G.L. McMichael, J.K. Eglinton, A.R. Barr and K.J. Chalmer

Barr, A.

Chalmers, K.

Eglinton, J.

McMichael, G.

Adelaide Research &amp; Scholarship

DEVELOPING MARKER-TRAIT ASSOCIATIONS IN THE POPULATION CHIEFTAN/BARQUE//MANLEY/VB9104, USING NOVEL TECHNIQUES  G.L. McMichael1, J.K. Eglinton1, A.R. Barr2 and K.J. Chalmers3  1School of Agriculture and Wine, The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia 2Australian Grain Technology, The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia 3Cooperative Research Centre for Molecular Plant Breeding (CRC-MPB), The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia  INTRODUCTION Genetic mapping in cereals has been exclusively applied to populations derived from simple crosses.  Genetic studies are now beginning to target other population structures to take advantage of association mapping and whole genome analysis techniques. This paper describes an experiment in which 841 double haploid (DH) lines derived from the complex cross Chieftan/Barque//Manley/VB9104 are being genotyped with microsatellite (SSR) markers. The germplasm used in this experiment is a significant departure from typical mapping population structures. Phenotypic information is available for all 841 DH lines. More extensive phenotypic data is available for sub groups of these lines that advanced to successive stages in the breeding program. This population has been extensively phenotyped for malt quality and adaptation characteristics, through evaluation as a mainstream breeding population within the South Australian Barley Improvement Program (SABIP).    MATERIALS AND METHODS Germplasm Initial crosses between Chieftan and Barque, Manley and VB9104 were made, resulting in ‘single cross’ F1 plants. These F1 plants were then intercrossed, and 841 DH lines were derived from 9 different intercross F1 plants. The number of these DH lines generated per plant ranged from 4 to 332 with 206 lines and 332 lines being the two largest sets.   Phenotyping 837 of the 841 DH lines were evaluated in double row trials in 1999, all lines were harvested, data collected on screenings and quality traits were assessed by near infra-red spectroscopy (NIR).  350 selected individuals were promoted to stage 1 yield trials in 1999, grown as 1 replicate at 3 sites, with 7 cultivars as grid checks. Agronomic observations were recorded, yield measured and IOB wet-chemistry quality data obtained.  Under the same growing conditions as for stage 1 trials, 70 individuals were advanced to stage 2 yield trials in 2000. Data was collected as in stage 1 but with more detailed quality analyses.  Ultimately 10 individuals were advanced to stage 3 yield trials in 2001.   Genotyping The DNA was extracted based on a standard lab protocol using ball bearing method to grind the frozen leaf material. An SSR based marker system was chosen to genotype this population using tagged and untagged primers. These primers were designed for a touchdown PCR reaction with an annealing temperature of either 50o C or 55 o C.  Three tags FAM, HEX and NED were used to facilitate multiplexing, or simultaneous analysis of these markers.  Labelled samples are electrophoresed on the ABI 3100 and analysed with Genotyper software. Unlabelled SSRs were analysed on 8% acrylamide gels, then visualised by staining with ethidium bromide and photographed using a gel documentation system. Due to the size of the population a 384 well plate format was used in conjunction with the Corbett CAS-3800 pipetting robot.   RESULTS The minimum density of SSR markers for linkage map construction have been selected based on their map positions around known QTL and specific traits of interest (Table 1).  Table 1.  Quality scores and chromosome locations for barley varieties, Chieftan, Barque, Manley and VB9104.   Chieftan Barque Manley VB9104 Chromosome  Scald R S S MR 3H SFNB S R S MR 7H Mildew R MR S S 2H,4H Height SD T T T 3H Maturity L E L E 2H Leaf rust R S S S 5H,7H Lodging R MS  MS 2H Extract H L H M 1H,2H,5H DP M L VH M 1H,4H,5H Viscosity L H L MH 1H Fermentability M L VH M 4H,6H CCN(Ha4) S R S S 5H Yield M H L H 2H,3H,4H,7H Grain size SM ML M VL 2H ppd  sens  sens 2H eps  E  E 2H  SFNB – spot form net bloch; DP - diastatic power; CCN – cereal cyst nematode; ppd – photo period response; eps – earliness per se; R – resistant; S – susceptible; MR – moderately resistant; MS – moderately susceptible SD – semi-dwarf; T – tall; L – late; E – early; H – high; L – low; M – moderate; VH – very high; MH – moderately high; SM – small to medium; ML – medium to large; M – medium; VL – very large; sens - sensitivivity  226 markers were screened across the parental germplasm of which 134 were polymorphic (88 fluorescent labelled and 46 unlabelled).  20 of these 134 polymorphic markers allowed us to distinguish between each parental barley genotype in this study i.e. produced 4 alleles (Figure 1). The polymorphism distribution between the four parents is detailed in Table 2.  We have been successful in optimising multiple PCR reactions, thus allowing up to 8 markers per reaction to be analysed simultaneously on the ABI 3100 (Figure 2). This group of 8 markers is referred to as a “kit” and each kit is formed based on the primers’ tag and size of product generated.  For this study we have organized the 88 labelled markers into 11 kits.  68 SSR markers were chosen, to form the basis of an initial skeletal map (48 fluorescent labelled, 20 unlabelled).  To date, we have completed 4 kits, which have resulted in 24 labelled primers producing reliable results.  Of the 20 unlabelled SSR markers, 10 have been completed and analysed. From these initial results it appears that there up to six alleles present for some markers, rather than the maximum of four that would be expected.                       Figure 1.  Results of parental screen using a fluorescent labelled SSR marker which distinguishes between the four barley genotypes.                     Figure 2.   Multiplexing results with 8 SSR markers on parental genotype Barque.          225 230 235 240 245 250 255 260  BCMVKit2p1-4_A01_B…1.fsa  1 Yellow  1000200030004000  BCMVKit2p1-4_A02_B…0.fsa 20 Yellow  500100015002000  BCMVKit2p1-4_A03_B…6.fsa 36 Yellow  1000200030004000  BCMVKit2p1-4_A04_B…2.fsa 52 Yellow  100020003000Barque Chieftan Manley VB9104 110 120 130 140 150 160 170 180 190 200 210  BCMVK1p5-8_A01_BCM…1.fsa  1 Blue    500100015002000  BCMVK1p5-8_A01_BCM…1.fsa  1 Green   100020003000  BCMVK1p5-8_A01_BCM…1.fsa  1 Yellow  10002000Ned Hex Fam Table 2.  The polymorphism distribution between the four parents.  No. of markers No. of alleles Chieftan Barque Manley VB9104 12 2 A b b b 13 2 b a b b 13 2 b b a b 14 2 b b b a 3 2 a a b b 8 2 a b a b 2 2 a b b a 3 3 a b c b 6 3 a b c b 9 3 a b a c 3 3 a b c a 7 3 a a b c 6 3 a b b c 20 4 a b c d  The same letter indicates no polymorphism.  15 polymorphic SSRs are yet to be fully analysed an are not included.  DISCUSSION The full set of DH lines is similar to a very large conventional mapping population except that it is derived from one or more double-cross F1 plants and from four parents that are not all necessarily homogeneous and homozygous. From the results to date, some markers appear to have more than four alleles present, this may suggest that one, or more than one of these parents could be heterogeneous and/or heterozygous. Preliminary analysis suggests the additional marker alleles are derived from Barque.  Using additional SSR markers, a high density map will be generated on a sub group of the initial 837 DH lines, comprising the 350 stage 1 lines.  This will facilitate measurement of frequency changes to alleles in response to pragmatic selection, and to identify conserved linkage blocks associated with superior agronomic or quality phenotypes.  This analysis will determine the genetic basis of the elite line WI3408, which was a member of the largest family of 332 lines. WI3408 is currently being considered for commercial release, targeting medium-low rainfall production environments and premium export markets.  ACKNOWLEDGEMENTS The authors would like to thank the South Australian Barley Improvement Program, and Tim Pawlowski for their contribution to this project.  This work was supported by Australian farmers and the Commonwealth Government through the Grains Research & Development Corporation (GRDC).                                       

Developing marker trait associations in the population Chieftan/Barque//Manley/VB9104 using novel techniques

https://digital.library.adelaide.edu.au/dspace/bitstream/2440/37399/1/097McMichaelG.pdf

Developing marker trait associations in the population Chieftan/Barque//Manley/VB9104 using novel techniques

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Adelaide Research & Scholarship

Developing marker trait associations in the population Chieftan/Barque//Manley/VB9104 using novel techniques

Abstract

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Adelaide Research &amp; Scholarship

Adelaide Research & Scholarship