USING AVENA MACROSTACHYA FOR IMPROVEMENT OF OAT WINTERHARDINESS IN POLAND

cultivars and with accessions of perennial wild species macrostachya The F 1 hybrids were intensely cloned, treated with colchicine and grown in vicinity of winter oat pollinators. Results and conclusions: Three F 1 hybrids were obtained, two of them set seed. Large differences were noticed in fertility level and chromosome constitution of progeny of the two hybrids. The next generation (B 1 F 1 or F 2 ) consisted of plants with chromosome numbers between 40 and 49, octoploids (2n = 56) and plants carrying 60 to 70 chromosomes. The quasi-hexaploids of the first group were generally male sterile, but after addi­ tional back-cross to A. sativa L. valuable hexaploid breeding strains were pro­ duced. In the dry seasons 2009 and 2013 they yielded nearly 100% higher than the spring cultivar standard. Test weight of the winter oat was also ca. 4 kg/hl higher than in the spring oat. Two of the husked strains, showing winterhardi­ ness level better than the starting A. sativa population, are in the Polish state variety trials. They could be recommended for the regions with stable snow cover, because they were not able to survive naked ground surface tempera­ tures below -14oC, which twice in Radzikow in the last 8 years. Es­ pecially the last winter (2015/2016) was destructive, even for the octoploids, which were earlier considered the most winter-hardy oats. The octoploids pro­ duce healthy green mass and very large plump grain, rich in protein, however they require a breeding effort to increase yield (now 50-67% of the best winter hexaploids) and to make ripening uniform. The last severe winter season was helpful for selection of several new hexaploid strains from the 6x x 8x cross combinations, which were transgressive in frost resistance to their octoploid parents. The 8x or 10x sativa-macrostachya alloploids proved to be effective sources of winterhardiness for hexaploid oat.


Introduction
Preliminary experiments with winteг oat in Poland at the end of the 20 century indicated its potential of high yielding especially in dry seasons, when foreign winter strains or culti vars doubled yields of local spring standards (Zygmynt Nita, personal comm.). However, a mild preceding winter season was condition for the success; winterhardiness of the oat lines from western countries was generally insuffi cient.
A stagnation was recorded in oat winter hardiness in the second half of the last century until a progress was reported in North Caroli na, USA, first as a result of crossing the winterhardy cultivars 'Wintok' and 'Norline' (Liv ingston, Elwinger, 1995;Livingston et al., 2004), and later after application of the wild species Avena macrostachya Bal. ex Coss et Dur. as a cross component to the cultivar Brooks (Santos et al., 2002). A cross with an alien species may introduce new and useful genetic variation when intraspecific variation of a crop is insufficient; there is also possible advantage of whole genome incorporation through alloploidy. It is relevant particularly for the quantitative traits including abiotic stress responses. Wide crossing of Avena sativa L. with A. macrostachya was the starting point for winter oat acclimation in Poland. Ini tiation of the work in IHAR-Radzikow was encouraged by the acceptable level of winter survival in the Uniform Oat Winter Hardiness Nursery (UOWHN) grown in the location since 1993, as well as by presence of a rela tively large A. macrostachya collection in the IHAR gene bank. The accessions were collect ed during expedition in the Atlas mountains elevated sites, next sown in Gubalowka, Po land, on foothills of the Tatra mountains, where clones of this perennial species survived nearly 10 years. Attractiveness of the variation source was strengthened by high resistance of A. macrostachya to biotic stresses. Herrmann (2006) reported introgression of powdery mil dew resistance to A. sativa. Loskutov (2007) listed other resistances potentially useful for breeders: to crown and stem rust, septoria blotch, helminthosporium blotch, myrothecium blotch, frit fly; Weibull (1988) announced re sistance to aphid Rhopalosiphum padi L.

Breaking the interspecific isolation barriers
The crossing between foreign winter oats and A. macrostachya was performed in Radzikow in 2002. Single hybrid seeds were obtained only in three of 51 cross combina tions, when common oat was used as a mother plant. Only two of the F1 plants recovered (af ter in vitro embryo culture) were sufficiently vigorous and produced abundantly tillers in greenhouse, which enabled establishment of large clonal populations. The clones were treated with colchicine and planted in field in early spring, among a winter and spring sown mixture of winter oats, which were the addi tional pollen donors for the F1 plants. The clone derived from the cross involving winterhardy oat cv. 'Wintok' (W-F1) consisted of finally 969 plants and produced ca. 2050 pani cles and 3 germinable seeds. The other clone of 2073 plants based on a German (Lochow-Petkus) non-winterhardy 'Mirabel/Pendragon' recombinant line (MP-F1), produced ca. 9890 panicles and 54 germinable seeds. The earlier publications of Lapinski et al. (2012Lapinski et al. ( , 2013 contain more detailed description of overcom ing the interspecific reproductive barriers. The results strongly confirmed the idea of starting from a broad variety of genotypes in a difficult interspecific cross. In addition to the known and expected differences in crossabil ity, F1 viability and sterility, a substantial dif ference occurred in toleration of aneuploidy between the successful W-and MP-cross combinations, which greatly determined the direction of further evolution of the progenies. The F1 generation had 35-chromosomes, 21 from A. sativa mother and 14 from A. macrostachya (autotetraploid, 4n = 28). Prog eny of the W-F1 hybrid consisted of three euploids: two octoploids (8x = 56) and one decaploid (10x = 70). Aneuploids prevailed in progeny of the MP-F1 : there were 32 plants with chromosome numbers between 40 and 49, one octoploid and 28 hypo-decaploids, with 60 to 69 chromosomes (table 1). The quasihexaploids of the first group showed various level of weakness and sterility, but in the fol lowing generations most of the progenies quickly regulated their chromosome comple ment and restored fertility, usually with help of additional spontaneous back-cross to A. sativa. Eleven of these cytogenetically unbalanced progenies produced valuable and stable hexaploid (6x = 42) breeding strains. The octoploids, less frequent than expected, gave also rise to cytogenetically stable stocks. The de scendants of the hypo-decaploid primary syn thetics were less vigorous than the octoploids or hexaploids and their propagation required verification of ploidy level at reproduction, as there was a tendency to spontaneous decrease of number of chromosomes.  2 3 4 3 4 4 1 3 1

The octoploids as a new species comparable to primary triticales
An abiotic stress response is usually deter mined by numerous interacting genes, thus, recombination itself, usually restricted by re duced meiotic pairing, may not be effective in transfer of all necessary genes. Creation of alloploids makes it possible to transfer a whole genetic system (set of chromosomes) control ling a trait(s) of interest. Triticale is a conven ient reference species to compare results of transfer of abiotic stress tolerance through whole genome transfer; it exceeds wheat and its introgressive interspecific recombinants in resistance to drought, soil salinity and acidity (Hede, 2001). It requires less nitrogen and wa ter than wheat to produce a similar yield. Triticale winterhardiness was a more difficult breeding objective but a few decades were suf ficient to restore expression to the level of pa rental species. No such incompatibility of win terhardiness genetic systems occurred in our sativa-macrostachya alloploids; first genera tions of the obtained octoploids exceeded their hexaploid common oat parents in winter hardi ness. The 8x line PR-4H8 had the best survival scores in UOWHN 2010. However, we did not observe in the octoploids any further progress in the trait expression compared to that record ed recently in the hexaploid wide hybrids. Probably, lack of appropriate variation is the cause. As yet, our attempts to produce second ary winter octoploids through crossing hexaploids with decaploids are unsuccessful: in 24 crosses of this type progenies shifted to the 6x ploidy level. Variation in our octoploids comes mainly from recombination initiated by the back-cross of interspecific F1 to A. sativa. The most interesting lines, including the PR-4H8, came from the cross of the W-F1 ('Wintok' based) with Welsh line 95-43Cn4 (the only line among the pollen parents which had a black hull marker enabling parentage control). The sativa-macrostachya octoploids produce large, elongated seed (in nurseries TKW up to 70 g), plump and rich in protein (up to 20%) but with poor beta-glucan content (ca. 2,7%). The seed quality in these octoploids was much better than shrivelled and frequently sprouted seed of early wheat-rye alloploids. Yield of grain of the oat octoploids approximated to 50-67% of the good hexaploid winter forms and its average value in the years with winter survival is similar to that of common spring oat. Yield potential of the octoploids is proba bly underestimated, because of an uncontrolled negative effect of wild animals selectively grazing on the trial plots, which occurs regu larly in early spring at our location. Leaf dis eases resistance is high and green mass abun dance is suggesting usefulness for green forage or silage. In cultivation of the octoploids for grain, late and non-uniform ripening may be problematic in some years (the wild compo nent is a perennial plant with prolonged tiller ing phase). Care must be taken regarding the disappearance of resistance to diseases in sin gle 8x lines. We noticed loose smut, which was never recorded before in the location, on a few of the W-F1 lines. The loss of some genes (or a loss of their function) may be attributed to recombination of heterologously translocat ed chromosomes or rapid sequence elimination and epigenetic modifications (Ma and Gus tafson 2008) which occur in early generations of new alloploids. Continuing the comparison with first wheat-rye hybrids, grain yield poten tial of early triticales from CIMMYT was re ported as ca. 2.4 t/ha. After three decades of breeding (nearly 60 generations) it reached 10 t/ha (Hede, 2001). The 2/3 of common oat yield in our raw octoploids makes for a much better starting point. Considering the most dis tant systematic position of A. macrostachya in relation to A. sativa and other oat species in the genus, the relatively high performance of the octoploids could not only justify their breeding as a separate crop, but should also encourage the use of other Avena species in oat improve ment through whole genome addition or sub stitution

Hexaploid progeny of the wide crosses
The unexpected 40-49 chromosome plants of the MP-F1 progeny resulted most probably from irregularly reduced female gametes ferti lized with normal 21-chomosomal male gam etes of common oat. Such back-crosses should facilitate homeologous recombination and pro duce more unique genotypes than the products of 'academic' back-crosses of hexaploids with octoploids, aimed at promoting introgression. Conjugation of A. macrostachya chromosomes with those of A. sativa is possible but poor (Leggett, 1985), thus, in the 6x x 8x F1, they are expected to remain unpaired, particularly when the sativa chromosomes form competi tive bivalents. Finally, the most probable result should be a loss of the alien chromosomes without any introgression. Forcing of homeologous recombination and better chances for maintenance of alien germplasm may occur in progeny created from a selection of gametes with random chromosome composition. There fore, high numbers of independent recombi nant lines were derived from the MP-F1 quasihexaploids in the next generations. Their selec tion and stabilization was accompanied with division into winter or spring types, according to response to the occurring low winter tem peratures. After four years in nurseries, the first winter hardy strains were directed to field trials. Some of them showed winterhardiness levels higher than the lines from the foreign oat collection or the UOWHN nursery objects. Two husked lines were sent to the international UOWHN nurseries in 2011 and 2012, where they reached top scores of winter survival. Moreover, investigation of the 2011 UOWHN nursery objects with the winterhardiness DNA markers set, developed in the University of North Carolina (Wooten et al., 2008), revealed the lowest number of the frost resistance markers in our 5Q5.2 line, in spite of the high est phenotypic winterhardiness record (Lapinski et al. 2013). It confirmed uniqueness of the resistance source. Yields of the both UOWHN studied lines were satisfactory in two-locations field trials, thus they were di rected to the Polish state variety trials in 2014 and 2015. The line 5Q5.2 (proposed cultivar name 'Radzikus') is of medium height, yield ing grain with relatively high test weight (in Radzikow ca.60 kg/hl, 2-3 kg/hl more than the average for winter oat and ca. 4 kg/hl better than the spring standard) and relatively high oil content (see table 4). The other line 5T8.A is 10-20 cm taller (but not less resistant to lodging), has a larger grain and shows re sistance to mildew. In the Polish weather con ditions, both lines are of facultative type, how ever late sowing in some environments may cause problems with delayed heading. Yield ing of these lines is shown in tables 2 and 3, in relation to winter barley and spring oat stand ards. In Radzikow, the average yield of 5Q5.2 in five seasons with good winter survival reached 90,4% of the winter barley standard ('Carola') and was much better (141,2%) than average yield of the spring oat standard ('Kre-zus'). Two years with winter killing (2012 and 2016) resulted in lower values at 64,6% and 100,8%, respectively. However, considering the ca. 7 dt/ha yield equivalent necessary to pay for re-sowing of a winter killed plantation, the winter oat remains economically still very competitive to spring one. In Radzikow, in a season with winter survival, average advantage of winter oat yield over the spring one is 20,1 dt/ha, which compensates almost for three years of winter killing losses. In the submountaineous station Grodkowice, where the 5Q5. On less fertile sandy soils, winter killing risk is much higher and yield compensation, in relation to spring form, is smaller, thus care fulness and local trials are recommended be fore large scale planting. The year 2011 was conducive for oat growth in Radzikow and the potential for yield quantity and quality was well expressed for both winter and spring forms. The basic quality parameters for some bulks and advanced lines, shown in table 4, confirmed good quality of hexaploid winter oats derived from the interspecific crosses, in comparison with the spring oat standard and the 'mix W ' protein rich bulk from a spring x winter A. sativa intraspecific cross.

Relevance of the alloploids in winterhardi ness improvement
The last winter 2015/2016 was the most de structive in Poland since last 25 years, in spite of relatively high average winter temperatures. In Radzikow the first significant frost of -14oC suddenly attacked, without snow cover, in Jan uary 2-6 and totally killed more than 1/3 of winter oats in nurseries and in field trials. The lines which survived the first attack of winter showed a wide scope of damage level. Howev er, during the following two months with tem peratures oscilating near 0oC the surviving plants lost green colour and were not able to grow and regenerate. Finally, all oats died, while the winter barley standard stayed alive. Oat survival in Poland in 2016 was only possi ble in the south-western part of the country, where snow cover supplied sufficient protec tion against frost (and, probably, solarization).
Variation in frost resistance scores (collect ed shortly after the January frost) is presented in Each cross combination is represented in the table by only one line with the best frost resistance. The group 1. of hexaploid lines or bulks derived from spontaneous pollinations of the interspecific MP-F1, containing the 5T8.A and 5Q5.2 cultivar candidates, showed no more the best winterhardiness. The octoploids (group 2) maintained their resistance level, but the most hardy four oat lines, with scores higher than 6, were hexaploid. They had the W-F1 derived octoploid parent or grandparent (best score 6) as a source of the resistance. The other octoploid (MP-F1) showed lower level of resistance (score 4), related to distinctly lower winterhardiness of its MP (Mirabel/Pendragon) initial wide cross parent. The difference at tributable to these two grand-parental 6x forms is maintained, however less distinct in the groups of 6x x 10x crosses. Considering the 9 best cross combinations with top scores higher than 5, presence of an alloploid (8x or 10x) in a line parentage seems essential for high ex pression of frost resistance, while the re sistance level of the other (hexaploid) parent was less important. It suggests a low number of 'strong' genetic loci involved. An oat object with the score 8 has the spring cultivar ('Bug gy') as mother cross component. Another spring line (from 'Strzelce' Breeding Co.) brought also, as a grandparent, a remarkable contribution to the other two transgressive genotypes with scores 9 and 7. The high pro portion of spring oat genes in the elite winter type materials is surprising, however, in gen eral, their influence on winterhardiness is neg ative, as shown for other spring parents in comparisons between groups 4 and 5 or 7 and 8. Winterhardiness alleles in spring oat were reported earlier by Amirshahi and Patterson (1956), Murphy (1958)  Таблица 5. Распределение по морозостойкости различных скрещиваний озимого овса. Радзиков, 2016 г. Шкала 1 -гибель, 2-9 -уровень устойчивости. Каждое скрещивание представлено наиболее зимостойкой линией. Table 5. Distributions of frost resistance scores for various groups of winter oat crosses. Radzikow, 2016. Score 1 means total killing, 2 to 9 describe ascending levels of resistance.
Each cross is represented by its most winterhardy line.
be excluded until molecular verification of the alien source hypothesis is produced. Even when the interspecific gene exchange may not be a major factor, alloploid back-cross seems still attractive as a prospective version of 'in corporation' breeding strategy (Simmonds, 1993), which is usually based on secondary intercrossing among independently introgressed lines of a distant cross, aimed at resto ration of a trait genetic architecture disrupted by interspecific recombination. The most frus trating restriction in breeding for winterhardi ness is a decrease of yielding potential associ ated with increase of resistance to winter stress. The physiological basis of this link, re lated to deepness and duration of dormancy period in winter, is also known in crops other than oat. The only way to keep yield high is breeding for cultivars with the lowest accepta ble level of winterkill resistance in the targeted area (Reynolds et al., 2001).
A year ago the 'Radzikus' 5Q5.2 cultivar candidate seemed to have the most optimal combination of winterhardiness and yielding capacity, reaching in 2015 70,0% and 98,7% of the barley check yield in Radzikow and Grodkowice, respectively. Three new lines were identified which were more productive than 5Q5.2 in 2015 while being also more winterhardy in the current season. The best of these lines combined 84,2% of the barley check yield (in Radzikow, 2015) with the highest possible frost resistance score 9. The corresponding 2016 score for 5Q5.2 was only 2. It opens new prospects to rise level of the yield-resistance compromise in oat. The elite includes also one line of naked oat with win terhardiness score 6. Hulless forms from earli er crosses were generally classified as insuffi ciently resistant to winter killing. The elite re sistant line comes from a cross involving a winter naked common oat and the 'Wintok' derived octoploid. Its yield in Radzikow in 2015 was very low (3 dt/ha, 39,8% of the win ter barley standard). Better result recorded for that line in Grodkowice (56,2 % of the barley check) could make it more competitive to spring naked oat.