Wesley T. Barber, Wei Zhang, Hlaing Win, Kranthi K. Varala, Jane E. Dorweiler, Matthew E. Hudson and Stephen P. Moose

Transcription

1 Repeat associated small RNAs vary among parents and following hybridization in maize Wesley T. Barber, Wei Zhang, Hlaing Win, Kranthi K. Varala, Jane E. Dorweiler, Matthew E. Hudson and Stephen P. Moose Abstract Small RNAs (srnas) are hypothesized to contribute to hybrid vigor because they maintain genome integrity, contribute to genetic diversity, and control gene expression. We used Illumina sequencing to assess how srna populations vary between two maize inbred lines (, ) and their hybrid. We sampled srnas from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids. We found that parental differences in sirnas primarily originate from repeat regions. Although the maize genome contains greater number and complexity of repeats compared to Arabidopsis or rice, we confirmed that like these simpler plant genomes, 24-nt sirnas whose abundance differs between maize parents also show a trend of downregulation following hybridization. Surprisingly, hybrid vigor is fully maintained when 24-nt sirnas are globally reduced by mutation of the RNA-dependent RNA polymerase2 (RDR2) encoded by modifier of paramutation1 (mop1). We also discovered that 21-22nt sirnas derived from a number of distinct retrotransposon families differentially accumulate between and as well as their hybrid. We found that significant variation exists for nt sirna accumulation for these families among a larger set of diverse maize inbred lines. Thus, maize possesses a novel source of genetic variation for regulating both transposons and genes at a genomic scale, which may contribute to its high degree of observed heterosis.

2 Repeat associated small RNAs vary among parents and following hybridization in maize Wesley T. Barber Matthew E. Hudson Stephen P. Moose 48 th Annual Illinois Corn Breeders School March 5, 2012

3 Outline Heterosis/hybrid vigor srnas 22-nt vs. 24-nt srnas Behavior of srnas following hybridization Variation in srnas between inbred lines What to learn from this talk the maize genome provides a significant opportunity for inbred lines to vary for srnas srnas are a source for genetic variation at the epigenetic and post-transcriptional levels

4 Hybrid vigor phenotype Offspring perform better than their parents Greater cell proliferation in the hybrid x (right) is taller than one of its parents, (left).

5 Genetic principles of hybrid vigor Multiple mechanisms Genetic variation between parents Variation in regulatory genes = large effects? Altered genetic states in hybrids Additivity is an altered state Genomics uncovers more ways for how parents may vary Phenotypic value parent a parent b Stupar et al hybrid axb

6 Non-additive possibilities from genes behaving additively Inbred 1 Inbred 2 F1 Hybrid = regulatory gene = proteins Possibility for something new in hybrid (non-additivity) even from factors behaving additively

7 srnas regulate crop phenotypes Soybean seed coat color Tuteja et al Flowering time, biomass in maize Lauter et al. 2005

8 srnas are major regulators of the genome Genetically encoded mirna sirna Aberrant transcript 1 sirna rasirnas RDR Antisense transcripts natsirna DCL Cleaved transcript tasirna 2 sirna RDR AGO AGO AGO CH3 CH3 CH3 rrna srna primed 2 sirna RDR Small interfering RNAs (sirnas) regulate genes and repeats RDR enzymes Maintain genome integrity 21/22-nt sirnas posttranscriptional 24-nt sirnas transcriptional DNA methylation Target cleavage Translational inhibition

9 How do srnas behave following hybridization in maize? Shoot apex (tips) at 11 DAS srna sequencing from,, and their hybrids Compared abundances (reads per million) of individual srnas and blocks of srnas between the genotypes sampled actively growing tissues that program greater growth of hybrids Ear at V12 growth stage Emerged Leaf Leaf primordia SAM Sampled tissue

10 Differences between parents and hybrids result from and passing on divergent sirna populations % of nt sirnas Shoot apex x x 0.1% 0.2% 0.4% 1% 2% 1% 0.1% 20% 19% 1% 51% 1% 1% 1% 0.2% Ear 10% 2% 4% 50% 15% 17% 3% x Genotypes x x

11 Parental differences in nt sirnas are inherited at levels below mid-parent in the ear A. Shoot apex Log2 (F1/mp) B. Ear 5 Log2 (F1/mp) > 4x > 8x > 4x > 8x Increasing parental fold-change 24-nt sirnas reduced in Arabidopsis, rice, wheat hybrids/polyploids Connected to epigenetic and gene expression changes (Groszman et al. 2011) Transgressive phenotypes of tomato RILs are influenced by sirnas (Shivaprasad et al. 2011)

12 MOP1 (RDR2) provides unique system to test importance of 24-nt sirnas to hybrid vigor Parent 1 - wild type - mop1-1 X Parent 2 - wild type - mop1-1 Nobuta et al Reciprocal hybrids Do the mutant hybrids still show as much heterosis as wild type hybrids?

13 Loss of MOP1 reduces 24-nt sirnas and height and delays flowering for each genotype Relative Expression ( Ct) Wild type mop nt sirna-1 x 24-nt sirna-4 x wt mop1-1 wt mop1-1 x wt x mop1-1 x wt x mop1-1 Height (cm) 50% anther (days)

14 Loss of MOP1 does not suppress hybrid vigor for x Wild type x mop1-1 mutant x

15 MOP1 mutant hybrids show the same degree of heterosis as wild type hybrids Ratio of trait in hybrids relative to inbreds * ** *** x mut x wt x mut x wt * p-value p-value p-value ** ** *** *** * 0 plant height days to silk days to shed cob weight stover biomass Agronomic trait

16 Conclusions on how srnas vary following hybridization Hybridization doesn t cause global changes to srna populations or core components of srna biogenesis MOP1 result questions the functional significance of 24-nt sirnas to hybrid vigor 24-nt sirnas may play a bigger role in other processes (inbreeding?)

17 Differences in nt sirnas between and primarily originate from repeat regions gene Genomic feature repeat within gene within ±1000-bp of gene repeat within ±1000- bp of gene repeat gene repeat within gene within ±1000-bp of gene repeat within ±1000- bp of gene repeat Repeats = DNA transposons, MITEs, helitrons, retrotransposons All 22-nt 24-nt >8X 22-nt 24-nt Shoot apex Developing ear Low High Number of sirna regions Majority of genomic intervals annotated as high copy retrotransposon families

18 Parental differences in retrotransposon sirna activity are driven by 21/22-nt sirnas, not 24-nt sirnas * * * * * cinful zeon rire1 giepum grande ji misfit huck1 milt opie Total Abundance x Shoot apex x x 21-nt 22-nt 24-nt sirna length Total Abundance Developing ear x x * Indicates that for the length of sirna investigated, and were found to significantly differ in their abundance of sirnas in the same direction in both tissues (χ 2 test, Bonferroni corrected p-value 0.01). x 21-nt 22-nt 24-nt sirna length Abundance (rpm) Low High

19 Retrotransposon derived sirnas and cellular proliferation Retrotransposon derived sirnas in human stem cells may regulate DNA repair, cell cycle control and chromatin (Wang et al. 2011) Active retrotransposons in pollen cells are dealt with at posttranscriptional level Slotkin et al seedling shoot apex x polyprotein LTR-retro RNA Degraded RNA 21,22-nt sirna

20 Relative expression ( Ct) Combining divergent 21/22-nt rasirna cinful populations in the hybrid doesn t lead to greater silencing Cinful expression in seedling shoot apex x 3 leaves emerged x 4 leaves emerged x 5 leaves emerged What are retrotransposon derived sirnas doing in proliferating tissues? Regulate themselves? Regulate genes? (Ohtsu et al. 2007) Recent evidence for gene regulation found in Arabidopsis (McCue et al. 2011)

21 Current work: How do rasirnas vary among a diverse set of inbred lines in maize? Characterizing 21/22-nt rasirna across diverse maize germplasm combined with genetic tests of hybrid performance Determining if genes are targeted by rasirnas Investigating inheritance/stability of rasirnas Genetic Material 36 inbred lines, 22 NAM founders 10 PVP lines IHP1 ILP1 From Liu et al PH207 Iodent LH82 PHZ51 Unrelated PHG35 PHG84 Oh07/Midland LH123Ht PHG47 Oh43 Lancaster Stiff Stalk LH1 Stiff Stalk PHG39 Maiz Amargo PHJ40 Stiff Stalk

22 Are heterotic subgroups defined by specific patterns of 21/22-nt rasirna activities? PH207 Iodent LH82 PHZ51 Unrelated PHG35 PHG84 Oh07/Midland LH123Ht PHG47 Oh43 PH nt Oh43-21-nt - 21-nt - 21-nt Lancaster PH nt LH1 PHG39 PHJ40 Stiff Stalk Stiff Stalk Maiz Amargo Stiff Stalk Oh43-22-nt - 22-nt - 22-nt cinful dagaf giepum grande ji machiavelli opie rire1

23 Conclusions for parental variation in sirnas Studies in Arabidopsis, rice, and wheat did not find significant variation for 21/22- nt sirnas derived from retrotransposons Heterosis cannot be compared across genera because the relative degree of genetic differences varies (East, 1936) Related to the high degree of heterosis observed in the species? Nobuta et al. 2008

24 Take home messages The maize genome provides a significant opportunity for inbred lines to vary for srnas 21/22-nt rasirnas from retrotransposons may allow inbred lines to differ in their posttranscriptional regulation of repeat elements and genes at a genomic scale srnas are a source for genetic variation at the epigenetic and post-transcriptional levels Could this genomics information be useful to guide breeding strategies?

25 Acknowledgements National Science Foundation Heterosis Challenge Grant Pioneer Hi-Bred Bob Seifert Honorary Plant Breeding Fellowship Moose Lab Qing Li Wei Zhang Hudson Lab Kranthi Varala Wendy Win Ying Li Jane Dorweiler EBI/Miscanthus Magdy Albady Chris Austin