Genetic Analysis of Cadmium Tolerance And Exploring Its Inheritance Nature In Bread Wheat

Cadmium (Cd) is a nonessential and extremely toxic element that destructively impacts agricultural production. Accordingly, developing tolerant-Cd as well as low-grain Cd genotypes is considered a promising approach to cope with the pollution problem. The current study aimed at understanding inheritance nature of Cd tolerance and detect Cd-tolerant and low-grain Cd genotypes in bread wheat. Six parents were selected based on their Cd tolerance and were genotyped using triple-RAPD and ISSR markers to investigate their genetic diversity. The selected parents were crossed and the realized F 1s were selfed to produce F 2 populations and were backcrossed with their own parents to produce BC 1 and BC 2 populations. Six populations for each cross comprised P 1 , P 2 , F 1 , F 2 , BC 1 and BC 2 were evaluated in two adjacent experiments under non-Cd stressed and Cd-stressed conditions. Signicant positive relative and standard heterosis were detected for ag leaf area, leaf chlorophyll content, proline content, Cd concentration and grain yield/plant under Cd-stressed condition. Dominance gene effect was more pronounced in controlling the evaluated traits in most cases. F values coupled with F/ √ H×D ratio were positive for Cd concentration and Cd sensitivity index in the three crosses under both conditions. Heritability estimates from offspring regression were high (< 50%) for ag leaf area, leaf chlorophyll content, proline content, Cd concentration while, moderately low for grain yield/plant and Cd sensitivity index. Prediction results revealed to high transgressive segregates and exceeding F 1 with best-inbred line (P max) that have all favorable alleles were obtained from 3rd cross for ag leaf area, low Cd concentration and Cd sensitivity index under Cd-stressed conditions. using triple RAPD and ISSR markers and assessing heterotic effects, genetic parameters, expected response from selection and prediction for low-Cd content and Cd tolerance. ratio was less than unity for proline content in 3rd cross and Cd concentration in 2nd and 3rd crosses under non-Cd stressed condition as well as for proline content in 1st cross and Cd concentration in the three crosses under Cd-stressed indicating partial dominance occurred. The additive and over-dominance type of genetic architecture are previously detected for ag leaf area; leaf chlorophyll content, Cd content, proline content and grain yield/plant by Awaad et al. (2013). Furthermore, EL-Gharbawy et al. (2015) disclosed that both additive and dominance gene effects were involved in controlling Cd and proline contents with a greater role for dominance and relatively high narrow-sense heritability in respect to proline content. On the other hand, Dunwei et al. (2012) manifested that Cd tolerance in wheat was governed by additive genetic variance. yield/plant Cd sensitivity index under both environments. to moderate heritability estimates registered in for traits under both conditions. Genetic advance percentage of the population high for proline content, Cd concentration under non-Cd and moderate under Cd-stress and detected to be high for Cd sensitivity index, and varied from low to moderate for the remaining traits, under both conditions. The simple inheritance based on high heritability and genetic advance observed herein indicates substantial progress could be achieved


Introduction
Heavy metals are one of the main obstacles that seriously threaten food safety (Konate et al., 2017;Rizvi et al., 2020). Cadmium (Cd) is one of the most prevalent toxic heavy metals that negatively impact plant growth and development (Tolcin, 2009; Sha q et al., 2019). Cd is usually released from industrial activities as plastic manufacturing, re ning and mining (Dong et al., 2019). Furthermore, increasing consumption of phosphate fertilizers and irrigation using wastewater leads to widespread Cd pollution in farmland (Zhang et al., 2015;Zaid et al., 2018). Cd causes numerous physiological disorders in lipid and protein synthesis, cell membrane and nutrient metabolism (Chugh and Sawhney 1999;Lesser, 2006). Moreover, it has destructive impacts on root growth and elongation (Atal et al., 1991;Black et al., 2014).
Wheat is the most important grain source for humans worldwide, and it is cultivated on more land areas than any other eld crop (FAOSTAT, 2021). Wheat Cd contamination poses a substantial health risk (Nordberg, 2009). The European Food Safety Authority has lowered the tolerable weekly intake of Cd from 7 to 2.5 μg Cd -1 kg -1 body-weight (Adeniji et al., 2010;Singh et al., 2011). Long-term human exposure to Cd even at a low rate causes impaired kidney function, bone demineralization, emphysema, and proteinuria, and increases the threat of lung cancer (Bernard, 2008;Nordberg, 2009).
The hazard of Cd contamination highlights the importance of breeding for Cd-tolerant and low-grain Cd wheat genotype. Noticeable genotypic differences are detected in grain-Cd accumulation in bread wheat (Clarke et al., 1997;Stolt et al., 2003;Arduini et al., 2014;Guo et al., 2018). Developing tolerant wheat genotypes to Cd-stress with low Cd uptake into grains is the most e cient approach to reduce health threats as well as mitigate its negative impacts on plant growth (Zaid et al., 2018). Breeding Cd-tolerant wheat genotypes with lower Cd content requires crossing among superior individuals followed by selection in succeeding generations based on related traits to Cd-stress (Oladzad-Abbasabadi et al., 2018). Exploitation of heterosis provides an e cient perspective for enhancing wheat Cd potential and tolerance to Cd-stressed. Nevertheless, there remain several restrictions to breeding low-Cd wheat cultivars as slow and high cost of genetic improvement process as well as consuming long time. Developing low-Cd content and Cd-tolerant requires reliable understanding of natural genetic variation and inheritance of associated traits which is less characterized for hexaploid bread wheat. Therefore, the present investigation aimed at investigating the genetic diversity among bread wheat genotypes using triple RAPD and ISSR markers and assessing heterotic effects, genetic parameters, expected response from selection and prediction for low-Cd content and Cd tolerance. Materials And Methods 2.1. Investigating the genetic diversity among selected parents Twenty diverse bread wheat genotypes were screened for Cd tolerance in a preliminary experiment. The highly-tolerant six genotypes were selected to be crossed; namely Giza-168, Sids-6, ACSAD-925, Gemmeiza-10, ACSAD-935 and Line-1 (Table S1). The genetic diversity among selected parents was investigated using triple-RAPD and ISSR markers. DNA was extracted from young and fresh leaves (0.1 g) of the selected parents by the CTAB (cetyltrimethylammonium bromide). The quantity and quality of extracted DNA were measured (2 µl) by a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Delaware, USA). The DNA samples were altered to a concentration of 50 ng µl − 1 with ddH 2 O and used for PCR ampli cation.
Triple-RAPD-PCR reaction was applied as described by Williams et al. (1990)  A set of fteen ISSR primers was obtained from Metabion, Germany (Table 1). PCR ampli cation was applied as outlined by Dangi et al. (2004). Twenty ng of DNA was mixed with 50 mM KCI, 10 mM Tris-HCI pH 7.5, 0.5 mM spermidine, 1.5 mM MgCl2, 0.1 mM dNTPs, 0.8 U of Taq DNA polymerase and 0.3 uM primer in a 25 μl reaction utilizing Perkin Elmer 2400 thermocycler. All used chemicals for the reaction were procured from Sigma-Aldrich, USA.
Agarose gel electrophoresis (1.6%) was used for separating the ampli ed fragments. The fragments were recorded using EG-Gel Analyzer V1 software. The genetic similarity among parents was investigated by Nei's genetic distance (Nei, 1987). The dendrogram was performed using the Unweighted Pair Group Method with Arithmetic averages (UPGMA). The estimates were applied using the NTSYS-pc 2.02 software package (Numerical Taxonomy System, Exeter Software, Rohlf, 2000).

2.2.Crossing among selected parents
Three crosses were performed between the selected genetically diverse six parents; Giza-168×Sids-6 (1st cross), ACSAD-925×Gemmeiza-10 (2nd cross) and ACSAD-935×Line-1 (3rd cross). The F 1 populations were selfed to produce F 2 populations and were backcrossed with their own parents to produce BC 1 and BC 2 populations. Six populations for each cross; P 1 , P 2 , F 1 , F 2 , BC 1 and BC 2 were evaluated in two adjacent experiments in a randomized complete blocks design with three replications at the experimental farm of the Faculty of Agriculture, Zagazig University, Egypt (30°34'10" N 31°34'20" E). The rst experiment was sprayed with cadmium solution at the beginning of heading stage by a concentration of 30 ppm Cd ion/liter of water (475 liter/ha). The second experiment was used as a control with pure water spraying. Seeds of six populations were sown in the fourth week of November. Rows were 2.5-m long and 20-cm apart, while a plant to plant space was 10-cm. The recommended agricultural practices for wheat production in the region were applied.

Measured traits
Data were recorded on individual guarded plants for the evaluated populations. Flag leaf area was determined at the time of full emergence of main spike. Flag leaf chlorophyll content was measured by SPAD-502 apparatus. Proline content in leaves was estimated as described by Bates et al. (1973)  also computed according to Falconer (1989). The components of the genetic variance i.e. additive VD, dominance VH and environmental VE variances were estimated as described by Mather and Jinks (1982) and were utilized further to calculate frequency between dominance and recessive alleles in the parental populations F = (VBC 2 + VBC 1 ) and the dominance at different loci (F/√ H × D).

Predicting properties of new recombinant lines
The properties of new recombinant lines that fall outside the parental range and exceeding F 1 hybrid following sel ng generations were calculated using Jinks ). In the current study, genetic diversity was investigated using triple-primer RAPD and ISSR markers. Triple-RAPD ampli cation reactions were applied using different combinations of three different decamer oligonucleotides that had been previously tested in the single-primer PCR (Table 1). In all cases, the combination of three primers (in 1: 1: 1 ratio) led to appearance of new bands that were not ampli ed using each primer independently (Fig. 1A). The sizes of produced fragments varied from 100 bp to 2 Kbp. A total of 440 bands were recorded, in average 35 ± 2% band per primer/gel, 12 ± 2 % polymorphic, 25 ± 2% unique bands and 36 ± 2% polymorphic (with unique), which revealed 70 to 80% polymorphism. While certain bands were monomorphic in all genotypes, there were speci c bands for each one (Fig. 1A). Genetic similarity was determined by Nei's index value for all genotypes considering Triple-RAPD results, then were employed to perform dendrogram using Unweighted Pair Group Method with Arithmetic averages (UPGMA) (Fig. 1B, C ). The dendrogram displayed genetic diversity among used wheat parents.
ISSR technique utilizes frequently 16-25 bp long primers in a single primer PCR reaction focusing on multiple genomic loci to amplify principally the inter-SSR sequences of different sizes (Ziêtkiewicz et al., 1994). In the current study, a set of 50 ISSR primers was applied for preliminary screening of six wheat genotypes (Fig. 1A). However, only fteen ISSR primers identi ed intraspeci c variation in wheat genotypes produced on average fteen bands per gel/primer in the range of 100 bp to 2 kbp. Among these bands, four were polymorphic bands and sixteen were unique bands revealing polymorphism. Based on ISSR gels patterns, the similarity index values were employed to create dendrogram utilizing UPGMA. The obtained dendrogram showed different clusters displaying variation in the frequencies of SSR motifs (  Inbreeding depression and dominance deviations displayed a similar trend and were found to be signi cantly positive for ag leaf area in the three crosses,  (Table 2). F 2 deviation exhibited signi cant positive estimates for ag leaf area, leaf chlorophyll content and Cd concentration in 1st cross; grain yield/plant in 2nd cross under both conditions; leaf chlorophyll content in 3rd cross under Cd-stressed. Otherwise, it was negative and signi cant for ag leaf area, Cd concentration in 3rd cross; proline content in 2nd and 3rd crosses under both conditions; leaf chlorophyll content in 3rd cross under non-Cd stressed as well as Cd sensitivity index in 1st and 2nd crosses.

Gene effect and heritability
The nature of gene action and heritability play an important role in identifying the appropriate breeding method to improve economic traits through breeding programs. Genetic parameters controlling cadmium stress tolerance and related traits are presented in Table (3  h 2 is heritability estimates from parent-offspring regression, RH is realized heritability and Gs% is genetic advance from selection   Heritability computed from parent-offspring regression (h 2 ) and realized heritability (RH) are shown in Tables (3) and ( under Cd-stressed, the best-inbred line (P max) was registered by 2nd cross for ag leaf area, leaf chlorophyll content, proline content and Cd concentration while 3rd cross for grain yield/plant. Similar interpretation was stated by Mather and Jinks (1982) and also Awaad (2002) elucidated high proportion of recombinants falling outside parental range and exceeding F1 for grain yield/plant and morpho-physiological traits.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.docx