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BREEDING, GENETICS, AND PHYSIOLOGY
Molecular Characterization/Purification
of a Working Germplasm Collection
V.A. Boyett, A.M. Stivers, and J.W. Gibbons
ABSTRACT
Since 2001, Rice Breeding and Genetics at the University of Arkansas Division
of Agriculture Rice Research and Extension Center (UA RREC) has had a technical
support project utilizing DNA marker analysis to aid in the genetic enhancement of rice
germplasm, specifically in the areas of disease resistance and cooking quality. Simple
sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers linked to
these specific traits are used to predict the cooking quality of milled grain and screen for
the presence of rice blast [Magnaportha grisea (T.T. Hebert) M.E. Barr] resistance genes.
More than 90% of the project’s effort each year is devoted to Marker-Assisted Selection
(MAS) screening of early generation segregating populations with these trait-linked
markers, increasing the efficiency of selection of progeny with desired characteristics
that has the potential for commercial success. Since the program’s inception, major
emphasis has been placed on using these markers to genotype the bank of elite breeding
material used as parents for these populations. Characterizing this Working Germplasm
Collection (WC) on a molecular level enables the determination of which populations
would benefit from MAS, purification of the entries, and more efficient design of cross
combinations to introgress desired traits. Molecular analysis was performed on 307
entries of the WC for genes linked to rice blast disease resistance, amylose content,
and plant height. In addition, MAS screening with the same markers was utilized to
correlate genotype and phenotype and confirm purity of 45 WC accessions that were
phenotypically purified in the field.
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INTRODUCTION
In any plant breeding program, its foundation is its collection of elite breeding
lines, and it is important that it be extensively characterized so that the breeder can
improve chances of success in developing lines for commercial release. Each year, the
WC receives 30 to 40 new entries, so the characterization is a continuous endeavor. The
collection is meticulously evaluated for approximately 40 phenotypic traits (IBPGRIRRI, 1980). Based upon this evaluation process, 45 entries of the WC were determined
to be segregating for one or more of these traits and in need of purification before further
use in the breeding program.
In addition to the phenotypic characterization, genotyping this WC gives the
breeder more information regarding the genetic background, diversity, and potential of
the parental material. In an effort to “deepen the gene pool” or widen the germplasm
base, and identify new resources of desirable traits, many entries in the WC are of diverse origin, and frequently possess agronomic traits that are undesirable. Using MAS
to eliminate those lines with undesirable traits means that only lines with the highest
probability of acceptance will be advanced to large plots in later generations, thus saving
valuable and limited land area in Stuttgart and Puerto Rico for the best material.
Molecular markers for screening were chosen on the basis that they were not only
informative markers that are in routine use for MAS in the program, but also that they
were linked to rice blast disease resistance, cooking quality, and plant height genes that
would significantly impact phenotype.
The objectives of this continuous study are to (i) increase the efficiency of applying
MAS to the crosses made by the breeding program at the UA RREC, thus improving
the chances for success in the development of new lines for commercial release, (ii)
determine the haplotype of the entries of the WC at the loci for important agronomic
traits, and (iii) strive to ensure purity and a correlation between genotype and phenotype
of the entries of the WC.
PROCEDURES
All entries of the WC were tested, initially by screening two bulked seed samples
of 10 seeds each for a total of 20 seeds for each entry. For the initial screening, dehulled seed was placed into 2-ml ScrewCap Mictrotubes along with about 20 1-mm
glass beads, processed in a BeadBeater-96 (BioSpec Products, Bartlesville, Okla.), and
DNA was extracted using a sodium hydroxide based method.
Criteria for a heterozygous score were that the smaller peak had to be at least
20% of the taller peak and the sample had to have a genotyping quality (GQ) score of
at least 0.4 units. Close alleles were scored manually. Any entry amplifying more than
one allele at a given locus was further screened as a leaf sample from an individual plant
so that the difference between heterozygous individuals and seed mixtures could be
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determined. Data anomalies and suspected cross-contaminated samples were repeated
for confirmations and correction, at least from the polymerase chain reaction (PCR) step.
In some cases new DNA samples were extracted and the marker analysis repeated.
There were 45 WC accessions in need of purification based on phenotypic evaluation and these were planted in rows of 10 plants each in a separate Phenotype observation
bay (PB). Each individual plant was assigned a number and the tissue sample collected
into a separate envelope so that the marker analysis data could be traced to the exact
plant from which the leaf tissue sample was taken. After phenotype evaluation, molecular analysis, and comparison of descriptors in the Germplasm Resource Information
Network (GRIN) (http://www.ars-grin.gov/npgs/searchgrin.html), each row in the PB
was purified of off-types and desired plants allowed to mature for seed increase.
Leaf tissue was harvested into manila coin envelopes and stored at -80 ºC until
sampled with a single hole-punch. DNA was extracted using Sodium hydroxide/Tween
20 and neutralized with 100mM Tris-HCl, 2mM EDTA. The DNA samples were arrayed
in a 96-well format and 2 µl of template used for each 25 µl PCR analysis.
Markers chosen were RM208 linked to Pi-b resistance; YL155, YL183, and Piindica for the rice blast resistance gene Pi-ta; and AP5659-1 for Pi-z resistance (Fjellstrom et al., 2004, 2006; Jia et al., 2004). A “Null” allele with the AP5659-1 marker
was confirmed with additional PCR at AP5659-5 (Fjellstrom et al., 2006). Waxy, a gene
influencing amylose content of the mature grain was evaluated with RM190 (Bergman
et al., 2001), and RM1339 was used to screen for sd1, the most common gene to determine semidwarf plant height (GRAMENE, Sharma et al., 2009).
PCR was performed with either HEX or FAM labeled primers by adding template
and enough bovine serum albumin and polyvinylpyrrolidone 40 to have final concentrations
of 0.1% and 1% respectively (Xin et al., 2003) and cycling the reactions in a Mastercycler
Gradient S thermal cycler (Eppendorf North America, Inc., Westbury, N.Y.). Resulting
PCR products were grouped according to allele sizes and dye colors and diluted together
with an epMotion 5070 liquid handling robot, also from Eppendorf North America. The
amplicons were resolved with an Applied Biosystems 3730 DNA Analyzer, and analyzed
using GeneMapper software (Applied Biosystems, Foster City, Calif.).
RESULTS AND DISCUSSION
Of the 307 accessions, the PCR analysis was repeated on 84 to confirm heterozygous scores, investigate reaction failure scores in GeneMapper, and increase amplification of those samples with GQ below 0.4 units and therefore not trustworthy. Leaf
tissue samples were collected from individual plants to confirm heterozygotes or seed
mixtures in the case of 63 entries. After analysis of individual plant samples 43 entries
were determined to be problematic, segregating at one or more loci tested. (Table 1)
Of the 45 entries in the PB, 26 were segregating by genotype (Table 1), and four
were random off-types. The remaining 15 entries appeared homozygous and uniform
with the markers used. Evidently, these entries were segregating for traits for which
no markers were used.
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B.R. Wells Rice Research Studies 2009
SIGNIFICANCE OF FINDINGS
Molecular characterization of the WC explained the incidence of non-parental
alleles being amplified in MAS projects involving crosses made prior to the use of DNA
marker analysis in the breeding program at the UA RREC, and some of the phenotypic
segregation observed in the Crossing Block planted each year to serve as parental material for crosses. The genotyping enabled rapid determination of which crosses would
benefit from MAS screening of early generation progeny. In addition, the data identified
the genetic profile at the five loci linked to rice blast disease resistance, cooking quality,
and plant height of each WC entry and assisted in determining which entries needed
purification, or, in the case of four entries, replacement. The entries of the PB were
selected because of observed phenotypic segregation, yet 26 of the 45 entries were also
segregating on a molecular level for these important agronomical traits.
ACKNOWLEDGMENTS
The authors thank the Arkansas Rice Research and Promotion Board and the Dale
Bumpers National Rice Research Center for their financial support of this research.
We thank Anna McClung and M. Jia for promoting the Rice Genomics Program and
allowing the use of equipment, facilities, and supplies at the DB NRRC. We thank V.
Booth, J. Bulloch, B. Lockwood, J. Lockwood, and V. Thompson for their excellent
technical assistance.
LITERATURE CITED
Bergman, C.J., J.T. Delgado, A.M. McClung, and R.G. Fjellstrom. 2001. An improved method for using a microsatellite in the rice Waxy gene to determine
amylose class. Cer. Chem. 78:257-260.
Fjellstrom, R., A.M. McClung, and A.R. Shank. 2006. SSR markers closely linked
to the Pi-z locus are useful for selection of blast resistance in a broad array of rice
germplasm. Molecular Breeding 17:1380-3743
Fjellstrom, R.G., C.A. Conaway-Bormans, A.M. McClung, M.A. Marchetti, A.R.
Shank, and W.D. Park. 2004. Development of DNA markers suitable for marker
assisted selection of three Pi genes conferring resistance to multiple Pyricularia
grisea pathotypes. Crop Sci. 44:1790-1798.
Germplasm Resources Information Network-Agricultural Research Service. 2009.
National Plant Germplasm System. [Online]. Available at http://www.ars-grin.
gov/npgs/searchgrin.html. (accessed Jan. 2010; verified Feb. 2010). United States
Department of Agriculture, Washington, D.C.
International Rice Research Institute and International Board for Plant Genetic
Resources. 1980. Descriptors for rice (Oryza sativa L.). Int. Rice Res. Inst. Los
Baños, Philippines.
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Jia, Y., Z. Wang, R.G. Fjellstrom, K.A. Moldenhauer, M.A. Azam, J. Correll, F.N.
Lee, Y. Xia, and J.N. Rutger. 2004. Rice Pi-ta gene confers resistance to the major
pathotypes of the rice blast fungus in the United States. Phytopathology 94:296301. DOI: 10.1094/PHYTO.2004.94.3.296.
Sharma, A., A. McClung, S. Pinson, J. Kepiro, A. Shank, R. Tabien, and R. Fjellstrom. 2009. Genetic mapping of sheath blight resistance QTLs within tropical
Japonica rice cultivars. Crop Sci. 49:256-264.
Xin, Z., J.P. Velten, M.J. Oliver, and J.J. Burke. 2003. High-throughput DNA extraction method suitable for PCR. BioTech. 34:820-826.
Table 1. WC and Phenotype Bay entries segregating at five
loci tested. (Some entries were segregating at multiple loci.)
Pi-ta
Pi-b
Pi-z
Waxy
sd1
13
17
14
11
13
9
24
14
11
10
(no. of entries)
WC (307 total)
PB (45 total)
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