\‘- l ’ “I GENETICS 0F FREEZING HARDINESS IN WINTER WHEAT (T riticum aestivum L) DIssertaIion for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY MAGNE GULLORD 1974 This is to certify that, the thesis entitled, Genetics of Freezing Hardiness in Winter Wheat (Triticum aestivum L.). ' presented by MAGNE GULLORD has been accepted towards fulfillment of the requirements for _IhD_.___degree in mm (-3 Major professor ABSTRACT GENETICS OF FREEZING HARDINESS IN WINTER WHEAT (Triticum aestivum L.) BY Magne Gullord The inheritance of freezing hardiness was studied in (l) the winter tender variety Genesee, the hardy variety Winoka and the populations derived from the cross between them, and (2) in two complete diallels, one with six and one with four parental genotypes. The plant material was tested in an artificial freezing procedure under both high and low intensity freezing conditions. Moisture content in young leaf sections close to the crown was studied in nine wheat varieties and in genotypes from the four parental diallel. Freezing hardiness was found to be a quantitative character. The genetic variation found in F2 and hackcross populations appeared to be only additive. Interaction be— tween F2 lines and the level of freezing intensity suggests that genes controlling freezing hardiness under high in- tensity freezing is different from the genes controlling freezing hardiness under low intensity. .Gfi Magne Gullord ‘1; L" Q? Analysis of both diallels showed that freezing hardiness is controlled by partially dominant genes which are mostly additive in their effect. No reciprocal differences with respect to freezing hardiness were identified in any of the diallels. Moisture content in leaf segments close to the crown was negative correlated with freezing hardiness. Moisture explained about 70 percent of the variation in freezing hardiness in nine winter wheat varieties. If kinetic inhibitor ratings was included as an additional independent variable, about 80 percent of the variation in freezing hardiness was explained. Low moisture content was found to be controlled by partially dominant genes mostly additive in their effect. GENETICS OF FREEZING HAROINESS IN WINTER WHEAT (Triticum aestivum L.) BY Magne Gullord A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of CrOp and Soil Sciences 1974 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. E. H. Everson for his encouragement and guidance as research advisor and for his critical appraisal of this manu— script. I also wish to express my gratitude to Dr. C. R. Olien for advise and valuable discussions during this study. Thanks are extended to the faculty for its time, advice and assistance, particularly those who served on the guidance comittee: Dr. R. L. Anderson and Dr. J. B. Beard, and to Dr. C. M. Harrison for his help in the linguistic improvement of this dissertation. I also wish to acknowledge the assistance of Mr. B. L. Marchetti with the freezing tests. The author thanks the students and the staff of this department who made this period interesting and rewarding. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . iv LIST OF FIGURES. . . . . . . . . . . . . Vii INTRODUCTION. . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . 3 MATERIALS AND METHODS. . . . . . . . . . . 11 Generation Materials . . . . . . . . . ll Diallels . . . . . . . . . . . . . 12 Leaf Moisture Content. . . . . . . . . 13 Genetic Variance Components. . . . . . . l4 Diallel Analysis . . . . . . . . . . 15 RESULTS AND DISCUSSION . . . . . . . . . . 18 Generation Material . . . . . . . . . l8 Diallels—-High Intensity Freezing. . . . . 26 Diallels--Low Intensity Freezing . . . . 33 Influence of the Severity of the Freezing Test on Gene Action. . . . . . 46 Relationship Between Freezing Hardiness and Leaf Moisture Content . . . . . . . . 54 SUMMARY AND CONCLUSIONS . . . . . . . . . . 62 LIST OF REFERENCES. . . . . . . . . . . . 65 iii LIST OF TABLES Winter wheat varieties used as parents in two complete diallels . . . . . . . . . . Frequency distributions of lower peripheral crown meristem ratingstm'FZ, backcross and parental pOpulations from the cross Genesee x Winoka, tested under high intensity freezing conditions . . . . . . . . . . . . Frequency distribution of lower peripheral crown meristem ratings for F2, backcross and parental populations from the cross Genesee x Winoka, tested under low intensity freezing conditions . . . . . . . . . . . . Means and variances of lower peripheral crown meristem ratings for Genesee, Winoka and populations derived from the cross between them, tested under both high and low intensity freezing . . . . . . . . . . . . . The analysis of variance of freezing hardiness measured as lower peripheral crown meristem ratings of a six parental diallel tested under high intensity freezing . . . . . . . . Lower peripheral crown meristem ratings of parents and Fl's from a six parental diallel tested under high intensity freezing, totals of three freezing tests after averaging over reciprocal crosses. . . . . . . . . Components of variation of freezing hardiness under high intensity freezing in a six parental diallel . . . . . . . . . . Analysis of variance of the lower peripheral crown meristem ratings of parents and Fl's from a four parental diallel tested under high intensity freezing . . . . . . . iv Page 12 19 20 21 27 30 30 32 Table Page 9. -Analysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and Fl's of a four parental diallel tested under high intensity freezing. . . . . . . . . 10. Lower peripheral crown meristem ratings of parents and F 's from a four parental diallel tested under high intensity freezing, means of 25 replications and reciprocal crosses . . . 35 11. The analysis of variance of freezing hardiness measured as percent survival (transformed to logarithms) of a six parental diallel tested under low intensity freezing . . . . . . 35 12. Percent survival (transformed to logarithms) of parents and Fl's from a six parental diallel tested under low intensity freezing, totals of three freezing tests after averaging over reciprocal crosses . . . . . . . . 39 13. Components of variation for freezing hardiness in a six parental diallel under low intensity freezing . . . . . . . . . . . . . 40 14. The analysis of variance of freezing hardiness measured as lower peripheral crown meristem ratings of a four parental diallel tested under low intensity freezing . . . . . . 42 15. Lower peripheral crown meristem ratings of parents and Fl's from a four parental diallel tested under low intensity freezing, totals of two freezing tests after averaging over reciprocal crosses. . . . . . . . . . 45 16. Components of variation for freezing hardi- ness in a four parental diallel under low intensity freezing. . . . . . . . . . 45 17. Analysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and Fl's of six parental diallel tested under low intensity freezing at a temperature of -18.2 C . . . . . . . . . . . . . 47 Table 18. 19. 20. 21. 22. 23. 24. 25. Lower peripheral crown meristem ratings of parents and Fl's from a six parental diallel tested under low intensity freezing at a temperature of -l8.2 C, averages over replica- tions and reciprocal crosses . . . . . . Analysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and Fl's of a six parental diallel tested under low intensity freezing at a temperature of -l7.5 C . . . . . . . . Lower peripheral crown meristem ratings of parents and Fl's from a six parental diallel tested under low intensity freezing at a temperature of -l7.5 C, averages over repli- cations and reciprocal crosses. . . . . Analysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and Fl's of a six parental diallel tested under low intensity freezing at a temperature of -l6.7 C . . . . . . . . . . . . . Lower peripheral crown meristem ratings of parents and Fl's from a six parental diallel tested under low intensity freezing at a temperature of -l6.7 C, averages over repli- cations and reciprocal crosses. . . . . . Moisture content of young leaves for nine winter wheat cultivars, averages over 14 replications. . . . . . . Analysis of variance of moisture content of young leaves near the crown of parents and Fl's of a four parental diallel Moisture content of young leaves close to the crown of Fl's and parent plants from a four parental diallel, averages of five replica- tions . . . . . . . . . . vi Page 47 48 48 49 49 55 58 59 Figure 1. LIST OF FIGURES Page Relationship between lower peripheral crown meristem ratings under high and low intensity freezing for 96 F families originating from a cross between Genesee and Winoka, averages over 8 and 5 freezing tests respectively . . 22 Relationship between lower peripheral crown meristem ratings under high and low intensity freezing for 23 BC families originating from a cross between Genesee and Winoka, averages over 8 and 5 freezing tests respectively . . 23 Relationship between lower peripheral crown meristem ratings under high and low intensity freezing for 23 BC families originating from a cross between Genesee and Winoka, averages over 8 and 5 freezing tests respectively . . 24 The regression of W on V for freezing . . r . hardiness in terms of lower peripheral crown meristem ratings of a six parental diallel tested under high intensity freezing. . . . 29 The regression of W on V for freezing . . r . hardiness in terms of lower peripheral crown meristem ratings of a four parental diallel tested under high intensity freezing. . . . 34 The regression of W on V for freezing hardiness in terms 5f percent survival (transformed to logarithms) of a six parental diallel tested under low intensity freezing . 36 The regression of W on V for freezing . . I" . hardiness in terms of lower peripheral crown meristem ratings of a four parental diallel tested under low intensity freezing . . . . 43 The regression of Wr on V for freezing hardiness in terms of lowgr peripheral crown meristem ratings of a six parental diallel tested under low intensity freezing at -l8.2C. . . . . . . . . . . . . . 50 vii Figure Page 9. The regression of W on V for freezing hardiness in terms 5f lower peripheral crown meristem ratings of a six parental diallel tested under low intensity freezing at —l7.5C. . 51 10. The regression of W on V for freezing hardiness in terms 5f low5r peripheral crown meristem ratings of a six parental diallel tested under low intensity freezing at -l6.7C. . 52 11. Relationship between freezing hardiness in terms of lower peripheral crown meristem ratings tested under high intensity freezing and leaf moisture content for nine winter wheat cultivars. . . . . . . . . . . . . . 56 12. Relationship between freezing hardiness in terms of lower peripheral crown meristem ratings tested under low intensity freezing and leaf moisture content for nine winter wheat cultivars. . . . . . . . . . . . . . 57 13. The regression of W on V for leaf moisture content of a four parentaI diallel . . . . . 61 viii INTRODUCTION Yield of winter wheat is 30-50 percent higher than that of spring wheat, when winter kill is not a factor. Winter wheat also has the advantage of aiding in the dis- tribution of farm labor. The level of freezing hardiness in soft winter wheat compared to hard red winter is low (10). According to Everson et a1. (7) 20 percent of the soft winter wheat in the state of Michigan suffers some winter injury every year. Injury varies from slight damage to occasional total loss in individual fields. A joint effort was initiated by the Agricultural Research Service, USDA and Michigan Agricultural Experi- ment Station in 1960 to increase freezing hardiness in soft winter wheat. An artificial freezing technique has been develOped for efficient screening of breeding mate- rials both under high and low intensity freezing. This study utilizes this freezing procedure, and the theoreti- 'cal work of Dr. C. R. Olien on the relationships between water content in the cereal crown and the intensity of the freezing process. Nine winter wheat cultivars varying in freezing hardiness were selected for a genetic study. The l objectives were to: (1) study the inheritance of freezing hardiness under high and low intensity freezing and (2) study the relationship between leaf moisture content and freezing hardiness, and the inheritance of moisture con- tent. REVIEW OF LITERATURE Winter survival of cereals depends mainly on three factors: (1) winter habit, (2) disease and insect resist- ance, and (3) resistance to freezing. Other factors and interactions may also influence survival (32). Winter habit is a necessary character for fall sown cereals. With winter habit, plants remain in a vegeta- tive growth stage during cold weather and resist freezing damage to varying degrees. Length of the vernalization process varies with variety and is not correlated with winter hardiness.* In crosses between spring and winter wheat, the spring habit is generally dominant over winter habit, and depending on the parents involved in the cross, one, two, or three independently inherited genes were found to control these characters (2, 16, 36, 50). In areas where the field is covered with snow during the winter, snowmolds (Fusarium niyale_Fr.), Cesati, Typhula idahoensis (Remsberg) and T. incarnata often damage winter cereals. Attacks by diseases and insects usually make the plants less resistant to freez- ing. *E. H. Everson. Unpublished data. The most important and frequent cause of damage in winter cereals is freezing. Because of the erratic nature of winter killing and high experimental error in field testing, breeders must usually depend on average perform- ance of lines over a wide range of locations and years to determine freezing hardiness. Development of rapid and more efficient laboratory methods for testing winter cereals has therefore been the object of much investigation (5, 10, ll, 17, 20, 23, 48, 49). In earlier studies, plants were test-frozen in pots or flats (5, 11), but moisture content was difficult to control. Olien (27) identified the crown as the critical region for damage to winter cereals. Severe destruction of these tissues result in death of the plant. A crown freezing technique was developed by Kretschmer (17, 18) and modified by Marshall (20) for evaluation of winter oats. Warnes and Johnson (48) modified Marshall's freezing procedure for evaluation of winter barley. Metcalf et_al. (23) developed a crown freezing technique where the moisture content in the crown could be controlled. The method was later modified by Gullord g£_§l. (10). High correlations were found between percent sur- vival of winter cereals under controlled freezing condi- tions and under field conditions when the latter were means of several years' testing over many locations (20, 37, 42). Several attempts have been made to find simple chemical or physical measurements of winter cereals which could give an indication of resistance to freezing. Moisture content in leaves of winter wheat was reported to be negatively correlated with winter hardiness (l, 21, 25). Shutt (46) found a similar relation in apple twigs. In an attempt to understand the nature of winter hardiness, Olien (28) studied the different types of stresses asso- ciated with different types of water redistribution in barley. He identified two main types of freezing pro- cesses in hardy cereal crowns: (l) equilibrium or low intensity freezing, and (2) non—equilibrium or high intensity freezing. Equilibrium freezing occurs when the liquid between protoplasts is closely associated with cell walls and pro- toplasts, and heat is slowly removed from tissues contain- ing ice crystals. The kill temperature in equilibrium freezing for wheat varies between -15 and 20C (10), but is not low enough to provide sufficient free energy to kill by frost desiccation (31). The free energy of freez— ing is dissipated by a shift in activation energies of water transition as adhesion and more complex bonding interactions develop between ice and plant components, whereas the free energy of desiccation is dissipated by a shift in vapor pressure as water evaporates (33, 34). The injury under equilibrium freezing is cytological, centered around ice crystals and involves most of the tissues in the crown (35). A non—equilibrium freezing occurs when moisture content in the crown is high (>70%) and heat is rapidly removed. Freezing occurs so rapidly that water molecules do not have time to diffuse to areas where crystal growth is unrestricted. A large amount of free energy is dis- sipated by formation of ice structures and disruption of tissues. Wheat is killed at higher temperatures 0 to 15C (10), depending on free energy of freezing, which is a function of crown moisture in a standard freezing test (23). The injury under high intensity freezing is histo- logical and takes place in specific tissues and plant regions (35). Toxication of adjacent tissues occur dur- ing degeneration of injured tissues. Different gene systems are very likely to Operate in cereal genotypes under two such different stresses as those described. Evidence to support this hypothesis comes from Gullord et_al. (10) and Metcalf e£_31. (23) who showed significant interactions between wheat geno- types and level of intensity under which the genotypes were frozen. Interaction in percent survival of F2 and F barley lines and field locations shown by Eunus g£_§1. 3 (6) may also support this hypothesis, though different levels of disease or insect attacks in the various loca- tions also could create the reported genotype x location interaction. The structure of ice crystals influences survival under high intensity freezing. Olien (29) identified cell wall carbohydrates that modified ice crystal structure in rye (kinetic inhibitors). The polymers had little effect on the freezing temperature, but interfered with liquid : solid reaction as a competitive inhibitor. Shearman gt_§1. (45) reported correlation between kinetic inhibitor rat- ings and survival of winter wheat under high intensity freezing. Stability of membranes under different types of freezing stresses is very likely to influence freezing hardiness in plants. Schmuetz (44) showed high correla- tion (r = .85) between freezing hardiness and the content of membrane stabilizing sulfhydryl groups (SH) in six day old unhardened seedlings of 87 wheat varieties differ- ing in winter hardiness. Most genetic studies of freezing hardiness have been conducted under natural field conditions. Since sur- vival is not only determined by freezing stresses, but also by disease and insect attacks, heaving, etc. and interactions of these, no simple genetic system can be expected to control freezing hardiness when studied in the field. Even resistance to artificial freezing may be complex according to Olien (30). Inheritance of freezing resistance was studied in winter cereals early in this century. Nilsson-Ehle (26) crossed two winter wheat varieties intermediate in winter hardiness and found transgressive segregation for the character. He concluded that winter hardiness behaved as other quantitative characters controlled by many genes. Similar results were later reported in winter wheat (12, 19, 21, 38, 42, 43, 49), in winter oats (3, 14, 24) and in winter barley (6, 39, 40, 41). An eighteen parental diallel in barley was tested for winter hardiness in six field locations and under controlled conditions and analyzed by Rhode and Pulham (40, 41) and reanalyzed by Eunus et_al. (6). Dominant and recessive genes both additive and non-additive in their effect controlled winter hardiness. Jenkins (14) tested a five parental oat diallel for freezing hardiness under controlled freezing conditions. In one severe freezing test, freezing resistance was largely determined by recessive genes, essentially additive in their effect. Under less severe conditions his data indicates that freezing hardiness was controlled by dominant genes. Muehlbauer et_al. (24), Quisenberry (37), and Worzella (49) similarly reported that dominant genes controlled winter hardiness under mild winter conditions while lack of dominance was found under more severe conditions. Schafer (43) reported that winter hardiness was probably controlled by recessive genes, since a majority of the F3 rows from a cross between Turkey and Jenkin showed severe winter injury. Significant reciprocal differences in winter hardi- ness were reported by Muehlbauer et a1. (24). The differ- ences were inconsistent over locations indicating that the cytoplasmic factors were influenced by environment. Heritability studies of winter hardiness in cereals indicate that heritability estimates (broad sense) are proportional to the range in winter hardiness between the parents. Rhode and Pulham (40) calculated 20 heritability estimates on the average survival of bulk F2 and bulk F3 progenies from 18 winter barley varieties crossed in a diallel series. The values ranged from 36 to 74 percent. When progenies from crosses involving the five most tender varieties were eliminated, the heritability estimates ranged from O to 48 percent. Amirshahi and Patterson (3) similarly found that the heritability was lower in crosses with related than with unrelated varieties. Cytogenetic studies in barley showed that winter hardiness was associated with V and B loci on chromosomes 2 and 5 respectively (41). Law and Jenkins (19) test 10 froze 21 possible substitution lines between the tender wheat variety Chinese Spring and the hardier variety Cappelle Desprez. They found that only the 4D, 5D and 7A chromosomes were involved in freezing resistance and that their effect was additive. It is reasonable that the D genome accounts for the major portion of freezing hardiness, since it enables the original hexaploid wheats to expand and colonize more northern latitudes than tetraploids. Simple inheritance of freezing hardiness has been reported in crosses between broccoli and kale x cabbage (4). Two dominant genes with epistatic effect were found to control freezing hardiness. MATERIALS AND METHODS Generation Materials A cross between Genesee and Winoka was made in the greenhouse during the winter of 1971. F1 and F2 seeds were space planted in the field in the fall of 1971 and 1972 respectively. A segregation for seed coat color not significantly different from 15:1 (red to white) in F3 seeds confirmed that we had a segregating population. 1597 F2 lines were classified in four classes according to morphological characters: (1) breadless - red chaff, (2) beardless - white chaff, (3) bearded - red chaff and (4) bearded — white chaff. A segregation not signif- icantly different from 9:3:3:1 indicated that beardless- ness and red chaff were controlled by two dominant inde- pendent genes. Backcrosses to both parents were made in the winter of 1972 and F plants were grown in the greenhouse in the 1 spring of the same year. Ninety-six F lines, forty-six 2 backcross lines, Fl of the Genesee-Winoka cross and Genesee were freeze tested in a 12 by 12 partially balanced lattice design. Each incomplete block consisted of two pots containing twelve test plants and two plants each of Genesee and Winoka. Each line was replicated four times 11 12 per test in each of eight high intensity freezing tests and five low intensity tests. Genesee and Winoka were replicated twenty-four times in each arrangement. The test temperature under high intensity freezing varied between -14.4 and -13.3C and between -17.8 and -16.9C for low intensity freezing. Diallels One four and one six complete parental diallel were made in the greenhouse in the winter of 1973. The winter wheat varieties used as parents had been tested for freezing hardiness HIM and are within diallels ranked in the order of increasing freezing hardiness (Table 1) Table 1. Winter wheat varieties used as parents in two complete diallels. Diallel II CI or CI or PI no. Common Name PI no. Common Name 15079 Arrow 13278 Monon 12653 Genessee 13083 Dual 326310 Mironovskaja 808 8033 Yogo 13083 Dual 6155 Minturki 14000 Winoka 6938 Kharkov 22 MC 13 Both diallels were tested by using the high and low intensity freezing methods described by Gullord et a1. (10). A six by seven partially balanced lattice with three arrangements was used to test diallel I. The parents were repeated two times within each arrangement, to achieve the required 42 treatment number. Each culture represented one incomplete block containing six test plants and two Winoka checks. The parents and the Fl's were replicated fifteen times in each of six freez- ing tests, three under high intensity conditions and three under low. The test temperature under high intensity freezing was -13.6C in all three tests whereas the temperatures were ~18.2, -17.5 and ~16.7C in the low intensity tests. Diallel II was tested in a 4 by 4 square balanced lattice. Each culture contained four test plants in addition to two Winoka checks. The parents and the Fl's were replicated twenty-five times in each of the three freezing tests, one under high and two under low intensity freezing. The test temperatures were —13.5C under high intensity freezing and -18.3 and 18.1C under low. Leaf Moisture Content Moisture content was determined in the nine varie- ties used as parents in the diallels and in the genotypes of diallel II, the reciprocal crosses not included. The 14 plants were randomized within pots and grown in growth chambers for five weeks at 15.5C and then hardened at 2C for three weeks. Tillers and older leaves were removed before a 2.5cm cylindric part of the young leaf tissue closest to the crown was cut off and put in a small air- tight drying vial. The samples were then weighed and dried at 70C for 24 hours before being weighed again. Moisture content was calculated by dividing the amount of water removed while drying, by the fresh weight of the leaf sample multiplied by one hundred. Genetic Variance Components The F2 and backcross variances were calculated on means of four observations in each of eight and five tests under high and low intensity freezing respectively. Twenty-four means of four observations were created for each of Genesee and Winoka in each test. The average variance of Winoka and Genesee was used as an estimate of error. Variances of F2, backcrosses and parents, were partitioned into additive and non—additive, and environmental variances according to Mather and Jinks (22). The formulas used are shown below. F2 = l/2D + l/4H + E BCl + BC2 = l/2D + l/2H + 2E l/2D Narrow heritability = l/2D + l/4H + E 15 1/2D + l/4H Broad heritability = l/2D + l/4H + E D = additive variance H = dominance variance error variance (31 II Diallel Analysis Average crown meristem ratings over fifteen and twenty-five replications respectively for diallel I and II were used in the analysis of variance of the two diallels. Diallel I tested under low intensity freezing was also analyzed by using percent survival (log trans- formed). Hayman's (13) approach was applied to test the diallels for additivity, non-additivity and reciprocal differences by using tests as replication, in the cases where the freezing tests were repeated more than once. In diallel I under low intensity freezing and freezing hardiness evaluated as meristem rating, each test was analyzed for general and specific combining ability and reciprocal differences by usimgGriffing's (9) method I (parents, one set of Fl's and their reciprocals are included) and model I (fixed model-selected parent lines). The same procedure was also used when analyzing the one test of diallel II under high intensity freezing. If no reciprocal differences were found the Wr/Vr approach described by Jinks (15) and Hayman (13) was 16 apmxlied to the diallels after averaging over reciprocal crosses in each test. ‘Vr (and Wk is the covariance of the rth recurring parents. Two methods described by Mather and Jinks is the variancewithin arrays array with the non— (22) ‘were used to test the adequacy of the simple additive— dominance model. Gene action and heritability were calcu- lated when the model was satisfactory. are shown below (22). The formulas used v = D + E p — _ _ N + 1 vr — l/4D + 1/4Hl 1/4F + 2N E fir = l/2D - 1/4F + 1/N E v" = 1/4D + l/4H - 1/4H - l/4F + N I l E r l 2 2 2N 1/2D + 1/2Hl — 1/2H2 l/2F Narrow heritability = l/2D + 1/2Hl _ 1/4H2 l/2F + E 1/2D + l/2H - 1/4H 1/2F Broad heritabilit - l 2 Y ‘ 1/2D + 1/2Hl — 1/4F2 1/2F + E N = number of parents in the diallel VO = parental variance Vr = mean variance of r arrays Wr = mean covariance of r arrays V; = variance of the array means E = pooled error variance 17 D = fixable additive variance Hl = dominance variance H2 = dominance variance F = non-fixable additive variance u = frequency of dominant genes v = frequency of recessive genes When the ranking of the array variances and the array covariances were consistent over the apprOpriate tests, wr/Vr graphs were calculated after averaging over tests within diallel and test intensity. Points coordi- nated on the plane made by Wr and Vr axis were confined by a limiting parabola w = (v-V) 1/2. r r p RESULTS AND DISCUSSION Generation Material The frequency distribution of meristem ratings under high and low intensity freezing are shown in Tables 2 and 3 respectively. Freezing hardiness was quantitatively inherited both under high and low intensity freezing. Under low intensity freezing Genesee was killed in most cases, the variation of Winoka was therefore used as the only estimate of error. The additive and non-additive genetic components and heritability for freezing hardiness are shown in Table 4. The non-additive component is much higher under low intensity freezing than under high intensity freezing. This could very likely be caused by deviation from normality of the BCl population under low intensity freezing (Table 3). The heritability found under high intensity freezing (57.7%) is therefore more reliable than the heritability found under low intensity freezing (2.2%). The relationship between meristem ratings under high and low intensity freezing for F2 and the backcross populations are shown in Figures 1-3. Low correlation coefficients between meristem ratings under high and low intensity freezing may be due to: (1) large standard error (range .23-.31) on the means and/or (2) dif- ferent genes controlling resistance to freezing under high 18 l9 .Amummp m x mama vv mcoflum>nmmno mm m0 cmmE m we coflusnfluumflp mocwovmuw esp mo was: comm .1 em m m m h exocflz mm m m m e m m mom N mm N 5 HH 0H WN ON m o H h mm H m s m m e H Hem «N m 0H m m «m ommmcow CC CC 2 z Z Z I TL .I. Tl. Z 0 L 9 Z 0 L 9 Z O L 9 Z no #HEHQ mmmau HDBOA 9 T... 9 I 9 TL 9 I 9 I 9 T. 9 . . Hence Co E CC Z Z Z Z TL TL T. TL . . . . . . . . . . . . . . HHEHA mmmHU HDQQD S Z O L S z 0 L S Z O L C.— Z 0 C... 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F W U Y F W U 9 T 0 DHEHQ mmmHU H®30A 9T9 TL 9 T 9 TL 9 TL 9 _.I_. 9 Hence pt Ft 8 Z Z Z Z I I I I C; so 0 L S F O L S Z O L 9 Z DHEflA mmmHU meQD OCJO S O C.— 0 S O S O S O S mcflpmm Emumflnmz CBOHU .mcofluflccoo mafimmmum MDHmCmDCH 30H mecca Umumeu .mxocHB x mmmmcmo mmouo one Eoum mcofipmHsmom Hmucmwmm cam mmouoxomn .mm MOM mmcfluwu Emumwuma CBOHU Hmumnmflumm HmBOH mo coausnfluumflp wocmsqmumll.m mqmde 21 TABLE 4.--Means and variances of lower peripheral crown meristem ratings for Genesee, Winoka and popula- tions derived from the cross between them, tested under both high and low intensity freezing. Low Intensity Freezing High Intensity Freezing Population N Means Variances Means Variances Genesee 24 .09 - 1.21 .63 BCl 23 1.14 1.81 1.57 .87 F2 96 1.42 1.34 1.92 1.37 BC2 23 2.03 .84 2.21 1.03 Winoka 24 2.74 .57 2.67 .53 D .06 1.58 H 2.96 0 Narrow heritability 2.2% 57 7 57.7% UT o\0 Broad heritability 57. 22 3 Y .E 3? 5“: g :20 9'18. a}? >§ I37 "9 I 90 -'°-'-' 122 '3' g :3; 9| I28 “'5 .3 I39 I43 Ian-:9 '33 E 2 ' l26 I27 92 53 .9 I CD .5 I? 92 57 E I" 8' so .93 .‘L’ ‘— o 2 C Y: .4388)( + I. 2946 3 r=.5485(P<.0l) 9 o 0 o I 2 3 x Crown Meristem Rating( Low intensity freezing) Figure l.*—Relationship between lower peripheral crown meristem ratings under high and low intensity freezing for 96 F families originating from a cross between Genesee and Winoka, averages over 8 and 5 freezing tests respectively. 23 (H Y: .4I |7X + LIOOZ f:=:75CK)(P.75 1.2265 .50—.25 Tests 7.4490 2 66.6876 <.001 Pooled error .1117 70 Tbtal .7541 107 * Each component of variation tested against its own test interaction. I . . . All components of variation tested against pooled test interaction mean square. 28 additive-dominance model with genes independently distri- buted among the parents is adequate to describe the varia- tion in freezing hardiness. The non-additive genetic variance can, therefore, be ascribed to the dominance effects of genes only. The joint regression analysis of Wr on Vr for the three tests are highly significant (P < .001) and the regression coefficient (.9914 i .0883) is signifi- cantly different from 0 but not different from unity. This also indicates that non-additive genetic variation is present as dominance only. The relative order of Wr and Vr values are nearly consistent over tests, and a wr/Vr graph calculated by using the mean meristem ratings over tests shows that Kharkov 22 MC has the lowest Wr and Vr values and contains the most dominant genes, while Genesee and Arrow have the highest values and hence the highest proportion of recessive genes (Figure 4). Table 6 shows that Winoka is the hardiest cultivar but not significantly better than Kharkov 22 MC, and Arrow the most tender cultivar with the other varieties in between. The gene actions and heritability estimates are shown in Table 7. H1 is smaller than D indicating incomplete dominance, the same conclusion can be drawn from the Wr/Vr graph intercepting Wr axis significantly (P < .01) above origin. The mean value of uv over all loci, estimated from the ratio 1/4 HZ/Hl’ is not significantly greater than the 29 Wr=.995| Vr + . l7l2 Sb=.|560 - Kharkov 22 Mc 0 Winoka A Dual 0 Mironovskaja 808 a Genesee A Arrow O is) b: 3:)- GI b) 1, Figure 4.~—The regression of W on V for freezing hardiness in terms of lower pgripheral crown meristem ratings of a six parental diallel tested under high inten- sity freezing. 30 TABLE 6.--Lower peripheral crown meristem ratings of parents and Fl's from a six parental diallel tested under high intensity freezing, totals of three freezing tests after averaging over reciprocal crosses. Panamal N I Parent 1 2 3 4 5 6 Vi Wi l Genesee 3.99 6.65 4.42 8.55 10.02 7.11 .6242 .7527 2 Dual 7.26 6.06 9.18 10.07 9.21 .2943 .5023 3 Arrow 2.10 7.37 8.45 5.90 .5704 .7476 4 Winoka 9.96 10.43 9.71 .1360 .3637 5 Kharkov 22 MC 9.33 10.98 .0870 .1710 6 Mironovskaja 808 7.65 .3889 .5731 SUM 40.54 48.43 34.10 55.20 59.28 50.56 2.1008 3.1104 TABLE 7.--Components of variation of freezing hardiness under high intensity freezing in a six parental diallel. Components Of Estimated Values Variation D .9819 t .0735 H1 .1421 i .1673 H2 .1876 i .1567 F .1413 i .1258 E .1117 /Hl/D .3803 uv .3301 Narrow Heritability 77.26% Broad Heritability 83.99% 31 maximum value of .25 which arises when u = v = .5. Narrow and broad heritability for the freezing hardiness under high intensity freezing is 77.26 and 83.99 percent respectively (Table 7). In this selected set of wheat genotypes freezing hardiness measured under high intensity freezing is on the average controlled by partially dominant genes mostly additive in their effect. The position of Winoka in relation to Kharkov 22 MC in the wr/Vr graph (Figure 4) indicates that the dominance is less complete in Winoka than in Kharkov 22 MC. This is in agreement with the lack of dominance found for freezing hardiness in the generation material (Table 4) originating from the cross between Genesee and Winoka. Only results from one freezing test under high inten- sity conditions is available for diallel II. Table 8 shows that genotypic differences exist for freezing hardiness. The combining ability analysis of variance according to Griffing (9) are shown in Table 9, in which both general and specific combining ability are significant. After averaging over reciprocal crosses, since there were no significant reciprocal differences, the Wr and Vr values and the regression line between the values were calculated. The regression coefficient is significantly (.01 < P < .05) different from zero but not (P < .75) from unity, indicating that non-allelic interaction is present as dominance only. 32 TABLE 8.--Ana1ysis of variance of the lower peripheral crown meristem ratings of parents and Fl's from a four parental diallel tested under high intensity freezing. _—_. mm..._.. .L,,_.___._.. __ ,____._~____, ». a- ‘ MW..._M._._.—w .L...__...~-~._.. M... H_._____.__.. ._ .—._. H2 . __ .~. . _.— . Source of Variation df MS F P Total Replication 24 1.9340 2.4175 <.001 Genotypes (adj.) 15 5.2985 6.6232 <.001 Effective error 285 .8000 TABLE 9.--Ana1ysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and Fl's of a four parental diallel tested under high intensity freezing. Source of Variation df MS F p General combining ability 3 .8680 26.7901 <.001 Specific combining ability 6 .0771 2.3796 .05-.025 Reciprocal differences 6 .0187 .5772 .75-.500 Error 285 .0324 33 The regression line intercepts the Wr axis a significant (P < .01) distance above origin which implies that dominant gene action under high intensity freezing is incomplete. Yogo has the lowest Wr and Vr values and Monon the highest (Figure 5). According to Table 10 Minturki is the most hardy variety and Monon the most tender. Freezing hardiness seems to be controlled by partially dominant genes mostly additive in their effect also in this set of genotypes. Diallels—~Low Intensity Freezing Inheritance of winter hardiness as measured by lower peripheral crown meristem ratings tested under low intensity freezing was studied in both diallel I and diallel II. The calculations in diallel I were based on percent survival transformed to logarithms to achieve normality and these results are presented first. The error variances are homogenous except for bl' which is significant only against the pooled error variance (Table 11). This discrepancy for bl is a result of its own test interaction being based on only two degrees of freedom. The error variances are pooled to give a test interaction mean square as a common error variance. As for the high intensity freezing, the material shows significant additive (a) and dominant (b) effects. The mean freezing hardiness of the Fl's is significantly higher than the mid-parental value (bl) and a significant dominance deviation that is 34 Wr Wr= I.O37’7 Vr+.|254 Sb=.|344 o Minturki e Yoga A Dual + Manon Figure 5.~~The regression of WV on Vr for freezing hardiness in terms of lower peripheral crown meristem ratings of a four parental diallel tested under high inten~ sity freezing. TABLE 10.-—Lower peripheral crown meristem ratings of parents and Fl's from a four parental diallel tested under high intensity freezing, means of 25 replications and reciprocal crosses. Parental Number Parent 1 2 3 4 Vr Wr 1 IDual. 3.27 3.69 3.80 3.10 .1093 .2657 2 Yogc> 3.71 3.91 3.58 .0189 .1101 3 Mirnnirki 4.36 3.64 .0954 .2440 4 Dhanorl 2.33 .3646 .4925 Sum 13.86 14.89 15.71 12.65 .5882 1.1123 TABLE 11.--The analysis of variance of freezing hardiness measured as percent survival (transformed to logarithms) of a six parental diallel tested under low intensity freezing. Source of * Variation df MS F P Ff P a 5 4.1139 25.4030 <.001 68.2239 <.001 b 15 .2434 4.3464 <.001 4.0365 <.001 b1 1 1.0383 17.1620 .25-.10 17.2189 <.001 b2 5 .1926 5.4887 .05-.01 3.1940 .05-.01 b3 9 .1832 2.7303 .05-.01 3.0381 .01—.001 c 5 .0498 1.5911 .50—.25 .8259 .75-.50 d 10 .0623 2.0393 .10-.05 1.0315 .50-.25 Tests 2 .5307 8.8010 <.001 Pooled error 70 .0603 Total 107 .2839 *Each component of variation tested against its own test interaction. IAll components of variation tested against pooled test interaction mean square. 36 Wr= I.OI96 Vr+.l006 Sb=.|224 Wr in I Kharkov 22 MC .2 o Winoka A Dual 0 Mironovskaja 808 -' I D Genesee A Arrow 0 l l J Figure 6.-~The regression of Wr on Vr for freezing hardiness in terms of percent survival (transformed to logarithms) of a six parental diallel tested under low intensity freezing. 37 unique to each Fl (b3) is found. The mean dominance deviation of the Fl's from their mid-parental values within each array differ significantly over_arrays (b No 2). average maternal differences (c) or other reciprocal dif- ferences not ascribed to c (d) were identified between reciprocal crosses. With no reciprocal differences, Vr and Wr were cal- culated for each array and freezing test after averaging over reciprocal crosses. Highly significant (.01 < P < .05) differences in the magnitude of (Wr + Vr)' and no signifi- cant (.25 < P 4 .5) differences in the magnitude of (WI. - Vr)' indicate that the additive dominance model is adequate for the variation in freezing hardiness. A highly significant (P “ .001) joint regression of WI. on Vr for the three tests, and the linear regression coefficient (1.0547 I .0779) which is significantly (P s .01) different from 0 but not (.5 4 P d .4) from unity, confirm the adequacy of the additive dominance model. The relative values of Wr and Vr are nearly con- sistent over tests, and a wr/Vr graph was calculated for the diallel after averaging over the three freezing tests (Figure 6). Kharkov 22 MC has the lowest Wr and Vr values and, therefore, contains the most dominant genes. Genesee, however, has the highest values and the greatest number of recessive genes. The other cultivars in order of decreasing number of dominant genes for freezing hardiness are: 38 Winoka, Dual, Mironovskaja 808 and Arrow. Table 12 shows that Kharkov 22 MC is the most hardy cultivar, and Genesee the most tender. Components of variation and heritability estimates are shown in Table 13. H1 is smaller than D, indicating that dominance is incomplete, the same conclusion is drawn from the wr/Vr graph, since the line intercepts the Wr axis at a point significantly (.01 < P < .025) above origin. The mean value of uv over all loci, estimated from the ratio 1/4 HZ/Hl is not significantly different from the maximum value of .25 indicating that recessive and dominant genes are in the same proportion. Narrow and broad heritability are 68.57 and 76.12 percent respectively (Table 13). Freezing hardiness measured under low intensity freezing is apparently controlled by partially dominant genes mostly additive in their effect. The relative order of the Wr and Vr values for the six genotypes are identical under high and low intensity freezing (Figures 4 and 5). This suggests that the same sets of genes may control freezing hardiness under both levels of freezing intensity. This conclusion disagrees with results from the segregating population presented earlier. The varieties used in this study have probably been selected under natural conditions over long periods of time and they may have adapted in a similar degree to both high and low intensity freezing. 39 mamm.a Nomm. mmmm.mm Nvam.mm Hmmm.am mvna.ma mmmm.mm mmam.ma HBSm emom. MSH. Esme mom mflmxm>oaofiz w some. 88. News mange. 02 mm >829; m 83. some. 886 Homes $86 8.9:: a Rem. NHmN. was; mmmH.m $8.4 Hmem. 38s,... m 32... E8. Hemofi ESE SSH 834m 384 H95 N 83. mam. $85 $26 ammse mmmmH mafia. Hmoo. @8880 H J5 n> w m w m m H usmumm HMWMMWMM .mmmmouo Hmooumfloeu Hm>o msflmmwm>m seems mummu mcHNeme mmunu mo mamuou .mCHmmmHm xeflmcmch 30H Mecca peummu Hmaamflp Hepcmumm me m Eoum m.Hm cam museumm mo AmELDHHmmoH OD meHmecmHuv Hm>fl>usm useonmmrl.ma mamme 40 TABLE 13.-—Components of variation for freezing hardiness in a six parental diallel under low intensity freezing. Components of Variation Estimated Values D .5866 i .2086 H1 .1369 t .1423 H2 .1196 i .1098 F .1507 t .1976 E .0603 «8175 .4831 uv .2708 Narrow heritability 68.57% Broad heritability 76.12% 41 If that is the case different gene systems controlling freezing hardiness under high and low intensity freezing can therefore be identified only in a segregating popula- tion. The error variances for diallel II under low intensity freezing are homogeneous and are, therefore, pooled to give a test interaction mean square as a common error variance (Table 14). Table 14 shows that there is a significant additive (a) and dominant (b) effect. The mean deviation of the F 's from their midparental value 1 (b ) is also significant. Neither the mean dominance 1 deviation of the Fl's from their midparental values within each array (b ) nor the dominance deviation unique to 2 each Fl(b ), specific combining ability, is significant. 3 No average maternal differences (c) or other reciprocal differences not ascribed to c (d) between reciprocal crosses were found to be significant (Table 14). Slightly significant (.05 < P < .1) differences in the magnitude of (Wr + Vr) over arrays and lack of signifi- cant differences in the magnitude of (Wr - Vr) over arrays indicate that the additive dominance model is adequate for describing the variation in freezing hardiness. The adequacy of the model is confirmed by a highly significant (P < .01) joint regression of Wr on Vr' with a regression coefficient (.9271 I .0586) significantly different from zero but not from unity. 42 TABLE 14.--The analysis of variance of freezing hardiness measured as lower peripheral crown meristem ratings of a four parental diallel tested under low intensity freezing. Source of * I Variation df MS F p F P a 3 4.3228 11.1470 .05-.01 22.7636 <.001 b 6 .6620 5.1199 .05-.01 3.4860 .05—.01 b1 1 3.0317 60.1528 .10-.05 15.9647 .01-.001 b2 3 .0767 .3639 >.75 .4039 >.75 b3 2 .3552 7.6387 .25-.10 1.8705 .25-.10 c 3 .0341 .1635 >.75 .1796 >.75 d 3 .0099 .1045 >.75 .0521 >.75 Tests 1 11.0921 58.4102 <.001 Pooled error 15 .1899 Total 31 1.0004 * Each component of variation tested against own test interaction. +All components of variation tested against pooled test interaction mean square. 43 .9 ' .8 " .7 " .6 ' .5 l' 3 .4 ' Wr=.9572 Vr+.0682 .3 Sb= .0569 .2 O Minturki 3 Yoga . I A Dual + Manon o_ 1 1 1 1 i 1 J O l .2 3 .4 .5 6 .7 8 Vr Figure 7.-—The regression of Wr on Vr fer freezing hardiness in terms of lower peripheral crown meristem ratings of a four parental diallel tested under low inten- sity freezing. 44 The relative order of the Wr and Vr values are almost consistent over the two tests. The wr/Vr graph calculated after averaging over tests shows that Minturki has the lowest Wr and Vr values and contains the most dominant genes while Monon has the highest value and hence the highest preportion of recessive genes (Figure 7). The meristem ratings in Table 15, shows that Minturki is the most hardy and Monon the most tender. The gene action of freezing hardiness is shown in Table 16. The term le/D is smaller than one, indicating that the dominance is incomplete. The wr/Vr graph inter- cepting the Wr axis, significant (P < .01) above origin also suggests partial dominance. The mean uv value over all loci is not significantly different from .25 indicating that the prOportion of dominant and recessive genes is equal to .5. The average narrow and broad heritability for freezing hardiness is 63.90 and 77.09 percent respect- ively (Table 16). Freezing hardiness evaluated under low intensity freezing in diallel II is also controlled by partially dominant genes mostly additive in their effect. The relative order of the Wr and Vr values are not consistent for high and low intensity freezing (Figures 5 and 7), Yogo and Minturki have changed position. Two explanations are possible: (1) differentgfinmasystems are controlling freezing hardiness under the two levels of freezing inten- 45 TABLE 15.--Lower peripheral crown meristem ratings of parents and Fl's from a four parental diallel tested under low intensity freezing, two freezing tests after averaging over reciprocal crosses. Parental totals of Number Parent 1 2 3 4 Vi Wr 1 IDual. 3.56 6.12 6.64 3.70 .6423 .7251 2 ‘Yogt> 5.50 7.56 5.96 .1979 .2518 3 Mirnnirki 6.92 6.70 .0443 .1050 4 Monon 2.76 .8605 .8630 Sunr 20.02 25.14 27.82 19.12 1.7450 1.9449 TABLE 16.--Components of variation for freezing hardiness in a four parental diallel under low intensity freezing. Components of Variation Estimated Values D .8309 i .0170 H1 .3896 i .1794 H2 .4369 i .1610 F -.2755 t .4945 E .1899 VHl/D .6847 uv .2804 Narrow heritability 63.90% Broad heritability 77.09% 46 sity, (2) because the freezing temperature under the high intensity was too high the frequency distributions of the meristem ratings of the hardiest genotypes are skewed toward the upper end of the scale. This explains the low Wr and Vr values for the hardiest varieties (Yogo, Minturki and Dual). A lower freezing temperature would be necessary to separate the three genotypes with respect to Wr and Vr values. Influence of the Severity of the Freezing Test on Gene Action Diallel I tested under low intensity freezing at three different temperatures, is used to show changes in gene action with the severity of freezing conditions. Significant (P < .001) genotypic differences in freezing hardiness evaluated as meristem ratings were found for all tests. Genotypic variation was separated into general and specific combining ability in addition to reciprocal dif- ferences according to Griffing (9). The two former com— ponents were significant in all three tests whereas the latter was not significant in any tests (Tables l7, l9, and 21). Wr/Vr graphs were calculated after averaging over reciprocal crosses. The slopes of the graphs were all significantly different from zero but not from unity (Figures 8, 9 and 10) indicating the adequacy of the additive-dominance model. The graphs intercept the Wr axis significantly above the origin which is an indication of 47 TABLE 17.——Ana1ysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and Fl's of a six parental diallel tested under low intensity freezing at a temperature of -18.2 C. General combining ability 5 7.1212 90.9476 <.001 Specific combining ability 15 .2600 3.3206 <.001 Reciprocal differences 15 .6337 .0422 >.750 Error 484 .0783 TABLE 18.--Lower peripheral parents and Fl's crown meristem ratings of from a six parental diallel tested under low intensity freezing at a tempera- ture of ~18.2 C, averages over replications and reciprocal crosses. Pamxtal N 1 Parent 1 2 3 4 5 6 Vr W; 1 Genesee .13 .13 .06 1.35 1.41 .16 .4248 .8083 2 Dual 1.13 .41 2.75 2.41 1.44 1.1015 1.2939 3 Arrow .06 .44 1.50 .32 .2828 .5209 4 Winoka 2.94 3.32 1.97 1.1893 1.2270 5 Kharkov 22 MC 2.81 2.28 .5489 .8939 6 Mironovskajaéth .63 .7942 1.1072 Sum 3.24 8.27 2.79 12.77 13.73 6.88 4.3435 5.8512 48 TABLE 19.--Ana1ysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and Fl's of a six parental diallel tested under low intensity freezing at a temperature of -l7.5 C. Source of Variance df MS F P General combining ability 5 6.2688 64.4938 <.001 Specific combining ability 15 .1910 1.9650 .025-.01 Reciprocal differences 15 .1547 1.5920 .100-.05 Error 484 .0972 TABLE 20.--Lower peripheral crown meristem ratings of from a six parental diallel tested under low intensity freezing at a tempera- ture of -17.5 C, averages over replications and reciprocal crosses. parents and Fl's Panamal W Number Parent 1 2 3 4 5 6 ‘Vr r l Genesee .14 .91 .41 1.20 2.37 .88 .6065 .9600 2 Dual 1.67 .56 2.93 3.28 1.49 1.1796 1.4475 3 Arrow .10 1.63 1.75 .45 .4825 .8830 4 Winoka 2.88 2.88 2.04 .5573 .8935 5 Kharkov 22 MC 3.33 2.38 .3738 .6705 6 Mironovskaja 808 1.32 .5078 .9445 Sum 5.91 10.84 4.90 13.56 15.99 8.56 3.7075 5.7990 49 TABLE 21.-—Analysis of variance of the lower peripheral crown meristem ratings for combining ability and reciprocal differences of parents and F1's of a six parental diallel tested under low intensity freezing at a temperature of -l6.7 C. . _ . . - ._ . . . -. . 1.. .-_._._ ___-_-_...___ - ..—_...—._ -_-__.. .. _. .. -HH _. . L“...— Source of Variation df MS F P General combining ability 5 4.9985 51.2667 <.001 Specific combining ability 15 .2594 2.6605 <.001 Reciprocal differences 15 .1503 1.5415 .10-.05 Error 484 .0975 TABLE 22.--Lower peripheral crown meristem ratings of parents and Fl's from a six parental diallel tested under low intensity freezing at a tempera- ture of -l6.7 C, averages over replications and reciprocal crosses. Panxmal Nmflxa‘ Ikuent 1 2 3 4 5 6 Vi Wk 1 Genesee .12 .94 .24 1.38 2.39 .35 .7578 .9969 2 Dual 1.55 .98 1.82 2.72 2.08 .4623 .6706 3 Arrow .17 1.34 2.18 .21 .6553 .9253 4 Winoka 2.69 2.36 1.93 .2842 .6090 5 Kharkov 22 MC 2.93 2.70 .0789 .1896 6 Mironovscfia808 .90 1.0391 1.1652 Sum 5.42 10.09 5.12 11.52 15.28 8.17 3.2776 4.5566 50 I.3" l.2 " l.l " LO" .9 ’ .8 Wr= .7539 Vr+.4295 7 Sb= .ll9l b . 3 .6 ~5 I Kharkov 22 Me .4 O Winoka 3 A Dual ' 0 Mironovskaja 808 .2 CI Genesee .l A Arrow 0 A J L l j 0 .2 .4 .6 .8 LG l.2 IA Vr Figure 8.-—The regression of W on V for freezing hardiness . r . r . . in terms of lower peripheral crown meristem ratings of a six parental diallel tested under low inten- sity freezing at -18.2C. 51 l.5" L4“ L3” l.2“ Ll" LO" .9“ .8” .. Wr= .8806 Vr+.4224 3 -7 n Sb=.0940 .6 '5 lKharkov 22 Mc .4 OWinoka .3 ADUGI oMironovskaja 808 -2 EIGenesee .l ‘AI'I'OW O_ l l l l l I l l l l J O I .2 .3 .4 .5 .6 .7 .8 .9 LO |.| l.2 Vr Figure 9.——The regression of WT on Vr for freezing hardiness in terms of ltwmntperiphera1.crown meristem ratings of a six parental diallel tested under low intensity freezing at —17.5C. 52 l.2r I.lr in 'o I I Wr= .9803 Vr + .224l Sb= .0597 Wr I Kharkov 22 Me 0 Winoka‘ A Dual 0 Mironovskaja 808 CI Genesee A Arrow o—ie'oals'orbisrin O .2 .4 .6 .8 LC Figure 10.-—The regression of Wr on Vr for freezing hardiness in terms of lower peripheral crown meristem ratings of a six parental diallel tested under low inten‘ sity freezing at -16.7C. 53 incomplete dominance. The dominance seems to be less com- plete under the two most severe freezing conditions (Figures 8 and 9) than under the mildest freezing condition. This is in agreement with results reported by Eunus et a1. (6). Arrow has the lowest Wr and Vr values and Winoka and Dual the highest values in the test with the lowest test tempera- ture (Figure 8). Under the mildest freezing condition of the three, Kharkov 22 MC has the lowest Wr and Vr values whereas Mironovskaja 808, Genesee and Arrow have the highest (Figure 10). The ranking of the cultivars according to the Wr and Vr values under intermediate freezing tempera- tures is intermediate to the extreme tests. Tables 18, 20, 'and 22 show that Kharkov 22 MC and Winoka are the most hardy cultivars in all tests, whereas Arrow and Genesee are the most tender and Dual and Mironovskaja 808 inter- mediate. This data indicates that freezing hardiness is controlled by the most dominant genes at the highest freez- ing temperature, the most recessive genes at the lowest freezing temperature and dominant and recessive genes in the same proportion at the intermediate freezing tempera- ture. This is in agreement with results obtained in winter oats (14, 24) and winter wheat (43). The meristem rating scale used in this study to evaluate freezing hardiness, varies between 1 and 4, 0 (dead) and 5 (undamaged) being off the scale. Under severe freezing conditions the frequency distribution of 54 the meristem ratings of the tender genotypes are skewed toward the lower limit of the scale, and the mean meristem rating will accordingly be overestimated. Assuming the range in fre zing hardiness within an array is less than the scale ixnxye, overestimated. means will result in reduced array variances and covariances. The low Wr and Vr values shown for Arrow and Genesee at the lowest freez- ing temperatures in Tables 18 and 20 are due to the reduced range in freezing hardiness for the arrays and not to recessive genes controlling the character. A freezing test is difficult to monitor so that a normal or close to normal frequency distribution for meristem ratings is achieved for all genotypes tested simultaneously. The interpretation of a diallel will, however, not be wrong, if the freezing temperature is adjusted so the frequency distributions for the hardy geno- types are skewed a little toward the upper limit of the scale. Too mild conditions may prevent separation of the most hardy genotypes when freezing hardiness is controlled by dominant genes. Relationship Between Freezing Hardiness and Leaf Moisture Content Moisture content was measured in young leaf tissue of nine winter wheat cultivars. Table 23 shows that there are significant differences between varieties for the character. TABLE 23.-—Moisture content of young leaves for nine winter wheat cultivars, averages over 14 replications. Genotype Moisture Content in Percent Minturki 73.33 a* Yogo 73.74 ab Winoka 74.22 ab Kharkov 22 MC 74.48 bc Mironovskaja 808 75.36 cd Genesee 75.99 de Monon 76.40 e Arrow 76.88 e Dual 76.92 e * Duncans Multiple Range test after Steel and Torri (47). A regression analysis between the mean moisture content and freezing hardiness under high and low intensity freezing evaluated as meristem ratings, showed a significant negative correlation in both cases, and that moisture con- tent explained 68.9 and 72.3 percent, respectively of the variation in freezing hardiness (Figures 11 and 12). These results are in agreement with the higher kill temperatures reported in wheat genotypes (10, 23) as a result of environ- mental induced higher crown moisture. Higher moisture content in crown tissues results in more free energy of ice crystal growth available for disrupting crown tissues (28). According to presented materials valid predictions 56 A-< Y= 755x + 43.68 r= '183l6(P<.0|l C” I I Kharkov 22 MC 0 Minturki o Winoka 9 Yoga 4 Dual D | - o Mironovskaja 808 + Manon u Genesee A Arrow Crown Meristem Rating to 0 we : . . , o 74 75 76 77x Leaf Moisture Content Figure 11.——Re1ationship between freezing hardiness in terms of lower peripheral crown meristem ratings tested under high intensity freezing and leaf moisture content for nine winter wheat cultivars. 57 Y Y-".62X + 48.65 3 . r318544 (P< .0|) 0 .E s 2- E I Kharkov 22 Me 23 o Minturki ': O Winoka g 0 Yoga ‘3, o Mironovskaja 808 5 + Monon D Genesee D ‘ A Arrow O "% J A l 1 0 74 75 76 77x Leaf Moisture Content Figure 12.--Re1ationship between freezing hardiness in terms of lower peripheral crown meristem ratings tested under low intensity freezing and leaf moisture content for nine minter wheat cultivars. 58 of the freezing hardiness of genotypes could be made by knowing the moisture content of the leaf tissue close to the crown. To make sure one is not working with linkage between moisture and freezing hardiness this relationship should first be confirmed in a segregating population. As can be seen in Figures 11 and 12, factors other than moisture content must account for the relatively high freezing resistance of Dual, or low freezing resistance of Genesee. Shearman et_al. (45) showed a good relation- ship between freezing hardiness and kinetic inhibitor ratings in winter wheat. If kinetic inhibitor ratings (data provided by Dr. Olien) were included as an independent variable in addition to moisture content, 79.5 and 83.0 percent of the variation in freezing hardiness were explained under high and low intensity freezing respectively. The inheritance of moisture content was studied in diallel II. Significant differences were found between genotypes (Table 24). TABLE 24.--Ana1ysis of variance of moisture content of young leaves near the crown of parents and Fl’s of a four parental diallel. Source of Variation df MS F P Replication 4 19.2483 16.2983 <.001 Genotypes 9 10.4349 8.8356 <.001 Error 36 1.1810 59 The regression coefficient of the Wr/Vr graph is signifi- cantly different from zero, but not from unity (Figure 13), indicating that non-allelic interaction is present as dominance only. The graph intercepts the Wr axis signifi- cantly (P < .001) above origin, suggesting that the domi- nance is only partial. Minturki and Yogo have the lowest Wr and Vr values and the most dominant genes whereas Monon has the highest Wr and Vr values and the highest proportion of recessive genes. According to Table 25, Yogo and Minturki have the lowest moisture whereas Monon has the highest. Low moisture appears to be controlled by partially dominant genes mostly additive in their effect. TABLE 25.-—Moisture content of young leaves close to the crown of Fl's and parent plants from a four parental diallel, averages of five replications. Panamal l 4 V W N r Parent 1 2 3 r r 1 Dual 75.62 1.1740 2.0983 2 Yogo 73.79 72.19 .8181 1.7095 3 Minturki 73.81 72.05 72.69 .6891 1.5609 4 Monon 75.73 73.56 73.65 75.95 1.6745 2.5116 Sum. 298.95 291.59 292.20 298.89 4.3557 7.8803 Measurements of cross sections of leaf and root tissues close totflmacrown was made of Winoka, Kharkov 22 MC 60 and Arrow. Moisture content seemed to be positively correlated with cell size. 61 Wr= .9633 Vr +.92l2 Sb= .0549 LC .8 6 O Minturki ’ e Yoga .4 A Dual .2 + Manon O 1 . 1 1 0 .4 .8 l.2 L6 2.0 Vr Figure 13.-~The regression of Wr on Vr for leaf moisture con- tent ef a four parental diallel. SUMMARY AND CONCLUSIONS Inheritance of freezing hardiness was studied in (1) F2 and backcross pOpulations originating from a cross between Genesee and Winoka and (2) in two complete diallels, one six and one four parental diallel. The material was tested under both high and low intensity freezing according to a procedure described by Gullord et a1. (10). Moisture content in young leaves close to the crown was studied in nine winter wheat genotypes varying in freezing hardiness, and in a four parental diallel. 1. Freezing hardiness was found to be a quanti- tative character controlled by many genes. 2. Significant interaction between F2 lines and the level of freezing intensity suggests that genes con- trolling freezing hardiness under low intensity freezing is different from the genes controlling freezing hardiness under high intensity. 3. The simple additive-dominance model with inde- pendent gene distribution described by Hyman (13) ade— quately describes the variation in freezing hardiness in both diallels under both levels of freezing intensity. 62 63 4. Analysis of both diallels showed that freezing hardiness is controlled by partially dominant genes which are mostly additive in their effect. Some or all of the genes found to control freezing hardiness under low intensity freezing may very likely be different from the dominant genes found to control freezing hardiness under high intensity freezing. 5. No reciprocal differences were found in either of the diallels tested under any level of freezing intensity. 6. Very severe freezing conditions resulted in lower array variances and covariances than expected for the tender varieties. The reason is not that recessive genes control freezing hardiness at severe freezing condi- tions, but that the frequency distributions of the crown meristem ratings were skewed toward the lower limit of the scale, resulting in overestimated crown meristem rating means and reduced array variances and covariances for the tender genotypes. 7. Highly significant negative correlation was found between moisture content in young wheat leaves and freezing hardiness evaluated under both high and low intensity freezing in nine winter wheat genotypes. 8. 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