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The objectives of this experiment (Experiment 3) were:

1 . To assess the I x S mapping population for variation in seed yield and y ield related traits.

2. To identify major component traits for seed yield in perennial ryegrass. 3 . T o provide a data set for QTL analysis.

5.3 Materials a nd methods

5.3.1 Plant material and experimental design

The experiment was carried out from July 2003 to February 2004 at the AgResearch Grasslands site in Palmerston North, New Zealand. Four cloned replicates of the I x S mapping population plants (n=200) and o f the two parents were transplanted to the field in a randomised complete block (RCB) design, one copy of each plant per replicate. At transplanting in August 2003, plants comprised 1 0- 1 5 tillers and a recommended spacing of 60 cm between plants was used (Bajwa et al. 1 996). The area occupied by the experiment was 302.4 m2

Chapter 5 Phenotypic assessment for variation in seed yield component traits in a perennial ryegrass population

5.3.2 Agronomic practices 5.3.2.1 Soil analysis

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The soil at the experimental site is alluvial Kairanga silt loam (Cowie 1 978), and analysis by E-Iab. Limited, Hamil ton, New Zealand, indicated the following nutrient levels: Calcium, 8 (MAF QT); Olsen Phosphate, 44 (�g/ml); Potassium, 8 (MAP QT); Sulphate Sulphur, 7(ppm); Magnesium, 22 (MAF QT) and Sodium, S (MAF QT). The pH was 5 . 5 . Based on these results, l ime and superphosphate fertil iser were appl ied at 3 tonnes/ha and 300kg/ha, respectively, 2 weeks after plants were transplanted to the field.

5.3.2.2 C u ltural practices and tech nologies

N itrogen appl ication from 1 50 kg/ha to 250 kg/ha has been reported to increase ryegrass seed yield to over 2000 kg/ha ( Rolston and McCloy 1 997; Cookson et at. 2000). In this experiment, a total nitrogen appl ication of 200 kgN/ha was appl ied as urea in three equal splits on 1 st September, pt October and 1 5th October.

Weeding was carried out in October, 8 weeks after transplanting. The base ( I 8 cm radius) of each clone was first weeded manual ly using a hand hoe, and the following herbicides were then applied; 2,4-D Ester (3 Llha) to kill broad weeds and Dicamba (2 Llha) to kill clover (Figure 5 . 1 ). Buster (glufosinate ammonia) (3 Llha) was appl ied with a roller between plants to kill other weed species. These herbicides provide effective control of weeds, but have no effect on ryegrass seed yield or seed quality ( Ivany et at. 2002).

The plant growth regulator Moddus® trinexapacethyl (TE) was appl ied at 800mLlha at first head emergence (5th November) and then 3 weeks later to prevent plants from

lodging. Moddus retards stem elongation by inhibiting hydroxylation of the growth inactive form of gibberell ic acid to the active form (King et at. 1 997). This results in stem shortening of reproductive tillers and increases their efficiency through increased

S tem rust (Puccinia graminis) infestation was observed on some plants after heading and Systhane 1 25 was applied at 20mlsl l OOL of waterlha in December 2003 and January 2004 to control rust.

Figure 5. 1 : Weed control in I x S perennial ryegrass mappi ng population plants grown as spaced-plants in the field in 2003 .

5.3.3 Data collection 5.3.3. 1 Plant development:

To monitor plant development, the following data were recorded:

Tiller number (STN): During September 2003, tiller numbers per plant were estimated by dividing the base of a plant into four equal portions and counting tiller number for one portion (selected at random), and multiplying by 4.

Heading date (DH)): This was recorded as the number of days from transplanting, to when three heads were visible on each plant. This measurement was repeated in 2004 and expressed as days after 3 1 st October.

Spread of heading (SOH): The number of days between the emergence of the first and

Chapter 5 _{Phenotypic assessment for variation in seed yield} component traits in a perennial ryegrass population

66

Plant growth habit (PGH) : This was recorded after heading using a subjective scale of

1 to 9 ( 1 =erect, 3= semi erect, 5=medium, 7= semi-prostrate and 9=prostrate) .

5.3.3.2 Spike characteristics:

Three spikes were harvested randomly from each plant 7 weeks after heading, placed in sealed plastic bags and kept in a refrigerator. The following were l ater recorded and averaged for the three selected spikes:

Spike length (SL): Average length (cm) measured from the first node to the tip.

Spikelets per head (SH): All spikelets on each spike were counted.

Florets per spikelet (FS) : The number of florets on the bottom, middle and top spikeiets of each spike were counted and averaged.

Florets per head (FH): estimated by multiplying values obtained for spikelets per head and florets per spikelet, as described above.

5.3.3.3 Seed yield per pla nt:

Plants were harvested with hand-shears to about 10 cm above ground level between 6 and 26 January 2004 as seed heads ripened. All harvested tilIers from a plant were put into an opened paper bag and stored in a field laboratory on tables for drying. After 3 weeks at ambient temperature, the folIowing data were colIected:

Reproductive tiller number (R T): All harvested tilIers that produced heads.

Tillers with matured heads (TMH) : Tillers with matured and ripened seed heads, indicated by brown or gold coloured florets and expressed as percentage of total reproductive tilIer.

Seed yield per plant (Sd YP) : Weight of seed (g) per plant measured after drying, threshing and cleaning o f seeds. Seed cleaning was done mechanicalIy using a South Dakota Seed Blower. The scale on the machine was set at 4.5 to clean seed to a TSW of over 1 .3g. Setting above 4.5 cleaned seeds to a TSW above 1 .6g which would have resulted in losing saleable seeds. The air flow rate of the machine at the 4.5 scale was measured with a LeA 6000 Airflow anemometer (Airflow Developments Ltd., England). The cross sectional area of the air stream with average velocity of 3 .86m1s was calculated to 0.00053m2 and the air flow rate was calculated to 0.002m3/s. Seed moisture content was determined (1ST A 2004) after seed cleaning on 1 00 seeds per

genotype for four genotypes using the high constant oven method at 1 300e (±2°C) for one hour. The average moisture content was 1 1 %.

Floret site utilization (FSU) : Calculated as the number of saleable seeds per plant

divided by the number of florets counted per plant post anthesis (Elgersma 1985 ).

This was determined by the formula:

FSU = (SdYP (g) x 1 000 / TSW (g))/ (number of spike per plant x FH) (2)

Seed yield per head (mg): This was calculated as fol lows:

Seed yield per head (SdYH) = ( 1 000 x SdYF)/ number of reproductive tillers with

mature heads per plant.

Seed weight (TSW): This was measured as 1 000 seed weight. Two samples of 1 00

filled seeds were randomly selected from each plant from three replicates, weighed, and the average weight multiplied by 1 0 to calculate TSW.

Figure 5.2 : Processing seed samples of the [ xS perennial ryegrass mapping population plants grown as spaced plants in the field in 2003 .

Chapter 5 _{Phenotypic assessment for variation in seed yield}

component traits in a perennial ryegrass population

5.3 .3.4 Climatic factors

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Data on temperature, rainfall and sunshine during the period of experimentation were recorded at a weather station 600 m distant from the experimental plots (Table 5. 1 ).

Month Air Temp

July (2003) 1 2.7 August (2003) 1 4. 8 September (2003 ) 1 5 .3 October (2003) 1 6.4 November (2003) 1 7. 1 December (2003) 20.3 January (2004) 20.5 Mean 1 6.7 Air Temp 1 .6 4.7 6.3 6.2 8.8 1 1 .6 8.4 6.8 Soil Temp 5.9 7.9 1 0.3 1 3 .2 1 6. 1 1 5. 8 1 1 .5 Rainfall 83.7 27.9 1 5.3 89.9 8 1 .0 80.6 27.9 58.0 Sun 5 . 5 5.5 4.3 4.3 4.4 6.9 5.2

Table 5.1 : Mean monthly temperature, rainfall and sunshine during July 2003 to January 2004

5.3.4 Data a nalysis

Analysis of variation was performed usmg GenStat version 8. 1 (Genstat 2005) to identify significant differences between genotypes and replicate structure for all analysed traits. Broad sense heritability estimates were calculated for each trait as

Hb = (ig/( (j2g+(j2c/r)) (3)

Where (j2 g = genotypic component of variance, (j2 c = residual variance of genotypes and

r = number of replications (Burton and De Vane 1 953). Principal Component Analysis

( PCA) in MINIT AB was also performed to identify patterns of correlation between measured variables.