mapping of resistance genes in barley (hordeum vulgare l

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2015 Nansamba, Moureen Wageningen UR 3/26/2015 Mapping of resistance genes in barley (Hordeum vulgare L.) to oat stem rust pathogen, Puccinia graminis f.sp. avenae

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Page 1: Mapping of resistance genes in barley (Hordeum vulgare L

2015

Nansamba, Moureen

Wageningen UR

3/26/2015

Mapping of resistance genes in barley

(Hordeum vulgare L.) to oat stem rust

pathogen, Puccinia graminis f. sp.

avenae

Mapping of resistance genes in barley

(Hordeum vulgare L.) to oat stem rust

pathogen, Puccinia graminis f.sp. avenae

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Mapping of resistance genes in barley (Hordeum vulgare L.) to oat

stem rust pathogen, Puccinia graminis f. sp. avenae

Moureen Nansamba

MSc. Plant Sciences

Specialisation, Plant breeding and Genetic Resources

Reg No. 88122595120

MSc. Thesis

Supervisor: Rients Niks

26th

March 2015

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Abstract Previous studies have mapped resistance genes in barley (Hordeum vulgare L.), to several

heterologous rust fungi. The present study mapped resistance genes in three segregating

barley populations, Vada x SusPtrit, Cebada Capa x SusPtrit and SusPtrit x Golden Promise

to three Swedish single pustule isolates of the oat stem rust pathogen Puccinia graminis f.sp.

avenae. Parental lines Vada and Golden Promise were immune to all three isolates whereas

Cebada Capa was immune to Evertsholm and Pattala but incompletely resistant to Ingeberga.

SusPtrit was susceptible to Pattala and Ingeberga but resistant to Evertsholm, only showing

pinpoint flecks. Transgressive segregation was observed in all mapping population/isolate

combination except in CCxS/Evertsholm and SxGP/Evertsholm. Quantitative Trait Loci

analysis identified ten QTLs, Rpgaq1- to Rpgaq10 spread over five chromosomes. Rpgaq1

contributed by Vada in V/S population and Golden Promise in S/GP was effective to all three

isolates used, which may suggest isolate nonspecific resistance at that QTL. The two cultivars

Vada and Golden Promise had two resistance genes (Rpgaq1 and Rpgaq5) in common. Other

resistance genes Rpgaq3- to Rpgaq10 were isolate specific i.e. they were effective to one or

two isolates. Co-location of resistance genes mapped in this study with QTLs that have

previously been mapped to other heterologous rusts in the same mapping populations

suggests that genes such as Rpgaq1- to Rpgaq7 are effective to at least two heterologous rust

species. Microscopic examination showed that resistance to P.graminis f.sp. avenae, is only

prehaustorial in Vada whereas in SusPtrit both pre- and posthaustorial mechanisms play a

role.

Keywords: Barley, Puccinia graminis f.sp. avenae, Near non-host resistance, Quantitative

Trait Loci

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Table of contents Abstract.................................................................................................................. Error! Bookmark not defined.

1. Introduction ........................................................................................................................................................ 5

2. Materials and methods ........................................................................................................................................ 8

2.1 Plant material ............................................................................................................................................... 8

2.2 Oat Stem Rust Pathogen ............................................................................................................................... 8

2.3 Inoculation Procedure .................................................................................................................................. 9

2.4 Observations ................................................................................................................................................. 9

2.5 QTL mapping ............................................................................................................................................... 9

2.6 Fine mapping .............................................................................................................................................. 10

2.7 Histological determination of the mechanism of resistance ....................................................................... 11

3. Results .............................................................................................................................................................. 13

3.1 Genetic analysis of resistance ..................................................................................................................... 13

3.2 QTL analysis of mapping populations ....................................................................................................... 15

3.2.1 Vada X SusPtrit, VxS ......................................................................................................................... 15

3.2.2 Cebada Capa X SusPtrit, CCxS .......................................................................................................... 16

3.2.3 Golden Promise X SusPtrit, SxGP ...................................................................................................... 17

3.3 Comparison of the resistance QTLs mapped in the three segregating populations .................................... 18

3.4 Pre- and Post-haustorial resistance to P.graminis f.sp. avenae .................................................................. 21

4. Discussion and Conclusion ............................................................................................................................... 23

Acknowledgement ................................................................................................................................................ 26

References ............................................................................................................................................................ 27

Appendices ........................................................................................................................................................... 30

Appendix 1: LOD profiles of Vada x SusPtrit Population ............................................................................... 30

Appendix 2: LOD profiles of Cebada Capa x SusPtrit Population .................................................................. 31

Appendix 3: LOD profiles of SusPtrit x Golden Promise Population .............................................................. 32

Appendix 4: Effect of Rpgaq5 contributed by SusPtrit to three P.graminis f.sp. avenae isolates used in the

present study..................................................................................................................................................... 33

Appendix 5: Linkage map of Cebada Capa x SusPtrit ..................................................................................... 34

Appendix 6: Linkage map of SusPtrit x Golden Promise ................................................................................ 35

Appendix 7: Linkage map of Vada x SusPtrit .................................................................................................. 36

Appendix 8: Summary of resistance QTLs effective to three isolates of oat stem rust pathogen compared to

QTLs previously mapped to other heterologous rusts ...................................................................................... 37

Appendix 9: List of Primers ............................................................................................................................. 38

Appendix 10: Phenotype and genotypes of progenies of cross 152x110 (1H gene) ........................................ 39

Appendix 11: Phenotype and genotypes of progenies of cross 143x152 (7H gene) ........................................ 42

Appendix 12: Statistical analysis for Histology ............................................................................................... 45

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1. Introduction

Disease resistance shown by all genotypes of a particular plant species to a specific pathogen

species is known as non-host resistance. It is the most common form of resistance exhibited

by plants, hence its importance in science. To fungi such as rusts, non-host resistance maybe

manifested as a local necrosis, which is a rapid cell death at the infection site that is

associated with restriction of pathogen growth as well as defence gene activation (Goodman

and Novacky, 1994). Such type of necrotic reaction is called a hypersensitive response (Niks,

1987). The differentiation between host and non-host status is sometimes unclear (Heath

1985; Niks 1987). Within a presumed non-host plant species, there may exist a few

moderately susceptible genotypes to a normally heterologous pathogen (heterologous is used

for rusts that normally cannot infect barley). Such intermediate host status is referred to as

near- non-host or marginal host status (Niks 1987; Atienza et al., 2004).

Barley (Hordeum vulgare L.) has been reported to be a marginal host to several heterologous

rust pathogens (Mains 1933; Niks 1987; Niks et al., 1996; Atienza et al., 2004). This

marginal host status has been used to study the genetics of non-host resistance of barley to

heterologous rusts. In 2004, Atienza and colleagues, particularly developed SusPtrit to be

exceptionally susceptible to wheat stem rust, Puccinia triticina, by accumulating

susceptibility alleles from unrelated four barley accessions that were each somewhat

susceptible at seedling stage. The line turned out not only to be exceptionally susceptible at

seedling stage to P. triticina (for which it was selected) but also to at least nine other

heterologous rust species. Inheritance of resistance to P.triticina was found to be quantitative,

with transgressive segregation and the progenies of crosses from which SusPtrit was selected

showed continuous variation for level of susceptibility. These results were confirmed by

Jafary et al (2006) who reported that non-host resistance is due to quantitative trait loci, each

with a relatively low effect. So, the QTLs together result in immunity in the other parent.

Oat stem rust caused by Puccinia graminis f.sp. avenae – P.graminis f.sp. avenae is a major

constraint to oat (Avena sativa) production throughout oat producing continents including

Europe and Australia. For instance, Mellqvist and Waern (2010) reported yield losses of up to

30% in untreated fields compared to treated fields in Sweden. Sexual reproduction of the

stem rust fungus on the alternate host, barberry (Berberis vulgaris L.), allows recombination

of factors resulting in virulence and hence increase the ability of the pathogen to overcome

resistance in the host population (McDonald and Linde, 2002). Nonetheless, in the absence

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of barberry, the pathogen survives and reproduces on wild oats, volunteer oat plants and

certain grass species (Burdon et al., 1992). Changes in the pathogen populations in such areas

with no alternate hosts are caused by mutations, genetic drift as well as migration of

individuals. The ability of P.graminis f. sp. avenae to develop virulence for deployed

resistance genes in commercial oat varieties is of particular concern to oat breeders who now

seek more durable sources of resistance.

In barley, the oat stem rust pathogen can infect a few accessions at seedling stage (as

exception to the rule that barley is perceived to be a non-host), suggesting that barley is a

marginal host (Martens et al., 1983; Niks 1987; Niks and Dracatos, personal communication).

In early investigation, Martens and colleagues (1977) found a volunteer barley plant on which

P.graminis f. sp. avenae infections were first noticed. Later on, a P.graminis f. sp. avenae

susceptible progeny of that volunteer plant (line 73-G1) was crossed in diallel with two

immune regular barley accessions, Parkland and Wolfe (Martens et al., 1983). The

𝐹3 progenies were tested and evaluated with four races of oat stem rust pathogen. Resistance

to P.graminis f. sp. avenae was found to be conferred by a single dominant gene. In a recent

study, Dracatos et al., (2014) found five minor effect resistance genes (Rpga1- to Rpga5) in

the Yerong x Franklin doubled haploid population, which were effective in response to all

three tested diverse Australian pathotypes of P.graminis f. sp. avenae, hence suggesting non-

pathotype specific resistance. There is an infinite number of P.graminis f.sp avenae

pathotypes although only three were tested. In a preliminary experiment, SusPtrit was

susceptible to two field isolates of P.graminis f. sp. avenae (collected near Pattala and

Ingeberga in Sweden; Niks personal communication), but resistant to a single pustule isolate

of P.graminis f. sp. avenae collected at Evertsholm. This confirms that some isolates of

P.graminis f. sp. avenae are capable of establishing successful infections on SusPtrit while

other isolates are avirulent. However, isolates like Evertsholm that are avirulent on SusPtrit

can infect a few other barley accessions (Niks and Dracatos, personal communication).

Previous studies have found both hypersensitive and non-hypersensitive response

mechanisms in cereal-rust interactions (Jafary et al., 2006, Figueroa et al., 2013, Wang et al.,

2014, Dracatos et al., 2014). Near non host resistance in barley is primarily based on

prehaustorial resistance in which germination and subsequent development of the urediospore

germling is normal until penetration of plant cell walls (Niks, 1982, 1986; Heath, 2000;

Mellersh and Heath, 2003; Dracatos et al., 2014). If the rust fungus succeeds in penetrating

the cell wall and produces a haustorium, it may be arrested by a hypersensitive reaction

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which can be seen as a local necrosis on the plant tissue (Niks, 1987; Niks et al., 2007; Niks

and Marcel, 2009). The latter mechanisms of plant defence which terminate fungal growth

after cell wall penetration are referred to as post penetration / post-haustorial resistance.

The mechanism and genetics of non-host resistance in barley to oat stem rust is currently not

fully understood. Understanding the genetic basis of the specificity of P. graminis f. sp.

avenae may not only provide insight into how non host resistance is organised but also an

explanation for its specificity. It is important to know which and whether the same or

different genes confer resistance in resistant barley genotypes like Vada, Golden Promise and

Cebada Capa. SusPtrit has hence been used to develop a barley – Puccinia rust fungus model

to study the inheritance of non-host resistance in plants (Jafary et al., 2006; Jafary et al.,

2008, Yeo et al., 2014).

The objectives of this research were quadruple: First, to determine and map genes underlying

resistance against three single pustule isolates of P. graminis f. sp. avenae in barley obtained

from three locations in Sweden i.e. Pattala, Ingeberga and Evertsholm. Isolates Pattala and

Ingeberga are virulent on SusPtrit whereas the Evertsholm isolate is avirulent. Three regular,

hence immune cultivars (Vada, Cebada capa and Golden Promise) were the other parent of

the SusPtrit mapping population. Second, to compare the Quantitative Trait Loci (QTLs)

found for resistance to P.graminis f. sp. avenae in the three mapping populations with the

QTLs that have previously been mapped in response to other heterologous rusts. Third, to

determine whether the resistance mechanisms of Vada and SusPtrit to the Evertsholm isolate,

at cellular level are based on hypersensitive or non-hypersensitive resistance. Finally, use

available segregating barley populations for fine mapping of some of the largest-effect genes

for resistance that segregate in the Vada x SusPtrit mapping population.

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2. Materials and methods

2.1 Plant material

Three mapping populations are available (Table 1); Vada x SusPtrit, Cebada Capa x SusPtrit

and SusPtrit x Golden Promise as well as the parental lines of each mapping population and

Alfred oat variety were subjected to oat stem rust pathogen infection experiments. Alfred oat

was used as a control to confirm whether the oat stem rust pathogen infection was successful

and at a reasonable density. Two seeds per line were sown in plastic boxes and allowed nine

days to develop into seedlings before inoculation of the first leaf. Each experiment was

repeated twice.

Table 1. Plant and pathogen materials used in the study

Mapping

population

Type of

population No. of lines Isolate Resistant parent

Susceptible

parent Studied

Vada x SusPtrit

Recombinant

Inbred Lines 152 Evertsholm Vada & SusPtrit None Yes

Ingeberga Vada SusPtrit Yes

Pattala Vada SusPtrit Yes

Cebada Capa x

SusPtrit

Recombinant

Inbred Lines 113 Evertsholm

Cebada Capa &

SusPtrit None No

Ingeberga Cebada Capa SusPtrit Yes

Pattala Cebada Capa SusPtrit Yes

SusPtrit x Golden

Promise

Doubled

Haploid 122 Evertsholm

Golden Promise &

SusPtrit None No

Ingeberga Golden Promise SusPtrit Yes

Pattala Golden Promise SusPtrit Yes

2.2 Oat Stem Rust Pathogen

Field isolates of Puccinia graminis f. sp. avenae were collected from three different locations

in Sweden (Figure 1). These include; Pattala, Ingeberga and Evertsholm. Ingeberga and

Pattala field isolates produced pustules on SusPtrit. For both isolates, an isolated pustule on

SusPtrit was collected and multiplied on a susceptible oat variety, Alfred, in order to obtain

single pustule isolates that were virulent on SusPtrit. The Evertsholm single pustule isolate

was developed from a random single pustule on Alfred oat, and hence, was not selected to be

virulent on SusPtrit. Indeed, testing of this single pustule isolate showed it to be avirulent on

SusPtrit. During sporulation on susceptible adult oat plants, the three single pustule isolates

were each collected separately, weighed and stored in a desiccator at room temperature.

Surplus spores were transmitted to liquid nitrogen, for possible future use.

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2.3 Inoculation Procedure

Nine days after sowing, the first lower leaf of each seedling was pinned horizontally flat with

the adaxial side facing up. The seedlings of each mapping population were then inoculated

with freshly collected urediospores in a settling tower. For every box, containing 30-40

seedlings, 6mg of urediospores mixed with 48mg of lycopodium were applied to ensure

uniform distribution of about 360 urediospores per 𝑐𝑚2. After inoculation, the seedlings were

incubated in a humidity chamber overnight at 100% relative humidity and temperature of

about 17-18𝑜C to allow germination of the spores. On the next day, the plants were moved to

a greenhouse compartment. Only one isolate inoculation was performed per day in order to

prevent cross contamination of isolates.

2.4 Observations

About twelve days after inoculation, the level of infection of each seedling was quantified.

The data collected from each seedling included; the number of flecks (>0.5mm) and number

of pustules per leaf appearing on the adaxial leaf surface. The average level of infection over

the two seedlings per line was an indication of the susceptibility of the lines in each mapping

population. Collectively, the phenotypic data obtained from each mapping population was

used to map QTLs in that particular mapping population.

2.5 QTL mapping

Quantitative Trait Loci analysis was performed on the three mapping populations using

MapQTL 6 software (Van Ooijen, 2009) to investigate whether chromosome regions,

represented by markers were associated with resistance in barley to P.graminis f. sp. avenae.

To obtain a complete and even coverage of the barley genome, marker intervals of 5cM were

selected. For each mapping population / isolate combination, three methods were used to

detect significant QTLs: First, Interval mapping (IM) to detect putative QTLs which was

followed by selection of markers as cofactors to represent nearby significant QTLs to be used

in subsequent Multiple QTL Model (MQM) mapping. To reduce residual variance and

enhance the power to find other segregating QTLs, a second analysis with MQM mapping

was done using all indicated cofactor markers from the IM. Lastly, Restricted MQM

mapping using pre-selected cofactor markers was done as the final analysis to find the QTLs

conferring resistance to the oat stem rust pathogen isolates. During each analysis, a LOD

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score of three was used as the minimum threshold to select peaks in the LOD profile that

indicated significant QTLs. From the output of the QTL mapping analysis, the following

statistics were noted: Peak marker, position and LOD score of peak marker, support interval,

additive (which is the size of effect of the gene underlying the QTL), percentage of

phenotypic variation explained by the QTL (Vp), mean VIS values of individuals carrying

alleles from each parent (mu_A & mu_B) and which parent contributed the resistance allele.

2.6 Fine mapping

Two large effect resistance genes (in Vada x SusPtrit) effective to Evertsholm isolate were

identified: One on 1H group and the second one on 7H group. Several RILs in the VxS

population were susceptible when infected with the oat stem rust pathogen because they had

neither of the two resistance gene, one of them being RIL 152. We found that two previously

made crossings were useful (Table 2), as each was segregating for either the 1H or 7H gene.

Both crosses were between a resistant and susceptible RIL. Fine mapping of the two genes

has been started but the process will be completed in a future experiment. The 𝐹2 progeny

seed from the cross were sown in boxes and after nine days, the second leaf of each seedling

was collected in a separate micro-tube for DNA isolation. On the same day as leaf sample

collection, the first leaves of the seedlings were inoculated with the single pustule isolate,

Evertsholm (following the same inoculation procedure as in section 2.3). Twelve days later,

the segregating plants were phenotyped for visible infection sites.

Table 2. Fine mapping crosses

Cross Linkage group Resistance allele donor

152 x 110 1H Vada

143 x 152 7H SusPtrit

Prior to DNA isolation, two sets of SNP markers were developed: the first set consisted of 13

markers for the region of the resistance gene at 1H; the second set of consisted 10 SNP

markers for the region of the resistance gene at 7H. (Table 3). Markers were developed using

the SNP consensus map, in which for selected SNPs the markers flanking the SNP were

given. On those flanking markers (about 60 bp on either side of the SNP), primers were

developed (Appendix 9), and the amplification product was measured in the light-scanner, to

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visualise the pattern for Vada, SusPtrit and the heterozygous segregants. Markers

polymorphic between Vada and SusPtrit were selected. A few of the markers from each set

were tested on 96 plants of their respective crosses, and the genotypes read in a light-scanner

(Appendix 10 & 11). Recombinant plants for each cross were identified and transplanted.

Table 3. SNP markers and their positions on 1H and 7H linkage groups of barley as mapped

on the consensus map of VxS, CCxS and SxGP

Marker name Linkage group Position (cM) Polymorphic* between

Vada & SusPtrit BOPA2_12_31276 1H 41.931 Polymorphic SCRI_RS_116548 1H 42.428 Polymorphic SCRI_RS_232660 1H 42.753 Non-polymorphic SCRI_RS_193392 1H 55.312 Non-polymorphic BOPA1_409-1643 1H 58.686 Polymorphic BOPA2_12_30562 1H 61.160 Polymorphic BOPA2_12_10198 1H 65.323 Polymorphic SCRI_RS_156506 1H 67.715 Polymorphic BOPA1_5768-469 1H 74.772 Polymorphic SCRI_RS_204611 1H 79.114 Polymorphic BOPA1_12492-541 1H 89.942 Polymorphic SCRI_RS_139690 1H 103.271 Polymorphic BOPA2_12_31177 1H 51.673 Polymorphic BOPA1_7172-1536 7H 1.733 Non-polymorphic SCRI_RS_201028 7H 1.818 Polymorphic SCRI_RS_229445 7H 3.024 Non-polymorphic SCRI_RS_207095 7H 3.460 Polymorphic SCRI_RS_160297 7H 3.917 Polymorphic SCRI_RS_12396 7H 5.886 Polymorphic SCRI_RS_172655 7H 6.085 Non-polymorphic SCRI_RS_13615 7H 6.174 Polymorphic SCRI_RS_230959 7H 7.839 Polymorphic SCRI_RS_42792 7H 8.055 Polymorphic

* As observed by lightscanner trials (REN)

2.7 Histological determination of the mechanism of resistance

SusPtrit, Vada, Cebeco (oat), and Alfred (oat) were inoculated with Evertsholm isolate

(single pustule). About 6 seedlings per accession were inoculated. Six days after inoculation,

four leaf samples per accession, each about 3cm long, were collected. The two remaining

seedlings were used to check for the macroscopic infection types. The collected leaf samples

were then stained with Uvitex method (Niks 1982; Hoogkamp et al., 1998) to determine the

percentage of non-penetrating infection units, early abortion, established colonies and the

degree of hypersensitive reaction associated with the infection units (visible as auto-

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fluorescence). On average, 50 infection units were evaluated on each of the four leaf samples

under a Zeiss Axiophot photo microscope with an aniline blue filter. The infection units were

classified in five groups as described by Niks and Kuiper (1983). The groups included; Non

penetrating, early abortion (with less than six hyphae) without necrosis, early abortion

associated with necrosis, established colonies (when at least one infection hyphae had more

than six branches) without necrosis and established colonies associated with necrosis. The

experiment was repeated twice. The results of early abortion, per accession, are based on

approximately 50 infection units x 4 leaf samples x 2 replicates = 400 infection units. The

proportion of non-penetrant infection units was calculated by dividing the number of non-

penetrants with the total number of infection units. The proportion of the early abortion class

was obtained by dividing the number of early aborted infection units with the sum of

infection units that penetrated the stomata (early aborted + established).

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3. Results

3.1 Genetic analysis of resistance

Parental lines Vada and Golden Promise were completely immune to the three single pustule

isolates of Puccinia graminis f.sp avenae whereas Cebada Capa was immune to Evertsholm

and Pattala but incompletely resistant to the Ingeberga as each individual Cebada Capa plant

developed a few visible infection sites (VIS = flecks plus pustules ) upon infection with that

pathotype (Table 4). SusPtrit on the other hand showed high susceptibility to both Pattala and

Ingeberga but resistance to the Evertsholm isolate, which was manifested by several pin point

flecks of less than 0.5mm diameter (Table 4; Figure 2). Susceptible Alfred oat cultivar which

was used as a control developed more pustules than SusPtrit and any other susceptible line

forming part of the mapping populations used. The pustules on Alfred oat were difficult to

count because they tended to merge together. Transgressive segregation was observed in the

progeny of all mapping population/isolate combinations except with Evertsholm / CCxS and

/SxGP. Infection levels ranged from completely immune to highly susceptible (Figure 1).

This continuous quantitative variation is an indication of polygenic inheritance of resistance

to the oat stem rust pathogen isolates in the populations. However, all lines were in the CCxS

and SxGP mapping populations were resistant when infected with Evertsholm SP isolate,

suggesting presence of the same resistance gene or QTLs in both SusPtrit and Golden

Promise or Cebada Capa parents.

Table 4: Reaction of Parental lines to three Swedish isolates of P.graminis f.sp. avenae

Parental line Isolate Reaction of parents

Vada Evertsholm Immune

Pattala Immune

Ingeberga Immune

SusPtrit Evertsholm Resistant

Pattala Susceptible

Ingeberga Susceptible

Cebada Capa Evertsholm Immune

Pattala Immune

Ingeberga Partially resistant

Golden Promise Evertsholm Immune

Pattala Immune

Ingeberga Immune

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Figure 1. Segregation of lines in the Vada x SusPtrit population to Puccinia graminis f.sp.

avenae - isolate Evertsholm. A, Vada (immune). B, SusPtrit (resistant with several pinpoint

flecks). C, RIL 38 (large flecks) D, RIL 106 (flecks and pustules)

Figure 2. Reaction of SusPtrit to single pustule isolates of oat stem rust pathogen.

Evertsholm (Left), Ingeberga (middle) and Pattala (Right).

A B C D

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3.2 QTL analysis of mapping populations

Previously, mapping populations of VxS (152 recombinant inbred lines, RILs), CCxS

comprising of 113 RILs and SxGP (122 doubled haploid lines, DHs) were genotyped with

5020 single nucleotide polymorphism (SNP) markers. The position of each polymorphic

marker was inferred from recombinations (Yeo et al., 2014; Martin-Sanz, unpublished).

Relative marker positions were calculated to form an integrated / consensus map. QTL

analysis using phenotypic data was performed to determine which loci in the barley genome

represented by markers are significantly associated with resistance to the single pustule oat

stem rust isolates; Pattala, Ingeberga and Evertsholm.

Prior to the QTL analysis, values of VIS for the first and second replicate were adjusted by

multiplying or dividing by a factor to make the average values approximately the same, so

that both replicates would weigh equally heavily. The correlation coefficients between

replicates were also calculated to establish the reliability of the phenotypic data. For each pair

of replicates, a positive correlation of at least 0.7 was achieved (Table 5).

Table 5: Correlation between replicates of each mapping population/isolate combination

Population Pathotype Replicates Correlation coefficient

VxS Evertsholm 1&2 0.700

Pattala 1&2 0.788

Ingeberga 1&2 0.682

CCxS Pattala 1&2 0.741

Ingeberga 1&2 0.712

SxGP Pattala 1&2 0.860

Ingeberga 1&2 0.741

1&3 0.770

2&3 0.756

3.2.1 Vada X SusPtrit, VxS

In this mapping population, a total of five QTLs were found to confer resistance to the oat

stem rust pathogen isolates used (Figure 3; Appendix 1; Appendix 7). One QTL mapped on

the short arm of 1H group (53cM) was effective to all three isolates whereas the remaining

four QTLs detected in this population were effective to two isolates. In addition, the

resistance alleles were contributed by Vada except for the QTL mapped at the top of the short

arm of 7H (6cM), where SusPtrit donated the resistance allele in response to both Evertsholm

and Pattala.

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Figure 3. Significant QTLs conferring resistance to three single pustule isolates of Puccinia

graminis f.sp. avenae in the Vada x SusPtrit population. mu_A and mu_B are mean VIS

values of RILs carrying alleles of SusPtrit and Vada parents respectively.

3.2.2 Cebada Capa X SusPtrit, CCxS

QTL analysis in the CCxS mapping population detected four resistance QTLs spread over

three barley chromosomes. (Figure 4; Appendix 5). Two separate QTLs were mapped on 6H

while the other two QTLs were each mapped separately on 2H and 3H. The QTL mapped on

2H was effective to both Pattala and Ingeberga while the QTLs on 3H and 6H were effective

to only Pattala or Ingeberga, There was a tendency for the 6H linkage group to have multiple

peaks (Appendix 2), which may suggest several small effect genes distributed in that

chromosomal region. At all four QTLs, the alleles for resistance were obtained from Cebada

Capa parent and susceptibility alleles from SusPtrit. (Figure 4).

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Figure 4. Significant QTLs conferring resistance to two single pustule isolates of Puccinia

graminis f.sp. avenae in the Cebada Capa x SusPtrit population. mu_A and mu_B are mean

VIS values of RILs carrying alleles of SusPtrit and Cebada Capa respectively.

3.2.3 Golden Promise X SusPtrit, SxGP

A total of three QTLs were mapped in the SxGP (Figure 5; Appendix 6) on genomic groups

1H, 2H & 6H. All three QTLs were effective to Ingeberga while only one QTL (1H) was

effective to Pattala. In addition to the QTL on 1H group, QTL analysis with average number

of pustules (AvPust) also detected a minor effect QTL on the short arm of 7H (Appendix 6)

effective to Pattala. Unlike the QTLs on 1H, 2H and 6H on which resistance originated from

Golden Promise, resistance to Pattala on 7H was contributed by SusPtrit parent (Figure 5).

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Figure 5. Significant QTLs conferring resistance to two single pustule isolates of Puccinia

graminis f.sp. avenae in the SusPtrit x Golden Promise population. mu_A and mu_B are

mean VIS values of double haploid lines carrying alleles of SusPtrit and Golden Promise

respectively.

3.3 Comparison of QTLs mapped in the three segregating populations

First, for each mapping population (VxS, CCxS and SxGP), the QTLs for resistance to the

two or three (in the case of VxS) isolates of P. graminis f.sp avenae were mapped in their

respective biparental linkage map (Figure 3; Figure 4; Figure 5). In the next step, the QTL

positions in the individual linkage maps were converted to the positions on the integrated

map of Aghnoum et.al (2010) (Figure 6). Unfortunately, the markers mapped in the

population of SxGP were not mapped in the integrated map used. Nonetheless, fair

calculations of the positions of QTLs in SxGP were made with the use of the consensus map

(including VxS, CCxS and SxGP) and the VxS map by Jafary et al. (2006). Resistance QTL

regions of the three isolates and / or in different populations were considered to be same if

there was overlap between their LOD-1 confidence intervals on the integrated map. In total,

there were ten chromosomal regions associated with resistance to at least one isolate of

P.graminis f.sp. avenae (Figure 6). Five resistance QTLs were effective to only isolate and

four were effective to two isolates. Only one QTL was effective to all the three isolates.

Seven out of the ten QTLs were found in only one of the three populations. Three QTLs

mapped in two populations: QTL Rpga1 mapped on 1H in V/S and S/GP was effective to all

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three isolates; QTL Rpgaq2 on the 2H was found in V/S and CC/S and was effective to two

isolates, Ingeberga and Pattala; QTL Rpgaq5, donated by SusPtrit, at the top of the short arm

of 7H group was effective to isolates Pattala and Evertsholm in V/S and S/GP (Figure 6).

This may suggest that the resistance to oat stem rust pathogen in V/S & CC/S and V/S &

S/GP at such mapped loci on 1H, 2H and 7H is due to the same gene, which is effective to

two or three (in the case of Rpga1) isolates.

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Figure 6. Locations of QTLs associated with resistance to three Swedish isolates of oat stem rust pathogen-Puccinia graminis f.sp avenae in three barley mapping

populations, VxS, CCxS and SxGP. This integrated map was constructed by Aghnoum et al.,2010. Initially, the QTLs for each mapping population were mapped in the

respective biparental linkage map. The length of the coloured solid bars indicates the LOD-1 confidence intervals (with their corresponding peak markers in the same colour)

while the QTL lines are extended to the LOD-2 confidence intervals. On each QTL label is the name of the parental accession which contributed the resistance allele, LOD

score value from rMQM and letters that represent the isolates of P.graminis f.sp. avenae (I for Ingebrga, P for Pattala and E for Evertsholm) . The distances on the ruler are in

centimorgans.

Vada Cebada Capa Golden Promise

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3.4 Pre- and Post-haustorial resistance to P.graminis f.sp. avenae

Microscopic examination of P.graminis f.sp. avenae infected leaf samples of barley

accessions, Vada and SusPtrit and two oat accessions, Cebeco and Alfred revealed that the

spores of were able to geminate, find stomata, produce appressoria, substomatal vesicles and

hyphae (Figure 7). However, some sporelings did not penetrate the stomata (Figure 7A). The

percentage of non-penetrating infection units varied from 20% to 77%, with Alfred oat

having significantly more non penetrant units than the other three accessions (Table 6).

Immunity in Vada was mainly due to prehaustorial resistance (Figure 7B), with relatively

15% of the penetrated infection units getting established (Table 6). Necrosis did not play a

role in Vada immunity but contributed to resistance in SusPtrit, with at least 64% of the

established infection units being associated with necrosis (10.19µm) (Figure 7E). Such

hypersensitive reaction associated with established infection units is called post-haustorial

resistance. Large colonies of established infection units, with diameter of at least 84µm,

covered the leaves of susceptible oats, Alfred and Cebeco (Figure 7D). Established colonies

on susceptible oats were much larger than the established colonises observed on either

resistant barley accession (Table 6).

Table 6. Average proportion of infection units of P.graminis f.sp. avenae at three

development stages on two barley lines and two oat lines. The values in brackets are average

proportions of early aborted sporelings associated with necrosis.

Colony diameter

(µm)

Rep Accession Phenotype Non-pen EA (+N) EST(+N) (-N) (+N)

1 Vada Immune 0.32a 0.85 (0)a 0 7.12 ….

1 SusPtrit Resistant 0.28a 0.69(0.04)b 0.64 8.31 11.4

1 Alfred

Very

susceptible 0.77b 0.11 (0)c 0 85.64 ….

1 Cebeco

Very

susceptible 0.35a 0.10 (0)c 0 77.12 ….

2 Vada Immune 0.27a 0.84 (0)a 0 6.33 ….

2 SusPtrit Resistant 0.2a 0.4 (0)b 0.71 9.13 8.97

2 Alfred

Very

susceptible 0.57b 0.05 (0)c 0 84.64 ….

2 Cebeco

Very

susceptible 0.42a 0.03 (0)c 0 84.72 …. Non-pen stands for non-penetrating infection units. EA stands for Early abortion, EST stands for

Established colonies. (+N) = Diameter of established colonies associated with necrosis. (-N) = size

of established colonies not associated with necrosis. Colony diameter was measured at magnification

of 40x. Rep stands for replicate. Values in each column followed by the same letter are not

significantly different (P ≤ 0.05) according to Duncan’s multiple range test.

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Figure 7. Development stages of Puccinia graminis f.sp. avenae - Evertsholm Isolate. A,

Non-penetrating infection unit on Alfred oat. B, Early abortion without necrosis on Vada. C,

Early abortion with necrosis on SusPtrit. D, Macroscopic Established colony without necrosis

on Cebeco oat. E, Macroscopic Established colony with necrosis on SusPtrit.

A

E

D

B

C

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4. Discussion and Conclusion

Previous studies have mapped resistance genes / QTLs in barley to several heterologous rust

fungi (Jafary et al., 2006, 2008; Dracatos et al., 2014). Results from these studies have shown

a high diversity of non-host resistance genes to unadapted Puccinia pathogens with different

but also overlapping specificities. To my knowledge, the present study is the second to report

on the mapping of resistance genes in barley to the oat stem rust pathogen after a recent

publication by Dracatos et al (2014). Their research identified five minor effect genes

effective to three diverse Australian pathotypes of P.graminis f.sp avenae in the Yerong x

Franklin population. In the current study, QTL mapping detected a total of ten QTLs,

Rpgaq1- to Rpgaq10, which segregated for resistance to three Swedish isolates of P.graminis

f. sp. avenae in three mapping populations i.e. VxS, CCxS and SxGP. Such loci were found

spread over five barley chromosomes viz. 1H, 2H, 3H, 6H and 7H (Figure 6).

Rpgaq1 (1H) mapped in V/S and S/GP co-localises with Rpga1 and Rpga2 previously

mapped by Dracatos and his colleagues (2014) and were effective to all three P.graminis f.sp.

avenae isolates tested in the Yerong x Franklin population (Appendix 8). Likewise, Rpgaq1

was also effective to all three isolates (Pattala, Ingeberga and Evertsholm) used in this study,

which may suggest that resistance at this QTL is not isolate specific. Rpgaq1 occurs in

several barley accessions for example Vada, Yerong and Golden Promise but not in Cebada

Capa and Franklin Resistance of Rpgaq2 (2H) and Rpgaq4 (7H) could have also been isolate

non-specific because analysis of QTLs effective to Evertsholm in V/S revealed a hint (on 2H

& 7H), which was similar to the significant QTL peaks detected for Pattala and Ingeberga in

that population (Appendix 1). Failure to detect a significant peak could have been a matter of

experimental error and that the data was not good enough. Alternatively, it could be that in

the QTL analysis, the number of RILs showing susceptibility to Evertsholm may have been

too low to result in a significant LOD value, relative to the probably small effect of the gene.

Therefore, the smaller an effect, the more observations needed to obtain a significant

indication for such a gene. The remaining QTLs, Rpgaq3 and Rpgaq5 to Rpgaq10 were

effective to one or two isolates, which suggests isolate specific resistance of these resistance

genes.

Isolate specific resistance in the present research was also demonstrated by the different

reaction of SusPtrit to the oat stem rust pathogen isolates used. SusPtrit was susceptible to

Pattala and Ingeberga but resistant to Evertsholm. SusPtrit was selected to be exceptionally

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susceptible to rust fungi (Atienza et al., 2004), therefore its resistance to Evertsholm in the

current experiments was remarkable. In fact, the resistance gene Rpgaq5, (mapped on top of

the short arm of 7H) originated from the SusPtrit parent in both V/S and S/GP populations.

The SusPrit allele at this QTL reduced the number of visible infection sites for two isolates

but much more for Evertsholm (in VxS) and less for Pattala (Appendix 4). Such kind of

differential isolate specific resistance is called quantitative isolate specificity, in which the

resistance gene has a stronger effect against one isolate and a weaker effect against another

isolate but it is effective to both isolates. Rpgaq5 is not effective to Ingeberga in both VxS

and SxGP populations. Rpgaq5 could be the same gene reported by Jafary et al. (2008)

effective to wheat leaf rust. In both studies, SusPtrit is the donor of the resistance allele. Still

on 7H, Rpgaq4 contributed by Vada was mapped on the second half of the long arm of 7H.

Rpga4 may co-locate with similar loci that have been mapped by Jafary et al (2006, 2008) in

Vada in response to P.hordei and P.triticina.

Histological examination demonstrated that Vada is completely resistant to the oat stem rust

pathogen with a very high proportion of early aborted infection units which are not associated

with necrosis. Such non-necrotic resistance mechanism to oat stem rust pathogen is referred

to as prehaustorial resistance. On the other hand, resistance in SusPtrit was both pre- and

posthaustorial with early aborted infection units (without necrosis) and established colonises

associated with necrosis. The lack of resistance genes in Alfred and Cebeco oats makes them

very susceptible to P.graminis f.sp. avenae, which can be seen as large macroscopic pustules

the leaves. The difference in infection frequency on Alfred and Cebeco was due to the low

stoma penetration on Alfred.

In summary, the current study has shown that resistance to P.graminis f.sp avenae in barley is

polygenic, involving two or more minor effect genes, each with a small effect. So, the QTLs

together result in the relative immunity of Vada, Cebada Capa and Golden promise. This

polygenic resistance contrasts with the monogenic resistance reported by Martens et al.

(1983). Furthermore, quantitative resistance in V/S, CC/S and S/GP was indicated by

transgressive segregation, as some lines were more susceptible than SusPtrit. The current

research has illustrated that resistance to oat stem rust pathogen could be isolate specific, as

SusPtrit was resistant to Evertsholm and susceptible to Pattala and Ingeberga. Also, this

research has shown non-isolate specific resistance at certain QTLs (for instance, Rpgaq1) in

barley as previously demonstrated by Martens et al (1983) and Dracatos et al. (2014). The

co-location of resistance genes mapped in this study with QTLs that have previously been

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mapped to other heterologous rusts (Appendix 8) suggests that such genes are effective to at

least two rust species. Histological analysis showed that resistance to P.graminis f.sp. avenae,

is only prehaustorial in Vada whereas in SusPtrit both pre and posthaustorial mechanisms

play a role. For future research, the two resistance genes, Rpgaq1&Rpgaq5, effective to

Evertsholm could be fine mapped and this is work that is already underway. I would also

recommend investigation into barley adult plant resistance to P.graminis f.sp. avenae to see if

the same or different QTLs are involved in both seedling and adult plant resistance.

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Acknowledgement

I wish to extend sincere gratitude to my thesis supervisor, Rients Niks for his continuous

guidance, timely feedback and advice that has enabled me to learn so much from this

research. I acknowledge Peter Michael Dracatos for his remote supervision. I would also like

to thank Anton Vels for the technical support and Yajun Wang for the guidance rendered

during laboratory experiments.

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resistance to Puccinia hordei and of defence gene homologues. Theoretical and Applied

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Appendices

Appendix 1: LOD profiles of Vada x SusPtrit Population

Ingeberga Pattala Evertsholm

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Appendix 2: LOD profiles of Cebada Capa x SusPtrit Population

Ingeberga Pattala

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Appendix 3: LOD profiles of SusPtrit x Golden Promise Population

Pattala Pattala (AvPust) Ingeberga

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Appendix 4: Effect of Rpgaq5 contributed by SusPtrit to three P.graminis f.sp.

avenae isolates used in the present study.

Isolate Marker name LOD mu_A mu_B % Expl. Additive

Evertsholm E37M50-435-61 5.37 5.50626 43.1783 13.4 -18.836

Pattala E37M50-435-61 5.04 15.2728 25.0837 8.8 -4.9

Ingeberga E37M50-435-61 0.08 44.89 47.46 0.2 -1.29

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Appendix 5: Linkage map of Cebada Capa x SusPtrit

Ingeberga Pattala

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Appendix 6: Linkage map of SusPtrit x Golden Promise

Pattala Ingeberga

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Appendix 7: Linkage map of Vada x SusPtrit

Pattala Ingeberga Evertsholm

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Appendix 8: Summary of resistance QTLs effective to three isolates of oat stem

rust pathogen compared to QTLs previously mapped to other heterologous rusts

Linkage group Position (cM) Proposed QTL

name

QTLs mapped previously to other

heterologous rusts

1H 48 - 68 Rpgaq1 Rpga1 & Rpga2 (Dracatos et al 2014)

2H 93 – 118

131 - 156

Rpgaq2

Rpgaq9

P.triticina (Jafary et al ., 2006)

LP_Rpcq5 (Niks et al., 2015)

Rphq2 (Jafary et al., 2006, 2008)

3H 130 - 145 Rpgaq6 Rpcq6 (Niks et al., 2015)

6H 39 – 67

49 – 76

77 – 111

72 - 107

Rpgaq3

Rpgaq7

Rpgaq8

Rpgaq10

P.graminis f.sp. tritici and P.graminis

f.sp. lolii) and P.triticina. (Jafary et

al.,2006)

Rphq3 (Jafary et al., 2008)

7H 0 – 43

103 – 132

Rpgaq5

Rpgaq4

P.triticina (Jafary et al.,2008)

P.hordei and P.triticina (Jafary et

al.,2006, 2008)

Rpsnhq2 (Niks et al., 2015)

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Appendix 9: List of Primers

Linkage group Name primer Sequence ( in 5'----> 3' order)

1H BOPA2_12_31177-F ATCATAGCAGGAGGCCAGAGG

1H BOPA2_12_31177-R AGGTTGGAACACCCCCTGT

1H SCRI_RS_116548-F GATGGAGTGACCTGTTAGACTGCA

1H SCRI_RS_116548-R ATGGCTCTTCAAGAACGTCATG

1H BOPA2_12_31276-F GTCTGCGCCATGACAGCC

1H BOPA2_12_31276-R CGCTGTTCTCTTTGCACTCATAG

1H SCRI_RS_232660-F GATCAAGCTGTTGCTGCAGC

1H SCRI_RS_232660-R ATAACAAGAATGCTAACTCCAGAGTTT

1H SCRI_RS_193392-F ATGAACTAATATTGCATCTAGACAACTTAC

1H SCRI_RS_193392-R ACTAAATGCAATTCTGTACCCATTATAG

1H BOPA1_409-1643-F CTTGAGGGCTTCACTCACAGTG

1H BOPA1_409-1643-R GCCCGGATATGCCATGCT

1H BOPA2_12_30562-F GCTGCTCATGTTATCCAATCTTG

1H BOPA2_12_30562-R GCAGGAACATGCCGGCTG

1H BOPA2_12_10198-F GAGTTCAGCAGCTTCAGCTGTAC

1H BOPA2_12_10198-R AGCACCTTCTACCGCTCCATC

1H BOPA1_5768-469-F TGTTCAAGCAAATATCACAGTCTCA

1H BOPA1_5768-469-R CATTGCTAACATCAGAAGGTGGA

1H SCRI_RS_204611-F ATCGAAGACCGAAAGTATTCGAG

1H SCRI_RS_204611-R GAGTAGGACTGGGAGATGCTAGTG

1H BOPA1_12492-541-F CACCATCAACGTTACACGGAAC

1H BOPA1_12492-541-R GTGTGTTAGTGTGAGGATGGTGAA

1H SCRI_RS_139690-F ATTGTTGGCACCCATAAAAAGTC

1H SCRI_RS_139690-R CCACTGGAACCAACCAAAAGA

1H SCRI_RS_156506-F CTCTTTCCTGAGCTTGTATAACATGT

1H SCRI_RS_156506-R CATGTTCGGCATGAGGCCT

7H BOPA1_7172-1536-F TAAGAAGCAGCTGATAAGCTTGATT

7H BOPA1_7172-1536-R ACGGCCAACTAGCAGCTAGTC

7H SCRI_RS_229445-F AACCGGCACTACCCTGAAATTA

7H SCRI_RS_229445-R AAGCACTAGAGGACTTCATCCAGTT

7H SCRI_RS_12396-F TGGTAGAAACATACACAAAGTTGTACTACT

7H SCRI_RS_12396-R CGTCCCAAAATAAGTGGCTCA

7H SCRI_RS_230959-F CGGAGGAATCGAGGATCGTA

7H SCRI_RS_230959-R GCTGGATCTGTGCCTTTGGT

7H SCRI_RS_42792-F GATCAGTTGGGAAAGCACACAA

7H SCRI_RS_42792-R GTGCATCTGTAGGTTCCTATGCTAA

7H SCRI_RS_172655-F GGCTCCTGGTGCACTATGGA

7H SCRI_RS_172655-R GACGAACCGCCTTGCTCA

7H SCRI_RS_13615-F CAAGCTGAAGAACCTCGCC

7H SCRI_RS_13615-R ATGGCAAAGTCCGCCCAG

7H SCRI_RS_207095-F CGCTGGCACGGGCCTCT

7H SCRI_RS_207095-R CTCCACTGGGGCATGTGG

7H SCRI_RS_160297-F AACGTGGTTGATTACAAACTGATCT

7H SCRI_RS_160297-R ACAGTAAAAATTATGGAGTCCACTGATATA

7H SCRI_RS_201028-F AGGCATTCAAGAGCTACCTTGAG

7H SCRI_RS_201028-R TCCTACAGCAGCATGGTCGTC

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Appendix 10: Phenotype and genotypes of progenies of cross 152x110 (1H gene)

MARKER NAME ( POSITION)

Plant

no.

Phenotype

(VIS)

BOPA2_12_3127

6 (41.9)

BOPA2_12_3117

7 (51.7)

BOPA1_409-

1643 (58.7)

BOPA1_12492

-541 (89.9)

2˗1 0 v v v u

2˗2 41 h h h h

2˗3 0 h h h v

2˗4 5 h h h h

2˗5 14 h s s h

2˗6 3 s h h s

2˗7 10 h s s s

2˗8 0 s h h s

2˗9 2 h h h v

2˗10 0 h

h h

2˗11 7 s

s s

2˗12 1 s h h h

2˗13 13 s s s h

2˗14 18 h h h v

2˗15 3 h h h v

2˗16 40 h h h v

2˗17 21 h

h h

2˗18 15 h h h v

2˗19 5 s s s h

2˗20 12 h

h h

2˗21 24 h h h v

2˗22 16 s s h h

2˗23 0 v v h h

2˗24 0 v v v h

2˗25 0 h

h h

2˗26 25 h h h v

2˗27 5 h

h h

2˗28 7 h u h v

2˗29 0 h h h v

2˗30 38 s s s h

2˗31 4 h

h h

2˗32 1 h v v v

2˗33 2 v

v v

2˗34 1 h

h h

2˗35 18 s

s s

2˗36 22 s s s v

2˗37 0 h h h s

2˗38 1 h h h v

2˗39 12 h v h

2˗40 2 v h v

2˗41 4 v h v

2˗42 59 s

s s

2˗43 0 h h h s

2˗44 4 h v h

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40

2˗45 26 h h h v

2˗46 7 v

v v

2˗47 24 s

s s

2˗48 0 v

v v

2˗49 15 v h h h

2˗50 13 s

s s

2˗51 1 v

v v

2˗52 6 h

h h

2˗53 9 h h h v

2˗54 24 h h h s

2˗55 0 v v v h

2˗56 23 h

h h

2˗57 1 v

v v

2˗58 21 h

h h

2˗59 2 h

h h

2˗60 8 h

h h

2˗61 0 v v h h

2˗62 48 s s s v

2˗63 18 v h v

2˗64 0 v

v v

2˗65 1 v v v h

2˗66 0 v

v v

2˗67 1 h

h h

2˗68 1 h h h v

2˗69 14 h h h s

2˗70 10 h h h v

1˗1 2 s h s

1˗2 7 u v h

1˗3 2 s

s s

1˗4 2 s s h h

1˗5 11 s s s v

1˗6 1 h v h v

1˗7 9 s u s v

1˗8 1 h s h

1˗9 2 u

s s

1˗10 5 u

h h

1˗11 16 u

h h

1˗12 0 v v v h

1˗13 8 s

s s

1˗14 10 h

h h

1˗15 11 h

h h

1˗16 5 v u u h

1˗17 5 s u s v

1˗18 5 s u s v

1˗19 4 v v v h

1˗20 6 v h h h

1˗21 35 h

h h

1˗22 4 u s h

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1˗23 6 u

u v

1˗24 0 h

u h

1˗25 2 u

h

1˗26 0

s

1˗27 25

1˗28 7

1˗29 4

1˗30 1

1˗31 13

1˗32 6

1˗33 7

1˗34 4

1˗35 9

1˗36 1

1˗37 2

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Appendix 11: Phenotype and genotypes of progenies of cross 143x152 (7H gene)

MARKER NAME (POSITION)

Plant

no.

Phenotype

(VIS) SCRI_RS_201028 (1.818) SCRI_RS_42792 (8.055)

2˗1 2 h h

2˗2 25 v v

2˗3 1 h h

2˗4 0 h h

2˗5 0 h h

2˗6 17 v v

2˗7 0 h h

2˗8 20 v v

2˗9 3 h h

2˗10 58 v v

2˗11 0 h h

2˗12 1 h h

2˗13 8 v v

2˗14 32 v v

2˗15 0 h h

2˗16 1 v v

2˗17 28 v v

2˗18 1 s s

2˗19 1 h h

2˗20 0 s s

2˗21 9 v v

2˗22 22 v v

2˗23 1 s s

2˗24 0 h h

2˗25 1 h h

2˗26 2 v v

2˗27 3 h h

2˗28 1 h h

2˗29 19 v v

2˗30 0 s s

2˗31 1 h h

2˗32 14 v v

2˗33 0 s s

2˗34 1 h h

2˗35 1 s s

2˗36 1 v v

2˗37 7 v v

2˗38 15 v v

2˗39 0 s s

2˗40 1 h h

2˗41 12 v v

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43

2˗42 0 h h

2˗43 1 s s

2˗44 2 v v

2˗45 23 v v

2˗46 0 h h

2˗47 0 s s

2˗48 0 h h

2˗49 2 h h

2˗50 0 h h

2˗51 0 h h

2˗52 9 v v

2˗53 0 h v

2˗54 2 h h

2˗55 1 h h

2˗56 0 v v

2˗57 2 h h

2˗58 2 s s

2˗59 3 s s

2˗60 54 v v

2˗61 2 h h

2˗62 1 s s

2˗63 39 v v

2˗64 1 h h

2˗65 0 h h

2˗66 0 h h

2˗67 1 v v

2˗68 2 s s

2˗69 1 v h

2˗70 1 h s

1˗1 9 v v

1˗2 1 s s

1˗3 3 v v

1˗4 0 h h

1˗5 1 h h

1˗6 2 h h

1˗7 0 h h

1˗8 0 h h

1˗9 0 s s

1˗10 0 h h

1˗11 5 v v

1˗12 0 u h

1˗13 0 u s

1˗14 1 h h

1˗15 1 h h

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44

1˗16 2 h h

1˗17 1 s s

1˗18 0 s s

1˗19 0 h h

1˗20 0 h h

1˗21 23 v v

1˗22 0 s s

1˗23 0 h h

1˗24 0 h v

1˗25 0 h h

1˗26 1 v v

1˗27 22

1˗28 0

1˗29 0

1˗30 7

1˗31 0

1˗32 0

1˗33 1

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Appendix 12: Statistical analysis for Histology

Early Abortion

Duncan's multiple range test

Accession

Difference

Lower

95%

Upper

95% t

Significant

Comparison

Cebeco vs Alfred -0.015 -0.3139 0.2839 -0.139 no

Cebeco vs

SusPtrit -0.48 -0.7854 -0.1746 -4.459 yes

Cebeco vs Vada -0.78 -1.087 -0.473 -7.246 yes

Alfred vs SusPtrit -0.465 -0.7639 -0.1661 -4.32 yes

Alfred vs Vada -0.765 -1.0704 -0.4596 -7.107 yes

SusPtrit vs Vada -0.3 -0.5989 -0.0011 -2.787 yes

Mean

Cebeco 0.065 a

Alfred 0.08 a

SusPtrit 0.545 b

Vada 0.845 c LSD= 0.2989

Non-Penetrating Infection units

Duncan's multiple range test

Accession

Difference

Lower

95%

Upper

95% t

Significant

Comparison

SusPtrit vs Vada -0.055 -0.2827 0.1727 -0.671 no

SusPtrit vs

Cebeco -0.145 -0.3777 0.0877 -1.768 no

SusPtrit vs Alfred -0.43 -0.6639 -0.1961 -5.244 yes

Vada vs Cebeco -0.09 -0.3177 0.1377 -1.097 no

Vada vs Alfred -0.375 -0.6077 -0.1423 -4.573 yes

Cebeco vs Alfred -0.285 -0.5127 -0.0573 -3.475 yes

Mean

SusPtrit 0.24 a

Vada 0.295 a

Cebeco 0.385 a

Alfred 0.67 b

LSD = 0.2277