Twelve linkage groups (LGs) were created with 163 polymorphic markers using the JoinMap 4.1 software. The LGs (Table 1) were created using 31 SRAP, 19 SSR, and 11 RAPD markers. Based on the results, a map of 929.6 cM length was obtained, with a total of 62 polymorphic bands and 12 LGs. The length of each LGs is given in Table 1. The average distance between the markers was 14.99 cM. The SSR markers were used to assign the LGs similar to Wu et al [12] in Solgenomics.net for the population of C. annum x C. frutescens.
Table 1
Distribution of markers on linkage groups developed from C. annum x C. frutescens F2 population
Chromosome (Chr) | Chr1 | Chr2 | Chr3 | Chr5 | Chr6 | Chr8 | Chr9 | Chr10 | Chr11 | ChrX | ChrXX | ChrXXX | Total |
Total | 5 | 3 | 3 | 7 | 5 | 8 | 7 | 7 | 5 | 4 | 5 | 3 | 31 |
SSR | 2 | 2 | 1 | 1 | 1 | 5 | 3 | 2 | 2 | - | - | - | 7 |
SRAP | 3 | - | 2 | 5 | 3 | 3 | 3 | 4 | 1 | 3 | 4 | 1 | 15 |
RAPD | - | 1 | - | 1 | 1 | - | 1 | 1 | 2 | 1 | 1 | 2 | 8 |
Length (cM) | 95.4 | 2.,1 | 83.7 | 113.5 | 59.3 | 95.5 | 114.4 | 104.9 | 94.3 | 88.5 | 39.3 | 19.7 | 461.1 |
Average interval (cM) | 19.1 | 7.03 | 27.6 | 16.2 | 11.9 | 11.9 | 16.3 | 14.9 | 18.9 | 22.1 | 7.86 | 6.6 | 14.99 |
QTL mapping
QTLs were mapped in the MapQTL.6 software after the LGs was created by JoinMap 4.1 software. The quantitative data obtained from replicated F2:3 families under Zn deficiency were mapped on LGs using “Kruskal Wallis” and “Internal Mapping” analyzes. The QTLs for nine traits and their mapped chromosome region are presented in Table 2 and Fig. 1.
Scores of Zn deficiency symptoms in the F3 population
Zn deficiency symptom scores were recorded once a week throughout a six week period, starting at the beginning of the flowering stage. The average scores of 12 plants of each family derived from 126 F2:3 populations was analyzed by the MapQTL software. The four QTLs were mapped on LGs 1, 8, 10 and X. The most important markers for this character were EM8ME7.270, OPAH2.290, EM14ME1.480, and EM5ME13.350 (Table 2 and Fig. 1.). The QTLs explained 15.4% of the phenotypic variance of the trait for Zn deficiency symptoms.
Table 2
The list of QTLs and associated markers identified for Zn deficiency symptoms in pepper
Trait Number | Trait name | QTL Symbol | Chr | Markera | QTL Position (cM) | R2b | Directiona |
1 | Scores of Zn deficiency symptoms in the F3 population | f3scor1.1 f3scorx.1 f3scor8.1 f3scor10.1 | 1 X 8 10 | EM8ME7.270 OPAH2.290 EM14ME1.480 EM5ME13.350 | 0.00 0.00 73.02 31.89 | 2.3 2.0 7.9 3.2 | PI 281420 PI 281420 PI 281420 PI 281420 |
2 | Zn effectiveness in terms of total dry matter weight | tdmznef3.1 tdmznef8.1 tdmznef11.1 tdmznefx.1 | 3 8 11 X | EM6ME6.260 EM14ME1.480 OP108.370 EM11ME8.380 | 83.67 73.02 59.53 50.04 | 4.7 6.9 6.4 5.1 | PI 281420 PI 281420 PI 281420 PI 281420 |
3 | Zn effectiveness in terms of plant length | plhtznef3.1 plhtznefx.1 plhtznef5.1 plhtznef6.1 plhtznef8.1 plhtznef9.1 plhtznef11.1 | 3 X 5 6 8 9 11 | EM6ME6.260 EM11ME8.380 OPAH2.290 OPAC10.330 At1G14810 GPMS171 OP108.370 | 83.68 50.04 0.00 33.78 95.53 41.41 59.53 | 6.2 9.2 2.5 3.2 5.6 6.0 6.7 | PI 281420 PI 281420 PI 281420 PI 281420 - - PI 281420 |
4 | Zn effectiveness in terms of Zn concentration in total dry matter | znconef3.1 znconef6.1 znconef8.1 znconef9.1 znconef9.2 znconefXXX..1 | 3 6 8 9 9 XXX | BM59622 C2At1g44760 GP20095 EM5ME11.280 GPMS171 OP108.210 | 0.00 0.00 47.79 0.00 41.41 0.00 | 5.8 4.0 5.1 5.1 5.0 3.2 | - - - PI 281420 - Alata 21A |
5 | Zn effectiveness in terms of leaf dry matter weight | ldmwef3.1 ldmwef8.1 ldmwef11.1 | 3 8 11 | EM6ME6.260 EM14ME1.480 OP108.370 | 83.68 73.02 59.53 | 4.5 5.0 6.3 | PI 281420 PI 281420 PI 281420 |
6 | Scores of Zn deficiency symptoms in the F2 population | f2scor8.1 f2scor8.2 f2scor10.1 | 8 8 10 | GP20095 HPMS1155 OPB01.490 | 47.79 31.88 70.34 | 6.8 6.0 5.1 | - - PI 281420 |
7 | Zn effectiveness in terms of Zn content in leaves | lzncntef6.1 lzncntef8.1 lzncntef11.1 | 6 8 11 | OPAC10.330 HPMS1155 OP108.370 | 33.78 31.88 59.53 | 3.0 5.6 6.2 | PI 281420 - PI 281420 |
8 | Zn effectiveness in terms of zn content in dry matter | tdmzncntx.1 tdmzncntx.2 tdmzncnt8.1 tdmzncnt8.2 tdmzncnt9.1 tdmzncnt11.1 | X X 8 8 9 11 | OPAH2.290 EM7ME6.200 EM14ME1.480 GP20095 OPI03.350 EM5ME11.220 | 0.00 88.54 73.02 47.79 114.6 21.31 | 5.8 3.8 9.9 4.1 4.3 5.3 | PI 281420 PI 281420 PI 281420 - PI 281420 - |
9 | Zn effectiveness in terms of Root / Shoot Ratio | rsref1.1 rsref5.1 rsref6.1 rsref10.1 rsref11.1 | 1 5 6 10 11 | CAEMS060 EM3ME10.290 EM8ME7.190 EM5ME13.260 GP20117 | 40.66 0.00 16.41 24.38 0.00 | 3.4 2.3 5.7 3.5 6.0 | - PI 281420 PI 281420 PI 281420 - |
a Indicates the parent which contributes to the increase in the numeric value of the trait |
Zn efficiency for total dry matter weight
Zn efficiency in terms of total dry matter (Leaf + stem + root) was calculated from 24 plants of each F3 family (12 plants under Zn supplied and 12 plants under the Zn deficiency).
As shown in Online Resource 2, Alata 21A had 59% higher Zn efficiency when compared with PI 281420. The F1 plants exhibited 81% Zn efficiency, exceeding parental averages (71.85%). This indicates that the tolerance to Zn deficiency is partially dominant. Even when Zn deficiency symptoms are mild, a significant decrease can occur in the dry matter of the plant. A wide variation was obtained from the parent, F1 and F3 plants regarding to their response to Zn deficiency and at least 4 QTLs on LGs 3, 8, 11, and X were identified.
Zn efficiency measured by plant height
The effect of Zn on plant height was measured using F3 families grown with or without Zn (Online Resource 2). Plant height showed a wide variation and a transgressive segregation was observed. The effect of Zn deficiency to plant height was mapped on LGs 3, 5, 6, 8, 9, 11 and X. The locations of 4 QTLs on LGs 3, 8, 11 and X were common to the QTLs of the Zn efficiency for total dry matter weight (Table 2 and Fig. 1).
Zn efficiency for Zn concentration in total dry matter
The critical Zn concentration in tissues can vary depending on the plant species, type, plant age, plant part, and environment. All shoots, roots, young leaves, and grains can be used for the determination of Zn content. The most appropriate sample taken for plant nutrient status is leaves [17–18]. Based on the results, even though Alata 21A was the tolerant parent to Zn deficiency, its total dry matter weight was decreased under Zn deficiency. However, total dry matter weight of F1 plant was higher than the parents and also dry matter weight of the F3 plant was higher than F1 plants and parents (Online Resource 2). Alata 21A grew faster than the other parent, and the increase in the number of leaves and area together with the increase in the roots causes to a dilution in Zn concentration in the plant and it can cause to a decrease in the Zn concentration in the total dry matter. However, the PI 281420 is a slow-growing genotype and it helps to provide more stable Zn accumulation within its organs. On the other hand, F1 plants were able to achieve a value above the average of two parents by providing both rapid development and Zn accumulation in the unit area, hence they can inherit the characteristics of both parents. The F3 families showed transgressive segregation due to inheriting the characteristics of both parents. Similarly, Sadeghzadeh et al [19] found that Zn concentration in seeds is a multigenic character. In this study, the Zn concentration values (Online Resource 2) in the root, stem, and leaves of the F2 and F3 population were used for QTL analysis, and 6 QTLs were mapped on LGs 3, 6, 8, 9 and XXX (Table 2 and Fig. 1). The two QTLs belonging to the Zn efficiency for total dry matter and plant height were located on the same position on LGs 3 and 8. For the Zn concentration of total dry matter, five QTLs explained 28.2% phenotypic variance of this trait (Table 2 and Fig. 1).
Zn efficiency for leaf dry matter weight
The leaves, stem, and root samples of the plants were harvested separately and analyzed. The distribution of Zn based on leaf dry matter weight is presented in Online Resource 2. The lowest Zn dry matter was observed in the PI 281420 and the highest in Alata 21A while F1 had a value close to the tolerant parent, indicating an incomplete dominance of the trait. The three QTLs were mapped on LGs 3, 8, and 11 for the trait and explained 15.3% of variance (Table 2 and Fig. 1). The three QTLs associated with total dry matter weight were also co-localized with the QTLs for the plant height.
Scores of Zn deficiency symptoms in the F2 population
The six times scoring of Zn deficiency symptom of 455 F2 plants was made according to the 1–5 scale, and the distribution of data from the final scoring was presented in Online Resource 2. Based on the results, some F2 plants had more severe symptoms than the sensitive parent (PI 281420) and also some seedlings were more tolerant than the tolerant parent (Alata 21A) under Zn deficiency, indicating a transgressive segregation for both ends. As presented in Online Resource 2, it was demonstrated that Zn deficiency is partially dominant. Three QTLs were found for the symptoms in F2, two on the LG8, and one on LG10. However, an additional QTL was determined for the Zn deficiency symptom scores of the F3 population (Online Resource 2). Furthermore, it was estimated that number of genes governing Zn deficiency symptom in the F2 population is about 1.61 and 1.54 [20–21], indicating two genes control the trait. Therefore, the QTLs on LGs8 and 10 confirmed that this trait is multigenic. The QTL on LG8 co-localized with QTLs for Zn concentration in total dry matter, Zn contents in leaves and dry matter. The tree QTLs explained 17.9% variance for the Zn deficiency symptom in the F2 population (Table 2 and Fig. 1).
Zn efficiency for Zn content in leaves
The segregating populations showed variation for leaf Zn content. The three QTLs were mapped on LGs 6, 8 and 11, and the QTLs on LGs 8 and 11 were co-localized with QTLs for total dry matter and leaf dry matter weights, and Zn deficiency symptoms in F2. The three QTLs explained 14.8% variance of leaf dry matter (Supplement 2 G and Table 2 and Fig. 1).
Zn efficiency for Zn content in dry matter
A very wide variation (14,0–89,7%) was found in terms of Zn content in total dry matter (Online Resource 2). The F1 plants showed a higher value than both parents, indicative of a heterosis or epistatic effect. Furthermore, as for Zn content in stem and root, F1 plants showed the higher value than both parents, while in leaves they responded similar to sensitive parent.
The six QTL were mapped for this trait; two QTLs on LGX, two on LG8, one on LG9, and one on LG11. Although there were 3 QTL effective on Zn content of leaf dry matter, 6 QTL was determined in total Zn content. The position of the QTLs located on the LGs 8 and 11 are close to each other. The six QTLs (Table 2 and Fig. 1) explain 33.2% of variation for Zn content in total dry matter.
Zn effectiveness in terms of root / shoot ratio
Root / shoot ratio is one of the most important parameters in plant growth. The water and nutrient uptake may be increased by enhanced root/shoot ratio. In the Zn deficiency of the soils, the Zn uptake will be increased by an increase in the amount of root and expansion of the root surface area. There was a large variation in the F3 population. The F1 plants has similar root / shoot ratio, similar to sensitive parent. The five QTLs were mapped on LGs 1, 5, 6, 10, and 11 (Table 2 and Fig. 1). The 5 QTLs explained 20.6% variance for the trait (Online Resource 2).