Accelerated Development of Papaya Ringspot Virus (PRSV) Resistance in Squash

Part 1: Swati Shrestha Interview

Duration: 00:05:35
Transcript

00:00:05:18 - Hi I'm Thi Nguyen with the Science Communication Lab.
00:00:08:12 I'm so excited to talk this year
00:00:10:09 to all of the Share Your Research participants and winners.
00:00:13:05 Today we're talking to Swati.
00:00:14:20 Swati would you like to introduce yourself?
00:00:16:18 - Hey everyone I'm Swati Shrestha
00:00:19:00 currently a PhD student
00:00:20:15 in Vegetable Breeding and Genetics Lab
00:00:22:22 at the University of Florida.
00:00:24:23 My research work at the University of Florida
00:00:27:19 focuses on development and application of molecular tools
00:00:31:12 for accelerated breeding
00:00:33:08 of disease resistance in squash
00:00:35:17 that would ultimately increase
00:00:37:16 squash growers' productivity and profitability.
00:00:41:07 - And what motivated you to get
00:00:43:02 into this field of science?
00:00:44:22 - Actually I'm originally from Nepal
00:00:47:08 where the agriculture is one of the major occupation
00:00:51:15 of a lot of people.
00:00:53:05 So growing up farmers and farming community
00:00:56:15 had a deep impact on me.
00:00:58:11 I could see growers worrying
00:01:00:06 about their crop and yield due to different factors
00:01:04:01 like diseases insects pest and whatnot.
00:01:07:07 So I was always interested in getting
00:01:09:16 into agricultural research
00:01:11:02 and here I am doing what I love.
00:01:13:02 - Helping the farmers with their
00:01:15:12 yield and productivity. - Yeah exactly.
00:01:19:05 So basically the research that I am doing
00:01:21:12 we hope that we'll be able to develop
00:01:23:16 squash varieties that are resistant to diseases
00:01:26:15 so that the growers will have
00:01:28:05 one less thing to worry about.
00:01:30:01 - Yeah and you're doing it faster too right?
00:01:32:02 How are you doing it faster than traditional breeding?
00:01:34:13 - In case of traditional breeding
00:01:36:11 the plants need to be grown until maturity
00:01:39:10 to select for the resistant individuals.
00:01:41:18 But now in this era of science
00:01:43:18 we have molecular breeding
00:01:45:10 so that we don't even need to grow
00:01:46:21 the plant in the field
00:01:47:23 to actually see they are resistant or not.
00:01:50:11 We just need to extract the DNA from the plants
00:01:53:00 and then just use some molecular markers
00:01:56:15 to see if the plants are resistant or not.
00:01:59:08 And in this way we can select the resistant plants
00:02:02:03 without even growing them in the field.
00:02:04:04 So that increases
00:02:09:03 the varietal development process.
00:02:12:08 And then we can get new varieties
00:02:14:06 just within four to five years
00:02:16:01 where in traditional breeding
00:02:17:07 it can take up to 10 years
00:02:18:18 for the development of new varieties.
00:02:20:19 - You seem so excited as you talk about this.
00:02:23:00 Were you always this excited about science?
00:02:24:19 What was your first science memory?
00:02:26:11 - Science has always been there with me
00:02:30:05 ever since childhood.
00:02:31:14 I would say specifically
00:02:33:07 I was interested in gardening and plant science
00:02:36:17 since early childhood.
00:02:38:17 I remember I was in grade two
00:02:41:11 when I had got some corn seed.
00:02:43:07 And without thinking anything
00:02:44:18 I had just planted them in the pot.
00:02:47:21 And then after a week or so
00:02:50:16 I just saw small plants coming out of the pots.
00:02:53:12 And I realized I was so excited.
00:02:55:19 I went to my mom and asked her how that happened.
00:02:59:01 So she explained me that a small seed
00:03:01:08 can give rise to a entire plant.
00:03:03:10 I was so proud of the plant.
00:03:05:08 I showed that plant to all my friends
00:03:07:19 and I even harvested real corn from that plant.
00:03:11:21 So observing that small seed grow
00:03:15:00 into a entire plant and then giving corn
00:03:17:21 was my scientific memory
00:03:20:15 of observing the plants grow.
00:03:24:01 And then that's how I got interested in plant science.
00:03:27:06 And here I am today doing a PhD
00:03:30:04 in plant breeding and genetics.
00:03:32:12 - Well what is one thing about being a scientist
00:03:35:02 that you wish other people knew?
00:03:37:04 - Well I think that when people hear the word scientist
00:03:40:05 they actually think of people
00:03:42:23 spending a lot of time in their labs
00:03:45:15 or in their workplace
00:03:46:21 or people spending time in front of their books.
00:03:50:02 But I think people should know
00:03:52:04 that being a scientist
00:03:53:12 is also about following one's passion
00:03:56:14 and for translating science into solution
00:04:01:03 and trying to make this world a better place to live.
00:04:04:00 - And for a young person going into your field
00:04:06:16 what're some of the greatest things
00:04:08:00 about working as a plant scientist
00:04:10:02 and what're some of the most challenging?
00:04:12:02 - For any young person
00:04:13:23 who is trying to enter into my field
00:04:16:01 I would say that the greatest thing
00:04:17:20 is we are working into something
00:04:20:03 that is one of the most important things
00:04:22:09 required for human civilization.
00:04:24:14 That is food.
00:04:25:19 So it gives me immense pleasure and satisfaction
00:04:29:08 working on that field.
00:04:31:03 And the biggest challenge I would say
00:04:33:19 would be that we don't know
00:04:37:03 when a new disease or pest would outbreak in a crop.
00:04:40:15 So we always need to be ready
00:04:42:14 to dive into the unknown
00:04:44:07 and find solution to the immediate problems.
00:04:47:12 - That's great advice
00:04:48:12 and I'm so excited for people to hear
00:04:50:06 about your research talk
00:04:51:15 that talks about bringing food to the table
00:04:56:11 your genetic analysis
00:04:57:19 your excellent experimentation
00:04:59:10 and the way that you tell the story.
00:05:00:18 Thank you so much for joining us Swati.
00:05:05:09 - Thank you for having me.
00:05:06:16 It was a pleasure and we learned a lot from iBiology.

Part 2: Accelerated Development of Papaya Ringspot Virus (PRSV) Resistance in Squash

Duration: 00:18:19
Transcript

00:00:13:06 - Imagine yourself at a barbecue
00:00:14:08 with your family and friends.
00:00:17:08 I am pretty sure that the barbecue
00:00:19:00 would be incomplete without some perfectly grilled squash
00:00:23:10 those low-calorie delicious squash
00:00:26:05 with some salt pepper and olive oil on top
00:00:30:05 would surely make your get-together healthier and better.
00:00:34:23 Or even if you're not a barbecue person
00:00:38:01 you definitely might have enjoyed a hot bowl
00:00:41:11 of squash soup at your home.
00:00:45:01 Well many of you might have enjoyed squash
00:00:47:21 in a way or another and only seen
00:00:51:11 healthy looking squash fruits at the grocery stores.
00:00:55:21 But did you know that squash plants in the grower's field
00:00:59:11 is infected by several diseases
00:01:02:07 and sometimes severe infection of these diseases
00:01:06:06 can even cause up to 100% yield loss?
00:01:11:19 Well just in case you're not familiar
00:01:13:17 with the squash field this is a picture
00:01:16:17 of a healthy squash field that would produce
00:01:19:22 acceptable yield profitable to the growers.
00:01:24:19 However this is a picture of a diseased squash field
00:01:28:11 and even though it might look similar
00:01:31:00 to any other squash field to the normal eyes
00:01:34:17 these yellow diseased squash plants here
00:01:37:21 significantly reduce the yield and quality
00:01:41:01 of squash fruits produced
00:01:42:23 thus reducing profitability to the growers
00:01:46:08 and quality to the consumers.
00:01:49:04 Now there are many viral fungal and bacterial diseases
00:01:54:15 infecting squash but my PhD research
00:01:58:15 at the University of Florida focuses
00:02:01:11 on one major viral disease of squash
00:02:05:04 named Papaya Ringspot Virus or PRSV.
00:02:11:04 Thus my talk today is gonna be
00:02:13:22 on accelerated development of Papaya Ringspot Virus
00:02:17:13 resistance in squash.
00:02:21:07 Now before I jump into the details of my work
00:02:24:03 I would like to talk a little bit more
00:02:26:11 on Papaya Ringspot Virus or PRSV.
00:02:32:08 As you can see in the figure here
00:02:34:05 Papaya Ringspot Virus infects both the vegetative
00:02:37:19 and reproductive part of the plant.
00:02:42:02 Infection on vegetative part of the plant
00:02:44:04 results in severe leaf mottling that can be seen here
00:02:48:07 and infection on reproductive parts on the plant
00:02:51:14 result in deformed and unmarketable fruits
00:02:55:06 as can be seen here.
00:02:57:17 Studies have shown that severe infection
00:03:00:22 of PRSV in squash can cause up to 80% yield loss.
00:03:08:02 Now just in case you are thinking
00:03:09:13 why not control these plant pathogenic viruses
00:03:12:16 by just spraying some chemicals
00:03:15:05 well that's not possible in case
00:03:18:02 of these plant pathogenic viruses
00:03:20:21 because viruses are obligate intracellular parasites
00:03:26:18 which means that they get into the plant's
00:03:30:00 cellular system and multiply
00:03:32:02 within the plant cellular system.
00:03:35:16 So any chemical that is used to kill the virus
00:03:38:04 will kill the plant as well
00:03:40:03 and we definitely don't want that.
00:03:43:19 So the best option for controlling
00:03:45:07 yield loss due to viral infection in squash
00:03:48:09 would be genetic control or use
00:03:51:15 of virus resistant-squash varieties.
00:03:55:21 However unfortunately squash varieties
00:03:58:19 with stable resistance to viruses are currently
00:04:02:02 unavailable in the market.
00:04:05:11 Now the virus-resistant squash varieties
00:04:07:10 can either be developed by using
00:04:09:06 traditional breeding approaches
00:04:11:04 and the process for traditional breeding
00:04:13:17 looks something like this.
00:04:16:21 In case of traditional breeding we would cross
00:04:18:20 the virus-susceptible commercial squash variety
00:04:22:12 with the virus-resistant wild squash genotype.
00:04:26:13 The progeny obtained from the cross
00:04:28:13 are then grown until maturity to select
00:04:31:01 for the virus-resistant individual.
00:04:34:10 Now the process of selecting virus-resistant individual
00:04:37:17 is continued in each generation until we get
00:04:41:13 high yielding virus-resistant squash plants
00:04:45:06 that can be released as a virus-resistant squash variety.
00:04:50:04 And this process of developing virus-resistant
00:04:52:18 squash variety by using traditional breeding approaches
00:04:56:11 can take up to eight to 10 years.
00:05:01:05 Or alternatively virus-resistant squash varieties
00:05:04:10 can be developed by using molecular breeding
00:05:07:15 or marker-assisted selection
00:05:10:18 and the process for molecular breeding
00:05:13:01 looks something like this.
00:05:15:04 Even in case of molecular breeding
00:05:17:18 the initial step of crossing the commercial
00:05:21:12 virus-susceptible squash variety
00:05:24:02 with the wild squash genotype is the same;
00:05:27:11 however in this case the progenies obtained
00:05:31:16 are not grown until maturity.
00:05:34:10 In case of molecular breeding virus-resistant individuals
00:05:38:15 are selected in each generation with the help
00:05:41:06 of DNA markers which can be done
00:05:44:02 by extracting DNA from the plants
00:05:46:12 when they are really young.
00:05:49:02 So as in case of molecular breeding
00:05:51:12 you don't need to grow the plant until maturity
00:05:54:10 to select for the virus-resistant individuals
00:05:57:07 in each generation development of new
00:06:00:06 virus-resistant squash varieties can be done
00:06:03:03 just within four to five years' time period
00:06:06:07 unlike in traditional breeding
00:06:08:10 where it takes almost eight to 10 years
00:06:11:22 for the development of new virus-resistant squash varieties.
00:06:16:06 However unfortunately the genomic regions
00:06:20:13 and DNA markers associated with PRSV resistance
00:06:24:10 in squash are currently unknown
00:06:26:18 thus making molecular breeding in squash impossible.
00:06:31:16 Thus keeping in mind this knowledge gap
00:06:34:01 in squash breeding program my study aimed
00:06:37:21 to identify the genomic region associated
00:06:41:04 with PRSV resistance in squash
00:06:43:23 and then develop DNA markers associated
00:06:47:11 with PRSV resistance that can be used
00:06:50:02 in molecular breeding of squash.
00:06:53:19 Now before I jump into the details
00:06:56:22 of all the methods used in the study
00:06:59:15 the slide here provides a brief overview
00:07:02:10 of all the steps used to achieve the study goal.
00:07:07:00 The first goal of the study as I mentioned
00:07:09:05 was identification of genomic regions
00:07:11:19 associated with PRSV resistance
00:07:14:21 and the first step for the process
00:07:17:11 was development of the mapping population.
00:07:21:01 Now just in case you're not familiar
00:07:23:02 with the plant science research
00:07:24:20 let me tell you that in any plant science
00:07:28:04 genomic region identification study
00:07:30:19 the first step is the development
00:07:32:22 of the mapping population and in my case also
00:07:36:10 it was the same so the first step
00:07:38:14 is development of the mapping population.
00:07:42:16 Now after first step we move to step two
00:07:45:16 which was identification of the genomic region
00:07:49:11 using the mapping population that was developed
00:07:52:00 in the step one.
00:07:54:02 The second goal of the study was to develop
00:07:56:15 DNA markers associated with resistance
00:07:59:22 which takes us to step three which was to develop
00:08:02:20 DNA markers from the genomic regions
00:08:05:18 identified in step two and then the final step
00:08:09:14 of the study was validation of the markers
00:08:12:11 developed in step three which was to check
00:08:15:11 if the markers are actually working or not
00:08:18:05 and differentiating the resistant
00:08:20:08 and the susceptible plants at the DNA level.
00:08:24:16 Now don't get worried if you didn't get a lot of terms
00:08:27:07 in this slide because I'm gonna explain
00:08:30:04 each of these steps in brief
00:08:33:01 in the upcoming slide starting with step one
00:08:36:16 that was development of the mapping population.
00:08:41:06 So for development of mapping population
00:08:43:00 we crossed the virus-resistant wild squash genotype
00:08:47:06 with the virus-susceptible commercial squash variety
00:08:51:02 and in breeding languages the progeny obtained
00:08:54:16 from this cross are known as F1.
00:08:58:04 The F1 were then selfed to produce the F2 population
00:09:01:20 and if you are familiar with Mendel's Law
00:09:04:18 then you would know that these F2 plants
00:09:08:07 would be segregating for virus resistance.
00:09:11:01 So the F2 plants are then artificially inoculated
00:09:15:03 with the virus and 10 most resistant
00:09:18:05 and 10 most susceptible individuals are selected
00:09:21:23 that would be our mapping population.
00:09:27:10 Now just in case you lost me when I said
00:09:29:00 the term artificial inoculation
00:09:31:07 and you're wondering how the plants
00:09:33:04 are artificially inoculated with the virus
00:09:36:01 well let me walk you through that.
00:09:39:07 For artificial inoculation
00:09:41:16 we maintain the live viral-infected plants
00:09:44:22 in the cages in the greenhouse which can be seen
00:09:47:17 in the figure here.
00:09:49:20 For the artificial inoculation process
00:09:52:20 we take the leaves from the viral-infected plant
00:09:56:00 as can be seen here in the figure A
00:09:59:02 and then we grind these viral-infected leaves
00:10:03:22 in the phosphate buffer as can be seen in the B
00:10:07:18 and then we get a clean viral solution
00:10:11:14 in the phosphate buffer as can be seen
00:10:14:03 in the figure C.
00:10:16:13 So after getting this clean viral solution
00:10:20:02 we apply this clean viral solution
00:10:23:18 in the plants as can be seen in the figure here
00:10:29:02 and if the plants are susceptible
00:10:30:08 they start showing the symptoms two weeks
00:10:33:02 after the inoculation.
00:10:36:04 Based on the response of the plants to the viruses
00:10:38:07 we select 10 most resistant and 10 most susceptible plants
00:10:43:02 and as I mentioned earlier these plants
00:10:46:02 would be then used as the mapping population.
00:10:50:19 Now moving to step two which is genomic region
00:10:53:08 identification using the mapping population
00:10:56:01 that was developed in step one.
00:10:59:20 So for genomic region identification
00:11:02:03 we extract the DNA from the parents
00:11:04:18 and the resistant and susceptible selected individuals.
00:11:10:03 The extracted DNA is then sent for sequencing
00:11:13:12 to whole genome sequencing labs
00:11:15:18 providing sequencing facilities.
00:11:20:01 Basically we get the sequencing results
00:11:21:14 within a month or so and as the genomic DNA
00:11:25:16 is made up of nucleotides the sequencing results
00:11:29:23 look something like this which is nothing
00:11:32:16 but long strings of nucleotides
00:11:35:02 arranged in specific order.
00:11:38:20 After getting the sequencing results
00:11:40:10 we do some bioinformatic analysis
00:11:44:11 where we compare the nucleotide arrangement
00:11:47:01 of the resistant and susceptible individuals.
00:11:51:03 Now this would be similar to comparing
00:11:53:23 two necklaces of different colored beads
00:11:58:06 arranged in specific order and then identifying
00:12:01:16 the regions that have significantly
00:12:03:18 different colored beads.
00:12:05:22 Thankfully we have some strong computer algorithms
00:12:09:14 to do the comparison and then by doing
00:12:12:13 the bioinformatic analysis we are able
00:12:16:01 to identify the regions between the resistant
00:12:18:20 and susceptible individuals that have
00:12:21:09 significantly different nucleotide arrangement.
00:12:26:07 Scientifically speaking the regions
00:12:28:06 between the resistant and susceptible individuals
00:12:31:08 that have significantly different
00:12:33:04 nucleotide arrangement would be
00:12:35:04 the genomic region associated with PRSV resistance.
00:12:39:11 So hooray after doing the bioinformatic analysis
00:12:43:12 we are able to identify the genomic region
00:12:46:12 associated with PRSV resistance.
00:12:50:21 This is the graph that shows how the output
00:12:53:14 of the genomic region identification study looks like.
00:12:58:06 Now as the squash genomic DNA is made up
00:13:00:15 of 20 chromosome the X axis here gives
00:13:04:07 the number of chromosomes that is the 20
00:13:07:00 and the Y axis here gives the difference
00:13:10:08 in nucleotide arrangement between the resistant
00:13:13:01 and the susceptible individuals.
00:13:17:15 Now I would like you to focus your attention
00:13:19:14 on the peak here on chromosome nine.
00:13:22:23 This peak here means that this region here
00:13:26:08 in chromosome nine is significantly different
00:13:29:16 between the resistant and susceptible individuals
00:13:32:20 at 95% confidence interval where this green line
00:13:37:02 here represents 95% confidence interval.
00:13:42:00 So basically it would mean that this is the region
00:13:44:13 that is significantly associated
00:13:46:14 with PRSV resistance in squash.
00:13:50:18 This graph here is just a zoomed in version
00:13:52:20 of what's happening in chromosome nine.
00:13:56:08 This top graph here the black dots represent
00:14:00:13 the nucleotide arrangement of the susceptible individuals.
00:14:05:20 In this middle graph here the black dot represents
00:14:08:05 the nucleotide arrangement of the resistant individuals.
00:14:12:12 And this bottom graph here the blue line represents
00:14:16:09 the difference in nucleotide arrangement
00:14:18:14 between the resistant and susceptible individuals.
00:14:23:01 I would like you all to focus your attention
00:14:25:03 on this region here inside the blue bar.
00:14:28:15 So this is the region where the nucleotide arrangement
00:14:31:14 is significantly different between the resistant
00:14:34:06 and susceptible individuals
00:14:36:11 so in other words again this is the genomic region
00:14:40:02 associated with PRSV resistance.
00:14:43:23 So after identification of genomic region
00:14:46:11 associated with PRSV resistance
00:14:49:06 our next step is marker development
00:14:52:10 from this genomic region.
00:14:54:13 And now I wouldn't go into details of how the marker
00:14:57:15 I developed from this genomic region
00:15:00:01 but it's a straightforward process and is done
00:15:03:22 by using a software called a spatch primer three.
00:15:08:16 Now after development of the marker
00:15:10:07 the next step is marker validation
00:15:12:23 which is to actually check if the marker developed
00:15:16:15 in our study are working or not
00:15:18:20 which means to check if they are able
00:15:21:19 to differentiate the resistant and susceptible plants
00:15:23:21 at the DNA level or not.
00:15:26:14 For marker validation we actually extract the DNA
00:15:29:09 from the resistant and susceptible parent
00:15:32:05 and then we mix the marker that was developed
00:15:35:06 in our study with the DNA.
00:15:39:01 Now this marker DNA combination is then fed
00:15:41:15 into a machine called a light cycler
00:15:44:15 which looks something like this.
00:15:47:23 Now if the marker developed in our study
00:15:49:22 are actually working then the output
00:15:53:01 of the light cycler machine should be able
00:15:55:06 to differentiate the resistant and susceptible plants' DNA.
00:15:59:16 And this is how the output of the light cycler study
00:16:03:21 looked like and here in this graph
00:16:06:19 we can see that there were two separate groupings
00:16:09:21 of the resistant and the susceptible individuals
00:16:12:23 which means that yes the marker developed
00:16:15:13 in our study are actually working
00:16:17:16 and they are able to differentiate
00:16:19:06 the resistant and susceptible plants
00:16:21:08 at the DNA level.
00:16:23:21 So yay from this graph we can see that yes
00:16:26:15 we have developed the markers
00:16:28:10 that are actually working.
00:16:31:12 So from the study we can conclude that we were able
00:16:34:15 to identify the genomic region associated
00:16:37:15 with PRSV resistance in chromosome nine of squash
00:16:41:14 and also we were able to develop the DNA markers
00:16:46:00 associated with PRSV resistance
00:16:48:12 that could differentiate the resistant
00:16:51:09 and susceptible plants at the DNA level.
00:16:55:14 So what's the implication of the study?
00:16:57:16 Well the DNA markers that were developed in the study
00:17:01:01 can be used for rapid selection
00:17:02:22 of PRSV-resistant individuals in each generation
00:17:06:23 and with the help of these DNA-marker-assisted
00:17:09:21 molecular breeding new PRSV-resistant squash varieties
00:17:14:09 can be developed just within four to five years'
00:17:17:16 time period unlike in traditional breeding
00:17:20:14 where it takes almost eight to 10 years
00:17:23:16 for the development of new virus-resistant squash varieties.
00:17:28:00 So with the rapid development
00:17:29:19 of PRSV-resistant squash varieties in the future
00:17:34:02 growers don't have to worry about yield loss
00:17:36:21 due to viral infections and the consumers
00:17:40:02 will get healthy and delicious squash
00:17:42:11 to enjoy in every meal.
00:17:45:00 With that I would like to thank you all
00:17:46:21 for attention and I would like to end my presentation.
00:17:50:22 Thank you.

Videos in this Talk

Part 1: Swati Shrestha Interview
Audience:
- Student
- Researcher
- Educators
- Educators of Adv. Undergrad / Grad
Part 2: Accelerated Development of Papaya Ringspot Virus (PRSV) Resistance in Squash
Audience:
- Student
- Researcher
- Educators
- Educators of Adv. Undergrad / Grad

Speaker: Swati Shrestha

Total Duration: 0:23:54

Recorded: May 2022

All Talks in Neuroscience

Talk Overview

Profitable production of squash in the growers field is threatened by a virus called Papaya Ringspot Virus (PRSV). Yet, manual phenotyping for PRSV-resistance in each generation is tedious and time-consuming, thus making breeding for PRSV-resistance difficult. In this 2022, Share Your Research Talk, Swati Shrestha describes her research on developing virus-resistant squash. As a researcher from Nepal which is an agricultural country, Swati has always been passionate about developing disease resistant crop varieties that would be beneficial to growers. She addresses the issue of yield loss due to viral disease in squash by using genetic, molecular and computational techniques to develop molecular markers associated with PRSV-resistance. These findings have implications in speeding up the identification, selection, and breeding of PRSV-resistant squash for the growers in the field.

Speaker Bio

Swati Shrestha

Swati Shrestha is a PhD candidate at the University of Florida, in the Vegetable Breeding and Genetics lab. For her thesis research, she develops and applies molecular tools for accelerated breeding of Potyvirus resistance in squash. Swati is interested in science communication as much as she is into research and development. In the future she… Continue Reading