Plant material.

Inheritances of the three traits were explored in F₁, F₂, BC_1P1, and BC_1P2 families derived from hybridization of C. canadensis ‘Ruby Falls’ (noncompact/weeping architecture/purple leaf) × ‘Ace of Hearts’ (compact/nonweeping architecture/green leaf). ‘Ruby Falls’ was released in 2009 (Werner and Snelling, 2010). ‘Ruby Falls’ carries the recessive weeping gene (wp1wp1) and the recessive purple gene (pl1pl1) derived from C. canadensis ‘Forest Pansy’ (USPP 2556), a purple-leaved redbud discovered in 1947 by Forest Nursery, McMinnville, TN (Dirr, 2009). ‘Ace of Hearts’ is a compact cultivar with small, glossy, heart-shaped leaves introduced in 2005 (Fantz and Woody, 2005).

Hybridizations of potted parental plants were conducted in the North Carolina State University Horticulture Field Laboratory greenhouse in Raleigh, NC, in 2011. Modified seed scarification treatment was used (Banner and Stein, 2008). Harvested seed was sown after acid scarification treatment and 2-month cold stratification at 4 °C. For acid scarification, seed was submerged in concentrated (18.4 M) sulfuric acid for 30 min. Seed was gently stirred briefly every 2 min for the first 10 min, and at 5-min intervals thereafter. Acid-scarified seed was then rinsed and cold stratified for ≈2 months. A total of 27 F₁ plants were ultimately recovered. F₁ plants were grown in the greenhouse in 2011 until being transplanted into a field isolation block in Winter 2012 at the Sandhills Research Station, Jackson Springs, NC.

To create the F₂ family, one F₁ tree was dug from the field, planted in a 95-L pot, and brought to the greenhouse for crossing. In Mar. 2015, branches containing flower buds were cut from multiple F₁ trees in the field and brought to the greenhouse for flowering. Mixed pollen from the cut branches was bulked and used to pollinate the single F₁ tree. F₂ seed was harvested in Fall 2015, and acid scarified and cold stratified before being sown in January 2016. Resultant seedlings were grown in the greenhouse in individual pots to a height of ≈15 cm and transplanted to the field in May 2016 at the Sandhills Research Station. Trees were evaluated for leaf color, weeping/nonweeping, and compact/noncompact characteristics in Oct. 2016 (Fig. 1).

View Full Size

Morphologies of parental cultivars of Cercis canadensis and their segregated F₂ progenies. (A) ‘Ruby Falls’, a noncompact, purple-leaved, weeping cultivar. (B) ‘Ace of Hearts’, a compact, green-leaved, nonweeping cultivar. (C) Left: a noncompact, purple-leaved, nonweeping F₂ plant. Right: a compact, purple-leaved, nonweeping F₂ plant. (D) A compact, green-leaved, nonweeping F₂ plant. (E) A compact, green-leaved, weeping F₂ plant.

Citation: HortScience 56, 12; 10.21273/HORTSCI16031-21

• Download Figure
• Download figure as PowerPoint slide

Hybridizations to create backcross families BC_1P1 and BC_1P2 were conducted in Mar. 2017. To create the BC_1P1 family, bulked pollen from two F₁ individuals was used to pollinate one ‘Ruby Falls’ tree in the greenhouse. To create the BC_1P2 family, pollen of ‘Ace of Hearts’ was used to pollinate one F₁ individual in the same greenhouse. BC_1P1 and BC_1P2 seeds were harvested in Sept. 2017, scarified and stratified as described previously, and sown in Jan. 2018. Seedlings were grown in the greenhouse in individual pots to a height of ≈15 cm before being transplanted into the field plots in May 2018. Trees were evaluated for leaf color, weeping/nonweeping, and compact/noncompact characters in Sept. 2018 (Fig. 1).

Phenotyping.

Phenotyping methods for leaf color and weeping/nonweeping characters were applied on 5- to 10-month-old plants as described by Roberts et al. (2015). Phenotyping of compact growth was conducted on 10-month-old plants. Plants showing compact growth had demonstrably smaller leaves, enhanced branching, shorter internodes, and reduced height compared with noncompact plants (Fig. 1C). Phenotypes of the three traits were readily identified, and no intermediate phenotypes were observed.

Statistical analysis.

Each plant in the F₂, BC_1P1, and BC_1P2 families was phenotyped, and expected segregation ratios in each family were then tested. For the F₂ family, the expected 3:1 ratio for each trait was tested by a χ² goodness-of-fit test with a threshold of 0.05. For the BC_1P1 and BC_1P2 populations, a 1:1 ratio of each trait was tested by a χ² goodness-of-fit test with a threshold of 0.05 when segregation was observed. Odds ratio independence tests between each pair of the three traits were tested to assess the potential linkage between any two genes. The 9:3:3:1 ratio of each of the two traits was further tested by a χ² goodness-of-fit test with a threshold of P > 0.05.

Inheritances of the three traits.

Segregation results indicated that the three traits are all controlled by single recessive genes (Tables 1–3). All 27 F₁ offspring of ‘Ruby Falls’ × ‘Ace of Hearts’ were noncompact, green-leaved, and nonweeping. Segregation data of the three traits characterized by noncompact/compact (Table 1), green/purple leaf (Table 2), and nonweeping/weeping (Table 3) are shown.

Table 1.

Segregation ratios and tests for goodness of fit for compact growth habit in F₁, F₂, and backcross families derived from hybridization of Cercis canadensis ‘Ruby Falls’ (noncompact) × ‘Ace of Hearts’ (compact).

View Table

Table 2.

Segregation ratios and tests for goodness of fit for leaf color in F₁, F₂, and backcross families derived from hybridization of Cercis canadensis ‘Ruby Falls’ (purple leaf) × ‘Ace of Hearts’ (green leaf).

View Table

Table 3.

Segregation ratios and tests for goodness of fit for weeping in F₁, F₂, and backcross families derived from hybridization of Cercis canadensis ‘Ruby Falls’ (weeping) × ‘Ace of Hearts’ (nonweeping).

View Table

Data for the compact trait (Table 1) in the F₂ family fit the expected 3:1 ratio based on segregation of a single recessive gene (P value = 0.053 > 0.05). The BCIPI family segregation also fit the expected 1:1 ratio based on control by a single recessive gene (P value = 0.36 > 0.05). Although we accepted the single recessive gene model, the P value of the F₂ family was borderline significant. The observed number of compact plants in the F₂ and BC_1P1 families was slightly less than expected. This phenomenon could be explained by the possibility of slightly reduced fitness and survival of compact progeny during germination or early seedling growth. We propose that ‘Ace of Hearts’ has a genotype cp1cp1 (compact) and ‘Ruby Falls’ has a genotype CP1CP1(noncompact).

For the green/purple leaf trait (Table 2), all F₁ progeny were green-leaved, consistent with a single recessive gene model. The segregation ratio of the F₂ families followed the expected 3:1 segregation ratio expected of a single recessive gene (P value = 0.92 > 0.05). In the 572 F₂ progeny, 142 plants were purple-leaved, almost the same as the expected 143 plants. The segregation ratio of the BC_1P1 fit the expected 1:1 segregation ratio expected for the single recessive gene model (P value = 1 > 0.05). In the 42 BC_1P1 progeny, 21 plants were purple-leaved, and 21 were green-leaved. All BC_1P2 progeny were expected to be green-leaved; however, five unexpected purple-leaved plants were observed among the 76 BC_1P2 plants. Without considering the five unexpected purple-leaved plants, all segregation ratios of F₁, F₂, BC_1P1, and BC_1P2 families followed the single recessive gene hypothesis. The purple leaf gene from the same ancestor that has been reported previously is controlled by a single recessive gene pl1 (Roberts et al., 2015), and our results confirmed the single recessive gene 3:1 segregation. The five unexpected purple-leaved plants in the BC_1P2 family can be explained by occasional self-pollination or pollen contamination.

In this research, phenotypes of F₁ and BC_1P2 progeny were all nonweeping, consistent with the single recessive gene model (Table 3). The F₂ and BC_1P1 families fit the expected 3:1 (P value = 0.77 > 0.05) and 1:1 (P value = 0.06 > 0.05) segregation ratios, respectively, again consistent with the single recessive gene model. Like the compact trait, the observed number of weeping plants in the F₂ and BC₁P₁ families was slightly lower than expected, which could be explained by reduced survival of weeping progeny in seed or during early seedling growth. Segregation ratios of the weeping trait in the F₁, F₂, BC_1P1, and BC_1P2 families are consistent with the earlier report that the weeping trait is controlled by a single recessive gene (Roberts et al., 2015).

Linkage of the three traits.

Results of plant numbers and the odds ratio independence tests between trait comparisons are listed in Table 4. Cosegregation of compact/weeping and purple/weeping genes passed the odd independence tests, and there was no significant deviation between the observed segregation numbers and the expected 9:3:3:1 segregation ratio, suggesting no linkage (Table 4). However, cosegregation of the compact and purple leaf traits deviated significantly from the expected 9:3:3:1 ratio (P value = 0.02 < 0.05); this deviation from the expected 9:3:3:1 ratio cannot be explained as due to linkage. In the present research, the two parental cultivars were Ruby Falls (noncompact/purple leaf) and Ace of Hearts (compact/green leaf), with the genes in the repulsion phase. One would expect that deviation due to linkage would result in greater numbers of parental combinations than expected; however, increased nonparental type and reduced parental type phenotypes were observed, arguing against linkage. Thus, our data for testing linkage between these two genes is inconclusive, and further studies are required to resolve this inconsistency.

Table 4.

Independence test analysis between each pair of the three genes, compact, weeping, and purple leaf, in the F₂ population derived from hybridization of Cercis canadensis ‘Ruby Falls’ (purple leaf/normal growth/weeping) × ‘Ace of Hearts’ (green leaf/compact/nonweeping).

View Table

Like many woody ornamental plants, redbud has a longer generation time than most herbaceous species, thus breeding and selection can be time-consuming and costly. The results of this study can be useful for future redbud breeding efforts, especially in programs focusing on the development of compact cultivars, by allowing the appropriate breeding strategies and population sizes to be determined. Future molecular studies on redbud can confirm the results of this study, and perhaps resolve the inconclusive results of linkage between the compact and purple leaf genes as described in this study. Whole genome sequence studies potentially would allow the identification of the exact gene sequences and locations of these three traits.