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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