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Analysis of SNP Array Data Frequency and Characteristics of Large • Data sets • 15 Array – about 700,000 SNPs called on ~220 samples Apparent Deletions in Citrus Germplasm • 56 Array – about 51,000 SNPs called on ~1500 samples • Data characteristics 1 1 2 Mikeal Roose , Yoko Hiraoka , and Tulsi Dey • Technical replicates (same DNA) – about 99.8% identical 1 Botany and Plant Sciences, University of California, Riverside • Biological replicates (different trees) – about 99.8% identical 2Kalyani Mahavidyalaya, City Centre Complex, Kalyani, Nadia 741235 India • Clonally-derived cultivars – typically at least 99% identical • Can we distinguish cultivars within clonally derived groups? • Only a few of the 871 germplasm accessions analyzed with the smaller array have replicate samples • Hypothesis – information on genome location of SNP differences among clonally-derived cultivars provides a type of replication • Test – Look for loss-of-heterozygosity (LOH) in sets of adjacent SNPs as signature of heterozygous deletions

Ancestry of Citrus Discovery of Loss-of-Heterozygosity in Clonal Citrus Array Data (Tulsi Dey) groups • Initial analysis – organize SNPs in spreadsheet by chromosome and position x cultivar group, look at patterns of heterozygosity among cultivars • Later analysis – ALICE program: detects LOH/CNV in array data from heterozygosity and signal intensity • Used data only from large array (~700,000 SNPs) • Compared 41 pairs of accessions • Similar to analysis of cancer cells • Discovered 17 LOH cases: 15 deletions, 2 copy neutral • Many validated by sequencing or qPCR

Wu et al., 2018, Nature

Applications of SNP Arrays Developed at UCR Genome sequence data (n=41) Linkage Mapping Extension to smaller array (~51,000 SNPs) Analysis of Citrus Genetic (~1000 - 15,000 markers) Diversity - > 900 varieties Gene-focused SNP arrays • ~56,000 or Arrange data by chromosome and position x cultivar type ~700,000 markers • Count # heterozygous SNPs in each 1 Mb interval • Identify 1 Mb intervals with at least 3 fewer heterozygous markers in an accession compared with other accessions of same cultivar type Detection of Deletion • Sensitivity – varies with heterozygosity Mutations (Moro) Genotype Single Pollen Grains to • % of 1 Mb intervals with > 7 heterozygous SNPs Infer Haplotypes Preselect informative SNPs • oranges: ~95% for specific trait genes • grapefruit: ~92% • lemons: ~98% • Clementines: ~73% • satsumas: ~65%

image: Dr. Aseel Alanbari

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Deletion in 3 Blood Oranges (homozygous intervals reduced to single loci)

Left end of Burris Bream Tarocco Malta Deletion Moro Smith Red Orange Origin: Riverside Origin: backyard Origin: Redlands Origin: Sicily Origin: backyard “sport of Prior: Malta? Valencia”

5 blood oranges that share a deletion on chromosome 1 (1.65-2.00 Mb)

34 homozygous SNPs

Right end of Deletion

LOH in Blood Oranges on Chromosome 1

Sarah Khalily Origin: Israel Origin: Egypt “sport of Shamouti” “sport of Shamouti” Shamouti Origin: Palestine

5 oranges that share Precoce de Valence deletion on chromosome 1 Perao Valencia Origin: Morocco (1.65-2.00 Mb) Origin: Brazil “similar to Shamouti” Orange – “messy” deletion “similar to Cadenera”

Chromosome 1 – Putative Unique Heterozygous Deletions Interesting Deletions Mb Lapithiotiki lemon. Origin – Cyprus. “Said to be a good 0 quality lemon which is mal secco resistant.” Many deletions – some large 5 Chr1 – 3.18 Mb, Chr3 – 1.42 Mb, Chr4 – 1.29 Mb, 10 Chr5 – 5.73Mb, Chr5 - 2.98 Mb, Chr6 – 0.76 Mb, Chr7 – 10.50 Mb 15 Seedless Valencia. Origin – UCR (Director’s residence) 20 “About half as many seeds as Olinda Valencia” Two unique deletions 25 Chr1 – 1.37 Mb, Chr6 – 0.41 Mb

30 Moro Cogan Seedless Macetera Sanguinelli Ricalate Berna, Pera Valencia Calabrese Miyamoto Star Ruby grapefruit. Origin – irradiated seed from 35 orange navel Valencia B. Macetera 3 other navel Verna orange oranges C. Ovale satsuma Hudson grapefruit. “Compared to other grapefruit 4 more oranges oranges oranges all 25 oranges varieties, Star Ruby is more difficult to grow well. ” 40 One unique deletion. Chr9 – 0.3 to 0.7 Mb

45 not shown Robyn navel, Workman navel, Lapithiotiki lemon 50 DaisySL mandarin. Origin – UCR. Irradiated budwood of “Lost” alleles are in the same haplotype within a group when haplotype is known. Daisy. One unique deletion. Chr9 – 1.8 Mb

Daisy DaisySL

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Summary of “Deletions” in Clonally-Derived Citrus Groups Number of Number of Accessions Largest Age of Group Accessions Deletions with Deletion Group In Group Deletions (Mb) (yrs) Blood Oranges 18 6 15 3.18 500? Clementines 16 1 1 1.67 130 Grapefruit 26 4 3 1.44 300 Lemons 56 12 6 10.5 3000 Mexican Limes 12 1 1 0.18 6000? Navel Oranges 67 12 11 7.66 350+ Other Oranges 74 17 24 3.86 Old Satsumas 45 2 2 8.37 <1000? Valencia Oranges 26 5 26 1.37 500? Total 340 60 89

Approximate chromosomal locations of all identified heterozygous deletions

Mb 1 2 3 4 5 6 7 8 9 0 5 10 15 20

25

30 35 40 45 Min. Size Mb 1 Mb ~ 70 genes 50 < 0.5 0.5-2.0 > 2.0

Questions • Deletions vs somatic recombination/replacement? • Are similar deletions really identical – resolution? • Are “messy” deletions due to chimerism? • Inheritance – extreme segregation distortion is common in citrus crosses – could deletions contribute? • Functional consequences – are heterozygous deletions basis of cultivar-specific traits? • Cause – chromosome breaks due to TE activity, fragile sites, etc. • Next • Develop ALICE method for 56K array • Sequencing cultivar pairs

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