Genetic Mapping of Root-knot Nematode Resistance Genes in Solanaceous Vegetables

Bingzheng JIANG Ziji LIU Zhenmu CAO Jie ZHU

Abstract Root-knot nematodes are becoming more and more harmful to solanaceous vegetables. The most economical and effective way to deal with root-knot nematode disease is to breed varieties with disease resistance. This paper reviewed the recent research progress and achievements of tomato， pepper， eggplant and potato on root-knot nematode disease resistance gene characteristics， disease resistance gene mapping and disease resistance gene molecular markers， and prospects for future research directions.

Key words Solanaceae; Root-knot nematode; Gene mapping; Molecular marker

Root-knot Nematode Species and Harm

Root-knot nematodes （Meloidogyne spp.） are a class of plant pathogens distributed worldwide. They are widely parasitic on various crops， and are one of the most serious soil-borne pathogens that endanger crop production. The average annual loss to world agriculture is as high as 100 billion U.S. dollars[1]. Root-knot nematodes mainly damage the roots of various vegetables， which is manifested in that the lateral roots and fibrous roots increase more than normal， and root knots of different sizes， which are star or cone-shaped， some of which are bead-shaped， are formed on the fibrous roots of young roots. The root-knot nematodes that damage vegetables mainly include M. incognita， M. hapla， M. arenaria and M. javanica. In the past few years， the damage of M. enterolobii has also become more and more serious， with a tendency to spread from south to north[2]. The host range of nematodes is wide， and there are more than 3 000 kinds of host plants that have been reported. They often damage more than 30 kinds of vegetables such as melon， solanaceous vegetables， bean and radish， carrot， lettuce， and cabbage. They can also induce mixed infection of soil-borne diseases such as blight and bacterial wilt， aggravating the disease condition. The aboveground parts of injured plants grow short， slow， and show abnormal leaf color and small fruits， resulting in a low yield， and even early death of the plants.

M. incognita is one of the four dominant species of Meloidigyne， which is very harmful to solanaceous plants， especially in long-term replanted protected areas. In the diseased fields， the yield is reduced by 20%-30% throughout the year， more than 70% in severe cases， and even total crop failure can be caused. Hainan has superior climatic conditions and is suitable for the reproduction of M. incognita throughout the year. M. incognita mainly damages the roots of crops， forming spherical or conical root knots at the ends of fibrous roots and lateral roots， destroying the hosts' ability to absorb nutrients and water， causing plant malnutrition， short plants， weak growth， few and small fruit， and even death of plants. It seriously affects the yield and quality of pepper[3]. The wounds left by nematode intrusion become a channel for soil-borne pathogens such as blight and wilt to invade， and a small root-knot nematode population often causes serious damage to hosts. The serious damage of root-knot nematodes has become one of the important problems that hinder the sustainable and stable development of Solanaceae crops such as peppers， tomatoes， and tobacco. Methyl bromide can be used to control root-knot nematodes， but the cost is high and it is easy to cause environmental pollution[4]. Compared with chemical pesticide control， disease resistance breeding is a more economical and effective method. However， conventional breeding requires a long time， and the identification of disease-resistant phenotypes is greatly affected by the environment. Therefore， identifying crop disease resistance genes and developing closely linked molecular markers to improve selection efficiency is the key to accelerating the process of disease resistance breeding.

Research Progress on Root-knot Nematode Resistance Genes in Tomato

At present， 9 root-knot nematode resistance genes have been found in wild tomatoes， named respectively Mi-1， Mi-2， Mi-3， Mi-4， Mi-5， Mi-6， Mi-7， Mi-8 and Mi-9， of which Mi-1 is researched most deeply and most widely used[5]. Mi-1 is a mono-dominantly inherited resistance gene with broad-spectrum resistance. It is resistant to M. incognita， M. arenaria and M. javanica， but is not resistant to M. hapla[6]. Mi-1， Mi-3， Mi-7， and Mi-8 are temperature sensitive， and when the temperature is higher than 28 ℃， their root-knot nematode resistance decreases or even disappears. Mi-2， Mi-4， Mi-5， Mi-6 and Mi-9 are of the non-temperature-sensitive type， which can still exert disease resistance under high temperature[7].

Genetic mapping of Mi genes

The Mi-1 gene was introduced from a wild species （Lycoperisicon peruvianum） into a cultivated tomato species （L. esculentum） through the embryo rescue method of interspecific hybridization. Milligan et al.[8]located Mi- in a small region of tomato chromosome 6 and isolate the gene by positional cloning. Wang et al.[9]bred cultivated tomato materials harboring a heat-stable root-knot nematode resistance gene and carried out accurately mapping of the heat-stable resistant gene， which was finally located on the short arm of tomato chromosome 6. Mi-3 is resistant to the toxic population of M. incognita， and is located at the far end of chromosome 12. It is closely linked to the heat-stable M. incognita-resistant gene Mi-5， and Mi-9 is also located at chromosome 6， and is linked to Mi-1[10]. In addition， Mi-2 is linked to Mi-8， and Mi-6 is linked to Mi-7， but none of them are accurately located[11]. Recently， the Mi-HT gene was newly named in the hybrid ZN17. This gene is located on the short arm of tomato chromosome 6， but the Mi-HT gene may be an allele of Mi-1 and Mi-9， or it may be a new one[12].

Development of molecular markers for Mi genes

Dai et al.[13]used three molecular labeling methods to develop Mi-1 functional markers and found that the CAPS method was prone to false positive， while the SCAR markers could directly identify the presence or absence of the Mi-1 gene and its genotypes through a single PCR reaction， and was thus the most convenient and quickest. The study of Yu et al.[14]showed that CAPS was not only faster and easier than RFLP， AFLP and other markers， but also more stable and specific than PCR markers such as PAPD and ISSR. Li et al.[4]used the BSA method to develop Mi gene molecular markers and obtained a RAPD marker OPD20/1454 linked to tomato root-knot nematode resistance genes， and transformed it into SCAR marker SCD20/1000. Seah et al.[15]developed a co-dominant SCAR marker that is linked to the Mi-1 gene and also to the Ty-1 gene. Kuroyanagi et al.[16]established a co-dominant molecular marker of the Mi-1 gene by specific primer amplification and restriction enzyme Msel digestion. Arens et al.[17]also developed a co-dominant SCAR marker that was closely linked to Mi-1.

Research Progress on Root-knot Nematode Resistance Genes in Pepper

Characteristics of root-knot nematode resistance genes in peppers

At present， at least 10 root-knot nematode resistance genes have been found in wild pepper. The N gene is a mono-dominant nematode resistance gene， which is discovered and named by Hare. It is mainly resistant to M. incognita， M. javanica and M. arenaria， etc.， but the resistance of the N gene is unstable under different temperature conditions， and will be partially lost in an environment above 28 ℃ and be significantly reduced or even disappear in an environment with higher temperature and humidity[18]. There are many members of the Me gene family， including Me1， Me2， Me3， Me4， Me5， Me6 and Me7， as well as Mech1， Mech2 and Cami. Among them， the resistance spectra of Me1 and Me3 are broad and similar， but the mode of action and temperature sensitivity are quite different. The resistance response mediated by the Me1 gene does not start until the nematodes have established feeding sites. Part of the resistance will be lost in the high temperature environment， while the Me3 gene-mediated resistance reaction occurs in the early stage of the contact between nematode and the host rhizosphere， which is mainly manifested as an allergic necrosis reaction， thereby preventing the nematode from continuing to infect， and the Me3 gene can maintain full activity even at a high temperature of 42 ℃. In addition， Me2 is a dominant gene and is sensitive to temperature response; the Me4 gene is a dominant gene and linked to the Me3 gene， and is relatively stable in response to temperature; Me5 has not been determined as dominant and recessive; and the Me7 gene exhibits thermal stability[19].

Mapping of root-knot nematode resistance genes in pepper

Wang[20]mapped the Me1 gene between the markers 16880-1 and 16830-H， with genetic distances of 1.44 and 0.63 cM， respectively. Xu[21]conducted a fine mapping study on the Me3 gene， and the two genetic markers developed were located on both sides of the Me3 gene， with the genetic distances reaching 0.56 and 1.33 cM， respectively. Research by Zhang et al.[22]showed that Me8 was located on chromosome 9 of pepper， and the four nearest markers SSCP-B322， COS710， SCAR-315 and COS970 were 0， 0.1， 1.3 and 3.3 cM away from the gene， respectively. The genes N， Me1， Me3 and Me7 resistant to M. incognita are also located in this chromosomal region[22].

Molecular markers for root-knot nematode resistance genes in pepper

Gu et al. used the BSA method and AFLP technology to screen polymorphic primers from the AFLP primer combinations of E/M， and developed a molecular marker closely linked to the pepper root-knot nematode resistance gene N. The linkage distance between the developed AFLP marker and the pepper root-knot nematode resistance gene N was 6.258 cM[23]. Zhang et al.[24]obtained EST-SSR markers closely linked to the dominant root-knot nematode resistance gene Me1 in pepper. Three EST-SSR marker loci that were linked to the root-knot nematode resistance gene Me1 were found， and the genetic distances of these 3 pairs of markers with Me1 were 6.984， 18.684， and 29.310 cM， respectively.

Research Progress on Root-knot Nematode Resistance Genes in Eggplant

Zhang et al.[25]introduced the Bt cry6A gene on the binary vector plasmid pBI 121 harboring the GUS gene and the Npt II gene into eggplant explants through the mediation of Agrobacterium， performed screening on a selective medium containing kanamycin， and obtained a batch of transgenic plants through molecular testing and field testing. It was preliminarily confirmed by GUS staining， PCR and RT-PCR that 5 plants were successfully transformed with the root-knot nematode gene. The resistance test after inoculation with root-knot nematodes showed that the 5 transgenic plants obtained showed resistance to root-knot nematodes.

Research Progress of Potato Root-knot Nematode Resistance Genes

Li et al.[26]searched the potato EST database and spliced 28714 contigs by the electronic cloning technology. Using the cloned root-knot nematode resistance genes in tomato and pepper as probes， the contigs were subjected to alignment and sequence analysis， and a total of 2 potato root-knot nematode resistance genes were obtained （contig_5164， contig_12247）[26].

Problems and Prospects

At present， studies at home and abroad on tomato， pepper and other solanaceous vegetables against root-knot nematodes has achieved initial results， but there are still many problems that need to be resolved： ① Most of the materials containing root-knot nematode resistance genes are wild species， and the problem of sterility exists in the process of hybridization with conventional cultivated varieties， which brings great difficulties to follow-up research. ② The research on the resistance mechanisms of Mi， Me and other resistance genes is not thorough enough. ③ Part of the root-knot nematode resistance genes have been successfully cloned， but few new varieties have been bred through transgenic technology， and their practicality is not high. ④ Some molecular labeling methods are poor in stability and specificity， and can only be applied to theoretical research， and it is difficult to apply them to the practical work of assisting breeding.

Root-knot nematode disease is one of the main diseases that endanger solanaceous vegetables. At present， many research results have been obtained in the resistance mechanisms of disease resistance genes， precise mapping of disease resistance genes and molecular markers at home and abroad， but there are still many aspects need to be studied in depth. We need to continue to search for genes with resistance to root-knot nematode disease， and to fully understand the resistance mechanisms of disease-resistant genes， use molecular marker technology and transgenic technology to cultivate more disease-resistant varieties， and accelerate the transformation of commercial varieties.

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