zyxwvutsrqponmlkjihgfedcbaZYXW zyxwvutsrq zyxwvutsrqp zy zyxw zyxwv Ann. uppl. Bid. ( I 999), 135:3540 Printed irz Great Britain 35 Evaluation under long-day conditions of 4x-2x progenies from crosses between potato cultivars and haploid Tuberosum-Solanurn chacoense hybrids By J A BUSO', L S BOITEUX' and S J PELOQUIN2* 'Centro Nacional de Pesquisa de HortaliGas (CNPH) - Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA), Caixa Postal 218, 70359-970 Brasilia-DF: Brazil 2DepartmentHorticulture, Plant Sciences I5 75 Linden Drive, University of Wisconsin-Madison, WI 53 706, USA (Accepted 6 December 1999) Summary Twelve 4x families (obtained from a sub-set of crosses between seven 4x-potato cultivars and three 2x haploid Tuberosum-Solanum chacoense hybrids) were evaluated at Hancock, Wisconsin (USA). The 4x-parents were elite cultivars selected for adaptation in three continents (Europe, South America, and North America). The 2x male clones were able to produce 2n-pollen grains by a mechanism akin to firstdivision restitution with crossover (FDR-CO). The estimation of the degree of heterosis for total tuber yield (TTY) was obtained by comparing the field performance of the progenies with their respective 2x and 4x parents. Haulm maturity (HM) and general tuber appearance (GTA) were also evaluated. For TTY, the 4x-2x families (as a group) outyielded both the 4x and 2x parental groups by 10.6% and 42.5%, respectively. In addition, 5 out of 12 famiIies outyielded their corresponding 4x-parents. These best five families outyielded the group of 4x-parents by 40.6%. A considerable variability was observed for HM but, in general, the families were later maturing than the 4x cultivars. The identification of 4x2x families with GTA within the range of the 4x commercial cultivars was another important observation. An overall lack of parent-offspring correlation was detected indicating that performance of the parents per se cannot provide a reliable prediction about the performance of the families. Therefore, progeny testing would be an imperative step for selection of parental clones at both ploidy levels. Our study indicated that haploid Tuberosum-S. chacoense hybrids are able to generate heterotic 4x-2x families for TTY in combination with good GTA. These results reinforce the view that selection of superior clones for the Northern Hemisphere can be feasible using germplasm with -25% genomic contribution of this wild South American species. zyxw zyxwvu Key words: Solanum chacoense, Solanum tuberosum, unilateral sexual polyploidisation, wild species, 2n-gametes Introduction Solanurn chacoense Bitter is a wild diploid (2x) species distributed across a large area of South America. Its natural occurrence has been reported in Argentina, Bolivia, Brazil, Paraguay, and Uruguay (Hawkes, 1990). Habitats of this species range widely, from sea level to over 3500 m in the Andes mountains (Miller & Spooner, 1996). S. chacoense is highly polymorphic and its germplasm is a potential source of genes for improvement of numerous traits in the cultivated tetraploid (4x) potato. These traits include insect and disease resistance, heat tolerance, high tuber starch content and strong vegetative vigour (BaniAameur, Lauer, Villeux & Hilali, 1991; Brown & Thomas, 1994; Lynch et al., 1997; Veilleux, Paz & Levy, 1997). Another useful characteristic of this species for plant breeding is that male-sterile Tuberosum haploids can hybridise with accessions of S. chacoense to produce male-fertile progeny. However, there are also some major problems associated with the adaptation of S. chacoense including the presence of very long stolons and late tuber initiation under field conditions in Northern temperate areas (Leue & Peloquin, 1977). Three major breeding strategies have been used to introgress S. chacoense genes into Tuberosum germplasm: (1) doubling the chromosome number of S. chacoense for subsequent use in 4 x 4 crosses; (2) 4x-2x mating with unilateral sexual polyploidisation (USP) and, (3) interdiploid crosses with bilateral sexual polyploidisation (Peloquin, Yerk, Werner & Darmo, 1989). Schemes (2) and (3) rely upon the production of 2n-gametes for recovery of the tetraploid level in the progenies. The frequency of 2n- *Corresponding Author: Tel. OOl(608) 262 1878; Fax: OOl(608) 262 4743 0 1999Association of Applied Biologists 36 zyxwvuts zyxwvu zyxwvutsrqponm zyxwvuts zyxwvutsrqp BY J A BUSO, L S BOITEUX AND S J PELOQUIN pollen grains formed by first-division restitution with crossover (FDR-CO)was found to be reasonably high in S. chacoense (Leue & Peloquin, 1980). The FDRCO gametes are considered to be more valuable than those obtained by the second-division restitution (SDR) mechanisms because a larger amount of the parental heterozygosity and epistatic interactions can be transferred to the progeny without disruption by recombination (Peloquin et al., 1989). Selection work was carried on S. chacoense germplasm for more effective tuberisation and 2ngamete formation (Leue & Peloquin, 1980). It was observed that hybridisation between 2n gameteproducers of S. chacoense was a good method of obtaining new 2n-gamete producing individuals. Similar selection programmes with 2x haploid Tuberosum- S. chacoense hybrids for 2n-gamete production have been carried out in Italy (Barone, Carputo & Frusciante, 1993)and Brazil (Cunha, Pinto & Davide 1994; Morais & Pinto, 1996). USP- and BSP-derived genetic materials have allowed a further and more systematic exploitation of this germplasm by incorporating genome segments from distinct S. chacoense accessions into Phureja and haploid Tuberosum clones (Leue & Peloquin, 1980; Werner & Peloquin, 1991; Morais & Pinto, 1996).In addition, 4x-2x (FDR-CO) crosses involving haploid Tuberosum -S. chacoensegermplasm have been also used in basic genetic studies to determine centromerelocus distances of important genes (Douches & Quiros, 1987; Wagenvoort & Zimnoch-Guzowska, 1992) and to ascertain the average heterozygosity transmitted through 2n-gametes to the progeny (Douches & Quiros, 1988; Barone, Gebhardt & Frusciante, 1995). The USP breeding scheme has generated valuable 4x-2x families derived from 2x Phureja-haploid Tuberosum hybrid clones (for review see Tai, 1994). Good results have been also obtained with 4x-2x families derived from crosses between haploid Tuberosum and S. berthaultii Hawkes, S. tarijense Hawkes, S. bukasovii Juz. and S. sparsipilum (Bitt.) Juz. et. Buk. (Buso, 1986; Darmo & Peloquin, 1991; Serquen & Peloquin, 1996). However, few field tests have been conducted to evaluate the breeding potential of 4x-2x (FDR-CO) families obtained from crosses between adapted 4x cultivars and 2x haploid Tuberosum-S. chacoense hybrids. Promising results were obtained with interdiploid families from 2x (SDR) - 2x (FDR-CO) crosses involving S. chacoense or S. chacoense x S. injiundibuliforme (Phil.) hybrids (Werner & Peloquin, 1991). In addition, three-way hybrids with 25% genomic contribution from S. chacoense outperformed other types of hybrids for tuber yield in assays conducted in Africa (Bani-Aameur et al., 1991). Complex hybrids with S. chacoense genome were also more tolerant to tuber heat necrosis than the standard 4x cultivar used for comparison in Virginia, USA (Veilleux, Paz & Levy, 1997). In Brazil, selected families and clones derived from crosses of S. tuberosum x S. chacoense showed very good performance for traits such as yield and specific gravity (Morais & Pinto, 1996). The aim of the present study was to extend the evaluation of the potential value of 4x-2x (FDR-CO) crosses using (as female parents) 4x potato cultivars and as staminate parents haploid Tuberosum-S. chacoense hybrids. Materials and Methods Plant material Three 2x haploid Tuberosum- S. chacoeme hybrids (‘T-480’, ‘T-704’, and ‘T-706’) were used. The hybrid ‘T-480’ was obtained from the cross [‘Chippewa’ haploid-W 1831 x S. chacoense 6-91. The 2x hybrids ‘T-704’ and ‘T-706’ were full-sibs derived from the cross [‘Merrimack’haploid-W3304 x S. chacoense 1-24-11. All 2x clones produce 2npollen grains due the presence of the ps (parallel spindles) gene in homozygous condition. Theps gene controls 2n-gamete formation by a mechanism genetically equivalent to FDR-CO (Mok & Peloquin, 1975). A group of seven 4x cultivars representing a cross-section of elite genetic materials selected for adaptationin three continents(Europe, SouthAmerica, and North America) were used as femaleparents. They were ‘Aracy’ (Brazil), ‘Baronesa’ (Brazil), ‘Chiquita’ (Brazil), ‘Mantiqueira’ (Brazil), ‘Atlantic’ (USA), ‘Elvira’ (Holland) and ‘Spunta’ (Holland). zyx 4x-2x crosses and interploidfamilies Altogether, twelve 4x-2x families were obtained for field evaluation. The haploid-species hybrid ‘T704’ was hybridised as male with all the seven 4x potato cultivars. ‘T-706’ was hybridised only to ‘Chiquita, ‘Elvira’, and ‘Spunta’. The hybrid ‘T480’ was hybridised as male only to ‘Aracy’and ‘Chiquita’. Crosses were made in the greenhouse (at MadisonWI, USA) using fresh or refrigerated pollen that was applied to previously emasculated flowers. After crossing, plants were kept in a greenhouse with supplementary light conditions. The seeds were extracted when the fruits were at least 5 wk old. All the seeds were treated with 1500 ppm of giberellic acid for 24 h and rinsed in water before planting. Seeds of the families produced in the greenhouse were sown in jiffy-mix in 13 cm x 13 cm plastic trays. The seedlingswere transplanted individually to 10-cm pots and kept under greenhouseconditions. The tubers were harvested about 158 days later. Two tubers of at least 20 plants per cross were used to constitute two familybags, with one tuber per genotype per bag. Tubers of the 4x-2~families were treated with Rindite (1 mg kg-’ of tubers for 3 days) to break dormancy. After treatment, the tubers were left at room temperature until sprouting. These tubers were utilised in the field trials. zyx zyxwvut zyxwvutsrq zyxwvutsr zy Evaluation of 4x-2x (haploid Tuberosum-Solanum chacoense) progenies Field experiment and trait assessments The field assay with the 4x-2x (FDR-C0)-derived families was set up at Hancock, WI in a randomised complete block design (RCBD) with two replications (20 hills per plot). For estimation of the degree of heterosis, a set of six 4x cultivars (used as female parents) and the 2x species-hybridclones (used as male parents) were also planted in RCBD (with two replications) in the same experimental field but using &hill plots. These different plot sizes were used since the comparison was done between identical clones (i.e, the parents) vs. a group of 20 distinct genotypes (i.e., 20 hybrid clones from 4x-2x crosses). Tubers of the parental 4x cultivar ‘Mantiqueira’ (Brazil) were not available for this assay. The parental tubers (used as seed) were field-grown and larger than the 4x-2x family tubers (see Discussion). Plants were spaced 0.9 I m between rows and 0.3 1 m between hills in a row. The total duration of the experiment was 110 days from planting to harvesting (June-October). Tubers were dug and harvested by hand. Three agronomic traits were evaluated. Total tuber yield (TTY) was estimated using only tubers over 2.5 cm in diameter. Tubers were weighed and plot means (in kg per hill) were used in the analysis of variance. Haulm maturity (HM) was based on a description of the vine appearance of the plot at 77 days from planting. The following scale was used for HM assessment 1 = completely senesced; 2 = partially senesced; 3 = prostrate, green, post-flowering; 4 = upright, green, post-flowering; 5 = upright, green, flowering or pre-flowering. General tuber appearance (GTA) was evaluated using a qualitative key from 1 = poor to 5 = excellent, with 0.5 increments. 37 mean squares. The means of the 2x and 4x parent groups were contrasted with those of the 4x-2x families for the three traits. Correlation analysis of parent mean and family mean were done for each individual trait. zyxwvuts Results Total tuber yield The ANOVA indicated the presence of a significant difference for TTY among the 4x-2x (haploid Tuberosum-S. chacoense) families (Table 1). The family as a group outyielded both the 4x and 2x parental groups by 10.6% and 42.5%’ respectively (Table 2). The overall mean of the hybrid families (I .04 kg per hill) was identical to that observed for ‘Atlantic’ (the only North American 4x cultivar involved in this set of crosses). Expressed as the percentage of the 4x parent, the yield of the families ranged from 48% to 2 10%. The average performance of the best five families was I .32 kg per hill (a value identical to that observed for the best 4x parent ‘Aracy’). Furthermore, the best five families outyielded the 4x parent as a group by 40.6%. About 42% of the families outyielded their respective 4x parent. In addition, three families [‘Spunta’ x ‘T704’1, [‘Spunta’ x ‘T-706’1, and [‘Baronesa’ x ‘T704’1 outyielded ‘Aracy’ which was the best for TTY among the 4x parents assessed (Table 2). zyxwvu zyxwvutsrqp Analysis of variance and parent-family correlation The analysis of variance (ANOVA) for this experiment was partitioned into the sources of variation due to blocks, families, and experimental error. Tests of significance were conducted by comparing their mean squares with the respective error Table 1. Analysis of variance of total tuberyield (TTY), haulm maturity (HM) and general tuber appearance (GTA) of families from 4x-2x crosses between 4x potato cultivars and haploid Tuberosum-S. chacoense hybrids evaluated at Hancock, Wisconsin, USA Mean squares Source of variation dP TTY Block Families Error CVb(%) 1 11 11 0.132 0.160* 0.052 22.13 ”df = degrees of freedom bCV= coefficient of variation * = Significant at 0.05 level HM GTA 3.375 0.496 0.193 10.87 0.260 0.579* 0.192 13.94 General tuber appearance The ANOVA indicated a significant difference among families for GTA (Table 1). The 4x-2x families had a slightly lower GTA mean score when compared with both the 4x and the 2x parents. A GTA score of three is the minimum limit of acceptance in many breeding programs. Therefore, 10 out of 12 families were considered phenotypically acceptable and would be saved for additional evaluation (Table 2). Haulm maturity The ANOVA indicated significant differences among families only at the 10% level for HM (Table 1). The two parental groups were quite similar in terms of HM scores indicatingthat previous selection against very late maturity was effective at the diploid level. However, the families (as a group) were later maturing than both the 4x and the 2x parent groups (Table 2). When considered individually, each family was later maturing than both groups of parents with only one exception, [‘Aracy’ x ‘T-704’](Table 2). Parent-family correlation No significantparent mean-family mean correlation could be observed for HM and GTA (r-value ranged from -0.12 to 0.16). For TTY, a significant and negative correlation (-0.7 1) between the 4x-parent mean and the family mean was observed (Table 3). zyxwvutsr zyxwvu zyxwvut zyxwvutsrqpo zyxwvutsrqp BY J A BUSO, L S BOITEUX AND S J PELOQUlN 38 Table 2. Means of totul tuber yield (TTY) in kg per hill, haulm maturib (HA@and general tuber appearance (GTA) of 4x-2x (Tuberosum-S. chacoense) families and their parents evaluated at Hancock, msconsin, USA Means Genotypes 2.-parents T480 T-704 T-706 4x-parents Aracy Atlantic Baronesa Chiquita Elvira Mantiqueira Spunta TTY HM” GTAb 0.71 0.72 0.77 3.0 3.0 2.5 3.00 3.50 3.00 1.32 1.04 0.65 1.06 0.64 4.0 3.0 3.5 3.5 3 .O 4.00 3.25 2.50 3.75 3.50 material as observed in painvise comparisons (Table 2). Except for ‘Atlantic’, all 4x cultivars were originally selected for adaptation to distinct environmental conditions, which differed from those under which the present trial was conducted. However, some of the 4x cultivars performed similarly or even better than ‘Atlantic’ (Table 2). Some 4x cultivars that had poorer performance such as ‘Baronesa’ (Brazil), ‘Elvira’ (Holland), and ‘Spunta’ (Holland) gave rise to heterotic 4x-2x families. Surprisingly, the two families with the highest means (higher than the mean of the best 4x cultivar) were originated from crosses involving ‘Baronesa’ and ‘Spunta’ (Table 2). These results suggest that complementary genetic factors for TTY may be present in certain pairs of parental clones. The TTY increase in 4x-2x families over their 4x parents can be attributed to an increase in allelic and non-allelic interactions. Preponderance of nonadditive genetic variance for tuber yield has been reported in potato (Mendiburu & Peloquin, 1977). In fact, as pointed out by Mendiburu, Peloquin & Mok (1974) losses and gains, respectively,of allelic and non-allelic interactions rather than inbreeding can explain haploid depression as well as vigour in tetraploid potatoes. The increase in yield of 4x-2x families over the 4x parent may also be explained by the introgression of allelic diversity and more favourable ‘linkats’ (i.e. linkage blocks of favourable genetic factors) (DeMarly, 1979). The identification of heterotic families for TTY is in agreement with previous investigations using the USP breeding scheme. The heterosis values reported here are difficult to compare with those reported in the literature since a large number of distinct haploidspecies and 4x parents have been evaluated as parents in 4x-2x crosses (Tai, 1994; Buso, Boiteux, Tai & Peloquin, 1999). Previous reports with genetic material similar to our families are those derived from interdiploid 2x(SDR)-2x(FDR-C0) crosses with S. chacoense or S. chacoense x S. infundibuliforrne hybrids. The results indicated that best five 4x families outyielded the best five 4x cultivars by 57% and 69%, at two distinct locations in Wisconsin (Werner & zyxwvutsrqpon 4x-2x families Aracy x T-480 Aracy x T-704 Atlantic x T-704 Baronesa x T-704 Chiquita x T-480 Chiquita x T-704 Chiquita x T-706 EIvira x T-704 Elvira x T-706 Mantiqueira x T-704 Spunta x T-704 Spunta x T-706 - - - 0.76 3.0 3.75 1.05 0.84 0.81 1.37 0.51 0.88 0.86 I .26 1.26 0.89 1.43 1.33 0.50 5.0 3.5 4.0 3.5 4.0 4.5 4.5 3.5 4.0 4.0 4.5 3.5 NS 3.00 2.75 4.00 2.75 2.00 3.50 3.00 3.50 3.75 3.00 3.00 3.50 0.96 zyxwvutsrqponml zyxwvutsrqponmlkjihgfe LSD 0 05 anlong tmlllca Haulm maturity (HM) based on a scale from I still flowering a = senesced to General tuber appearance (GTA) based on a scale from I 5 =excellent = 5= poor to NS = Non-significant at 0.05 level Discussion Total tuber yield The results suggest that 2x haploid Tuberosum - S. chacoense hybrids are promising genetic material with potential to increase total tuber yield in potatoes. It is important to mention that the yield potential of the 4x-2x families in our experiment was probably underestimated due to the lower weight of the greenhouse-grown tubers of the families (1 5-50 g) in comparison with those of the field-grown seed tubers of the parental clones (50 g or more). The fact that seed tubers were not homogenous between parental group and families, penalizing the 4x-2x progenies, may give some erroneous conclusions about the yield potential of these crosses. The levels of high-parent heterosis were variable and seemed to be dependent upon both the 4x cultivar and the 2x haploid Tuberosum-S. chacoense hybrid used as parental Table 3. Parent mean-family mean correlation coeflcients (r) of total tuber yield (TTr), haulm maturity (HW,andgeneral tuber appearance (GTA) of I1families from 4x-2x crosses between 4x potato cultivars and haploid Tuberosum - S. chacoense hybrids evalu ated at Hancock, Wisconsin, USA TTY Parent r HM 2x 4x 0.27 4.71** ** = significant at the 1% level GTA 2x 4x 2x 4x 0.05 0.15 0.16 -0.12 zyxwvutsrq zyxwvutsrqpo zy Evaluation of 4x-2x (haploid Tuherosum-Solanum chacoense) progenws Peloquin, 1991). In the experiment reported here, the best five families outyielded the 4x-parent group by 40.6%. In another set of experiments in North Africa, three-way 4x hybrids (with 25% genomic contribution of S. chacoense)were able to outperform other hybrids for tuber yield in assays conducted in the fall (cooler) season (Bani-Aameur et al., 1991). The same promising results were observed for yield and specific gravity in field trials in Brazil (Morais & Pinto, 1996). It is important to highlight that the results reported here considered the families as a group (i.e. degree of heterosis at family level). Therefore, superior clones with a degree of heterosis even higher than that reported here could potentially be identified in progenies derived from this set of crosses. Heterosis, however, is not the only expected outcome in crosses involving wild species germplasm. There are several examples in the potato breeding literature where the lack of adaptation of a given wild species could erode field performance even in highly heterozygous genetic materials in which epistatic interactions have been preserved (Bani-Aameur et al., 1991). However, this seems to be not the case for most of the 2x haploid Tuberosum - S. chacoense hybrids used in our study. This would indicate that previous selection work for adaptation and tuberisation under long days conditions was relatively effective (Leue & Peloquin, 1980). 39 done to cross S. chacoense introductions to earlymaturing haploids in order to generate genetic variability for this trait. This could be done more efficiently via development of new early-maturing haploid-species hybrids. zyxwvut Parent-family relationships The lack of correlation found between the 4x and the 2x parents and their respective offspring for total tuber yield is in agreement with previous results with other 4x-2x crosses (Tai, 1994). This indicates that when selecting for tuber yield, test crossing and progeny testing will be necessary since the performance of the parents per se will be uninformative. Likewise, unsatisfactory predictions have been reported for TTY in 4 x 4 crosses ~ using correlation coefficients between mid-parent value and progeny performance (Neele, Hab & Louwes, 199 1). A somewhat surprising outcome was the significant and negative 4x parent-family correlation observed for TTY (Table 3). A possible explanation for this result is the relatively large number of unadapted 4x cultivars that were employed as female parents in our study. These elite cultivars were originally developed and selected for cultivation under distinct environmental conditions. The improved performance of the hybrid families with genomic contribution from more adapted Tuberosum germplasm ( e g the cultivars ‘Chippewa’ and ‘Merrimack’) in the male parents could explain, in part, this negative correlation. zyxwvutsrq General tuber appearance The identification of 4x-2x families with GTA similar to commercial cultivars is a very important observation. Families with GTA scores above our selection limit (3) were observed in five out 11 families. Some 4x-2x families with significantly higher ratings for general tuber appearance have been observed in other haploid Tuberosum-wild species hybrids (Darmo & Peloquin, 1991). For improved results, it seems that selection of 2x hybrid parents for important GTAcomponents (e.g. eye depth) would be a necessary step before using them as staminate parents in 4x-2x crosses. Results indicating that relatively few genes contribute to GTA suggest that selection for and fixation of desirable tuber characteristics will not be a difficult breeding task (Ortiz & Huaman, 1994). Conclusions The overall results with haploid Tuberosum-S. chacoense hybrids pointed out the ability of the USP breeding scheme to produce families with higher TTY than their 4x parents. The results highlight the importance of the USP breeding scheme in introgressing high yield factors from wild species into the gene pool of the cultivated potato. Selection of clones from crosses between closely related but highly adapted cultivars has been the major strategy of several breeding programs. In this context, the resources present in the 2x wild germplasm have been used predominantly in breeding for simply inherited traits (Plaisted & Hoopes, 1989). This may be caused by the previous erroneous notion that yield-enhancing genes are not available in wild species (Tanksley & McCouch, 1997). The results with 4x-2x and 2x-2x crosses in potatoes clearly demonstrate that heterotic families could be obtained using wild and cultivated diploid germplasm (for a recent review see Tai, 1994). Likewise, 4x-2x families with 25% genomic contribution from S. chacoense had (as a group) a higher tuber yield than seven elite 4x cultivars obtained from different breeding programmes around the world. In addition, our results suggest that superior clone(s) for cultivation in temperate regions of the world can be obtained from this South American potato species. zyxwvut HauEm maturity Even though there is considerable variability for this trait, the families were later maturing than both parental groups. HM score higher than the parental groups could be some manifestation of hybrid vigour in the families. It is important to note that the two Tuberosum haploids (‘Chippewa’ haploid-W 1831 and ‘Merrimack’ haploid-W3304) used to generate our 2x clones are also relatively late maturing (Buso, 1986). The 2x haploid-species hybrids used in the present set of crosses were medium-late maturing clones. Therefore, further breeding effort needs to be 40 zyxwvuts zyxwvuts zyxwvutsrqp zyxwvutsrqp zyxwvuts zyxwvuts zyxwvutsrqp zyxwvuts zyxwv BY J A BUSO, L S BOITEUX AND S J PELOQUIN References Bani-Aameur F, Lauer F I, Veilleux R E, Hilali A. 1991. 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