Validation and implementation of lentil molecular markers for boron tolerance and ascochyta blight resistance

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1 Validation and implementation of lentil molecular markers for boron tolerance and ascochyta blight resistance Matthew Rodda PBA Lentil Breeder S. Sudheesh, G. Rosewarne, M. Javid, J. Davidson, A. Slater, S. Kaur

2 Overview Breeding for tolerance to boron (B) in lentil 1. Rationale; Sources of tolerance; Screening methods 2. A molecular marker for B tolerance 3. Validation and implementation of B markers in the breeding program Breeding for ascochyta blight (AB) resistance in lentil 1. Current resistance to Ascochyta lentis 2. Three QTLs for AB resistance 3. Genetic basis for AB resistance in Australian lentil germplasm

3 Toxic soil boron in Southern Australia In SA, 35% of cleared land affected by boron toxicity (>15 mg/kg boron in top 100 cm of soil)

4 ILL2024: Boron tolerant landrace Hobson et al Euphytica 151:

5 ILL2024: Salt tolerant landrace Salt screen in gravel pots (6 ds/m) ILL2024 Nipper Nugget T MT Intolerant

6 Boron tolerance screening methods Soil Hydroponic

7 % necrosis, HYDROPONIC ASSAY B tolerance screening methods R² = % necrosis, POTTED SOIL Comparison of phenotypic screening for B tolerance in potted soil versus a simple hydroponic assay

8 A marker for boron tolerance Two recombinant inbred line (RIL) populations ILL2024 x cv. Cassab ILL2024 x ILL6788 One major gene for B tolerance gene, with flanking markers, found in both populations: 93% & 80% phenotypic variance explained Gene also present in ILL0213A LG2 0.0 SNP_ Lc_ Lc_ Lc_ LcC01233p Lc_ SNP_ LcC08594p Lc_ SNP_ Lc_ Lc_ Lc_ Lc_ PBA_LC_ Lc_ Lc_ Lc_ Lc_ SSR Lc_ LcC24967p Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ Lc_ SNP_ B_tolerance_QTL

9 Validation in diverse lentil germplasm Category No. entries Australian lentil cultivars 15 Australian breeding lines 129 Canadian breeding lines or cultivars 11 USA breeding lines or cultivars 10 ICARDA sourced germplasm 23 ICRISAT accession 1 Total 189 Utility of B markers Tolerant Intermediate Intolerant Correct Tolerant allele % Intolerant allele % Total % (Shaded = correct prediction)

10 Validation in diverse lentil germplasm Category No. entries Australian lentil cultivars 15 Australian breeding lines 129 Canadian breeding lines or cultivars 11 USA breeding lines or cultivars 10 ICARDA sourced germplasm 23 ICRISAT accession 1 Total 189 Utility of B markers Tolerant Intermediate Intolerant Correct Tolerant allele % Intolerant allele % Total % Correct prediction of phenotype in about 50% of lines Significant false positives and false negatives

11 Validation in selected populations Germplasm derived from B tolerant donor ILL2024 Genetic background from ILL2024 No. lines 1/16 (6.25%) 27 1/8 (12.5%) 89 1/4 (25%) 6 Total 122 Utility of B markers in ILL2024-derived germplasm Tolerant Intermediate Intolerant Correct Tolerant allele % Intolerant allele % Segregating % Total % Correct prediction of phenotype in 88% of lines with tolerant allele

12 Conclusions Breeding for boron tolerance Markers for B tolerance gene were effective in tracking tolerance within populations derived from ILL2024 Can be utilised for selected breeding populations Poor marker-trait association in unrelated germplasm (Canadian etc.) Trait has had a yield penalty & linkage to seed shattering There is an additional type of intermediate B tolerance present in Australian lentil breeding germplasm PBA Flash and PBA Bolt Now in a wide range of advanced germplasm, e.g. CIPAL1301 Our low-cost screening method is an effective, and necessary, alternative to B markers

13 Ascochyta blight of lentil Thresholds for staining at Grade 1 receivals = 0.02%

14 Ascochyta blight resistance Long-term emphasis on resistance to AB Current status of AB resistance in PBA Lentil germplasm: R MR MR-MS MS or S Stage 2 Germplasm 45% 32% 24% 0% Stage 3 Germplasm 63% 30% 7% 0%

15 Ascochyta blight resistance Two RIL populations IH x NF LG2 IH x DIG LG2 Indianhead x Northfield Indianhead x Digger SNP_1 SNP_2 SSR_3 SNP_4 SNP_5 q_ab_ih SNP_1 SNP_4 SSR_3 SNP_6 SNP_5 q_ab_ih1 LG2 Major resistance gene from cv. Indianhead (AB_IH1) Identified in both populations independently In a diverse panel of lentil germplasm, alone correctly predicted resistance for 86%

16 Ascochyta blight resistance Linkage Grp R Source Vp (IHxNF) Vp (IHxD) SNP marker AB_IH1 LG 2 Indianhead 47% 30% SNP_4 AB_IH1.2 LG 3 Indianhead 15% 22% SNP_10 AB_NF1 LG 6 Northfield 7% - SNP_20 IH x NF LG3 IH x DIG LG3 IH x NF LG SNP_7 SNP_8 SNP_9 SNP_10 SNP_12 SSR_13 q_ab_ih SNP_7 SNP_14 SNP_15 SSR_16 SNP_11 SNP_12 SSR_13 q_ab_ih SNP_17 SNP_18 SNP_19 SNP_20 SNP_21 q_ab_nf1

17 Phenotyping and genotyping for resistance to Ascochyta lentis isolates in Australian lentil breeding germplasm (foliar disease in controlled environment assay) Field R rating Kewell, historical isolate CE screen recent isolate Putative "Nipper" gene AB_IH1 AB_IH1.2 AB_NF1 Name ILL7537 R - - NF_pR IH1 IH1.2 PBA JUMBO2 R - - NF_pR IH1 CIPAL1501 R - - NF_pR IH1 NF1 CIPAL1301 R - - NF_pR IH1 NF1 CIPAL1502 R - - NF_pR IH1 NF1 INDIANHEAD R + - IH1 IH1.2 PBA HERALD XT R + - IH1 IH1.2 NF1 CIPAL1522 R + - IH1 IH1.2 NF1 PBA ACE R + - IH1 IH1.2 NF1 CIPAL1521 R + - IH1 NF1 PBA BOLT MR + - IH1 NF1 CIPAL1523 MR + - IH1 NF1 CIPAL1504 MR IH1 IH1.2 NF1 PBA HURRICANE XT MR IH1 CIPAL1422 R IH1 NF1 BOOMER MR - - NF_pR IH1.2 NIPPER MR-MS - + NF_pR IH1.2 NORTHFIELD MR-MS - + NF_pR NF1 PBA BLITZ MR-MS? + NF_pR PBA GREENFIELD MR-MS NF_pR PBA JUMBO MR-MS NF_pR PBA GIANT MR-MS + + IH1.2 NF1 NUGGET MR-MS + + IH1.2 NF1 PBA FLASH MS NF1 CUMRA S SNP_4 SNP_10 SNP_20 = susc. allele

18 Phenotyping and genotyping for resistance to Ascochyta lentis isolates in Australian lentil breeding germplasm (foliar disease in controlled environment assay) Field R rating Kewell, historical isolate CE screen recent isolate Putative "Nipper" gene AB_IH1 AB_IH1.2 AB_NF1 Name ILL7537 R - - NF_pR IH1 IH1.2 PBA JUMBO2 R - - NF_pR IH1 CIPAL1501 R - - NF_pR IH1 NF1 CIPAL1301 R - - NF_pR IH1 NF1 CIPAL1502 R - - NF_pR IH1 NF1 INDIANHEAD R + - IH1 IH1.2 PBA HERALD XT R + - IH1 IH1.2 NF1 CIPAL1522 R + - IH1 IH1.2 NF1 PBA ACE R + - IH1 IH1.2 NF1 CIPAL1521 R + - IH1 NF1 PBA BOLT MR + - IH1 NF1 CIPAL1523 MR + - IH1 NF1 CIPAL1504 MR IH1 IH1.2 NF1 PBA HURRICANE XT MR IH1 CIPAL1422 R IH1 NF1 BOOMER MR - - NF_pR IH1.2 NIPPER MR-MS - + NF_pR IH1.2 NORTHFIELD MR-MS - + NF_pR NF1 PBA BLITZ MR-MS? + NF_pR PBA GREENFIELD MR-MS NF_pR PBA JUMBO MR-MS NF_pR PBA GIANT MR-MS + + IH1.2 NF1 NUGGET MR-MS + + IH1.2 NF1 PBA FLASH MS NF1 CUMRA S SNP_4 SNP_10 SNP_20 = susc. allele

19 Conclusions Breeding for ascochyta resistance The AB_IH1 resistance gene, from Indianhead, currently confers strong field resistance There are a number of partial resistance genes providing backup resistance However, Some current field isolates are able to overcome AB_IH1 resistance Certain current cultivars are particularly at risk, as they contain only one known resistance gene, namely PBA Hurricane XT In 2016, 70% of the Yorke Peninsula was planted to this variety A new pathotype is likely to emerge that will overcome both the major IH and NF resistance genes

20 Conclusions Breeding for ascochyta resistance Additional sources of ascochyta resistance are required Two current candidates: ILL7537 and Boomer A genetic marker would most effectively select this resistance; work is underway An existing Boomer x ILL7537 RIL population is being tested Resistance in breeding progeny of ILL7537 is being tracked by phenotyping

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