Modern farming is dependent on continual development of improved cultivars and efficient cultural management practice. In addition, dissecting genetic components of heritable traits also relies on the development of large mapping populations. Artificial hybridization is the critical initial step in these processes. Peanut is a self-pollinating crop with a typical yield of less than three seeds per flower; therefore, significant effort is required to produce sufficient hybrid seeds for subsequent trait selection and/or establishment of mapping populations. A study was conducted to evaluate the effect of multiple factors on the success rate of artificial hybridization assessed by transmission of molecular markers unique to the paternal parent. Multiple peanut genotypes were crossed with a breeding line homozygous for both high oleic acid and nematode resistance. The impacts of operator, pollination time, flower integrity, genotype and environment on hybridization were evaluated. Data indicated that operator, pollination time and environment significantly affected the success rate of peanut hybridization. Pods from runner type parental plants that contain hybrid seeds were more likely to contain single seeds than those derived from self-pollination. Hybrid seed loss due to seed rot and peg damage reduced yield. Improving hybridization success rate by increasing humidity, decreasing temperature, personnel training and greenhouse management is recommended.
Peanut is an important cash crop valued for its oil, protein, and flavor content. In 2016, 1.7 million acres of peanut were planted in the U.S. (National Agricultural Statistics Service, 2016). U.S. peanut yield achieved a six-fold increase from 739 kg ha−1 in 1909 to 4695 kg ha−1 in 2012 (
As is typical of species in the legume family, peanut flowers self-pollinate. Each flower has one large standard, two lateral wings, and a keel, which encloses the staminal tube at the distal end of which extends eight anthers surrounding a club-shaped stigma (
In order to create artificial hybrids, flowers from the female plants must be emasculated prior to anther dehiscence. Mature pollen released from male plants should be applied to the stigma of female plants. Hybrid pegs produced from the cross need to be identified and tagged before harvest. Since peanut artificial hybridization is low yielding and time consuming, reported to cost 10 minutes per flower (
Success rates of artificial hybridization can be affected by multiple factors such as humidity, temperature, crossing schedule, peanut genotype, operators and integrity of emasculated flowers etc. High humidity had also been shown to enhance peanut flowering and peg formation (
In the first experiment, common male parents for all female parents were selected from F3 plants of C1976 [Tifguard ((
Parental information of crosses
Parental seeds were sown in 12 inch pots with a mix of 50% Promix (Premier Tech Horticulture, Quaker, PA) and 50% steam-sterilized sandy soil from the Coastal Plain Experiment Station in Tifton, GA. The six female genotypes were arranged on the bench top in a greenhouse as indicated in
Arrangement of female pots on the greenhouse bench for experiment 1 (A) and experiment 2 (B). Position 1 and 2 indicate the location of HOBO data loggers.
To separate the effects of location and genotype on the success of crossing, a second experiment was performed with three breeding lines (
In a third experiment, all pods from three crosses (
Emasculation and pollination procedures followed a previously published method (
Data analysis was performed by ANOVA using SAS 8.2 statistical software (SAS, Cary, NC). Tukey's test was performed to separate the means. Significant differences were detected at a P-value less than 0.05.
Parents of crosses were genotyped using molecular markers prior to crossing. In the first experiment, 19 potential male plants from F3 plants of C1976 [Tifguard × (Tifguard × (Tifguard × (Tifguard × Florida-07))] were genotyped and all of them were homozygous for both high O/L and nematode resistance markers. Tifguard is the donor of the nematode resistance trait and Florida-07 is the donor of the high oleic trait. Earlier generations of these plants had been selected for both traits by molecular markers; therefore, consistent inheritance of both traits was expected. In the second experiment, 2 out of 28 potential male plants from F4 lines of C1805 (Tifguard x Florida-07) were eliminated from the study since they lacked both molecular markers. In the third experiment, all parents demonstrated expected genotypes with our markers. Although the presence of off-type parents in a breeding program is rare, excluding off-type parents is critical to preserve the purity of hybrid lineages. Currently, limited markers are available to peanut breeders. Advances in peanut genome sequencing (
In the first experiment, a total of 119 putative hybrid pegs were tagged. Thirty-two pegs failed to produce viable seeds. Peg damage, pod rot and seed immaturity contributed to the loss. Upon pollination, it takes approximately 5-14 days for a hybrid peg to emerge and another 3-4 days before the peg can be permanently wired. It takes 10 to 14 days from wiring of the peg until the peg tip enlarges sufficiently to secure its position in the soil. Wired pegs can be damaged incidentally while emasculating or checking for newly formed pegs. A few rotten seeds were found and several pods yielded extremely shriveled seeds. One hundred ten putative hybrid seeds were treated to break dormancy and nine seeds failed to produce viable plants. All of the germinated plants were genotyped with both high O/L and nematode resistance markers. Eighty-nine F1 plants (81%) were unequivocally identified as hybrids. Twelve plants (10.9%) were identified as self-pollination derived. During harvest, two wires were noted as having two pods each. It was apparent that pegs from self-pollinated flowers grew into the holes of the wires marking hybrid pegs. Since we have molecular markers to distinguish self-pollination derived versus hybrid genotypes, they were harvested to maximize the chance of recovering hybrids. The distribution of pod losses and self-pollination derived seeds was random among treatment groups, thus data analysis of crossing success rate was based on wired peg numbers.
The “separate” pollination schedule produced significantly more pegs than that of the “concurrent” pollination schedule (
Success rate of concurrent and separate pollination schedules (A); success rate of operators (B).
Significant differences were found among the three groups of operators in which group 2 had the highest success rate of 22% followed by group 3 and group 1 (
The average success rate of emasculated-full-blooms was higher than, but not significantly different from that of emasculated-but-unopened flowers (
Success rate of emasculated-full-blooms and emasculated-unopened flowers (A); Hypanthium length of emasculated-full-blooms, emasculated-unopened and self-pollinated flowers (B)
Crossing success rates between parental pairs were also found to be significantly different in which C2244 and C2247 were the lowest in experiment 1 (data not shown). These two least successful crosses had been placed furthest away from the cooling pads in this experiment (
Success rate of crossing between parent plants clustered at data logger 1 and 2 positions in experiment 2 (A); Average daily temperature from 9:00 to 14:00 hr recorded by data loggers (B); Average daily relative humidity from 9:00 to 14:00 hr recorded by data loggers (C).
A high frequency of single pods was observed among the hybrids in our breeding program. In the third experiment, all pods were harvested from three crosses whose female plants are runner type, typically producing double pods. After genotyping all seeds for hybridity, it was found that the self-pollination derived pods from these runner-type female plants produced an average of 30% single-seeded pods whereas 80% of pods containing hybrid seed were single-seeded (
Comparison of percentage of single-seeded pods developing from cross-pollinated or self-pollinated flowers.
In the present study, multiple factors including operator, crossing schedule, temperature and humidity were found to significantly affect the success of artificial hybridization in peanut. The conventional breeding schedule is suitable for our breeding program using a greenhouse facility. Decreased temperature and increased humidity, as well as proper training of operators, are likely to improve the success rate of crossing. Watering the benches and floor immediately after pollination improves the crossing condition and is routinely practiced in our program. Improvement of greenhouse management should reduce hybrid loss due to fungal disease and water logging. Given the already low yield and labor intensiveness of peanut crossing, optimizing the factors presented in this study will improve the efficiency of crossing programs.
We acknowledge funding from the Cultivar Development Research Program of the University of Georgia Research Foundation. We greatly appreciate Betty Tylor, Shannon Atkinson, and Rattan Gill for their participation of this project.
First, second, and fourth authors: Research Professional, Technician, and Professor, Department of Horticulture and NESPAL, University of Georgia Tifton Campus, Tifton, GA 31793; Third author: Research Geneticist, USDA-ARS, P.O. Box 748, Tifton, GA 31793.
Current address of C. L. Wu: Center for Applied Genetic Technologies, 111 Riverbend Rd, Athens, GA 30602