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Comparison of root staining and root elongation in predicting aluminium tolerance using SSR markers in barley

Junping Wang1,2, Harsh Raman2, 3, Barbara Read2, Meixue Zhou1, Neville Mendham1 and Shoba Venkatanagappa2

1 School of Agricultural Science, University of Tasmania, Hobart, Tasmania, 7001, Australia
NSW Agriculture, Wagga Wagga Agricultural Institute, PMB, Wagga Wagga, NSW 2650, Australia
Corresponding author E-mail:


Hematoxylin staining of root tips and root elongation measurements under aluminium (Al) stress are two criteria which have been used widely for screening tolerance to Al toxicity in barley (Hordeum vulgare L.). Our results suggest that Eriochrome Cyanine R staining can be used to identify Al tolerant and Al sensitive barley lines. Root staining data confirmed that Al tolerance was controlled by single gene (Alp) derived from ‘Dayton’ in five F2 populations comprising 987 plants from a complex cross, ‘WB238’// ‘Dayton’/ ‘F6ant28B48-16’/3/ ‘F6ant28B48-16’. The correlation coefficients between root elongation and root staining were 0.90, 0.52, 0.66, 0.25, and 0.45 in five populations XB2340-1, XB2340-2, XB2340-3, XB2340-4, and XB2340-5 respectively. The correlations were significant at the 0.001 level. Linkage analysis identified that Bmag353 was the closest marker linked to the Al tolerance gene Alp as shown by root staining. However, Bmag353 explained 10-78 % of the variation for root elongation in 48 hours under 15μM Al stress. F3 progeny testing is being carried out to confirm F2 genotypes.

Media summary

Eriochrome Cyanine R staining can be used in selection of barley lines for aluminium tolerance. SSR marker Bmag353 is closely linked to an Al tolerance gene as indicated by root staining pattern and can be used for marker assisted selection in barley breeding programs.


Acidic soil, hydroponic culture, microsatellite, MAS, PCR


Aluminium (Al) toxicity is a major constraint to barley growth in acidic soils. Al tolerance in barley is controlled by a single gene (Minella and Sorrells 1992; Raman et al. 2002). Cultivar ‘Dayton’ is one of the most tolerant genotypes to Al toxicity (Raman et al. 2003a). The gene Alp conferring Al tolerance in cv. ‘Dayton’ has been mapped on chromosome 4H by RFLP markers (Tang et al. 2000). Subsequently, Simple sequence repeat (SSR) markers linked with Alp were developed in the same population, as they are more suitable for marker-assisted selection (Raman et al. 2003a).

A hematoxylin staining method was used to screen an F2 population from ‘Dayton’/ ‘Harlan hybrid’ for Al tolerance (Tang et al. 2000). However, this method was not suitable for definitive discrimination of Al tolerant genotypes from sensitive genotypes and therefore a root elongation method was used (Raman et al. 2001, 2003a). In addition to hematoxylin, Eriochrome Cyanine R staining pattern was used to screen barley cultivars for Al tolerance by Ma et al. (1997).

In this study, two methods, (a) root staining using Eriochrome Cyanine R and (b) root elongation measurements under Al stress were compared to determine whether these methods would identify the same Al tolerant/Al sensitive barley lines. The efficiency of SSR markers closely linked with Alp locus was also tested in predicting Al tolerance in five F2 segregating populations.

Materials and Methods

Plant materials

Five F2 populations with a total of 987 individuals derived from a complex cross ‘WB238’// ‘Dayton’/ ‘F6ant28B48-16’/3/ ‘F6ant28B48-16’ were used is this study. Parents of the populations ‘Dayton’, ‘WB238’ and ‘F6ant28B48-16’ were used as checks.

Al tolerance evaluation

Hydroponic culture was performed as described by Raman et al. (2003a). The longest root length per plant was measured before and after 48 hours under 15 μM Al stress. Root staining was conducted using 0.1% Eriochrome Cyanine R (Ma et al. 1997) after 3 days Al stress. Heavily stained roots with no lateral root growth were scored as sensitive. Roots with no staining and good lateral roots growth similar to ‘Dayton’ were scored as tolerant. Roots with partial staining and limited lateral root growth were scored as intermediate. The correlation of root elongation under Al stress and root staining was statistically analysed.

DNA extraction, PCR and genotyping

DNA was extracted from the young leaves of the 987 F2 plants from ‘WB238’// ‘Dayton’/ ‘F6ant28B48-16’/3/ ‘F6ant28B48-16’. Eight markers (Bmac181, Bmac186, Bmac310, Bmag353, Bmag375, Bmag490, HVM3, HVM68) previously mapped on the long arm of chromosome 4H (Ramsay et al. 2000) and reported to be linked with Al tolerance (Raman et al. 2001, 2002, 2003a) were screened for polymorphism among parents of the F2 populations, ‘Dayton’, ‘F6ant28B48-16’ and ‘WB238’. The primers pairs were synthesised from published sequences (Liu et al., 1996; Ramsay et al., 2000) and were tailed with M13 generic sequence at 5’ end and M13 fluorescent dyes were added in PCR reactions (Raman et al. 2003b). After amplification, samples were loaded in Beckman Coulter CEQ 8000 Genetic Analysis System for capillary electrophoresis.

Linkage analysis

SSR marker alleles from ‘Dayton’ (Al tolerant) were scored as ‘A’ type, SSR alleles from sensitive parents (‘WB238’ and ‘F6ant28B48-16’) were scored as ‘B’ type, and SSR alleles from both ‘Dayton’ and ‘F6ant28B48-16’ or ‘WB238’ were scored as ‘H’ type. Linkage analysis was performed in one population XB2340-4. Then the closest linked marker, Bmag353 was used to validate Al tolerance as indicated by root staining pattern in all five F2 populations. Root elongation measurements were used as trait values to determine the linkage between the trait and Bmag353 by marker regression analysis using software Map Manager QTX version 19b.


The qualitative assay based on Eriochrome cyanine R root staining patterns of the parents used to develop F2 populations exhibited marked differences (Fig. 1). The roots of tolerant ‘Dayton’ showed no staining and had normal lateral roots, while roots of sensitive ‘WB238’ and ‘F6ant28B48-16’ accumulated stain (purple) and did not develop lateral roots but were replaced by a number of small, heavily stained nubs along the seminal roots (Fig. 1).

Figure 1. Root stain pattern of ‘Dayton’ (left), ‘F6ant28B48-16’ (middle) and ‘WB238’ (right)

The results of Al tolerance classification by root staining and the range and average of root elongation under Al stress in each F2 population are shown in Table 1. A chi-square test showed that the segregation of four out of the five populations fitted into 1:2:1 pattern, indicating a single gene controlling Al tolerance. Only population XB2340-2 exhibited a distorted segregation ratio, with less tolerant plants than expected. Root elongation under Al stress and root staining were highly correlated in XB2340-1(r = 0.90, t = 26.28), but poorly correlated in XB2340-4 (r = 0.25, t = 3.71). In other populations the correlations were moderate (Table 1). Significance tests showed that all correlations were significant at the 0.001 level.

Table 1.Phenotypic expression of Al tolerance using root staining assay and correlation with root elongation (mm) under Al stress







Number of plants












Range of Root Elongation






Average of Root Elongation












Range of Root Elongation






Average of Root Elongation












Range of Root Elongation






Average of Root Elongation






χ2-test (p) for 1:2:1






Correlation Coefficient (r)
Significance test of
Correlation (t)






* Lines scored on the basis of root staining test

** Significant at the 0.05 level

*** Significant at the 0.001 level

Among the eight markers, Bmac181 and Bmac186 were not polymorphic. HVM3 exhibited distorted segregation and was delimited from linkage analysis. The location of the gene for aluminium tolerance in XB2340-4 was: Alp-Bmag353 (5.01.1) -Bmag490 (3.20.9)-Bmag375 (3.81.0). Bmag353 was the closest linked marker and it was detected in all other populations. The linkage distance between Alp conferring Al tolerance indicated by root staining assay and Bmag353 varied from 0.9 to 15.5 cM depending on the population (Table 2).

Table 2. Map distance and variance in root elongation under Al stress explained by Bmag353


Map distance Standard error (cM)

Root elongation explained by Bmag353 (%)
















Marker regression analysis revealed that Bmag353 accounted for 10 to 78% variation of the root elongation measurements under Al stress in five populations (Table 2).


In previous studies, the hematoxylin method was used for screening segregating populations derived from improved germplasm (Minella and Sorrells 1992; Tang et al. 2000; Echart et al. 2002). However, this method was unsatisfactory for definitive screening for Al tolerance in barley (Raman et al., 2003a). Eriochrome cyanine R staining was previously used to assess root re-growth after Al stress in wheat (Aniol 1995). In our study, the Eriochrome cyanine R staining method was successfully employed to screen barley segregating populations. When the Al did not destroy the root apical meristem, the part of the root which grew after Al stress was white (unstained). When the root apical meristem had been irreversibly damaged, the root tips remained intensively stained (Aniol 1995). In the present study, we have made similar observations in F2 populations derived from the complex cross ‘WB238’// ‘Dayton’/ ‘F6ant28B48-16’/3/ ‘F6ant28B48-16’.

In this study, data on the root staining assay fitted a co-dominant monogenic segregation ratio (1:2:1), corresponding to Al tolerant, intermediate/heterozygote and Al sensitive phenotypes respectively. Echart et al. (2002) observed a 3:1 segregation ratio in an F2 population from barley cross between ‘FM-404’ (Al tolerant) and ‘Harrington’ (Al sensitive) using hematoxylin staining and this ratio fitted a dominant monogenic model. Minella and Sorrells (1997) noted that dominance or recessivity was dependant on Al concentration. Even though the correlation coefficients were from 0.25 to 0.90, significant test showed that the correlations of root elongation and Eriochrome Cyanine R were significant at the 0.001 level. Marker regression showed that Al tolerance evaluation based on root elongation measurements under Al stress and root staining was not consistent in the five F2 populations. Poorer correlation could be attributed to the involvement of genes that affect root growth rate, differences in root vigour and lack of replication in experiments based on single F2 plants. However, root staining is suitable for screening segregating populations and the root growth assay was able to identify Al tolerance in different genotypes (Hede et al. 2002).

In earlier studies, Alp and Bmag353 linkage was reported to be 3.1 cM in ‘Dayton’/ ‘Harlan Hybrid’ population, based on only 48 F2 individuals from a primary cross (Raman et al. 2003a). In the present investigation, we used larger numbers of F2 plants (987) to confirm the Al tolerance-SSR association. Mapping distances between Bmag353 and Alp were observed to range from 0.9 to 15.5 cM in these five populations. F3 progeny testing is currently underway to confirm F2 genotypes.


This work is a part of PhD research of Mrs Junping Wang. The authors would like to thank NSW Agricultural Genomic Centre and University of Tasmania for providing financial support for this project.


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