TOWARD MAP-BASED CLONING OF ROOT-KNOT NEMATODE RESISTANT GENES IN COTTON

¹ Department of Soil & Crop Sciences and Crop Biotechnology Center, Texas A&M University, College Station, TX 77843-2474 USA

² USDA-ARS, 2765 F & B Road, College Station, TX 77845 USA

³ USDA-ARS, 2413 E. Hwy 83, Weslaco, TX 78596 USA

The root-knot nematode is a major constraint to maintaining and increasing cotton fiber productivity and quality. It not only seriously impairs cotton roots but also significantly increases the incidence and severity of Fusarium wilt, a major cotton disease that often occurs in association with the root-knot nematode. To tag, map and clone the root-knot nematode resistant gene (RNR), several approaches have been used. First, we developed ten pairs of DNA pools from nearly-isogenic lines (NILs) for the RNR and used as templates in PCR analysis of the NILs. A total of 700 random decamer primers were screened, from which 6 DNA fragments showing apparent difference between the susceptible and resistant lines were identified; one of them is present in all resistant lines but not in the susceptible lines. This DNA fragment was shown single-copy in the cotton genome by Southern analysis. Second, a partial BAC library (2.3x genome coverages) was previously generated from the G. hirsutum TAMCOT. The library was screened using the cloned sugar beet cyst nematode resistant gene Hs1^pro-1 cDNA as a probe and two positive BACs were isolated. Third, studies have demonstrated that some domains of the genes resistant to nematodes, bacteria, fungi, and viruses are highly conserved at the animo acid sequence level among a variety of plant species. According to the P-loop and the membrane spinning domain consensus sequences of several cloned disease resistant genes, we have designed a pair of degenerate primers and analyzed the highly RNR line Auburn 634 genomic DNA with these primers. One 500 bp and one 700 bp bands were obtained and cloned. Three random clones from the 500 bp band were sequenced. The results showed that the nucleotide seqeunces of these clones have similarities of 61 - 63% to the soybean disease resistance protein homolog genes (RLGs), Nicotiana glutinosa virus resistance gene (N) and the Arabidopsis downy mildew resistance gene. To determine the relationships of these DNA markers and clones with the RNR genes, three mapping populations have been or are being developed from Deltapine 16 (susceptible) x Auburn 623 (RNR), Deltapine 16 x Wild Mexican Jack Jones (RNR), and G. hirsutum Auburn 623 x G. barbadense. To clone the RNR genes by map-based cloning, a large-insert BAC library is being developed from the Auburn 623.