Introduction
Perennial ryegrass (Lolium perenne L) is an important turf species. It has a fast establishment rate, strong seedling vigor, good tolerance to both traffic and low mowing which makes it a good choice for use on golf course fairways and athletic fields. However, perennial ryegrass has a poor ability to survive in the severe winter, which limits its use in the far northern areas of United States, including Iowa. One important breeding objective for perennial ryegrass is to improve its winter hardiness.
Winter hardiness is a complex quantitative trait that is
controlled by multiple genes with each having minor genetic effect. In
addition, the expression of such genes is often affected by the environment,
which makes it difficult to identify such genes. With the development of DNA
marker techniques, it is now possible to locate these genes (quantitative trait
loci, QTLs) that are associated with winter hardiness. There are abundant DNA
marker variations present in natural population; some of these markers are in
the same chromosome as the genes responsible for winter hardiness. These
markers often transmit together with the winter hardiness genes into their
progenies. The DNA markers are stable and relatively easy to identify compared
to winter hardiness genes that are influenced by the environment and difficult
to identify with classic genetics. The long-term goal of this project is to
facilitate germplasm improvement of perennial ryegrass with enhanced winter
hardiness through marker-assisted selection (MAS). The specific objectives of
this research are to identify the QTLs that are associated with winter
hardiness in perennial ryegrass.
Materials and methods
Plant materials:A segregating population of 174
genotypes was created by crossing a perennial ryegrass cultivar ‘Manhattan’ with an annual ryegrass cultivar ‘Floregon.’ While Manhattan has good winter
hardiness, ‘Floregon’ is very sensitive to winter killing. This population was
maintained in our research greenhouse at 20-21°C and was fertilized with
Miracle Gro to prevent nutrient deficiency. Irrigation was provided as needed.
In May 2003, four clones of each genotype were planted in the field within each
replication in an a lattice design with
three replications. The distance between individual clones of a genotype is 30
cm, and the distance between each genotype is 60 cm. The distance between rows
is 90 cm.
Data collection
Fall regrowth: Plants were mowed on July 17, August
14, and September 22, respectively. Fall regrowth was measured as the vertical
height of regrowth in centimeters on November 14. Fall regrowth may be
correlated with winter hardiness.
Freezing tolerance: Freezing tolerance was assessed
by measuring ion leakage. One clone of each genotype was removed from the
field on November 30, 2003, and was placed into a sealed plastic bag with a wet
paper towel in a corner and then stored at a 4 °C in a walk-in cold room.
Individual tillers of similar size were separated from sampled plants, and were
then washed quickly in deionized distilled water to remove the soil and blotted
dry in a paper towel. The aerial parts and roots were trimmed to 2 cm and 0.5
cm respectively. For each genotype, 18 individual tillers were prepared and
then placed into glass tubes (16 x 125mm) with 2 tillers in each tube. Eight
different temperature treatments (-6°C, -10°C, -14°C, -18°C, -20°C, -24°C,
-28°C, -32°C) were applied to every two trimmed tillers using a ScienTemp programmable
freezer (Model:8.5-3.1), and the 4°C treatment was used as a control. The
freezer was first equilibrated at -2°C, -3 °C and -4 °C for 30 minutes,
respectively. The duration at each test temperature is 15 minutes. The
temperature was cooled at a rate of 2°C per hour until -10°C when the
temperature started to lower at a rate of 4°C per hour until it reached -32°C.
Samples were taken out at each test temperature and thawed on ice overnight. The
conductivity of these samples was measured to calculate ion leakage. The freeze-treated
samples were infiltrated twice for 4 min each after adding 7ml ddH2O
and shaken horizontally for 1 hour at 250 rpm. The conductivity was then measured
with an YSI conductance meter (model 3403). Total conductivity for each sample
was determined by measuring the autoclaved samples. Percentage of ion leakage
was plotted as a function of the freezing temperatures. LT 50 value
was determined from the midpoint between the maximum and minimum (control) ion
leakage obtained for each genotype.
Winter survival: Winter survival was evaluated at the
end of April using a scale of l - 5 with 1 being completely dead and 5 being no
injury.
QTLs analysis: Data for the same characters will be
measured again in 2004. Once the second-year data becomes available, QTL
analysis will be conducted. First, a single-factor Analysis of Variance
(ANOVA) analysis will be computed for each pair wise combination of
quantitative traits and maker loci. The trait values of all individuals having
a marker will be compared with those without this marker by using an F test.
Then the interval mapping method will be used to find the more robust position
of QTL’s.
Contents ©1995-2004, ISU Horticulture Department
ISU Turfgrass:2004 Turfgrass Report
College of Agriculture