Gazza L

Pogna NE

Zanella L

English

Sydney eScholarship

 1Development of durum wheat (Triticum turgidum ssp durum) lines with soft kernel texture by chromosome engineering Gazza L, Zanella L, Pogna NE C.R.A. - Research Unit on Cereal Quality Improvement, Via Cassia 176, Rome, Italy  ABSTRACT Puroindolines A (Pin-A) and B (Pin-B) are the principal determinant factors of endosperm texture in common wheat and, as a consequence, a main  determinant in end product quality. They are encoded by the Pina-D1 and Pinb-D1 loci on chromosome 5DS and, therefore, are absent in durum wheat (Triticum turgidum ssp. durum). In order to introduce puroindolines A and B  into durum wheat through allosyndetic recombination, the 5D(5B) substitution line of durum wheat cv. Langdon was  crossed  with durum wheat line Cappelli M lacking the Ph1 locus on chromosome 5BL.  Amongst the resulting progeny, 11 F6 recombinant inbred lines (RILs)  with soft kernels were crossed as the male parent with durum wheat cv. Colosseo, and 81 F5 progeny from this cross  were analyzed for the presence and expression of puroindoline genes. Moreover,  16 F5 lines containing  Pin-A and Pin-B were compared for their kernel texture  and storage protein composition. Finally, ten F6 RILs were analysed for their chromosome structure using microsatellite sequences located on homoeologous group 5 chromosomes. Soft grain texture (SKCS value = 34.9 ± 11.2), vitreous kernels, high protein content (15.7 ± 2.15%) and good gluten quality as determined by the SDS sedimentation volume (58.4 ± 9.1 mL) were found in 16 F6 RILs possessing a small terminal fragment of chromosome 5DS carrying Pina-D1 and Pinb-D1.  INTRODUCTION  Puroindolines A (Pin-A) and B (Pin-B) are the principal determinant factor of endosperm texture in common wheat (Greenwell and Schofield 1989; Morris et al. 1994). Pin-A and Pin-B are basic polypeptides, 13 kDa in size, with a characteristic tryptophan-rich domain, which makes them unique amongst  plant proteins (Blochet et al., 1993; Gautier et al., 1994). Variation in endosperm texture is probably due to affinity of the tryptophan-rich domain of puroindolines to the polar lipids of the amyloplast membranes. Because of their lipid-binding properties, puroindolines are claimed to  influence loaf volume and crumb structure of bread, and protect beer against foam destabilisation (Blochet et al., 1993; Dubreil et al., 1998). Puroindolines are encoded by the Pina-D1 and Pinb-D1 genes closely linked to each other at the Ha locus in the distal part of the short arm of chromosome 5D and, therefore, are absent in durum wheat (Triticum turgidum ssp. durum) (Giroux and Morris 1997). In order to introduce the Pina-D1 and Pinb-D1 loci into durum wheat through allosyndetic recombination, the 5D(5B) substitution line of durum wheat cv. Langdon was  crossed  with durum wheat Cappelli M, a deletion line lacking the Ph1 locus (allele ph1c), which prevents pairing of homoeologous chromosomes at meiosis (Giorgi, 1978). In the resulting progeny, the absence of the Ph1 locus was found to promote pairing and allosyndetic recombination between homoeologous chromosomes 5B and 5D. Eleven F6 recombinant inbred lines (RILs) with soft kernels obtained from the cross mentioned above were crossed as the male parent with durum wheat cv. Colosseo.  Here, 81 F5 progeny from this latter  cross are screened for the presence and  expression of puroindoline genes,  and several F6 recombinant lines  are compared for their (i) chromosome structure using microsatellite (SSR)  and (ii) kernel texture, storage protein composition and technological properties.   MATERIALS AND METHODS  Plant material The 5D(5B) substitution line of cv. Langdon was crossed with durum wheat line Cappelli M lacking the Ph1 locus (allele ph1c) , and the F1 progeny was selfed for five generations. Amongst the resulting F6 progeny, 11 recombinant inbred lines (RILs) with soft kernels, as measured by the SKCS  method, were crossed as the male parent with durum wheat cv. Colosseo. The 81 F5 progeny from this latter cross  were used for biochemical and technological analyses.  DNA  extraction and PCR amplification The presence of the Pina-D1 and Pinb-D1 loci was determined in DNAs extracted from leaves by the  CTAB method. Puroindoline genes were amplified by PCR as described by Gautier et al., (1994). SSR (Simple Sequence Repeats) sequences on 5A, 5B and 5D chromosomes (Song et al., 2005) were used for microsatellite marker characterization.  Protein analyses Puroindoline fractionation by A-PAGE (Acid-PAGE)  and western blotting were performed as described previously (Gazza et al., 2006). SDS-PAGE and A-PAGE fractionations of storage proteins were carried out according to Pogna et al. (1990).  Kernel hardness and biochemical analyses Kernel hardness was measured by the Perten Single Kernel Characterization System (SKCS 4100) following the manufacturer’s operating procedure. Protein content of the F5 progeny was determined by semi-automatic micro-Kjeldhal (N x 5.7). Gluten content and gluten index were determined according to UNI 10689 and UNI 10690, respectively. SDS sedimentation volume  2was measured according to AACC Method 56-70, using a 3% SDS solution.  RESULTS AND DISCUSSION  Production of durum wheat Recombinant Inbred Lines (RILs) with soft texture Amongst the F2 progeny of the cross Cappelli M x  Langdon 5D(5B) substitution line, 42 plants devoid of the Iodine Binding Factor (IBF) encoded by  the  Ibf-D1 locus  on chromosome 5DL were found to be   homozygous for  IBF encoded by the Ibf-B1 locus on chromosome 5BL inherited from line Cappelli M (Gazza et al., 2002). Upon PCR amplification with puroindoline-specific primers, 127 F3 progeny from those plants were shown to contain Pina-D1 and Pinb-D1, suggesting that allosyndetic recombination occurred between chromosomes 5B and 5D. Single kernel hardness readings obtained by the SKCS method revealed that the selfed progeny of 116 out of 127 F3 plants was heterogeneous, their SKCS values ranging from 20 to 90. However, the progeny of 11 F3 plants turned out to be homogeneous for soft grain texture, their average SKCS value being as low as 33. Those 11 plants were assumed to be homozygous at the Pina-D1 and Pinb-D1 loci and crossed as the male parent with durum wheat cv. Colosseo in order to reintroduce the Ph1 locus into the resulting progeny and  improve their  technological (gluten quality) and morphological (reduced plant height) traits (Gazza et al., 2002). The 81 F5 Recombinant Inbred Lines (RILs) obtained form this latter cross were compared for their chromosome structure, and biochemical and technological properties.    Presence and expression of puroindoline genes Upon PCR amplification with  primers specific  for Pina-D1 and Pinb-D1,  DNAs  extracted  from 49 out of  81 F5  RILs were positive for both loci (Fig.1). Direct sequencing of the amplicons demonstrated the presence of wild-type alleles Pina-D1a and Pinb-D1a.           Nevertheless, when starch-bound proteins extracted from those 49 RILs were fractionated by A-PAGE, only 19 lines revealed a couple of bands that co-migrate with puroindolines in soft-textured common wheat cv. Lontra (Fig.2). The polyclonal antiserum developed against a 16-mer sequence in the C-terminal region of mature Pin-A (Gazza et al., 2006) reacted strongly with  protein marked with A in Figure 2, confirming that this protein  is encoded by allele Pina-D1a. On the other hand, gene silencing in 30 RILs could be accounted for by a positional effect at the site of insertion of puroindoline genes on chromosome 5B.           Kernel texture, storage protein composition and technological properties of  durum wheat RILs A-PAGE fractionation of gliadin from 16 F5 RILs  showed the presence of both γ-gliadin 45 and ω-gliadin 35 associated with good gluten quality (Pogna et al., 1990). Upon SDS-PAGE fractionation of HMW glutenin subunits (Fig.3), these 16 lines turned out to be homozygous for either subunit pair “6+8” inherited from Langdon 5D(5B) substitution line or subunit pair “13+16” inherited from cv. Colosseo.                  The SKCS values of these RILs were typical of a population segregating for kernel texture, showing a bimodal distribution of the single kernel hardness readings. However, amongst the F6 progeny of  36 single plants belonging to two F5 RILs (L482 and L504), 16 lines showed a mean  SKCS value of  34.9 ± 11.2, which is typical of soft kernels homozygous for the puroindoline genes. Moreover, 10 F6 lines  produced  extra-hard kernels (mean  SKCS value = 89.6 ± 11.8), whereas  the remaining 10 lines  showed a bimodal distribution with a mean SKCS value of 58.5 ± 24.1. All the 16 F6 RILs with soft kernels exhibited very high protein content (15.7 ± 2.1%)  and a mean SDS sedimentation volume as high as 58.4 ± 9.1 mL. Furthermore, they  showed vitreous kernels, confirming that kernel texture and vitreousness are independent traits.    Figure 1. PCR amplicons  obtained with Pinb-D1 specific primers in eight F5 RILs  of durum wheat. The first lane on the left shows DNA size markers.          A           B Figure 2. Fractionation by A-PAGE of starch-bound proteins from (from left to right) durum wheat cv. Colosseo, common wheat cv. Lontra and 10 F5 RILs of durum wheat. (A) Pin-A; (B) Pin-B.  Figure 3. Fractionation by SDS-PAGE of total endosperm proteins from (1) cv. Langdon, (2)  5D (5B)  substitution line,  (3) common wheat cv. Chinese Spring, (4) Cappelli M,  (5) cv. Colosseo and (6-13, 15-19) F5 RILs of durum wheat. Lane 14 shows  molecular weight markers.  1   2    3    4    5   6    7   8    9  10  11  12 13  14  15 16  17  18 19  3SSR analysis  Molecular marker analysis was  carried out on 10 F6 progeny of  lines L482 and L504. PCR amplification with primers specific for microsatellite Xbarc130 localized at 0 cM on the  short arm of chromosome 5D produced amplicons  in eight soft-textured progeny, all of them possessing  the Pina-D1 and Pinb-D1 loci (Table 1). The remaining two F6 progeny (L482-5 and L504-5) with hard kernels gave no amplification product for both Xbarc130 and puroindoline genes. Microsatellites Xbarc205 (18.4 cM), Xbarc322 (126.0 cM),  Xbarc110 (140.4 cM) and  Xgmw169 (169.0 cM) were absent in all the progeny analysed except line L482-1. This latter soft line revealed the presence of xbarc110 and Xgmw169 localized at the distal end of the long arm of chromosome 5D, suggesting that this genotype inherited two intergenomic exchanges between chromosomes 5B and 5D.  Seven soft lines were found to contain Xgmw190 located at 8.6 cM  on the short arm of chromosome 5D (Table 1). On the contrary,  soft line L504-1 gave no amplification product with primers specific for  Xgmw190, suggesting that a very short fragment of chromosome 5DS  occurs in this genotype.    + present; - absent  Several SSR sequences located on chromosomes 5A and 5B were polymorphous, giving amplicons  of different sizes in the parental genotypes Cappelli M, Langdon 5B(5D) substitution line and Colosseo (Fig. 4). PCR amplifications with primers specific for polymorphous SSR sequences  revealed that the F6 progeny of  RILs  L482 and L504 inherited  chromosome arms 5AL and 5BL from  cv. Colosseo (data not shown).           Previous findings have shown that the presence  of wild-type alleles Pina-D1a and Pinb-D1a is the principal determinant factor of soft kernel texture in common wheat  (Greenwell and Schofield 1989; Morris et al. 1994; Corona et al.  2001). Puroindolines modulate grain texture and, as a consequence, they exert a strong indirect influence on several technological parameters such as flour yield, starch damage, water absorption, farinograph peak time and stability, alveograph P, L and W parameters. Moreover, they influence directly loaf volume and crumb structure of bread. Protein content and kernel weight were found to explain a large proportion of the variation in kernel hardness due to environment and genotype x environment interaction (Gazza et al., 2008). Here, evidence has been provided that introgression of wild-type Pin-A and Pin-B  through  a non-transgenic approach resulted in durum wheat genotypes with soft kernel texture without any negative effect on grain vitrousness. These genotypes are currently being analysed for their pastamaking and breadmaking properties.   REFERENCES  Blochet J.E., Chevalier C., Forest E., Pebay-Peyroula E., Gautier M.F., Joudrier P., Pezole M., Marion D. (1993). FEBS Lett.329 :336-340. Corona V., Gazza L., Boggini G. and Pogna N.E. (2001). J. Cereal Sci. 34:243-250. Dubreil L., Meliande S., Chiron H., Compoint J.P., Quillen L. and Branlard G. (1998). Cereal Chem. 75 :222-229. Gautier M.F., Aleman M.E., Guirao A., Marion D. and Joudrier P. (1994). Plant Mol.Biol. 25:43-57. Gazza L., A. Niglio, E. Mei, E. De Stefanis, D. Sgrulletta and N.E. Pogna. (2002). In: Proceedings of 2nd International Workshop Durum Wheat and Pasta Quality. Recent Achievements and New Trends. Rome 19-20 Nov.pp.285-288. Giorgi B. (1978). Mutation Breeding Newsletter 11: 4-5. Gazza L., Conti S., Taddei F. and Pogna N.E. (2006). Mol. Breed. 17:191-200. Gazza L., Taddei F., Corbellini M., Cacciatori P. and Pogna N.E. (2008). J. Cereal Sci. 47: 52-58. Giroux M.J. and Morris C.F. (1997). Theor. Appl. Genet. 95: 857-864. Greenwell P.  and Schofield J.D.(1989.In: Proceedings ICC’89 Symposium (Lathi, Finland,13-15 June 1989),pp.59-72. Morris, C.F., Greenblatt G.A., Bettge A.D. and I.Malkawi (1994). J.Cereal Sci. 20:167-174. Morris, C.F. (2002).Plant Mol.Biol. 48:633-647. Pogna N.E., Autran J.C., Mellini F., La Fiandra D. and Feillet P. (1990). J. Ceral Sci. 11: 15-34. Song Q.J., Shi J.R., Singh S., Fickus E.W., Costa J.M., Lewis J., Gill B.S., Ward R. and Cregan P.B. (2005). Theor Appl. Genet. 110: 550-560. SSR L482           L504 1 2 3 4 5 1 2 3 4 5 Chromosome  5DS Xbarc130 (0 cM)  +  +  +  +  -  +  + + + - Xgmw190 (8.6 cM) + + + + - - + + + - Xbarc205 (18.4 cM) - - - - - - - - - - Chromosome 5DL Xbarc322 (126.0 cM)  -  -  -  -  -  -  -  -  -  - Xbarc110 (140.4 cM) + - - - - - - - - - Xgmw169 (169.0 cM) + - - - - - - - - - Figure 4. PCR amplification with primers specific for polymorphous microsatellite xbarc4 on chromosome 5B. Lanes show (from left to right) DNA size markers, common wheat cv. Chinese Spring, Langdon 5D (5B) substitution line,  F6  line 482-1, Cappelli M and durum wheat cv. Colosseo. Table 1. PCR amplification of  the selfed progeny of  lines L482 and L 504  with primers specific for six SSRs on chromosome 5D. 

Development of durum wheat (Triticum turgidum ssp durum ) lines with soft kernel texture by chromosome engineering

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Development of durum wheat (Triticum turgidum ssp durum ) lines with soft kernel texture by chromosome engineering

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