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Crop Science 40:370-374 (2000)
© 2000 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Association of Puroindoline Sequence Type and Grain Hardness in Hard Red Spring Wheat

Michael J. Girouxa, Luther Talberta, Debrah K. Habernichta, Susan Lanninga, Amber Hemphilla and John M. Martina

a Dep. of Plant Sciences, P.O. Box 173150, Montana State University, Bozeman, MT 59717-3150 USA

mgiroux{at}montana.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
Wheat (Triticum aestivum L.) endosperm texture is a primary determinant of milling and end-product quality. Friabilin, a marker protein for grain hardness, is composed of two proteins, puroindoline a and b (pinA and pinB, respectively). Hard-textured wheats have variant alleles consisting of a glycine-to-serine change in pinB (pinB-D1b) or the complete absence of pinA (pinA-D1b). Our objectives were to examine the influence of pinA and pinB alterations on grain hardness from populations among elite hard red spring wheat cultivars differing in puroindoline alteration, and to measure associations of grain hardness with kernel weight and grain protein concentration. Fifty F3:6 progenies from three pinA-D1b x pinB-D1b, one pinB-D1b x pinB-D1b, and one pinA-D1b x pinA-D1b type crosses were evaluated with their parents in two field experiments. Lines classified as pinA-D1b were significantly harder than lines classified as pinB-D1b when averaged across the three segregating populations. This difference was significant (P < 0.05) in one of the three populations. Significant genetic variation existed for grain hardness, protein concentration, and kernel weight within puroindoline classes and among lines from crosses not segregating for pinA-D1b vs. pinB-D1b. Significant positive correlations were observed in all five populations for grain hardness and protein concentration ( ). Our results indicate that most of the genetic variation in grain hardness among the populations studied was due to factors other than pinA and pinB, as the pinA-D1b vs. pinB-D1b difference explained <12% of the variation in grain hardness in these hard wheat populations.

Abbreviations: Ha, hardness gene • NIR, near-infrared reflectance • NIT, near-infrared transmission • pinA, puroindoline A • pinB, puroindoline B • pinB-D1b, glycine-to-serine change in pinB allele • pinA-D1b, absence of pinA allele • 5DS, short arm of chromosome 5D


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results and discussion
 REFERENCES
 
WHEAT ENDOSPERM TEXTURE is a primary determinant of both milling and end-product quality (reviewed in Pomeranz and Williams, 1990; Anjum and Walker, 1991). Endosperm texture difference between hard and soft classes in wheat is simply inherited and is believed to be controlled by one or two major genes with one or more modifying genes (Symes, 1965; Baker, 1977). One major gene governing texture, designated Hardness (Ha), resides on the short arm of chromosome 5D (5DS) (Mattern et al., 1973; Law et al., 1978). However, Baker and Sutherland (1991) observed significant genetic variation in grain texture within crosses of the same textural class.

Greenwell and Schofield (1986) reported friabilin, a marker protein for grain hardness. This protein is abundant on starch of soft wheat in comparison with hard wheat starch (Bettge et al., 1995; Greenblatt et al., 1995; Morris et al., 1994; Oda et al., 1992). Friabilin is controlled by genes on 5DS (Jolly et al., 1993) and is composed of a 1:1 ratio of the polypeptides pinA and pinB. Recent results indicate two linked genes, pinA and pinB, are involved in controlling grain texture. Mutations in pinA or pinB have been found in all hard-textured wheats examined (Giroux and Morris, 1997, 1998). PinA and pinB are unique among plant proteins in having a tryptophan-rich, hydrophobic domain (Blochet et al., 1993; Gautier et al., 1994). A hydrophobic domain would have a strong affinity for lipids (Schiffer et al., 1992) and may hold these two proteins to the lipid-rich surface of the amyloplast membrane, transiently attached to the starch granule. This idea is consistent with the predicted protein structure of pinA and pinB that suggests the hydrophobic-rich region of pinA and pinB would be physically available for binding to lipids (Marion et al., 1994). The ability of pinA and pinB to bind starch granule surface lipids in soft and not hard wheats is consistent with the report that much higher levels of membrane lipids and pinA and pinB are bound to soft wheat starch than hard (Greenblatt et al., 1995). This information suggests a causative role for these proteins found at the surface of starch granules in affecting grain texture. Therefore, differences in the structure of the tryptophan-rich region of pinA or pinB, or the amount of either protein, may be expected to influence grain texture.

Physical linkage between pinA and Ha was first demonstrated in a report that a pinA probe detected a restriction fragment length polymorphism linked to grain hardness (Sourdille et al., 1996). This result suggested that Ha and pinA were closely linked, but it did not establish a puroindoline sequence difference between soft and hard wheats. Recently, mutations in either pinA or pinB were shown to be inseparably linked to hard-textured grain. A glycine-to-serine change in the tryptophan-rich domain of pinB (allele pinB-D1b) that would probably decrease the hydrophobicity and lipid-binding properties of pinB, or the complete lack of pinA (allele pinA-D1b), has been observed in each hard wheat cultivar examined (Giroux and Morris, 1997, 1998). In recombinant populations or near-isogenic lines, the altered pinA or pinB gene was inseparably linked to hard texture (Giroux and Morris, 1997, 1998). These results suggest a direct role for the puroindolines in affecting grain texture and suggests that the Ha locus is functionally composed of just two proteins, pinA and pinB. However, the results do not preclude the involvement of additional genes linked to Ha.

Hard wheat cultivars that possess only one of the two known pinA and pinB mutant alleles may differ in grain hardness. Those having the pinA-D1b null allele might be expected to have harder texture than those carrying the pinB-D1b allele. Those carrying the pinB-D1b allele may not be as hard since some activity of functional friabilin may be possible assuming that Ha function requires the presence of both pinA and pinB. Our objectives were to examine the influence of pinA and pinB alterations on grain hardness, using recombinant lines from populations among elite hard red spring wheat cultivars differing in puroindoline alteration, and to measure associations of grain hardness with kernel weight and grain protein concentration.


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results and discussion
 REFERENCES
 
Plant Culture and Measurement of Grain Characters
A pinB polymerase chain reaction (PCR) primer specific for the serine sequence change present in numerous hard wheat cultivars (i.e., the pinB-D1b allele) (Giroux and Morris, 1997) was used to identify elite hard red cultivars containing this alteration. Hard red cultivars lacking pinA (the pinA-D1b allele) were confirmed by separating pinA and pinB on SDS-PAGE gels (Giroux and Morris, 1998). No recombination was observed between pinA and pinB in 83 homozygous chromosome 5D recombinant substitution lines (Giroux and Morris, 1997). For this reason, identification of one altered allele assumes the presence of a linked unaltered version of the other puroindoline gene. Therefore, cultivars lacking the pinA protein (pinA-D1b) have a soft-type pinB protein (pinB-D1a) and will be referred to simply as pinA-D1b. Those having an altered pinB protein (pinB-D1b) have a soft-type pinA protein (pinA-D1a) (Giroux and Morris, 1998) and will be referred to as pinB-D1b. Hard red spring wheat cultivars Amidon, Fortuna, and Glenman were confirmed as pinA-D1b, while cultivars Newana, Hi-Line, and Marberg were pinB-D1b types. Three populations were created by crossing pinA-D1b and pinB-D1b type (Amidon x Newana, Fortuna x Hi-Line, and Glenman x Marberg), while one population each of pinA-D1b x pinA-D1b (Glenman x Amidon) and pinB-D1b x pinB-D1b (Hi-Line x Newana) was created.

Five populations comprised of fifty random F3:6 lines per population were developed from crosses among the six hard red spring wheat parents via single seed descent followed by seed increase from a single F3 plant. These populations are a subset of those used by Burkhamer et al. (1998). About 25% of these lines would be expected to derive from heterozygous F3 plants, assuming a single genetic factor. These populations plus the six parents were planted in two replications of a randomized complete block split-plot design with populations as main plots and random lines plus parents as subplots. Each subplot was a single 3-m row with 30 cm between rows. Two adjacent experiments were grown near Bozeman, MT in 1996. One experiment was irrigated, receiving 17.5 cm of additional water. Field husbandry for the two environments was the same except for the added irrigation.

Grain hardness and protein concentration were measured from a sample of grain from each plot using an Infratec 1225 NIT (near-infrared transmission) Whole Grain Analyzer (Tecator, Höganäs, Sweden). The NIT hardness predictive equation was derived from 55 lines, which included the parents plus four additional hard red spring wheat cultivars and nine random lines from each of the five populations. Grain samples were ground on an UDY Cyclone mill (UDY Co., Fort Collins, CO) with a 0.5-mm screen. Hardness was measured on this calibration set with a Technicon InfraAnalyzer 400 near-infrared reflectance (NIR) (Technicon Industrial Systems, Tarrytown, NY). The NIR hardness is dimensionless, hard wheats generally having values of 50 to 100 and soft wheats values of 20 to 50, and is obtained via a calibration derived using a set of wheats of known hardness values (Approved Method 39-70A; American Association of Cereal Chemists, 1995). Kernel weight was measured from a sample of grain from each plot where {approx}175 kernels were counted and weighed.

DNA Isolation and Polymerase Chain Reaction Amplification of Puroindoline b
DNA was isolated from a sample of 10 to 20 coleoptiles of each entry following the procedure of Dellaporta et al. (1983). The PCR amplification of pinB sequences specific for the Gly-46 or Ser-46 primers was performed as described by Giroux and Morris (1997). Annealing temperature was maintained at 58°C and PCR reactions were replicated three or more times. Lines whose DNA gave PCR products with both the Gly-46 (soft-type pinB) and Ser-46 (hard-type pinB) primers were classified as heterogeneous (mixtures) and lines that gave PCR products with only the Ser-46 primer were classified as pinB-D1b allele types. Lines whose DNA gave PCR products only with the Gly-46 were labeled as pinA-D1b allele types since no recombination between pinA and pinB has been observed. The PCR reactions were replicated for each primer pair three or more times.

Fractionation of Puroindoline A and B by TX114 Extraction and SDS-PAGE
Fractionation of puroindolines and separation via SDS-PAGE was done via a Triton X-114 phase extraction as described previously (Giroux and Morris, 1998). Protein samples were prepared from a subsample (>=20 kernels) of whole grain ground in a UDY mill.

Data Analysis
Analysis of variance was first performed for individual populations combined across the two environments. Environments were considered fixed and replications and progeny lines were considered as random effects. The analysis was then combined across populations (fixed effects) and environments. Progeny line sum of squares for each of the three populations segregating for pinA-D1b vs. pinB-D1b was partitioned into a sum of squares among class means (pinA-D1b, pinB-D1b, and heterogeneous) and lines within classes. Puroindoline mutation class means were averaged across the three populations segregating for pinA-D1b vs. pinB-D1b. All analyses were performed with PROC GLM in SAS (SAS Institute, 1988) using a RANDOM statement with the TEST option. Denominator mean squares for F ratios were computed using the appropriate linear combinations of mean squares, and degrees of freedom were computed using the Satterthwaite (1946) approximation. Population means were compared using an LSD where the error term equaled the mean square for population by replications within environments. Means for the pinA-D1b, pinB-D1b, and heterogeneous classes for each segregating population were compared using a t statistic where the error mean square was the progeny lines within classes mean square. These classes were averaged across the three segregating populations, and t tests were performed using the pooled lines within-classes mean square as the error term. The proportion of within-population variation accounted for by puroindoline class was estimated as the sum of squares for classes as a percentage of the among-lines sum of squares. Phenotypic correlations between traits were obtained from entry means averaged across environments.


   Results and discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
REFERENCES
 
A significant correlation ( ) was obtained between grain hardness measured on whole-grain samples and UDY-ground material, indicating that the hardness calibration was adequate for predicting grain hardness on the remaining samples. The feasibility of measuring grain texture using whole-grain NIT has been demonstrated previously for maize (Zea mays L.) (Eyherabide et al., 1996). While the correlation between ground NIR and whole-grain predicted NIT was not extremely high, the calibration equation relies on a subset of the samples where grain hardness was measured directly. Therefore, a close relationship is expected between the predicted and actual readings.

Parents within each cross differed in grain hardness (Table 1) . The pinA-D1b parents averaged 7.1 units higher than the pinB-D1b parents (Table 1). Glenman (pinA-D1b) and Marberg (pinB-D1b) were exceptions to this trend. This mean difference is consistent with initial surveys of hard wheat cultivars that differ in puroindoline mutation (Giroux and Morris, 1997, unpublished results) where not all pinA-D1b cultivars are harder-textured than pinB-D1b, most likely due to the presence of numerous modifying genes. A difference in both protein concentration and kernel weight was also observed between the pinA-D1b and pinB-D1b type parents (Table 1). The pinB-D1b parents averaged 7.1 mg lower in kernel weight than the pinA-D1b parents and 4 g kg-1 higher in protein concentration. While the difference in grain hardness is probably associated with a particular puroindoline alteration, no previous report suggests the Ha locus is associated with kernel weight or protein concentration differences.


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  		Table 1 Parental means and puroindoline mutation type of five hard red spring wheat populations

 
The pinA-D1b x pinA-D1b population mean was higher and the pinB-D1b x pinB-D1b population mean was lower in grain hardness than the three pinA-D1b x pinB-D1b population means (Table 2) . The three pinA-D1b x pinB-D1b population means did not differ in grain hardness. F tests did not detect differences among puroindoline mutation class means, except for grain hardness in the Glenman x Marberg population (Table 3) . The lines classified as pinA-D1b were on average harder than lines classified as pinB-D1b for each of the three segregating populations (Table 2), though the difference was not statistically significant for two of the three populations. The inability to detect a difference in two of the three populations may be because of significant (P < 0.01) residual genetic variation within classes in each cross (Table 3). It may also be related to the method used to measure grain hardness. However, this difference was consistent in direction for the three populations and ranged from 2.8 hardness units for Glenman x Marberg to 6.3 hardness units for Fortuna x Hi-Line. Lines with a mixture of pinB-D1b and pinA-D1b types, presumably derived from heterozygous F3 plants, had grain hardness between the two homozygous classes for each population, except for the Glenman x Marberg population. Although population means differed for grain protein concentration and kernel weight, no significant differences were observed among the puroindoline class means for protein concentration and kernel weight in the three segregating populations. This is not unexpected as there have been no reports indicating that Ha or 5DS is associated with these types of kernel traits. Significant (P < 0.01) genetic variation for kernel weight and grain protein concentration was observed within classes, and among progeny lines in crosses not segregating for pinA-D1b vs. pinB-D1b (Table 3).


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  		Table 2 Puroindoline mutation class means for 50 random F3-6 progeny for five spring wheat populations grown in two Montana environments

 

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  		Table 3 Analysis of variance for grain hardness, protein concentration, and kernel weight combined across five spring wheat populations with 50 random lines per cross, and parents having either pinA-Db1 or pinB-Db1 puroindoline alteration type

 
When the puroindoline class means were averaged across the three populations, a 4.5 hardness-unit difference (P < 0.05) was found between pinA-D1b and pinB-D1b genotypes (Table 2). However, the total range of hardness values for either homogeneous group was more than 30 units. Differences in puroindoline genes or genes linked to them accounted for 12, 9, and 6% of the total genetic variation in grain hardness in the Glenman x Marberg, Fortuna x Hi-Line, and Amidon x Newana populations, respectively. Puroindoline mutation class means did not differ for grain protein concentration or kernel weight when averaged across populations.

Populations and progeny lines within populations interacted with environments (P < 0.05) (Table 3). The interaction tended to reflect greater differences in the irrigated environment compared with the nonirrigated environment. Differences in puroindoline mutation class means were consistent across environments, even though significant environment x line interactions were present.

Grain hardness, kernel weight, and grain protein concentration tended to be positively related (Table 4) . Significant positive correlations were observed in all five populations for grain hardness and grain protein concentration, in four populations for kernel weight and grain protein concentration, and in three populations for grain hardness and kernel weight. Baker and Dyck (1975) reported a positive correlation between grain hardness and grain protein concentration, and Wells and Kofoid (1986) found kernel weight and grain protein concentration were positively related in spring wheat. Correlated response from long-term selection suggests that one or more of these traits may share genetic factors in common. Selection for increased grain protein concentration resulted in a linear increase in grain hardness but no correlated change in kernel weight in spring wheat (Delzer et al., 1995). Similarly, direct selection for kernel weight produced a positive correlated response in grain protein concentration in spring wheat (Busch and Kofoid, 1982). The reason for the positive associations among these traits is not known. However, the pinA-D1b vs. pinB-D1b difference is probably not a factor because the correlations were also observed in populations not segregating for the mutation.


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  		Table 4 Correlations between three quality traits for five spring wheat populations

 
In conclusion, an association between puroindoline alteration type and grain hardness indicates that pinA-D1b lines have harder texture than pinB-D1b lines. This allelic difference was manifested as 4.5 hardness units between hard wheats having a mutant pinA-D1b allele and those having an altered pinB-D1b allele. Significant genetic variation was observed within classes and among lines in crosses not segregating for the pinA-D1b vs. pinB-D1b difference. This allelic difference explained, at most, 12% of the variation in grain hardness in these hard wheat populations. While the pinA-D1b vs. pinB-D1b difference observed here is relatively small, this study compared only hard wheat x hard wheat populations. In a cross involving soft and hard wheat parents the puroindoline locus on 5DS explained more than 60% of variation in grain texture (Campbell et al., 1999). Our results indicate that most of the genetic variation in grain hardness among the populations studied was a result of factors other than pinA and pinB. While differences in grain hardness associated with puroindoline alteration is consistent with pinA and pinB having some direct control of wheat grain hardness, it is not direct evidence for control of grain softness by pinA and pinB. It is also possible that the effect described here may be related to one or more genes linked to the puroindoline genes. Further studies are needed to determine the effect of the pinA-D1b or pinB-D1b mutations on the quality of wheat and to further clarify the role of puroindolines in wheat endosperm texture.


   ACKNOWLEDGMENTS
 
This research was funded in part by grants from the Montana Wheat and Barley Committee, USDA-ARS-NRICGP (9504374), and the Montana Agricultural Experiment Station.

Received for publication October 15, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 




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