 2 ). Seed yield per plant ranged from 0.1 to 51.2 g in F 4 , while mean seed yield per plant was 5.3 and 12 g in the Sierra and CA 2969, respectively. The 100-seed weight ranged from 10 to 69.6 g in F 4 , while the 100-seed weight was 46.3 g and 28.9 g in the Sierra and CA 2969, respectively. Biological yield in F 4 was 0.3-110.4 g, while harvest index was 0.8-59.3%. Biological yield and harvest index were 39 g and 38% in Sierra, while in CA 2969, they were 30 g and 48%, respectively (Table 2, Fig. 3 ). Superior lines for agro-morphological traits in the F 3:4 . As for the selected F 3:4 superior lines for 100-seed weight and double/multi-podded traits, transgressive segregations for all agro-morphological traits are shown in Table 3 . Days to first flowering and 50% flowering were 43-66 days and 59-74 days, respectively. Table 1 . Means ± standard errors and range for agro-morphological traits in F 3 population derived from intraspecific crosses between Sierra (single-podded and unifoliolate-leafed) and CA 2969 (double-podded and imparipinnate-leafed). I-S: imparipinnate-leafed and single-podded progeny, I-D: imparipinnate-leafed and double-podded progeny, I-M: imparipinnate-leafed and multi-podded progeny, U-S: unifoliolate-leafed and single-podded progeny, U-D: unifoliolate-leafed and double-podded progeny, U-M: unifoliolate-leafed and multi-podded progeny. X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X Range Table 2 . Means ± standard errors and range for agro-morphological traits in F 4 population derived from intraspecific crosses between Sierra (single-podded and unifoliolate-leafed) and CA 2969 (double-podded and imparipinnate-leafed). I-S: imparipinnate-leafed and single-podded progeny, I-D: imparipinnate-leafed and double-podded progeny, I-M: imparipinnate-leafed and multi-podded progeny, U-S: unifoliolate-leafed and single-podded progeny, U-D: unifoliolate-leafed and double-podded progeny, U-M: unifoliolate-leafed and multi-podded progeny. www.nature.com/scientificreports/ 100-seed weight in the F 3 and F 4 populations. The F 3 and F 4 populations were evaluated for seed size based on 100-seed weights and transgressive segregations were observed (Fig. 4a ,b). In F 3 and F 4 , a large number of genotypes had 100-seed weight ≥ 45 g (as selection criteria) and were larger than the parent Sierra (46.9 g) (Fig. 4c) . Distribution of 100-seed weight depending on the pods per axil (single, double or multi podded) and leaf shapes (unifoliolate or imparipinnate) of genotypes with 100-seed weight ≥ 45 g is given in Fig. 4c -f. In single and double/multi-podded genotypes, a larger number of extra-large-seeded chickpeas (as large as Sierra) were available in more imparipinnate leaves than unifoliolate leaves, in both F 3 and F 4 ( Fig. 4c-f ). In F 3 , 24 single-podded genotypes and 22 double/multi-podded genotypes had 100-seed weight ≥ 54 g and among them, the highest value was 64 g in an imparipinnate-leafed and single-podded genotype (Fig. 4c,e) . In F 4 , 10 singlepodded genotypes and 25 double/multi-podded genotypes had 100-seed weight ≥ 54 g (Fig. 4d ,f) and among www.nature.com/scientificreports/ them, the largest seed was 69.6 g in a unifoliolate-leafed and multi-podded genotype (Fig. 4f ,h). The selection was performed from F 3 to F 3:4 generations based on 100-seed weight ≥ 45 g in double/multi-podded genotypes and 152 F 3:4 lines were selected. Table S1 ). Nine of them had homozygous resistant alleles in both markers. In the remaining five lines, two lines were heterozygous and homozygous for QTL AR2 and QTL AR1 , respectively. Two other lines were homozygous and heterozygous for QTL AR2 and QTL AR1 , respectively, while one line was heterozygous for both QTLs. Six lines having 100-seed weight ≥ 50 g had resistant alleles for both QTLs and among them, the highest 100-seed weight was 59.8 g in an imparipinnate-leafed and double-podded line. On the other hand, 100-seed weight of a line found to be resistant only in SCY17 590 marker was determined as 62.5 g (Supplementary Table S1 ). I-M U-S U-D U-M F 4 X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X X ± S X Range Heat tolerance. Plants were subjected to maximum temperatures over 30 °C during flowering in the first year, while they were exposed to heat stress during the pod setting stage of as high as about 40 °C in the first year. In the second year, plants tried to keep standing with heat stress of 43.1 °C during flowering and about 39.5 °C during pod setting (Fig. 6 ). Six superior lines that were double-or multi-podded, imparipinnate-leafed, suitable for combine harvest, and heat-tolerant, and that had extra-large seeds as high as 50-60 g per 100-seed weight were integrated with resistance to ascochyta blight having both of two resistance markers (Tables 1, 2 Table S1 ). Double flowers or pods per axil were governed by a recessive single gene 14, 42, 44, 47 , while imparipinnate leaf shape in cultivated chickpea was controlled by a dominant gene 14, [57] [58] [59] . In early generations such as F 2 and F 3 , after the genotypes with double pods per axil trait are selected, as it is controlled by a single recessive gene 14 , this trait is not expected to segregate in subsequent generations. Genetic studies have shown that transgressive segregations are mostly due to the occurrence of combinations of alleles from both parents with the same effect (complementary gene effect) 60, 61 . It is possible that hybrid individuals combining the 'desired' or 'unwanted' alleles of both parents show superior (unexpected or extreme) phenotypes. Similarly, in this study, transgressive segregations were determined for all agro-morphological traits and 100-seed weight not only in F 3 but also in F 4 population (Tables 1, 2, 3, Figs. 1, 2, 3, 4) . Among the progeny, 209 progeny in F 3 population had higher 100-seed weight than that of the best parent Sierra with 46.9 g and of these, a total of 131 progeny in F 3 population had higher 100-seed weight than 50 g (Fig. 4a) . It was also determined that some of them could produce double pods and be larger seeded than expected up to 62.5 g (Fig. 4e) . Transgressive segregations are considered to be due to the complementary effect of genes and activity of suppressed recessive genes in the parents and to occur especially in crosses with wild origin plants 35, 45, [60] [61] [62] . Moreover, additional explanations have been outlined in relation to observations of transgressive segregations in segregating generations (see detail in Rieseberg et al. 61 ). These consist of: (i) an elevated mutation rate, (ii) reduced developmental stability; (iii) epistasis or non-additivity of allelic effects among loci; (iv) overdominance or non-additivity of allelic effects within a locus; (v) unmasking of recessive alleles that are heterozygous in the parents; (vi) different number of chromosomes; and (vii) complementary action of additive alleles that are dispersed between parents. As promising chickpea genotypes, extra-large seeded genotypes containing double or multiple pods were determined not only in F 3 but also in F 4 (Fig. 4e,f) . X ± S X X ± S X X ± S X Range X ± S X X ± S X Days to first flowering 56.4 ± 0.14 59.5 ± 0. 16 . Distribution of 100-seed weight of genotypes with single-poddded and 100-seed weight ≥ 45 g according to leaf shape in F 3 (c) and F 4 (d). Distribution of 100-seed weight of genotypes with double/multi-poddded and 100-seed weight ≥ 45 g according to leaf shape in F 3 (e) and F 4 (f). A line in F 4 (multi-podded per axil, imparipinnate-leafed) derived from intraspecific crosses between Sierra (single-podded and unifoliolate-leafed) and CA 2969 (double-podded and imparipinnate-leafed) (g). Seeds of a superior line (multi-podded, unifoliolate-leafed and 69.6 g per 100-seeds, left side) in F 4 and its large-seeded parent Sierra (single-podded, unifoliolate-leafed and 46.9 g per 100-seeds, right side) (h). www.nature.com/scientificreports/ The double-podded trait in chickpeas is one of the important traits since it provides yield increase and yield stability compared to its single-podded counterparts [41] [42] [43] 46, 47 . While most cultivated chickpeas in the world have a single flower and therefore a single pod in the flower cluster 63,64 , double-podded chickpeas were first recorded by Khan and Akhtar 65 and governed by a single recessive gene 's' or 'sfl' 14 . As a result of the epistatic effect of the double-podded trait on a different background, multiple-flowered (more than two flowers) or multiple-podded progeny were first observed in some genotypes in F 3 and then F 4 (Fig. 4g) . Single-podded genotypes had larger seed size than double-and multi-podded genotypes, while double-and multi-podded genotypes had higher seed yield, numbers of pods and seeds per plant than single-podded genotypes. The triple-flowered trait (sfl t ) is controlled by a single recessive gene in the cultivated chickpeas 66 . The double-flowered trait (sfl d ) is dominant on the triple-flowered trait, and the dominance relationship of these alleles in this locus has been reported to be sfl (single flower) > sfl d (double flower) > sfl t (triple flower) 66 . In our study, the definition of 'multi-flowered' was used since the formation of four and five flowers per axil besides the formation of triple flowers was observed. When the genotypes with four and five flowers were examined, these formations were observed together with 'triple flowers' . These findings are similar to the triple-multi-flowered phenotypes resulting from crossing of doubleflowered and multi-flowered genotypes in the allelism study for the sfl and cym genes conducted by Srinivasan et al. 66 . Therefore, it was interpreted that the formation of four or five flowers may also be due to the presence of the cym (multi flowers) gene 66, 67 . In addition, it was determined that multi-flowered gene was expressed more phenotypically in unifoliolate leaf genotypes when compared to imparipinnate leaf genotypes (Tables 1, 2, 3) . There may be a relationship between multi-flowered and unifoliolate leaf traits. However, in the genotypes with unifoliolate leaf, it was determined that 'the third flower did not fill in pods' , while it was observed that pod filling was proportionally higher in multi-flowered plants in genotypes having imparipinnate leaf (Fig. 4g) . It was stated that greater photosynthetically leaf area in the genotypes with imparipinnate leaf could be the reason for higher seed yield 39 . Multi-podded plants had 23% and 7.6% more seed yield than that of single-podded and double-podded plants in F 4 population ( Table 2 ). The seed yield advantage of multi-podded plants was greater in F 3 than in F 4 population (Tables 1, 2) . Cho et al. 68 concluded that QTLs for 100-seed weight and number of seeds per plant were determined on LG 4. Also, these two traits were found to be associated negatively 68 . Genotypes having imparipinnate leaf had a higher seed yield than genotypes with unifoliolate leaf (Tables 1, 2 ). This result bore a resemblance to the findings of Abbo et al. 39 in chickpea having imparipinnate leaf. In addition, it was determined that genotypes having imparipinnate leaf had higher 100-seed weight, number of pods and seeds than genotypes having unifoliolate leaf (Tables 1, 2) . Higher seed yield and larger seed size in genotypes having imparipinnate leaf compared to unifoliolate-leafed genotypes were considered to be due to larger photosynthetic area (Tables 1, 2, 3, Fig. 4 ). Abbo et al. 39 stated that chickpeas reach higher leaf area indices in both www.nature.com/scientificreports/ program 72, 73 . Furthermore, the area of a single imparipinnate leaf during seed development is about 2 or 3 times larger than that of a single unifoliolate leaf 39 . Imparipinnate-leafed progeny attained 35% higher yield than unifoliolate-leafed progeny ( Table 2 ). In the chickpea trade, seed size and color characteristics are important criteria although its acceptability varies according to cultural preferences in different parts of the world. Large-seeded chickpeas are preferred by farmers to produce these chickpeas, as they are sold at high prices in local and international markets due to consumer preference 31, 32, 34, 46, [74] [75] [76] . Additionally, seed size is an important part of yield and adaptation 77 . This is also considered as an important factor for subsequent plant growth parameters, including germination, seedling vigor and seedling biomass 78, 79 . Therefore, large seed size provides an advantage to cope with drought stress compared to small seeds when planted deeper into the soil 22, 80 . In chickpeas, it is known that studies on genetic control of large seed size have been carried out since the 1950s [81] [82] [83] [84] [85] . Inheritance of seed size was demonstrated to be monogenic 86 and polygenic 84,87-89 through different studies. Although seed size in chickpeas has a high heritability 84,90-92 , it is not only affected by genetic factors but also by the environment 14, 17, 44, 93 . In terms of seed size, many genotypes (209 genotypes) with larger seeds than large-seeded parents were determined (Tables 2, 3, Fig. 4) . The additive gene effect has been reported for seed size in chickpeas 84, 94 and shows that selection for this trait will be effective in early generations. Seed size in chickpea was mapped using inter-and intraspecific recombinant inbred lines (RILs) and two quantitative trait loci (QTL) were located in linkage groups: L1, L2, L4, L5, L7 and L8 [95] [96] [97] [98] . Resistance to ascochyta blight has been one of the most important aims of chickpea breeding programs. Ascochyta blight is the most devastating disease of chickpea, attacking all upper parts of the plant (stems, leaves, pods and seeds) and the loss of seed yield can reach up to 100% under epidemic conditions in many chickpea growing regions around the world 48, 99, 100 . The introduction of winter sowing in order to increase the yield in the Mediterranean basin 101 has led to the need to develop varieties that are resistant to blight disease, which is one of the main factors that reduce the yield during this growing season. Resistance to ascochyta blight is a quantitative trait and numerous QTLs have been located on the chickpea genetic map [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] . As a requirement of conducting breeding studies on quantitative traits, the breeding process is complicated by trying to combine genes or QTLs in www.nature.com/scientificreports/ a new variety to increase the level and durability of resistance. Therefore, marker-assisted selection for ascochyta blight resistance has been successfully and effectively used in recent years to increase the selection efficiency in the breeding process 50, 51, 103, [115] [116] [117] [118] [119] . In recent years, several cultivars having an acceptable resistance level have been developed 38, 117, [120] [121] [122] . However, a significant portion of these cultivated varieties have small to medium seed sizes and do not meet the demands of the markets where large and extra-large seeded chickpea varieties are preferred. Since extra-large seeded kabuli chickpeas can find buyers at higher prices in the market, there is a need to develop extra-large seeded white/white-cream colored chickpeas. Resistance sources to blight have been identified by ICARDA from chickpea lines with medium seed size 123 . Some of these lines were included in breeding programs with large-seeded chickpeas. However, this allowed the selection of medium-sized chickpeas that were determined to be resistant to blight disease 117 . Resistance to ascochyta blight in chickpea is known to be controlled by more than two genes with small genes having an additive effect 124 . Knowledge on ascochyta blight was updated six years ago by Sharma and Ghosh 49 and about 50 markers/QTLs were listed. Madrid et al. 118 reported that the phenotype of 36 of 40 resistant genotypes (90%) and 14 susceptible genotypes could be accurately predicted by using the markers CaETR and SCY17 590 . The same markers were effectively used by Bouhadida et al. 125 to select genotypes resistant to blight. In the present study, both parents were selected as resistant to blight disease according to the evaluations in the local field conditions of the countries where they were registered. Although chickpea is tolerant to drought and heat stresses, it will be forced to be exposed to higher temperatures and more drought conditions owing to global warming as a result of climate change 29, 30 . In the present study, plants were subjected to high temperatures as high as 40 °C during flowering and pod setting stages (Fig. 6) . Despite the mentioned high temperatures, plants had more seed yield in F 3 than in F 4 population (Tables 1, 2, Fig. 6 ), meaning that yield could be decreased in homozygosity. High plant height is an important morphological trait for mechanized harvest in chickpea 22, 24, 25 , and plant height of 50-60 cm is considered as necessary for combine harvesting. In the present study, plant height ranged from 58 cm in double/multiple-podded plants having imparipinnate leaf to 62 cm double/multiple-podded plants having unifoliolate leaf (Table 3 ) when we considered superior lines. Days to first flowering in these superior lines were recorded to be 57 days in imparipinnate-leafed and double/multi-podded lines and 55 days in unifoliolate-leafed and double/multi-podded lines (Table 3 ). In spite of a crucial escape mechanism of earliness for drought and heat stress conditions, tolerance is the other vital mechanism in chickpea for drought and heat stresses 22, [26] [27] [28] . In the present study, plants had a sufficient tolerance mechanism for heat stresses since they were exposed to considerable heat stress (Fig. 6) . According to the findings, the following ideotype was defined in kabuli chickpea: • It should have imparipinnate-leafed traits, because these have more photosynthetic area than unifoliolate leaf type, and imparipinnate-leafed chickpeas attained 35% more seed yield than that of unifoliolate-leafed chickpeas under heat stress conditions. • It should be double/multi-podded, since these plants had the highest number of pods and seeds per plant and also the highest yield. Multi-podded plants not only produced 23% more seed yield than that of singlepodded plants, but also, multi-podded plants produced 7.6% more seed yield than that of double-podded plants under heat stress conditions. • It should be extra-large-seeded as large as 50-60 g per 100-seed weight, because extra-large seeds are preferred by consumers and producers due to their high price in national and international markets and advantage during germination, related with drought. • It should have enough plant height for combine harvest. Superior lines had 58-62 cm plant height. • It should be heat tolerant, since heat stress is forecast to increase in the near future due to global warming as a result of climate change. Superior lines had a six times higher seed yield that that of the best parent under heat stress conditions. • It should carry different resistance genes or QTLs for resistance to ascochyta blight, which is one of the widespread diseases of chickpea in the world, as minor genes from different backgrounds provide durable resistance. Two resistance markers (SCY17 590 and CaETR-1 markers) were integrated into six superior lines via marker-assisted selection. • Yield increases on a single plant basis will lead to significant yield increases per hectare. Thus, millions of tons of productivity of chickpea can be achieved worldwide with the ideotype improvement under heat stress, playing a crucial role for future demands of population and food security. • These approaches seem to be applicable in ideotype breeding for other important crop plants. Plant materials. Sierra (PI 631078) was crossed with CA 2969 (PI 632396) and the traits of the parents were previously described 38, 126 . Sierra and CA 2969 chickpea genotypes/cultivars were registered by USDA-ARS in cooperation with the Washington Agricultural Research Center, Pullman, WA and CIFA, Cordoba, Spain, respectively. The procurement of seeds of Sierra and CA 2969 used in the present study complies with relevant institutional, national, and international guidelines and legislation. From F 1 to F 3 population, advancement of the progeny was detailed by studying inheritance of large seed size and qualitative traits 32 . A total of 626 F 3 and 485 F 4 lines were used in the present study. Also, 152 F 3:4 lines with double-/multi-podded and 100-seed weight over 45 g were evaluated by marker-assisted selection (MAS) for resistance to ascochyta blight. These 152 lines were independently evaluated for agro-morphological traits. Experimental area and seasons. The study was conducted in fields at Akdeniz University, Antalya, Turkey (30° 38′ E, 36° 53′ N, 51 m above sea level) for two years during the crop seasons of 2019 and 2020. Plants were sown in April 2019 and March 2020, and harvested in July in both years. www.nature.com/scientificreports/ Agronomic applications. F 3 and F 4 lines were grown as progeny rows, while the parent plants were grown as four replicates (10 plants in a row) in the spring of 2019 and 2020. The selected 152 F 3:4 lines were grown to have 20 siblings from each line and distributed randomly into two blocks in the spring of 2020. Plants were sown in rows of 2 m length and spaced 50 cm apart between rows with a within-row plant spacing of 10 cm. Drip irrigation was used in both growing seasons. Weeds were plucked by hand during planting and before flowering. No fertilizer application was made. Soil properties. Soil in the experimental area was sampled between 0 and 30 cm and then analyzed to define the experimental soil characteristics. Some plant nutrient elements were found to be at an adequate level, while organic matter and nitrogen, iron and zinc levels were defined to be at a low level. Soil texture was loam with CaCO 3 of 26.5%, whereas pH was high with 7.69. Weather conditions. Plants were grown in the April-July period of 2019 and March-July period of 2020. Climatic data for the periods were provided by the T.C. Ministry of Agriculture and Forestry 4th Regional Directorate of Meteorology (Fig. 6) . (Fig. 6 ). Agro-morphological data. Qualitative traits such as leaf shape (as unifoliate or imparipinnate) and flower/pod per peduncle (as single/double/multiple flowers/pods) were recorded for each parent and progeny. In addition to the qualitative traits, the following quantitative traits, namely, days to first flowering (day) and days to 50% flowering (day), plant height (cm), first pod height (cm), number of main stems per plant (no), canopy width (cm), number of pods per plant (no), number of seeds per plant (no), seed yield per plant (g), biological yield per plant (g), 100-seed weight per plant (g) and harvest index (%) were evaluated. Seed size that is 100-seed weight was determined by using the following formula 32 : Transgressive segregations and superior lines. Transgressive segregation was coined as the occurrence of progeny with values greater or less than the values of their parents in segregated generations 60 . Superior lines are referred to as progeny with better values than that of the best parent 127, 128 . Molecular data for resistance to ascochyta blight. Young leaves were harvested from the F 3:4 plants and their parents, and then they were stored in a − 20 °C freezer until the date of DNA extraction. Genomic DNA was extracted according to the cetyl-trimethyl bromide (CTAB) protocol 129 . In order to determine the presence of QTLs associated with blight resistance, 152 F 3:4 lines and their parents were screened by using the markers CaETR-1 and SCY17 590 . While both markers are located on the LG4, CaETR-1 is an ASAP (allele specific associated primers) marker 118 and linked to QTL AR1 ; SCY17 590 is a SCAR (sequence characterized amplified region) marker linked to QTL AR2 108 . The polymerase chain reaction (PCR) for these analyses contained 1.5 µl of 20 ng/ µl DNA, 7.05 µl ddH2O, 1.5 µl 10× buffer, 1.5 µl dNTP's, 1 µl of 10 pmol primer and 0.2 µl of 5 U/µl Taq DNA polymerase. Amplification was provided in a Blue-Ray Turbo Cycler ® , which was used throughout this study, programmed for 35 cycles with the following temperature profile: 3 min at 95 °C, 40 s at 50 °C, and 50 s at 72 °C. Cycling was accomplished with a final extension at 72 °C for 5 min. The amplification products were separated on a 3% agarose gel and visualized with ethidium bromide staining. Data analyses. All agro-morphological data were analyzed for descriptive statistics using IBM SPSS Statistics 130 software. F 3 and F 4 populations were first divided into two groups according to the leaf shape as imparipinnate (compound, fern-like or normal) and unifoliolate (simple). Then, each group was divided into three groups as single-podded, double-podded and multi-podded according to pods per axil. In this way, six groups were formed. When selecting for the chickpea ideotype, each group was compared with the others. Agromorphological traits were analyzed using SPSS 22 (IBM SPSS Statistics 2014). 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This study was a part of MSc thesis of T.E. We are thankful to Dr. Fred J. Muehlbauer (Washington State University, Pullman,WA) and Dr. J. Gil (Universidad de Cordoba, Cordoba, Spain) for providing seeds of chickpeas. T.E. advanced generations in populations, carried out the investigations. T.E. and C.T. wrote the manuscript. T.E., H.S. and H.S.T. extracted DNA for the marker-assisted selection analysis. T.E. genotyped the parents and progeny used in the study. C.T. conceived the idea, supervised the research analysis, and edited the manuscript. D.S. read and reviewed the manuscript. All authors contributed and approved the final manuscript. The authors declare no competing interests. The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-022-05559-3.Correspondence and requests for materials should be addressed to T.E.Reprints and permissions information is available at www.nature.com/reprints.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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