Walbot Lab at Stanford University

Collaborations

Blake Meyers' Lab at University of Delaware

New NSF Project starting soon with Blake Meyers' lab at the University of Delaware. Here they are frolicking outdoors -- with children and pets! Kids and dogs are not working in the lab, but everyone else is! We really enjoy our collaboration involving analysis of small RNAs, phasiRNAs, and gene expression in pre-meiotic maize anthers.

Jixian Zhai and Blake Meyers, modeling the Walbot lab 2013 field hats! Welcome to the world of corn. For non-scientists, the hat depicts the wild ride of a research ear of corn, husks raised into the air to heighten the thrill, on the roller coaster of a DNA double helix. WHEEEEEE!!

Cal Poly San Luis Obispo

We maintain our annual summer project at Cal Poly SLO, now in its sixth year in 2010. This involves 3 or 4 undergraduates each summer in our field screen for new Mu-tagged mutants of key genes in anther development. See the Methods section for a handbook describing this summer research experience.

Dave Skibbe of Stanford (second from left) smiles for the camera, reflecting the excellent help he received from Sarah (far left), Eric, Cedric, and Kristen in dissecting tens of thousands of staged anthers from Mutator tassels. Multiple replicate samples of each stage from four pairs of sister active and inactive Mutator lines are being used in proteomics and transcriptome profiling to pinpoint gene expression changes during anther development and to discover proteins whose abundance or post-translational status is affected by Mutator activity.

Our 2005 Stanford field hat commemorates the drive to Cal Poly -- 3 hours on Highway 101 -- well worth every minute to work with our collaborators at SLO.

Anther Cell Fate Setting Projects:
Cell Fate Acquisition Project

Table 1. Cytological analysis, allelism results
Table 2. Transcriptome profiling, BSA mapping, Mu tagging, cloning progress
Table 3. Double mutant analysis

Table 1. Cytological analysis, allelism results

Locus	#	Alleles we found	Cloned	Transverse cytology	Confocal cytology
*am1*	1		yes	Meiotic entry failure am1-praI l/z failure
*msca1*	2	EMS63131 = ms6064* = 3 alleles Multiple Mu tagged alleles were all deletions	yes	Leaf cell types in anther; excess cell layers; no archesporial cells	Yes
*mac1*	3	mac1-MuDR = 2 alleles	yes	Excess PMC	Yes; early defect in archesporial proliferation rate
*ocl4*	4	mtm99-66 = 2 alleles	yes	Extra subepidermal anther layer ?? from epidermis??
*ms7*		Used in allelism testing
*ms8*	5	mtm99-56 = 2 alleles	yes	PMC failure; too few tapetal cells	Epidermal cells fail to elongate; too few tapetal cells; excess callose
*ms9*	6	Multiple Coop allleles	yes	Irregular tapetal layer; partial double middle layer.	ML & T more active cell division than normal but Ratio of ML/T is normal
*ms10*		Late mutant Used in allelism testing		Late uninucleate failure Dong Xue has extensive cytology
*ms11*		Late mutant Used in allelism testing		Tapetal failure
*ms13*	7	Multiple Coop alleles		Additional anti and periclinal tapetal divisions; all tapetal cells are binucleate	Extra divisions confirmed Cell walls don’t look good – Maybe a wall defect?
*ms14*	8	Multiple Coop alleles		Small meiocytes disappear
*ms17*		Late mutant Used in allelism testing		Irregular first and second meiotic divisions; no defects in anther wall detected	Check by confocal?
*ms23*	9	Placed on chromosomes 6 and 8?; Coop and Mu tagged alleles		Extra periclinal division in tapetal cells	Yes Cell division defect at 900 µm
*ms25*		3 Mu-tagged alleles Late Used in allelism testing		Post-meiotic Vacuolate tapetal cells; microspores die
*ms26*		Used in allelism testing
*ms32*	10	Same as ms60662 Now cloned	yes	Extra periclinal division in tapetal cells	Cell division defect at ~1200 µm
*tcl1*	11	11 Mu-tagged alleles plus EMS72063 is the same		deranged middle and tapetal cell divisions
*ms45*		msN2499 = EMS64409 Used in some allelism testing Late mutant = 3 alleles		Different descriptions for these alleles
*ms775 = csmd1*	12	Mu tagged alleles; mapping down to just a few genes by Dongxue	close	Dongxue Excess callose pre-meiotically; uninucleate failure & vascular tissue senescence coincide
*mtm00-06*	13			Very small anthers
*RescueMu A60-35A*	14			Collapse of tapetal cells before meiosis
*EMS63089*	15	Tagging populations built for 2012		Multiple defects in wall layers – parenchyma like
**ms-si-355***	16	3 putons with Mu tags recovered in summer 2011		No anthers at 2 mm
**ms-6015***	17	Placed by the Schnable lab for us		Multiple number of cells in tapetal layer	Very thin ML; multiple tapetal layers SPL div are asymmetric Tapetal cells smaller initially but catch up
*RescueMu C17-32*	18			Callose deficiency around meiocytes? Meiocytes lose their borders and degrade
*RescueMu E03-23*	19			Irregular middle and tapetal layers. Multinucleate tapetum
*RescueMu A60-22b*	20			Tapetum layer separates from other layers
*EMS72032*	21	Allelic to 71924 = 2 alleles		Two lobed anther structure. All layers present in the abaxial lobes; Meiosis is not affected
*EMS71990*	22	To be crossed with ms-si355*		Few spikelets on the tassel, ? with no anthers. Stronger phenotype than si-355*
*EMS72091*	23			Deranged middle and tapetal cell divisions and vacuolization
		Late Mutants	=	Lower Priority
*EMS63265*		looks similar to EMS71777		Defective at late stage (3mm): few hypersized multi-nucleate tapetal cells lose their contact with the tapetal layer
*EMS71777*		looks similar to EMS63265		Same description as above
*EMS71787*				Vacuolization of middle and tapetal layers at tetrad stage; premature tapetal cell degradation
		Not defective	after all
*EMS72098*				No anther defects detected – maybe special field conditions?

Key genetic results: 23 loci with pre-meiotic defects. Six are cloned. Mapping in progress for csmd1, ms13, ms*6015, tcl1, ms23

Allelism testing ~90% done we have two 3 allele cases, and seven 2 allele cases, and 14 “unique” loci (3 of these have multiple alleles from the Coop).

Table 2. Transcriptome profiling, BSA mapping, Mu tagging, cloning progress

Locus	Mapped?	Cloning progress	Transcriptome	Mu-tagged or other alleles	Published?
*am1*	Done	Pioneer protein	Gillian: am1-489 & am1-praI	N/A	Cande lab PNAS Gillian poster and ms. in progress
*msca1 = ms22 on chr7*	Done	glutaredoxin	Leaf cell types in anther		Pioneer patent application 20090038028
*mac1*	Done	tpd1-like ?secreted	22K array and 44K PMC & early stages by Tim	N/A	Rachel writing ms. Long overdue.
*ocl4*	Done	HD zipIV transcription factor			Vernoud 2009 Plant J 59: 883-894.
*ms7*	Used in allelism testing
*ms8*	Dave – cloned Sept 2010	1,3 beta galactosyl transferase best match	1.0 1.5 2.0 mm precocious expression of 40% genes; many changes	Historic and 8 Mu-tagged alleles	Dong Xue pub 2010 Plant J Strasser lab in Vienna unlikely match to Lewis epitope gal transferase
*ms9*				Historic Mu tag 1,350 pilot screen 2011; more 2012
*ms10*	Dongxue chr10L bin04			Historic 2 Mu putons 2010	Definitely not allelic to csmd1
*ms11*	Late			1 Mu puton 2010 Mu tag
*ms13*	Ch5			Mu tag to screen 2011
*ms14*				Failed to tag in large 2010 population
*ms17*	Late
*ms23*	Dave 8S small interval		OId 22K for 1, 1.5, 2.0 mm Lan summer 2010 1.5 mm	Multiple Mu and historic
*ms25*	Late			3 Mu tag plus historic
*ms26*	Used in allelism testing			historic
*ms32*	Dave 2L Sm interval Jihyun cloned Feb 2011	bHLH factor	Lan summer 2010 1.0 & 1.5 mm array	2 Mu tag plus historic	Write up cloning, expression, arrays & confocal ??
*ms45*	Done	Possible strictosidin synthase		5,478,369 patent number	Sexual Plant Reproduction 14, 135-142 Pioneer
*ms775* *= csmd1*	Dong Xue small interval chr10L bin04			2 Mu tag Mu tag to screen 2012	Dongxue 2011 Sex. Plant Reprod.
*mtm00-06*				Mu tag 19,200 screen 2011
*RescueMu* *A60-35A*
*EMS63089***	Karl, Sidae			Mu tag to Set up 2011
**ms-si-355***	Newly tagged			Mu tag 10,500 screen 2011
*tcl1*	Tim 3L BSA –location confirmed by Gillian via Schnable lab			2011 alleism scoring testing mac1-Mu likely to be tcl1-mu
**ms-6015***				Mu tag 7300 seed screen 2011
*RescueMu* *C17-32*
*RescueMu* *E03-23*
*RescueMu* A60-22b
*EMS72063***
*EMS72032***	Karl, Sidae			EMS71924 is allelic
*EMS71990***
*EMS72091***
Lower priority late mutants
*EMS71777***	Late
*EMS71787***	Late
*EMS63265***	Late
*EMS72098***	Drop it

Set-up for screening in summer 2011 at Webb Ranch 1.5 acres
ms*6015 microscopy makes a strong case for this locus = 7300 seed No putons
ms si*355 10,500 3 putons
mtm00-06 19,200 No putons
ms9 1,350 pilot population No putons

Generate tagging populations in summer 2011 at Stanford University for 2012 screen on 4 acres at Webb Ranch
ms9 (transverse & confocal imaging look very interesting) 20K seed goal
ms*6015 another 13K seed goal
EMS63089 20K goal has been very hard to get seed
ms si*355 11K goal
ms14 20K goal (second attempt to get tagged alleles)
ms13 10K goal

Tagging populations already available for 2012 when there is more field space
csmd1, if cloned we can look for another allele if we want to 10,200 seed
ms10, outside our developmental window 10,000 seed lower priority
ms13, 9,400 seed
ms17, 2,850 low priority

Table 3. Double mutant analysis

The crosses used in allelism testing yield plants suitable for double mutant construction.

msca1//msca1 male-sterile x mac1//Mac1 male-fertile

20 to 30 individuals are evaluated in the following season. If all are fertile, two loci are defined as in this example. The individuals are of two genotypes

Single carriers Mac1//Mac1 Msca1//msca1
Double carriers Mac1//mac1 Msca1//msca1

Eight to ten fertile individuals are self-pollinated.

Single carriers segregate 3:1 F:sterile Double carriers segregate 9:7 F:sterile

The two types of families are distinguished in growouts of 60 or more individuals. Within the 9:7 families, 1/7 of the plants are double mutants. If one or both of the genes of interest has been cloned or fine-mapped, PCR markers are used to genotype plants before the tassels develop to identify the homozygous double mutants and each of the single mutants for confocal or molecular analysis.

To check if we have made the double mutant stock, do a search in GENETIC RECORDS from the Walbot lab website (click on the tag on the left margin of the home page). After the genetic records load, click on “search” and put in both gene abbreviations and click “and” to make it a both required search. This search should take you to the cross that combined the two defective alleles and the subsequent progeny, if analyzed.