MS INTA 416 : A new Argentinean wheat cultivar carrying Fhb 1 and Lr 47 resistance genes

MS INTA 416 is a hard red winter wheat selected for high yield potential and good bread-making quality, combined with moderate resistance to Fusarium-head-blight and high resistance to leaf-rust, due mainly to presence of resistance genes Fhb1 and Lr47. MS INTA 416 is adapted to main production areas of Central-Argentina.


INTRODUCTION
The diseases Fusarium Head Blight (FHB), caused by Fusarium graminearum and leaf rust, caused by Puccinia triticina, are widespread and devastating for bread wheat production in the Southern cone of South America (Germán et al. 2007, Bainotti et al. 2013).Damage caused by FHB includes reductions in yield and seed quality, and toxin contamination by deoxynivalenol, threatening human health.Every year, according to the climatic conditions and the area in which susceptible cultivars are planted, leaf rust causes widespread epidemics if no chemical control is applied.Host resistance is considered an efficient and eco-friendly way to manage both diseases; however, progress in breeding for FHB resistance has been limited by the complex inheritance of the partial resistance currently available in wheat.Resistance to the spread of the disease within a spike (Type II resistance) is considered a stable form of FHB resistance, and one of the worldwide best known and most reliable sources of Type II FHB resistance is Fhb1 from Sumai 3, a major QTL mapped on chromosome 3BS (Anderson et al. 2001, Buerstmayr et al. 2009), still not widely used in Argentina.In the case of leaf rust, several races are generally present in the P. triticina populations in the Southern cone of Latin America, probably due to a high acreage where wheat cultivars susceptible or moderately susceptible to leaf rust were sown (Germán and Kolmer 2014).Host resistance is governed by seedling and adult-plant resistance genes, among which the seedling resistance gene Lr47 is particularly effective (Vanzetti et al. 2011).
MS INTA 416 is a hard red winter wheat (Triticum aestivum L.), developed and released by the INTA EEA Marcos Juárez, in 2016.MS INTA 416, previously designated JN12009, was selected for its high yield potential and good bread-making quality, aside from moderate resistance to Fusarium head blight and high resistance to leaf rust, conferred mainly by the resistance genes Fhb1 and Lr47 introgressed by artificial crosses and selected by marker-assisted selection (MAS).MS INTA 416 is adapted to rainfed and irrigated production areas in the sub-humid and humid plains of the provinces Córdoba, Santa Fé, Buenos Aires, and La Pampa, Argentina.

BREEDING METHODS
MS INTA 416 (JN12009) was selected from a population derived from two backcrosses, using the breeding line R4004 as recurrent parent and Sumai 3 as Fhb1 donor.Breeding line R4004 was obtained from a population derived from six backcrosses using ProINTA Oasis (pedigree OASIS/TORIM-73) as recurrent parent and PI 603918 (pedigree Pavon 76 *8//T7AS-7S#1S-7S#1S/ph1b) as donor of the leaf rust resistance gene Lr47.ProINTA Oasis is a hard red spring wheat developed by INTA EEA Saenz Peña, released in 1989.Since the agronomic performance of this cultivar was very good and yields were high, it was readily adopted in the main wheat-producing areas of Argentina until 1997, when it became highly susceptible to leaf rust by the breakdown of resistance gene Lr26 (Antonelli 2003).The development of line PI 603918 including a Triticum speltoides interstitial translocation carrying Lr47 was previously described (Bainotti et al. 2009).Lr47 is effective against field leaf rust infection in Argentina, according to information obtained from the commercial cultivar BIOINTA2004 released in Argentina in 2009, which carries this gene (Bainotti et al. 2009, Vanzetti et al. 2011, Campos 2013, Campos and Lopez 2015).For the development of R4004 in 1996, in greenhouse facilities of the Instituto de Recursos Biológicos (IRB) INTA, Hurlingham, PI 603918 was crossed with PROINTA Oasis and then backcrossed with the same cultivar for six generations.RFLP marker Xabc465 (Dubcovsky et al. 1998) was used to select Lr47 heterozygous plants from BC 1 to BC 3 , and PCR markers (Helguera et al. 2000) from BC 4 to BC 6 (Figure 1).Then, at least three Lr47-heterozygous plants were self-pollinated producing BC 6 F 2 seeds.In 2000, BC 6 F 2 seeds were planted at INTA EEA Marcos Juárez for the selection of Lr47 homozygous plants, using PCR markers as before.About 30 selected BC 6 F 3 head rows were planted in June 2001 in single 1-m rows, and evaluated in a non-replicated leaf rust screening nursery.
In June 2002, 18 BC 4 F 5 lines were advanced to a non-replicated observation plot trial in Marcos Juárez (plots of six 3-m rows) and in 2003, the same lines were advanced to a Multilocation Trial (MLT) at Pergamino, Corral de Bustos and Marcos Juárez.At this point, R4004 was selected for further backcrossing with Sumai 3 on the basis of grain yield, leaf rust resistance, uniformity, and general agronomic appearance.For the development of MS INTA 416, in 2004, by IRB INTA, R4004 was crossed with cultivar Sumai 3 (Funo/Taiwan Xiaomai), the Fhb1 donor, kindly provided by Dr Jim Anderson (Dpt. of Agronomy and Plant Genetics, University of Minnesota), and backcrossed with R4004 for three generations.In heterozygous plants, locus Fhb1 was traced with SSRs Xgwm533 and Xgwm493 (Figure 2).Then, at least three Fhb1heterozygous plants were self-pollinated, producing BC 3 F 2 seeds.During 2007, the BC 3 F 3 plants were planted in Marcos Juárez for selection of homozygous Fhb1 plants using SSRs Xgwm533 and Xgwm493, as before.About 30 selected BC3 F4 head rows were planted in Marcos Juárez in June 2008 and 2009, in single 1-m rows under 15x2x1.5-mgreenhouses, covered by nylon mesh 35, and the agronomic appearance of the plants was evaluated.

CT Bainotti et al.
In 2010, 28 BC 3 F 6 lines were planted at the same location in non-replicated observation plot trials (six 5-m rows) and, in 2011, 27 lines were advanced to the Preliminary Yield Trials (PYT) in Marcos Juárez.The PYT were arranged in a 10×9 alpha lattice design with two replications and the above plot size.In 2012, based on its yield potential, JN12009 was advanced to the Regional Yield Trials (RYT) in the provinces Buenos Aires (five locations), Córdoba (two locations), Entre Ríos (one location) and Chaco (one location), for three years (2012, 2013 and 2014), under rainfed conditions.A 6x8 alpha lattice design was used for the RYT at all locations, with three replications per trial (plots with seven 5-m rows).Seeding rates were standardized based on the seed size (300 seeds per m 2 ).The RYT trials at Marcos Juárez were also used to measure: (1) days to heading (days from emergence until 50% of the spikes emerged from the boot), (2) plant height (measured at maturity as the mean stem length from the soil to the tip of the spike, excluding the awns), (3) leaf rust (Puccinia triticina) and stem rust (Puccinia graminis tritici) severities based on the modified Cobb Scale (Peterson et al. 1948), (4) head blight (Fusarium graminearum spp), tan spot (Drechslera tritici spp) and bacterial stripe (Xanthomonas translucens p.v. undulosa) incidence and severity, both on 0-9 scales (Stubbs et al. 1986).
MS INTA 416 in the seedling stage was also evaluated for resistance to stem rust and leaf rust at the Cereal Disease Laboratory in INTA Bordenave, in 2013 and 2014.Local leaf rust races MDP 10-20, MFP 10, MKT 10-20 were inoculated on seedlings as described by Long and Kolmer (1989).Leaf rust severity was evaluated on a 0 to 4 scale, as proposed by Stakman et al. (1962).The infection types identified by the symbols 0, 1, 2, or combinations were considered low infection types, indicating resistance, while 3 and 4 were considered high infection types, indicating susceptibility.Seedlings were also inoculated with the local stem rust races QHFTC, QRFTF, QRFTC, and evaluated according to Stakman et al. (1962).The molecular basis of vernalization and photoperiod response were determined using Vrn-A1, Vrn-B1, Vrn-D1, Ppd-B1, and Ppd-D1 allele-specific PCR markers, as previously described (Yan et al. 2004, Fu et al. 2005, Beales et al. 2007, Díaz et al. 2012).To assess the bread-making quality, grain harvested in RYT trials in 2012, 2013 and 2014, in Marcos Juárez (unreplicated samples) was used.Samples were analyzed by standard AACC methods at the Quality Laboratory of INTA Marcos Juárez, for milling, volume weight, protein content, Chopin Alveograph, and bread baking as previously described (Bainotti et al. 2009).High-molecular-weight subunits of glutenin (HMWGs) in the composition of MS INTA 416 were determined by SDS-PAGE as before (Lawrence and Shepherd 1980).Juvenile plants of MS INTA 416 (JN12009) (growth stages 22 to 29, according to Zadoks et al. (1974)) are semi-erect, with a recurved flag leaf at the beginning of inflorescence emergence (GS 52).At maturity (GS 90), the spikes are semishort (81-100 mm), white yellow and dense, with inclined position.The glumes are white, long (9mm), have medium width (3.5 mm) and a straight shoulder shape.The kernel is red, vitreous, and ovate; the germ is medium-sized; the brush is medium-sized and has no collar.According to data from the RYT trials between 2012 and 2014 in Marcos Juárez, MS INTA 416 had a mean plant height of 91.6 cm and developed within 114.6 days from emergence to heading (HD).

AGRONOMIC AND BOTANICAL DESCRIPTION
Comparisons of these values with other frequently grown varieties in Argentina are presented in Table 1.As expected, the HD values observed in our study were significantly longer than those in cooler regions of Brazil (Marchioro et al. 2007, Franco et al. 2014, Franco et al. 2015, Marchioro et al. 2016).MS INTA 416 is uniform for plant type, without obvious phenotypic variants, and remained stable over five generations of evaluation (2011)(2012)(2013)(2014)(2015).Molecular data obtained from Vrn-1 and Ppd-1 adaptation genes defined MS INTA 416 as a winter (carrying the triple combination of "winter" -recessive-alleles within Vrn-1 homoeologs) -insensitive (carrying at least one "insensitive" allele within Ppd-1 homoeologs, with low photoperiod response -in this case Ppd-D1) wheat.Local cultivars with the same combination of Vrn-1 /Ppd-1 adaptation genes (winter insensitive) are Baguette 21, BIOINTA 2004, Buck Ranquel, PROINTA Puntal, SRM Nogal, and Themix, among others (Vanzetti et al. 2013, Gomez et al. 2014).Phenological data (heading time) obtained from a subset of local winter-insensitive wheats sown on six dates between April 29 (mid-autumn) and August 11 (midwinter), at approximately fortnightly intervals between sowings, indicated two clear groups: BIOINTA 3003, BIOINTA 3005 and Baguette 31, with high vernalization requirements (did not flower when planted after June 26), and, SRM Nogal and MS INTA 416 with milder vernalization requirements, as they did not flower when planted on August 11, the last tested sowing date (Table 2).Variation in the duration of cold requirements to complete vernalization has been described previously for Vrn-1 copy number variation (Díaz et al. 2012).Related with the photoperiod response, the relatively low frequency of winter-sensitive (1/11) compared to winter-insensitive (10/11) commercial wheats released in Argentina (Vanzetti et al. 2013) suggests a better adaptation of the second than the first group.Typical winter-sensitive wheats are normally grown in environments with a longer growing season than is being explored by most of the wheat cultivars sown in the wheat belt of Argentina (Gomez et al. 2014). 1 HD = days from emergence to heading; PH = plant height without awns in cm; LR = leaf rust incidence and severity based on modified Cobb scale (Peterson et al. 1948); FHB = Fusarium head blight incidence and severity based on the scale of Stubbs et al. (1986); LS = leaf spot diseases incidence and severity based on the scale of Stubbs et al. (1986); TW = test weight in kg hl-1 ; GP = grain protein content in g kg -1 ; LV = loaf volume in cm 3 ; W = general gluten strength by the Chopin Alveograph and GW = 1000-grain weight in grams.trials, in 2012, 2013 and 2014).In this analysis, the yield of MS INTA 416 was ranked highest or second highest in relation to the test control varieties Baguette Premium 11, Baguette 17 and Klein Yarará in 11 trials, including in Marcos Juárez in 2012, 2013, 2014;Pergamino in 2012Pergamino in , 2014;;Balcarce in 2013, 2014, Bordenave in 2013, 2014, Paraná in 2014and Tres Arroyos in 2014 (Table 3).In view of this good performance, cultivar MS INTA 416 was indicated for cultivation in rainfed and irrigated production areas in the sub-humid and humid plains of the provinces Córdoba, Santa Fé, Buenos Aires, and La Pampa, Argentina.

Disease resistance
Evaluations of dominant leaf rust races on seedlings in Argentina showed that MS INTA 416 is highly resistant, with an infection type (IT) of 0 (Table 4).The IT indicating high resistance observed in MS INTA 416 can be explained by the presence of Lr47, from R4004, confirmed by a gene-specific PCR marker (Helguera et al. 2000).In line with this hypothesis, in advanced breeding lines carrying Lr47 J12012, J13013 and JN12015, IT was 0. Our data agree with previous studies describing no virulence of the major P. triticina populations in relation to Lr47 in Argentina, México and the Southern cone (Huerta-Espino et al. 2011, Vanzetti et al. 2011, Campos 2013, Campos and Lopez 2015).Evaluations of seedlings infected with dominant stem rust races in Argentina also showed that MS INTA 416 is highly resistant, with ITs between 0 and 1+ (Table 4), based on an unknown source of genetic resistance.As expected, field observations for disease resistance showed that MS INTA 416 was highly resistant to the prevalent leaf rust races in Marcos Juárez in 2012, 2013 and 2014.A similar situation was observed for stem rust in Marcos Juárez in 2014.Leaf spot diseases (tan spot and Septoria leaf blotch) were detected in Marcos Juárez, in 2012, and MS INTA 416 showed moderate resistance (7/2), similar to Baguette Premium 11 (7/2), Baguette 17 (8 /2) and Klein Yarará (7/3).Bacterial stripe severity indices were intermediate to high (9/6), classifying the cultivar as moderately susceptible.In 2012, FHB was observed in Marcos Juárez and MS INTA 416 showed moderate resistance (0.5/1).Fusarium head blight was also evaluated in Marcos Juárez, in 2014, in a nursery with natural infection, where MS INTA proved moderately resistant (2/2), similar to Baguette Premium 11 (2/2) and Klein Yarará (4/2) (data not shown).The good performance of MS INTA 416 in response to FHB can be explained by the

Milling and baking quality
Since 1998, bread wheat cultivars from Argentina are being classified based on their commercial and industrial quality performance as Quality Group (QG)1, cultivars with extra strong gluten suitable for blending; QG 2, cultivars adapted to traditional baking (fermentation time longer than 8 hours); and QG 3, cultivars suitable for direct baking methods (fermentation time less than 8 hours) (Cuniberti and Ottamendi 2004).MS INTA 416 belongs to QG 2, with W values of 264, similarly to Baguette Premium 11 (QG 2 check), lower than Klein Yarará (W=336, QG 1 check) but higher than Baguette 17 (W= 257, QG 3 check).The mean test weight of MS INTA 416 and Klein Yarará was 74.73 kg hL -1 , higher than that of Baguette Premium 11 and Baguette 17 (72.27and 68.83 kg hL -1 , respectively).The mean grain protein content (137 g kg -1 ) was higher than of the check cultivars Klein Yarará, Baguette Premium 11 and Baguette 17 (134, 128 and 125 g kg -1 , respectively).Additional quality parameters for MS INTA 416 and check cultivars are listed in Table 1.

BASIC SEED PRODUCTION
The Argentinian National Institute of Agricultural Technology -INTA (Rivadavia 1439, Ciudad Autónoma de Buenos Aires, Argentina) has licensed the seed company LDC Semillas SA (Olga Cossettini 240, Ciudad Autónoma de Buenos Aires, Argentina) to multiply and sell protected wheat cultivars developed by the INTA Wheat Breeding Program for 10 years, as of June 14, 2014.Cultivar MS INTA 416 will be released on the market in 2018.

Table 2 .
Heading date of Argentinian winter wheat cultivars considering eight successive sowing dates (in bold).Data obtained at the experimental station of Marcos Juárez, 2014 dnf: did not flowerCT Bainotti et al.Yield performanceMS INTA 416 was tested at 18 trial locations of the provinces Buenos Aires, Córdoba, Santa Fé and Entre Ríos (RYT

Table 3 .
(Bainotti et al. 2013, Bernardo et al. 2014, Anderson et al. 2015)cations (RYT), in 2012, 2013 and 2014.In bold, grain yields of MS INTA 416 ranked highest or second highest Fhb1 confirmed by the flanking markers Xgwm533 and Xgwm493.Marker-assisted selection of Fhb1 has been used successfully used in the development of germplasm adapted to specific environments(Bainotti et al. 2013, Bernardo et al. 2014, Anderson et al. 2015).MS INTA 416 is the first wheat cultivar released in Argentina carrying the genetic resistance sources Fhb1 and Lr47 against FHB and leaf rust, respectively.