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Australian hard wheat cultivars are limited in their genetic variability in puroindoline genes

P. Pickering and M. Bhave

Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, Melbourne, Vic 3122, Australia

Introduction

Grain hardness defines the primary commercially important characteristics of wheat, its major grades, uses and export market. The genes Pina-D1 and Pinb-D1, located only on chromosome 5D of common wheat (Triticum aestivum, AABBDD), encode small lipid-binding proteins, puroindoline-a (PIN-A) and puroindoline-b (PIN-B) (Gautier et al. 1994; Sourdille et al. 1996). These are considered to be the key genes that determine grain texture, ‘soft’ texture being the dominant, wild type and the hard texture being determined by either Pina-D1 gene deletion (allele Pina-D1b) or six separate point mutations in Pinb-D1a (alleles Pinb-D1b to Pinb-D1g)(Giroux and Morris 1997; Lillemo et al. 2000; Morris et al. 2001). The Pina-D1b mutation produces harder grains than the latter group, wherein certain point mutations confer different degrees of hardness (Giroux and Morris 1997; Morris et al. 2001).

A considerable amount of work has been carried out on identifying puroindoline mutations in wheat cultivars of Northern America and Northern Europe (Lillemo et al. 2000; Morris et al. 2001). As wheat is the most important crop of Australia, it is necessary to identify the genetic basis of grain hardness and the extent of genetically determined variability in this phenotype of Australian wheat. However, this analysis of Australian wheat cultivars has been rather limited, and the two previous investigations show that all hard cultivars tested exhibit either the Pina-D1b or the Pinb-D1b mutation (Turnbull et al. 2000; Cane et al. 2004). In this study, we have expanded the range of cultivars studied and analysed the Pina-D1 and Pinb-D1 genes of fifty Australian hard wheat cultivars. In a search for new genetic resources of grain hardness, preliminary investigations have also been conducted on a number of land-race lines of common wheat (T. aestivum) from countries of early wheat cultivation, as well as other members of the Triticeae family. Twenty-five land-race accessions and twenty assorted Triticeae accessions (T. timopheevi; AAGG, T. zhukovskyi; AAAAGG, and T. dicoccon; AABB) have been examined. The work involved Pina-D1 and Pinb-D1gene isolation by the polymerase chain reaction (PCR), determination of diagnostic restriction enzyme sites for certain mutations in Pinb-D1, and/or DNA sequencing. These studies will aid in identification of the genetic basis of grain hardness in Australian wheats and identification of new mutations for breeding purposes.

Materials and methods

Plant Material

Seeds of all material used were obtained from the Australian Winter Cereals Collection (Tamworth, NSW). Eight seeds of each cultivar/accession were grown at 18-hour day lengths, 75% humidity and a temperature of 25C. Genomic DNA was extracted from leaf tissue using the Wizard Genomic DNA Purification Kit (Promega).

PCR amplification

Amplifications of the full length Pina-D1 and Pinb-D1 gene sequences were performed using primers yielding products ~450 base pairs (bp) in length for both genes (Gautier et al. 1994). All PCRs were performed with PCR Supermix (Invitrogen) using the calculated annealing temperatures (NetPrimer) and the products were separated on 1.7% agarose gels.

Site specific cleavage of PCR amplified puroindoline-b

Identifications of the glycine-46 to serine (Pinb-D1b), and the leucine-60 to proline (Pinb-D1c) mutations in Pinb-D1 were made through digestion of the Pinb-D1 PCR products from individual cultivars with the restriction enzymes BsrB1 and PvuII respectively (Tranquilli et al.1999; Lillemo et al. 2000). A single cleavage of the PCR product by BsrB1 (identified by a 318bp fragment) is indicative of the soft allele of Pinb-D1, while a double cleavage with it (identified by 222bp fragment) suggests the Pinb-D1b mutation. PvuII causes a cleavage of wild type Pinb-D1 PCR products, while those of the Pinb-D1c motif will remain uncut.

Sequencing

Puroindoline-b genes of the Australian common wheat and the land-race lines that could not be categorized using the restriction enzymes, as well as the puroindoline-a genes of the various Triticeae accessions, were purified then sequenced using Big Dye Terminator Chemistry 3.1 at the sequencing facility of the Baker Heart Research Institute.

Results

Of the fifty Australian hard wheat cultivars tested, sixteen failed to successfully amplify the Pina-D1 gene (see Figure 1a for an example), indicating the presence of the Pina-D1 null allele (Pina-D1b). Using the BsrB1 restriction enzyme digestions, a further twenty-nine cultivars were identified as having the Pinb-D1b mutation (see Figure 1b for an example). Five cultivars were identified as a mixture of seed lots due to exhibiting both the hard (Pinb-D1b) and soft (Pinb-D1a) motifs of Pinb-D1, as well as the presence of the Pina-D1a allele. Soft wheats were used as control cultivars for the PCR and restriction enzyme digestions. Of the twenty-five overseas land races investigated, sixteen had the wild type ‘soft’ motifs for both Pina-D1 and Pinb-D1, four were found to have the Pina-D1b allele, while four others were found to have the Pinb-D1b allele. Through use of the PvuII restriction enzyme, the remaining land race was determined to have the Pinb-D1c mutation (Figure 1c). These results are summarized in Table 2.

Figure 1. (a) PCR amplification of full-length Pina-D1 (L2, L4, L6) and Pinb-D1 (L3, L5, L7) genes. Note the lack of the Pina-D1 product (L2), representative of the Pina-D1b allele. (b) Restriction Digestion of Pinb-D1 PCR products with BsrB1. Note the digest yielding the 222bp fragment (L3) indicative of the Pinb-D1b mutation. Pinb-D1 products of the soft motif are identified by the 318bp fragment (L2, L4). (c) Restriction Digestion of Pinb-D1 PCR products with PvuII. Note the lack of digestion indicative of the Pinb-D1c mutation (L3).

Table 2. Puroindoline genotypes of wheat cultivars surveyed.

Australian cultivars

Pina-D1

Pinb-D1

Australian cultivars

Pina-D1

Pinb-D1

Overseas
land-race

Pina-D1

Pinb-D1

Amery

a

b

Kalyansona

b

a

Afghan 49

a

a

Barunga#

a

b

Katepwa

a

b

Afghan 51

a

b

Batavia*

a

a/b

Kennedy

b

a

Afghan 77

a

a

Baxter

a

b

Kite

a

b

Algeria 8

a

a

Blade*

a

a/b

Krichauff#

a

b

Azerbaijan

b

a

Brookton

a

b

Machete#

b

a

Burma 7

a

a

BT-Schomburgk

a

b

Meering#

a

b

Burma 8

a

a

Camm

a

b

Ouyen#

b

a

Crete 4

a

a

Carnamah

b

a

Owlet

a

b

Cyprus 3

a

a

Condor

a

b

Raven

a

b

Greece 5

a

a

Cranbrook#

b

a

Shrike#

a

b

India 113

a

a

Cunningham

a

b

Spear#

a

b

India 210

a

b

Diaz#

a

b

Stiletto#

a

b

India 223

a

b

Diamondbird#

b

a

Sunbri

a

b

Iraq 42

a

c

Dollarbird#

b

a

Sunbrook

b

a

Iraq 55

a

a

Dundee

a

b

Sunlin

a

b

Leon 4

b

a

Eagle#

b

a

Sunstate

b

a

Palestine 3

a

a

Frame* #

a

a/b

Sunvale

a

b

Persia 6

a

a

Gabo

b

a

Tasman

a

b

Rumania 4

a

a

Galaxy H45

a

b

Tailorbird #

b

a

Rumania 10

b

a

Goldmark#

a

b

Thatcher

a

b

Sinai 8

a

a

Gular

b

a

Westonia

b

a

Smyrna 2

a

a

Hartog

b

a

Whistler* #

a

a/b

Smyrna 8

a

a

Harrier

a

b

Wylah* #

a

a/b

Turkestan

a

b

Kalannie

a

b

Yitpi#

a

b

Varna 7

b

a

*Cultivars with mixed seed lots, containing both the Pinb-D1a and Pinb-D1b alleles.
#
Australian cultivars investigated elsewhere (Turnbull et al. 2000; Cane et al. 2004); some of these were investigated while our studies were underway, with our results confirming the published observations.

In the search for novel genotypes for grain hardness, twenty accessions of various Triticeae members were analysed. Of the seven T. zhukovskyi accessions, six lacked both the puroindoline genes, while one contained only puroindoline-a. Both accessions of T. dicoccon also lack both of the puroindoline genes. All eleven accessions of T. timopheevi analysed were found to contain the puroindoline-a gene but were devoid of puroindoline-b. Sequencing of the PCR products from both T. timopheevi and T. zhukovskyi ascertained five single-base mutations resulting in five amino acid changes throughout the Pina-D1 genes (Figure 2).

Figure 2. Alignment of deduced primary structures of puroindoline-a (# indicates ten cysteine residues; * indicates tryptophan-rich domain)

Discussion

In this study fifty Australian hard wheat cultivars were analysed, of which sixteen failed to amplify Pina-D1, indicative of the Pina-D1 null allele (pinA-D1b), while twenty-nine exhibited the glycine 46-to-serine mutation in Pinb-D1 (Pinb-D1b). Both of these mutations have been commonly associated with hard grain texture. The restriction enzymes Mnl1+Sty1, BstN1 and Hph1 were used to randomly test ten cultivars each of Pina-D1b and Pinb-D1b genotypes for additional mutations, i.e., Pinb-D1d, Pinb-D1e and Pinb-D1f respectively; however, none were detected (results not shown). Through restriction digest analysis, five cultivars were identified as a mix of two different types of seed, with the both Pinb-D1a and Pinb-D1b alleles; hence the Pina-D1 genotype of this mixture could not be addressed with certainty by PCR (as the Pina-D1 PCR products observed could come from only some of the seeds, while those, if any in the mixture, of the Pina-D1b type would not generate this PCR product). Single plant analyses are being conducted on these to determine whether they are a mixture of seeds, or heterozygous for the alternative forms of Pina-D1 and Pinb-D1 alleles. The above results, together with published data on >40 hard wheats (Turnbull et al. 2000; Cane et al. 2004), show that the Australian gene pool possibly offers only the two major types of hardness alleles, and supports the previous suggestion (Cane et al. 2004) that the possible explanation for this limited genetic variability could be the small number of shared parental lines, e.g., Falcon, Gabo and WW15. In contrast, out of twenty-five overseas land races analysed, one exhibited a different mutation (Pinb-D1c) that is known to provide a different degree of hardness (Lillemo et al. 2000). Such new genetic resources can be utilised in the Australian breeding programs to create a range of grain textures.

For the other members of the Triticeae family, DNA sequencing indicated five base changes, resulting in five amino acid changes, in the puroindoline-a genes of the T. timopheevi, in comparison to the T. aestivum Pina-D1a sequence; these accessions all lacked puroindoline-b. The tetraploid T. diccocon were lacking in both the puroindoline genes, similar to T. turgidum (AABB). Majority of the T. zhukovskyi accessions tested also lacked both the puroindoline genes, with only one having only the puroindoline-a gene, with the same five point mutations as those in T. timopheevi. One of these changes, arginine-85 to glutamine, has previously been reported in Ae. tauschii (DD) (Gedye et al. 2004) and is thought to increase grain softness. This sequence diversity of puroindoline-a genes within members of the Triticeae family may help broaden the range of endosperm textures in wheat through means such as transgenics. A comprehensive understanding of the underlying genetic control of endosperm texture will assist wheat breeders and growers in providing the industry with grain possessing desired levels of hardness.

References

Cane, K., Spackman, M. and Eagles, H. (2004) Aus. J. Agric. Res. 55: 89-95.

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Gedye, K.R., Morris, C.F. and Bettge, A.D. (2004) Theor. Appl. Genet. 109: 1597-1603.

Giroux, M.J. and Morris, C.F. (1997) Theor. Appl. Genet. 95: 857-864.

Lillemo, M. and Morris, C.F. (2000) Theor. Appl. Genet. 100: 1100-1107

Morris, C.F., Lillemo, M., Simeone, M.C., Giroux, M.J., Babb, S.L. and Kidwell, K.K. (2001) Crop Sci. 41, 218-228.

Sourdille, P., Perretant, M.R., Charmet, G., Leroy, P., Gauteir, M.F., Jourdrier, P., Nelson, J.C., Sorrells, M.E. and Bernard, M. (1996) Theor. Appl. Genet. 93: 580-586.

Tranquilli, G., Lijavetzky, D., Muzzi, G. and Dubcovsky J (1999) Mol. Gen. Genet. 262: 846-850.

Turnbull, K-M., Gaborit, T., Marion, D. and Rahman, S. (2000) Aust. J. Plant. Physiol. 55: 89-95.

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