Validation of SNP markers for selection of semi-dwarf and peduncle extension in barley

doi:10.21203/rs.3.rs-1649919/v1

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Validation of SNP markers for selection of semi-dwarf and peduncle extension in barley

https://doi.org/10.21203/rs.3.rs-1649919/v1

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published 13 Apr, 2023

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This study aimed to validate the use of two SNP markers associated to a sdw1 allele identified previously in a short barley genotype (ND23049) with an adequate peduncle extension which reduces predisposition to fungal disease development. First, the GBS SNP were converted in a KASP marker but only one of them, named TP4712, correctly amplified all allelic variations and had a mendelian segregation in a F₂ population. To corroborate the association between TP4712 allele with plant height and peduncle extension a total of 842 genotypes were genotyped and evaluated for both traits. Out of the 842 genotypes, 199 were F₄lines, 79 were a diverse panel and 564 were a complete breeding cohort of stage 1 yield trials. To corroborate the association between the sdw1 allele and the short plant height with adequate peduncle extension, contingency tables were created, grouping the 1954 data points into categories. Based on the contingency analysis it was demonstrated that the higher proportion of short plants with adequate peduncle extension were present in genotypes carrying the SNP allele present in ND23049 regardless the population and the sowing date. This study develops a marker assisted selection tool to accelerate the introgression of favourable alleles for plant height and peduncle extension in adapted germplasm.

Breeding population

marker assisted selection

plant height

peduncle extension

SNP marker

The abrupt increase in yield of the main crops, named as green revolution, went hand in hand with semi-dwarf varieties. In barley, there are many genes described as causing semi-dwarf and dwarf phenotypes, but the semi-dwarf 1 gene (sdw1) is one of the most widely used in modern barley breeding of Europe, North America, South America, and Australia. (Kuczynska et al. 2013; Xu et al. 2017). The sdw1 locus is located on chromosome 3H and encodes a gibberellin 20-oxidase enzyme or HvGA20ox2 (Jia et al. 2009; Xu et al. 2017). So far, there are four alleles of sdw1 described; one of them generated spontaneously (sdw1.c) and three induced by mutagenesis (sdw1.a, sdw1.d and sdw1.e) (Franckowiak et al. 2012). Sdw1.d has a 7-bp deletion in exon 1, sdw1.c has a 1-bp deletion and a 4-bp insertion in the 5’ untranslated region, and sdw1.a and sdw1.e present a total deletion of the HvGA20ox2 gene (Xu et al. 2017).

The main phenotypic effect associated with sdw1 gene is a decreased plant height (PH), compared to the wild type. However, it is well known that sdw1 gene not only affects PH but also influences various agronomic and physiological characteristics, as maturity, grain size and grain yield (Kuczyńska et al. 2013). It has been reported that the sdw1.d allele (the most used allele of the sdw1 gene in malting barley breeding) is associated with reduced expression of Hv20ox2, resulting in a lower GA level in the apical meristem, which inhibits apical growth, reduces internode length, plant hight and promotes the development of more tillers (Jia et al. 2011). In certain barley productive regions of South America, the reduced internode length determines that genotypes carrying sdw1.d have a phenotype with limited peduncle extension (PE). A limited PE means that the lower fraction of the spike reminds within the flag leaf sheath, which causes moisture to be retained around the developing grains and generates a microenvironment highly conductive for fusarium head blight disease (FHB) caused by Fusarium sp.. FHB disease is a major threat for the barley production where frequent rains and worm temperatures are likely to occur during flowering and grain fill. FHB can lead to several economic loses all along the barley agro-industrial chain as a consequence of grain yield loses, decrease of physical and industrial quality and less safety on the final product due to toxins produced by the fungus that are harmful to human and animal health (Pereyra and Dill-Macky 2010). For this reason, a main barley breeding objective in these regions is to develop cultivars with reduced PH but with adequate PE.

The semi-dwarf barley line ND23049, when planted in Uruguay (South America) combines a short PH with adequate PE. Belanger et al. (2018) demonstrated that the semi-dwarf phenotype in ND23049 is caused by a complete deletion of the sdw1 gene, although it is not known if this deletion corresponds to sdw1.a, sdw1.e or a new mutation. The gene association was determined by QTL mapping, identifying a very significant QTL in chromosome 3H, flanked by two single nucleotide polymorphisms (SNP): TP51167 and TP4712. This QTL explained more than 75% of phenotypic variation in two different seasons.

This study aimed to corroborate the genotype-phenotype association of reduced PH and adequate PE with the sdw1 allele on ND23049 barley line, and to validate SNP generated by Belanger et al. (2018) to implement marker assisted selection (MAS) as a routine methodology to introgress semidwarf phenotype with adequate PE in breeding lines.

The validation of a marker requires the verification of mendelian segregation by genotyping a segregant population and the corroboration of association of marker polymorphism with the objective traits, in this case PH and PE (Dubiela et al. 2019). First, a Kompetitive Allele-Specific Polymerase Chain Reaction (KASP) assays were designed from TP51167 and TP4712; SNP markers identified by Belanger et al. (2018). Afterward, mendelian segregation was evaluated in a F₂ population and marker-phenotypes association in three breeding populations.

Breeding populations for SNP marker and phenotype-genotype validation

To evaluate mendelian segregation, a F₂ population of 242 plants was analysed, derived from the cross of CLE 321, a breeding line developed by the Barley Breeding Program of the National Institute for Agricultural Research of Uruguay (BBP-INIA) with the sdw1 allele of ND23049, and Nahara, a European variety developed by Ackermann carrying sdw1.d allele.

To corroborate the genotype-phenotype association of sdw1 allele coming from ND23049 with the adequate PE and reduced PH phenotype, a total of 842 genotypes were genotyped and evaluated for both traits. Out of the 842 genotypes, 199 were F₄lines derived from the cross of CLE 321 and Danielle, a European variety developed by Ackermann carrying the sdw1.d allele; 79 were a diverse panel (DP), conformed by commercial cultivars and advanced breeding lines of the BBP-INIA, differing for the traits of interest; and 564 were a complete breeding cohort (BC) of stage 1 yield trials of the BBP-INIA which contains several families derived from ND23049. In contrast to the DP and BC populations, there was no previous selection done on the F₄ population.

Plant height and peduncle extension phenotyping

Phenotyping for PH and PE was carried out at INIA’s research farm “Estanzuela,” located in Colonia, Uruguay (34˚20′ S, 57˚41′ W; 81 m asl) in different breeding trials for each of the populations. The 199 F₄ genotypes were phenotyped in single-row plots in 2019. The DP genotypes were phenotyped at four different sowing times in 2019 and 2020, and at two sowing times in 2021, in two-row plots with one single replication for each of the 10 sowing dates. The BC genotypes were phenotyped in 2021 at INIA and in an additional location at Young, Rio Negro (north-west of Uruguay, 32°68´ S, 57°64´ W). For the BC genotypes phenotyping was done on one replication of the 5.1 m² yield plots at each location.

The PH was determined in centimetres (cm) based on the mean of two measures per plot. The level of PE was determined in each plot based on a 1 to 5 visual scale, as shown in figure 1, where values <3 are favorable and ≥3 are unfavourable.

Fig. 1

SNP marker development

The sequence of TP51167 and TP4712 GBS reads are shown in table 1. The SNP within the sequence is shown between brackets. According to information provided by Dr. Francois Belzile, ND23049 carry the G allele of TP51167 and the T allele of TP4712.

Using the read sequence information of TP51167 and TP4712, obtained from Dr. Francois Belzile, primers for KASP amplification were designed by LGC Biosearch Technologies (Hoddesdon, UK). SNP assays, including call rate and genotype scoring and performance was assessed using the DP population. The assays were performed in an Applied BiosystemTM QuantStudio™ 3 Real-Time PCR System with the default parameter established by the manufacturer. After running the standard PCR cycle a recycle was performed. The PCR cycle was as follow: a stage 1 (pre-read stage 1 of 30°C for 1 min); stage 2 (hold stage of 94°C for 15 minutes); stage 3 (touchdown of 94°C for 20 seconds, 61°C 60 seconds -decrement -0.6°C per cycle- repeated 9 times, achieving a final annealing temperature of 55°C); stage 4 (94°C for 20 seconds, 55°C for 60 seconds repeated 25 times); stage 5 (post-read stage 30°C for 1 minute). The recycle was as follow: stage 1 (94°C for 20 seconds and 57°C for 60 seconds, repeated twice); stage 2 (reading stage 30°C for 1 minute).

The genotype scoring of both cycles were recorded and compared. When both results matched, the SNP were assigned to the genotype unequivocally. In case that both results differed or one of them was unclassified, the samples were identified as undetermined.

In order to determine the genome location of both GBS sequences, TP4712 and TP51167 read sequenceswere blasted against H. vulgare Morex 3.v2. genome available at GrainGenes (https://wheat.pw.usda.gov/GG3/) tools. The sdw1 genes sequence was obtained from ncbi genbank (https://www.ncbi.nlm.nih.gov/genbank/).

Data analysis

Contingency tables and distribution plots for the genotype/phenotype association corroborations were built using the base package in statistical R Development Core Team (2014). For contingency analysis, phenotypic data was assigned into categories. Phenotypes with PH < 90 cm were considered as “short”, whereas PH ≥ 90 cm were considered as “tall”. Additionally, phenotypes with PE < 3 were considered as “favourable”, whereas PE ≥ 3 were considered as “unfavourable”.

Development of KASP marker assay associated to sdw1 locus

The GBS sequences of TP4712 and TP51167 (Table 1) were blasted against H. vulgare Morex 3.v2. genome available at GrainGenes (https://wheat.pw.usda.gov/GG3/) tools. TP51167 GBS read sequence blasted between 558.940.158 and 558.939.900 bp of chromosome 3H and TP4712 sequence between 563.182.837 and 563.182.580 bp of the same chromosome. After a search of sdw1 or gibberellin-20 oxidase-2-like (GA20ox2) gene in GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) the sequence of exon 1 assigned as KX789375.1 was found. This sequence was retrieved from GenBank and blasted against H. vulgare Morex 3.v2. genome. Blast results located the partial gene sequence around 563.926.764 and 563.926.279 of chromosome 3H. Therefore, the TP4712 SNP is closer to sdw1 gene than TP51167.

GBS sequences were used to design KASP primers to genotype the DP population. Taking in consideration the genotype score criterium, 5 lines and 38 lines were defined as unclassified for TP4712 and TP51167, respectively. For TP51167 all genotypes different to unclassified were scores as allele A (41 lines) and was unavailable to amplify allele G. On the other hand, using the TP4712 it was possible to amplify both homozygous alleles and heterozygous (Fig. 2). Considering these results, only marker TP4712 was chosen for further population genotyping.

Figure 2

Mendelian Segregation Of Tp4712 Kasp Marker

The 242 plants of the F₂ population were genotyped with TP4712 marker. Out of the 242 genotypes, 45 were homozygous for allele T (21,4%), 67 were homozygous for allele G (31,9%), 98 were heterozygous (46,6%) and 32 were unclassified. Considering the 210 genotyped plants, the chi square value was 5,54, which was bellow to 5,99 (2 df, p-value < 0,05). The chi-squared test showed that there is not statistically significant difference between the expected frequencies according to mendelian segregation and the observed frequencies.

Genotyping Of Tp4712 Marker In Breeding Populations

Out of the 842 genotypes of the DP, F₄ and BC populations genotyped with TP4712, 118 were homozygous carrying the T allele, 594 were homozygous carrying the G allele, 82 were heterozygous (TG) and 48 were unclassified (Table 2). Unclassified and heterozygous genotypes were excluded for the phenotypic analysis. In total, considering only the 712 homozygous genotypes, 1954 data points were collected for PH and PE.

Table 2

Genotyping results for the TP4712 marker
	Homozygous T		Homozygous G		Heterozygous TG		Unclassified		Total
Population	Number	%	Number	%	Number	%	Number	%	Number
DP	16	20.3	61	77.2	2	2.5	0	0.0	79
F4	61	30.7	95	47.7	39	19.6	4	2.0	199
BC	41	7.3	438	77.7	41	7.3	44	7.8	564
Total	118	14.0	594	70.5	82	9.7	48	5.7	842
DP: Diverse panel population, F4: F₄ population, BC: breeding cohort population

Phenotype-genotype Association Of Tp4712 With Plant Height And Peduncle Extension

A high dispersion of phenotypic data was recorded for both traits of interest. PH ranged from 53.0 cm to 105.0 cm, with a mean of 80.2 cm and a standard deviation of 8.4. PE values ranged from 1.0 to 4.5, with a mean of 2.9 and a standard deviation of 0.4.

To corroborate the association between the sdw1 allele of ND23049 and the short PH with adequate PE phenotype, contingency tables were created, grouping the 1954 data points into categories. Based on the contingency analysis, 22% of the total phenotypes were tall and 78% were short, whereas 58% had favourable PE and 42% had unfavourable PE. Of the short phenotypes, 56% had favourable PE and 44% had unfavourable PE, whereas for tall phenotypes, 67% had favourable PE and 33% had unfavourable PE. When the phenotypes were further classified based on the SNP allele at the TP4712 marker, the proportion of short phenotypes with favourable PE changed notoriously when comparing genotypes with the T or G SNP. In total, of the phenotypes classified as short carrying the G SNP, 48% had favourable PE and 52% had unfavourable PE, whereas 96% of the phenotypes classified as short carrying the T SNP had favourable PE and only 4% had unfavourable PE. This analysis was carried out for each population separately and the higher proportion of short plants with adequate PE when genotypes carried the T SNP was confirmed for all three of them (Fig. 3).

Figure 3

When analysing only the 77 homozygous genotypes of the DP population planted in different sowing times, it was observed that on the consecutive planting dates, the PH decreased and consequently the proportion of unfavourable PE phenotypes increased. However, this was not the case for the phenotypes carrying the sdw1 allele coming from ND23049 (Fig. 4). For all planting dates, the proportion of short phenotypes was larger for genotypes with the T SNP, and in all cases, the short PH was combined with favourable PE phenotype. On consecutive four sowing times in 2020, 100%, 93%, 93% and 100% of the short phenotypes carrying the T allele had favourable PE, compared to 60%, 35%, 29% and 51% of short phenotypes carrying the G SNP.

Figure 4

Use Of Tp4712 For Marker Assisted Selection In A Barley Breeding Programs

A shift in the PH and PE phenotype mean was observed if marker assisted selection with TP4712 marker is done over the 712 genotypes analysed in this study. The PH phenotype of genotypes carrying the G SNP allele of TP4712 marker ranged from 53 to 105 cm, with a mean of 84 cm. The PE for these genotypes ranged from 1 to 4.5, with a mean of 3.1. The PH mean for genotypes carrying the T SNP allele of TP4712 marker ranged from 60 to 95 cm, with a mean of 79 cm. The PE for these genotypes ranged from 2 to 3.2, with a mean of 2.5 (Fig. 5).

Figure 5

Belanger et al. (2018) reported two SNP within the GBS sequences TP4712 and TP51167 associated to semidwarf phenotypes and determining a QTL region close to sdw1 gene. In addition, they demonstrated a deletion which could correspond to sdw1.e allele. Their results were confirmed in this study with three additional breeding populations, but only for marker TP4712 due to it was the only able to amplify both alleles using KASP technology. Fortunately, this marker is located closer to the sdw.1 gene, compared to TP51167 marker. It was confirmed through the analysis of an F₂ population that the marker TP4712 segregated according to mendelian expectation.

Given that high proportion of the genotypes used for the phenotype/genotype corroboration analysis belong to breeding populations with previous selection, it was expected to have quite low PH phenotypic values. The category described as “tall” in the contingency analysis, refers to phenotypes ranging from 90 to 105 cm, which is still considered acceptable for variety release at commercial level. Even considering this, it was expected to have a higher proportion of shorter phenotypes as the sdw1.d and the sdw1 allele coming from ND23049 are in higher frequency in the INIA-BBP than the dominant wild type Sdw1 allele. Only in the F₄ population, where no previous selection was done, the proportion of short and tall phenotypes was more equally distributed.

Based on breeder observations, taller phenotypes have a good plant development with longer stems, which are likely associated with favorable PE. On the other hand, shorter genotypes tend to have less favorable PE. Phenotyping results confirmed these observations, as a higher proportion of the tall phenotypes had favorable PE compared to the short phenotypes, in which the PE trait was equally divided between favorable and unfavorable.

Marker TP4712 was useful to classify genotypes carrying the sdw1 allele coming from ND23049 (SNP allele T) from genotypes without it (SNP allele G). This means that genotypes with the G SNP allele at the TP4712 marker may carry one of the other sdw1 alleles (different to the one coming from ND23049) or the Sdw1 allele corresponding to the wild type. It was expected to have a lower proportion of T genotypes in the DP and BC populations, as the sdw1 allele coming from ND23049 has been recently introduced in the breeding program and it is in lower frequency than the highly used sdw1.d allele. Only in the homozygous genotypes of the F₄ population, the proportion of T and G was equally distributed.

It can be inferred that most of the tall genotypes with the G SNP allele correspond to the Sdw1 wild type, and short genotypes correspond to the sdw1.d allele. If this inference is considered, results of this study give evidence that the sdw1.d allele generates short phenotypes (Kuczyńska et al. 2013) with unfavorable PE, as a much higher proportion of short genotypes with the G SNP allele had unfavorable PE compared to the tall genotypes.

In all three populations studied, genotypes carrying the ND23049 sdw1 allele were always associated with a shorter phenotype (much higher proportion of short phenotypes than tall genotypes), and in all cases, the proportion of short phenotypes with favorable PE was much higher compared to the short genotypes carrying the G SNP. Generally, at favorable environments, barley plants have a good vegetative development and PE is likely to be favorable. When environmental conditions are poorer (for example, in later sowing times in Uruguay) the vegetative development is not ideal, and problems on PE are more likely to occur. By analyzing the results of the DP population planted in several years in consecutive sowing times, it was possible to confirm that later planting times lead to limited vegetation development (short PH) and consequently, higher proportion of unfavorable PE. However, for genotypes carrying the sdw1 allele of ND23049, even though the PH decreased with consecutive sowing times, the proportion of favorable PE phenotypes was consistently high, never lower than 93%.

In conclusion, the TP4712 KASP marker used in this study enables breeders to select for genotypes carrying the sdw1 allele coming from ND23049. It has been proved that this alternative sdw1 allele provides a short PH phenotype associated with a favorable PE phenotype. Even when environmental conditions lead to unfavorable PE phenotype, genotypes with the sdw1 allele coming from ND23049 are highly probable to be associated with a favorable PE phenotype. TP4712 KASP marker can be used by breeders for selection, driving to a shift on the PH and PE distribution of the breeding population toward shorter PH and favorable PE values.

ACKNOWLEDGEMENTS

We want to thank Dr. Francois Belzile for providing the marker information (read sequences) necessaries to achieve this work and Andrés Bidart and Pablo Soulier for their contribution on field work.

Funding

This work was funded by INIA (Instituto Nacional de Investigación Agropecuaria) Projects: Mejoramiento de cultivo de secano and Mejoramiento molecular de cultivos.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

S. Baraibar and V. Bonnecarrere contributed to the study conception and design. KASP genotyping was performed by Wanda Iriarte and DNA sampling and field phenotyping was done by Wilmar De Leon. The manuscript was written by S. Baraibar and V. Bonnecarrere. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Compliance with Ethical Standards

There are not conflict of interest. This study did not involve human participant and /or animals.

Ethics approval and consent to participate

The authors accept principles of ethical and professional conduct of this Journal.

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Table 1 is available in the Supplementary Files section

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You are reading this latest preprint version

Validation of SNP markers for selection of semi-dwarf and peduncle extension in barley

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Material And Methods

Results

Mendelian Segregation Of Tp4712 Kasp Marker

Genotyping Of Tp4712 Marker In Breeding Populations

Phenotype-genotype Association Of Tp4712 With Plant Height And Peduncle Extension

Use Of Tp4712 For Marker Assisted Selection In A Barley Breeding Programs

Discussion

Statements And Declarations

References

Table

Supplementary Files

Status:

Journal Publication

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