In peanut hybridization, distinguishing inadvertent selfs from the true hybrids may be difficult. In this study, to differentiate between selfs and hybrids, DNA was extracted from leaf tissue of F1 or F2 plants, and SSR markers were amplified and bands separated by a novel submarine horizontal polyacrylamide gel electrophoresis (H-PAGE). By comparing the resulting banding patterns to those of the parents, 70% of the putative hybrids were shown to be true hybrids on the basis of possessing a marker allele from the male parent. The H-PAGE gels gave better band separation and differentiation of selfed progenies than agarose gels, and were compatible with the common horizontal agarose gel units. This method provides a quick assay to distinguish hybrids from inadvertent selfs, and should result in greater efficiency and more effective use of resources in peanut breeding programs.
The identification of true hybrids is important for peanut breeding programs. Crossing involves removal of ten stamens in the evening, followed by cross-pollination in the morning. It is easy to miss a stamen, which can remain hidden in the keel and cause self pollination. In addition, the peanut inflorescence is compound, with two or three flowers produced at the same axil within approximately one week. Selfed flowers must be removed early in the morning, and if any are missed or pulled late, a selfed peg may emerge at the same site as a hybrid peg or where an unsuccessful attempt at cross-pollination had occurred.
Several methods are used to distinguish hybrid versus selfed progenies, including observing morphological differences among progenies, segregation for disease resistance or differences in the oleic:linoleic ratio (López and Burow, unpublished results). Identifying hybrids in the F1 generation can be difficult because the F1 may not be readily distinguishable from the parents, especially in the greenhouse where plants cannot grow to full size due to limited space. In the field, it is often possible to distinguish F2 plants by segregation for morphological traits. However, this may not be the case for closely-related parents and may not be useful in the case of attempted three-way crosses, where failure to cross hybrids produces segregating progeny. In addition, planting of F2 plants to identify hybrids by appearance is an inefficient use of field space and labor. Finally, discovery of selfs typically occurs a year after the crosses are made, resulting in potential delays to improvement programs.
Identification of hybrids can be performed through use of DNA markers. Codominant markers are preferable because they produce different alleles (markers) for each parent, and F1 hybrids will possess an allele from each parent. Of the major DNA marker types, restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers are usually codominant. The SSR-based markers require smaller quantities of DNA than do RFLP-based markers (
Currently, several separation methods are employed to determine the length of amplification products; among the methods are agarose gels and non-denaturing polyacrylamide gel electrophoresis (PAGE) (
This paper describes an inexpensive and simple method (H-PAGE) for identification of peanut hybrids in the F1 or F2 generation. SSR markers were used to distinguish parents of cultivated × cultivated crosses, or of crosses involving one cultivated and one wild species introgression line parent. After this, putative progeny were tested for presence of the male parent allele. Additionally, the use of horizontal polyacrylamide gel electrophoresis provided a simple and inexpensive method of separation of alleles differing by a few base pairs.
Experimental materials were comprised of F1 and F2 populations for development of heat stress-tolerant and leaf spot-resistant lines, respectively. Lines developed for heat stress tolerance included putative F1 plants derived from
All parents and F1 and F2 progenies were grown in potting soil (Sunshine SB-300) in plastic trays in the greenhouse at the Texas Agricultural Experimental Station (Lubbock, TX) greenhouse for 25 to 28 days to allow for collection of tissue. After confirmation of hybridization, plants were transplanted to larger pots for seed production. For putative F1 crosses, tissue was collected from only one plant per pod. For putative F2 populations, from 12 to 36 seeds were sown from each F1, and six randomly selected plants from each cross were used for marker analysis.
Unopened tetrafoliate leaves from 20 to 25 day-old parents or putative hybrids were used for DNA isolation. Leaves were stored at -80°C, or were collected fresh. Leaves were ground in a mortar and pestle using liquid nitrogen. Genomic DNA was isolated as per
A total of 24 oligonucleotide primer pairs flanking microsatellite repeat sequences were used in the present study. Fifteen primer pairs (PM3, PM15, PM32, PM35, PM36, PM45, PM50, PM53, PM65, PM137, PM145, PM183, PM188, PM200, and PM210) were from the published sequences of
The PCR reaction condition used was as follows: DNA samples (30 ng) were amplified in a 10 μl reaction volume containing 1× Polymerase Chain Reaction buffer (10 mM Tris HCl pH 8.3, 50 mM KCl, 0.1% Triton X-100, and 0.01% gelatin), 0.2 mM each of the four dNTPs, 2 mM MgCl2, 0.5 μM of each forward and reverse primer (synthesized by Integrated DNA Technologies, Coralville, IA), and 0.5 U of Hot Start
Non-denaturing polyacrylamide gels were cast in a horizontal gel casting plate designed for agarose gels. A 6% polyacrylamide gel was prepared using an acrylamide/bisacrylamide ratio of 19:1, 0.5× TBE (Tris boric acid ethylenediamine tetraacetic acid) buffer (
After electrophoresis, the gel was stained in 500 ml of water containing 15 μl ethidium bromide (100 mg/ml) for 15 to 20 minutes, followed by destaining for 15 minutes in distilled water. The staining solution was stored in the dark and could be used up to three times. Alternatively, ethidium bromide (25 μl for one liter of running buffer) could be added to the running buffer. The same running buffer was reused two additional times effectively. After staining, the gel was visualized either on a UV transilluminator (Model FBTV-816, Fisher Biotech, Pittsburgh PA), photographed using a Kodak DC-290 camera with a deep yellow 15 filter (Tiffen, Inc., Glendale, CA) connected to a PC running Slackware Linux v 10.2 (
A method for identification of true hybrids in peanut was developed. To our knowledge, this is the first use of DNA markers for this purpose in cultivated peanut. In this paper, we detail a new, simple, low cost method which could be used in peanut breeding programs worldwide.
Chemotypic heterogeneity among species may not allow optimal DNA yield with a single isolation protocol. Thus, even closely-related species may require different DNA extraction protocols (
Of the 24 microsatellite loci analyzed, eight were observed to be polymorphic (PM3, PM32, PM50, PM137, PM188, PM210, Ah193 and PGS12A07) for the lines screened. Four SSR markers (PM210, PM42, PM3 and PGS12A07) showed clear polymorphism for most of the crosses (
Crosses tested for hybrid production and results of SSR analysis.
The present study revealed 14 and 27% polymorphism in cultivated × cultivated and interspecific crosses, respectively. Although polymorphism was lower in cultivated × cultivated crosses than using interspecifically-derived lines as one parent, the set of 24 primer pairs used was adequate for identification of polymorphism in all crosses used (see
Horizontal PAGE has good resolving potential for distinguishing the heterozygote from the homozygote (
Use of the Kodak DC-290 camera also allows for inexpensive visualization of results. This camera, or similar models, is inexpensive, and has the ability to take close-up photos. Connection to a personal computer running Slackware Linux provided all the needed software at no cost. With the help of the free Image J software (
This system is ideal for small-scale breeding or newly-established laboratories with very limited facilities. For the Dellaporta DNA extraction method, a low-speed (3500 rpm) centrifuge, microcentrifuge, and heated conventional water bath are needed, but for the Qiagen DNA Easy kit protocol, a micro centrifuge and heated water bath are the only major pieces of equipment needed for DNA extraction. For detection, a UV transilluminator to visualize the DNA fragments is needed, and a camera is desirable to reproduce images. An inexpensive PC and printer using open source software can be used for long-term storage of images and printing of results. Also, the same horizontal gel unit can be used for agarose gel electrophoresis. The cost of using this method is low as this method does not require any sophisticated vertical apparatus. The gel ingredients cost less than a dollar, and a gel can be used to obtain 52 data points (two 26 well combs in 16 × 14 cm gel plate) without multiplexing. This system may be compared favorably with high-resolution agarose gels that are widely used in many laboratories for genotyping with microsatellite markers. This method is cheaper than the high-resolution SFR or Metaphor (Lonza Inc, Rockland, ME) agaroses used for SSR work, and amplified bands are clearer and sharper than those on SFR agarose gels. Currently we are using this method to enrich the tetraploid peanut map using microsatellite markers.
The horizontal PAGE method was used successfully to verify hybrids in F1 and F2 populations of peanuts using microsatellite markers. The high discriminating power of SSR markers and inexpensive setup should allow this to be affordable for many peanut breeding laboratories.
The authors would like to express thanks to Jamie Ayers and Yolanda López for assistance with greenhouse work. Funding for these studies was provided by the National Peanut Board, the Southwest Consortium for Plant Genetics and Water Resources (#2004-34186-14533), and the Peanut Collaborative Research Support Project.
First and second authors: Res. Agron. , USDA-ARS and Agric. Res. Statistician; Coastal Plain Exp. Sta., Tifton, GA 31793-0748. Third author: President and CEO, Hebert Green AgroEcology; Asheville, NC 28801.
Texas Tech University, Department of Plant and Soil Science, Lubbock, TX 79409
Savanna Agricultural Research Institute, Tamale, Ghana
Central Research Institute for Jute and Allied Fibers, Kolkata-700 120, India
Texas A&M University Agricultural Experiment Station, Stephenville, TX 76401
USDA-ARS, Cropping Systems Research Lab, Lubbock, Texas 79415
New Mexico State University, Agricultural Science Center, Clovis, NM 88101
Texas A&M Agricultural Experiment Station, 1102 East FM 1294, Lubbock, TX 79403