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WELCOMWELCOMEE
Root Knot Nematode Resistance Breeding in Solanaceous & Root Vegetables: Progress,
Challenges & Prospects
DIVISION OF VEGETABLE SCIENCEINDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI-110012
Seminar In-charge: Dr. A.K. Sureja Chairman: Dr. Anil Khar
Contents
IntroductionBreeding for RKN ResistanceProgress/Achievements Challenges in breeding Prospects Case studies Seminar conclusion
Kingdom: Animalia Phylum: NematodaRoundworms, Eel worms Microbial Feeder Animal Parasitic Human Parasitic Insect Parasitic Plant Parasitic
Nematodes
Most numerous animalSecond most numerous
species (1 million)Existence on the planet: 4
billion year agoLongest is 26 feet (in Blue
whale)No circulatory systemNo respiratory systemNo skeletonNo eyes
Interesting Interesting FactsFacts
Lambshead, (1993)
Over 4,100 species of plant-parasitic nematodes have been identified CosmopolitanAttacks almost all plantsOverall average loss
World – 12.3% India - 20-30%
Annual yield losses among Solanaceous & Roots Vegetables
Plant-Parasitic Nematodes
National loss -Rs 21,068.73 millionWorldwide loss - nearly $78 billion.
I. MeloidogyneII. PratylenchusIII. HeteroderaIV. DitylenchusV. Globodera
VI. TylenchulusVII. Xiphinema
VIII. RadopholusIX. Rotylenchulus
X. Halicotylenchus
Top Ten Genera
Ravichandra, (2014)
Decraemer and Hunt,( 2006)
RKN (RKN (Meloidogyne Meloidogyne sppspp.).)
Root-knot
Class: SecernenteaOrder: TylenchidaFamily: HeteroderidaeSub family :MeloidogynidaeGenus: Meloidogyne67 spp. causing root knot disease
Responsible for 95% total crop loss attributed to nematodes
Cause approximately 5% of global crop lossSymptoms
Stunted growth, yellowing and wilting Root knot or root gall in the roots Forking , branching in carrot
Meloidogyne acroneaMeloidogyne arenariaMeloidogyne artiellia
Meloidogyne brevicaudaMeloidogyne chitwoodiMeloidogyne coffeicola
Meloidogine exiguaMeloidogyne fruglia
Meloidogyne gajuscusMeloidogyne hapla
Meloidogyne incognitaMeloidogyne javanica
Meloidogyne enterolobiiMeloidogyne naasi
Meloidogyne partitylaMeloidogyne thamesi
Sasser and Carter, (1995)
RKN DistributionRKN Distribution
M. incognita -M. javanica M.hapla - Meloidogyne other spp –
RKN- Interaction with plants
Life cycle of a mitotic parthenogenetic root-knot nematode. (a) Longitudinal section of a root tip showing second-stage juveniles ( J2s) (stained with acid fuschin) turning around at the root meristem to migrate into the vascular cylinder. (b) Typical symptoms (i.e., galls) on tomato roots. (c) Longitudinal section of an infested root showing a mature female and five giants cells ( )∗ constituting the nematode feeding site.
One practice will control nematodes, One practice will control nematodes, so two or more control methods must so two or more control methods must be used.be used.Only resistance singly effectiveOnly resistance singly effective
Fallow
Sanitation
Solarization
Resistance
Chemicals
Rotation
SuccessfulSuccessfulNematodeNematode
ControlControl
Fallow
Solarization
Rotation
SuccessfuSuccessful l Nematode Nematode ControlControl
Sanitation
Chemicals
Resistance
Management SummaryManagement Summary
When We Call Resistant or Susceptible ?
RKN Resistance Tomato
Resistance to root-knot nematodes originates from the wild relatives, S.arcanum and S. peruvianum
Mi genes are inherited as single dominant genes.
Gene Map position Temperature limit ReferencesMi-1 Chromosome 6 28ºC Cap et al., 1993Mi-2 Heat stable Yaghoobi et al. 1995Mi-3 Chromosome 12 28ºC Yaghoobi et al. 1995Mi-4 Heat stable Veremis and Robert, 1996Mi-5 Chromosome 12 Heat stable Yaghoobi et al. 1995Mi-6 Heat stable Cap et al., 1993Mi-7 28ºC Roberts et al., 1990Mi-8 28ºC Yaghoobi et al. 1995Mi-9 Chromosome 6 Heat stable Jacquet et al., 2005Mi-LA2157 Chromosome 6 Heat stable Veremis et al.,1999
Peppers
Me gene inheritance- single dominance
One recessive gene has been hypothesised in the pepper cultivar ‘Carolina wonder’ (Fery and Dukes,1996)
Resistance mostly identified from the cultivated capsicum annuum
One C. chinense accession was considered to be resistant to M. enterolobii
Four QTLs have also been identified in pepper
The N and the Me genes (i.e. Me1, Me3, Me4, Mech1 and Mech2) conferring resistance to RKNs have been mapped on the 9 chromosome.
Djian-Caporalino et al., 2007
Sugar beet
Resistance source wild beet Beta vulgaris ssp. maritima and B. procumbens (Yu et al. 1999). Inheritance - single dominant genes
Gene located on chromosome 4 and designated as R6m-1 (Yu et al., 1999).
Carrot
The Mj-1 gene was discovered in a ‘Brasilia’ cultivar, imparts resistance to M. javanica (Boiteux et al. 2000; Simon et al. 2000). Mj-2 mapped in PI652188 from China (Ali et al. 2014).Inheritance- single dominant Chromosomal location- both on chromosome 8
Breeding programmes
Conventional or Traditional Breeding Approaches
Breeding Method for resistance Mass Selection Line breeding Pure line Hybridization followed by Pedigree
Bulk selection method Back cross
Recurrent SelectionInterspecific hybridization followed by embryo rescue methods have
recently allowed the introgression of Mi-3, Mi-1.2 resistance into a S. lycopersicum genetic background (Moretti et al. 2002)
BIOTECHNOLOGICAL APPROACHESMarker-assisted Selection (MAS)
The first resistance gene marker isozyme acid phosphatase linked With Mi-1 (APS-1) (Medina-Filho and Stevens 1980; Rick and Fobes 1974)
Phosphoglucomutase (PGM) - isozyme marker for RKN resistance in BeetUseful markers for MAS
For Mi-1 of tomato CAPS_ REX-1 and Cor-Mi and SCAR marker Mi-23, PMi For Mi-3 of tomato Scar_N22R, Scar_P22L and Scar_E21L at less than 0.25 cM ,
For Mj-1 of carrot one STS markers were developed (Boiteux et al.2004)A CAPS (NEM06) marker with resistance to the RKN gene in beet roots (Weiland and Yu ,2003).
Transgenesis
Intragenesis
CisgenesisBarbary et al., 2015
Achievements/ Host Resistance to RKN
Ravichandra, (2014)
Resistance or Tolerance Varieties in India
SL-120, Hisar Lalit, PNR-7, Hisar N-1,Hisar N-2, Hisar N-3, NT-3, NT-8NT-12,RonitaPatriot, PAU-5,ManglaKarnataka Hybrids
Parvatha Reddy ( 2008 )
CHERRY TOMATO Small Fry
RKN spp Resistant Genotype M. incognita Pusa Jwala, 579, CAP 63 Suryamukhi Black, Jwala, Bull
Nose, Hungarian Wax, Chinese Giant, Chilli NP-46-a, Chilli G-3 Suryamukhi, California Wonder
M. incognita race 1 Pusa Jwala, Jwala, Wonder Hot,Teja, Utkal Abha, Utkal Rashmi
M. incognita race 2 JwalaM. incognita race 3 Pusa Jwala, Jwala
M. incognita race 4 Jwala
CAPSICUM Mississippi-68, Santanka, AnaheimChile, and Italian Pickling
Pimento Pepper :Mississippi NemaheartHot pepper: Carolina Cayenne, Charleston Hot
Ravichandra, (2014)
Pusa Jwala
RKN spp Resistant Genotype M. incognita Vijaya , Black Beauty (Highly
Tolerant), Giant of Banaras,Mysore Green, Pusa PurpleLong , Maroo Marvel, Gulla, Gachha Baigan, Pbr-91-2, IC-95-13,HOE-101, Red Wonder
Meloidogyne spp. S. sisymbriifolium
M. javanica Black Beauty,Bhanta, Muktakeshi, Round Red,Coolie, Mathis B, Mysore Green,America Big Round, Arka Sheel,R-34, Sonepat, BR-112, Gulla
Ravichandra, (2014)
S. sisymbriifolium
RKN spp Resistant Genotype References Meloidogyne hapla PI 164461, 187236, 193506, 225867, 234623,
267091 & 269316Clark, 1969
M. hapla Daucus carota ssp. hispanicus Frese, 1983
CARROT
RADISH
RKN spp Resistant genotype References M. incognita Ramgo Reyes, 1981
Challenges in RKN Resistance breeding
Hidden enemies (Ravichandra , 2014)
Broad range of plant hosts ( Jones et al., 2013)
Most of these genes originate from wild relatives and their introgression and elimination of linkage drag , is a laborious and time-consuming Exploitation of QTLs is much more complex in breeding programmesNone of the currently known R genes in Solanaceae confers resistance to all RKN speciesThe resistance conferred by Mi-1 is broken at temperatures above 28°C (Williamson, 1998).
Prospects in RKN Resistance breeding
Need to developed varieties which confers resistance to all RKN species
Breeding for RKN resistant rootstocks would be considered as a possible alternative (Schwarz et al.,2010)
Pyramiding QTLs with the major R gene(s) into one cultivar is expected to provide a complete and durable resistance
SNPs will be useful in marker-assisted breeding very accurately for RKN resistance
RNAi to silence nematode susceptable genes
Develop subgenic crops resistance to RKN
Case Study -1
Objectives
To determine inheritance patterns of RKN resistance
To map chromosomal regions responsible for RKN resistance
Materials & MethodsPlant materials
M. incognita resistance cvs ‘‘Brasilia’’ (Br1091) ,‘‘Homs’’ (HM), and ‘‘Scarlet Fancy’’and ‘‘Favourite’’ (SFF). Br1091 × HM1 F2 population, SFF × HM2 F2 population, F 3 populations of
HMSusceptible control cv. ‘‘Imperator 58’’ DNA extraction and marker evaluations
DNA extracted according to Murray and Thompson (1980)AFLP reactions were performed according to Vivek and Simon (1999)Gene Marker version 1.5 was used to score allelesSNPs were evaluated using the KASPar system (http://www.KBioscience.co.uk).
Genetic map construction Linkage maps were constructed with JoinMap 3.0 softwareQTL mappingQTL analysis by the using of multiple imputations method QTL detection by using scanone followed by QTL modeling
Fig- QTL location on different chromosomes
Results and Discussion
Inference
QTLs for M. incognita resistance in Carrot were located on chromosomes 1, 2,4, 8, and 9
There were no significant interaction effects among QTLs Over-dominance noted for Mi-HM3-C9-Q1, all QTL effects were additive
Each population accounted for 55.5, 34.8, and 35.7 % of the variation in Br1091 × HM1, SFF × HM2, and HM3, respectively .
QTLs may be introgressed into susceptible germplasm with Mj genes to develop more durable resistance against both M. incognita and M. javanica
Case Study -2
Objectives
To identify a marker tightly linked to the N gene for MAS
To found R gene hotspots in the Me-gene cluster on chromosome P9
Materials & Methods
The location of the Me-gene cluster was identified by aligning flanking markers
Flanking markers were selected from the linkage map of Djian-Caporalino et al. (2007)
Total of 34,899 CDS generated by Kim et al. (2014) were downloaded from the pepper genome platform(PGP) database
All the CDS were functionally annotated using Blast2go software
For genetic linkage mapping, an F2 population derived from the cross between RKN resistant cv. Carolina Wonder and RKN susceptible cv. AZN-1 was developed.
The marker linked to the N gene was tested in an F4 population
F1 hybrids and F2 and F4 populations were tested for RKN resistance @ suspension of 1000 J2s at the fourth true leaf stage.
Results and Discussion
Mapping showed that the cluster contained three loci (lociA, B and C) hotspots for R genes.
The A, B and C loci were 1.46 Mb, 265.87 and 390.35 kb in length, respectively.
The Me1 gene was mapped to locus B
SSR and CAPS markers were developed from all the three hotspot lociMarker CASSR37 was found to be tightly linked to N at a distance of 0.8 cM
A Physical map of the Me-gene cluster, B genetic linkage map of N gene constructed using F2 population
Inference
The Me-gene cluster was contain three resistance gene hotspots
A SSR maker (CASSR37) was found tightly linked to the N gene (0.8 cM away) can be used in MAS for RKN resistance breeding in pepper
Me-gene cluster-specific SSR markers can be useful in mapping of other resistance genes (Me7 and Me3)
Seminar Recapitulation
Resistance is the eco-friendly & sempiternal RKN management strategy
Not only monogenic dominant R-genes have been identified for RKN resistance but also QTL
Pyramiding QTLs with the major R gene(s) into one cultivar is expected to provide a complete and eternal resistance to RKN
In traditional breeding introgression of R-genes is cumbersome due to linkage drag
Breeding of RKN resistant cultivars is quick and easy with the aid of biotechnological approaches like MAS, transgenic, cisgenic, intragenic, subgenic & RNAi technology