Greenhouse evaluation and inheritance analysis of transgenic cotton expressing <Emphasis Type="Italic">PTM3</Emphasis>, a MADS-box gene of Aspen

The PTM3 (MADS-box) gene characterized from flower of Aspen was ectopically expressed in cotton and was found to result in desirable agronomic traits. Among several transgenic lines, the PTM3 cotton event-10 was found to flower significantly earlier than the control and yield better [Ramachandran et al. (2011)]. We report our findings on performance based on greenhouse evaluation and inheritance of PTM3 cotton event-10. The T₁ progeny of event-10 were grown in pots under RBD design. The progeny were confirmed by kanamycin imbibition test, PCR, and GUS assay which showed the segregation of transgene at about 3:1 ratio. GUS assay was performed with pollen from all transgenic plants; plants that expressed gus in all pollen versus those that showed segregation for expression were found to be at a ratio of 1:2. Similar to observations made in the T₀ generation of event-10, the agronomic evaluation of T₁ progeny exhibited, on an average, earliness of 13 days in flowering, 13.5 days in crop maturity, and 22% of yield enhancement. The T₂ progeny of homozygous lines were grown on field soil in the greenhouse under strip trial design to see the effect as observed from potted plants of T₁ progeny. Inheritance of transgene cassette to T₂ progeny was confirmed by the same tests used to analyze T₁ progeny. As exhibited by T₀ and T₁ progenies, the T₂ progeny also showed earliness of 8 days in flowering, 12 days in crop maturity, and 17% of yield enhancement. The data generated from the progeny over two generations confirms that the PTM3 cotton event-10 is superior in agronomic characters as compared to non-transgenic cotton and is of interest for breeding shorter duration varieties with improved yield.     

Agrobacterium-mediated genetic transformation and production of semilooper resistant transgenic castor (Ricinus communis L.)

Summary Semilooper resistant transgenic castor plants were produced through Agrobacterium-mediated genetic transformation method. Two castor cultivars, Jyothi and VP1 were transformed using the super-binary vector pTOK233 carrying gus A and hpt genes. Putative transformants were regenerated following selection on the hygromycin containing medium. GUS positive primary transformants, when subjected to Southern analysis, revealed stable integration of gus A into their genomes. In the T₁ generation, a monogenic segregation ratio of 3 GUS positive: 1 GUS negative plants was observed. Furthermore, transformation experiments were carried out with the Agrobacterium pSB111 super-binary vector carrying a synthetic delta endotoxin gene cryIAb and the herbicide resistance gene bar both driven by cauliflower mosaic virus 35S promoter. Putative transformants were regenerated through selection on the phosphinothricin containing medium and Basta tolerant transformants were subjected to molecular analysis. PCR analysis revealed the presence of both bar and cryIAb genes in the Basta tolerant primary transformants. Southern analysis of PCR positive plants with cryIAb probe showed a 3 Kb band upon HindIII digestion and a > 6 Kb band with BamHI digestion, thus suggesting stable integration of cryIAb intact expression cassette and independent nature of the transformants. The primary transformants subjected to ELISA disclosed varied levels of Cry protein. These transgenics expressing cryIAb – when bioassayed against freshly hatched semilooper larvae – induced substantial (> 88%) insect mortality. Southern analysis of 2T₁ plants revealed the presence of cryIAb gene, indicating stable inheritance of the transgene into the next generation. In T₁, all the Southern-positive plants for cryIAb invariably exhibited tolerance to Basta, denoting co-segregation of both bar and cryIAb genes. Transgenics, expressing cryIAb exhibited ample resistance against the castor semilooper.     

Amenability of castor to an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation strategy using a <Emphasis Type="Italic">cry1AcF</Emphasis> gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T₁ generation seedlings on 300 mg L⁻¹ kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T₂ generation plants proved the stability of the transgene indicating the feasibility of the method.     

Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants

Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T₀). This chimeric plant passed the transferred nptII gene to its T₁ progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T₀ spikes and 15 T₁ offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T₂ and T₃ plants were produced by isolating embryos from green grains of transgenic T₁ and T₂ plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T₂ offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin ^R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.     

Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants

The agronomic and morphological characteristics of Agrobacterium-transformed rice plants carrying the synthetic cry1Ab or cry1Ac gene were investigated. Tremendous variations in plant height, seed fertility, grain size and other traits were seen in 80 T₁ lines, derived from 80 T₀ plants of 9 rice varieties. On average, about 33% T₁ lines had either morphological or agronomic variant plants. Most of the variations in T₁ plants had no significant correlation with transgene insertion and were proved heritable to their progenies. Genetic analysis in T₃ or T₄ generations showed some simple mutations such as chlorophyll deficiency and stunted plants were independent of transgene insertion and seemed to be controlled by a pair of single genes. However, in two independent transgenic progenies of Xiushui 11, all plants homozygous for transgenes showed dwarfism while all hemizygous and null segregants had normal plant heights. Two advanced homozygous Bt lines, KMD1 and KMD2, were developed from these two progenies. Comparison of the agronomic traits of KMD1 and KMD2 with their parent displayed marked differences among them in terms of seedling growth, tillering ability, yield components and yield potential. The genetic variation observed was generally not linked to the transgene locus and was ascribed to somaclonal variation, but other causes might also exist in particular cases. The results are discussed in the context of choosing appropriate transformation methodology for rice breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of a backcross population from a multi-copy transgenic Brassica napus line

Brassica napus is one of the crops at the forefront of biotechnological development. The procedures used to produce transgenic varieties are all prone to generating plants with multiple transgene copies. There has been considereable interest in the behaviour of such multi-copy lines because gene dosage rarely appears to correlate simply with gene expression. Here we report the analysis of a population of 107 progeny from a B. napus transformant containing multiple copies of a GUS marker gene construct. A total of 12 GUS sequence copies were identified including one that was non-functional. The expression of GUS increased with increasing copy number but this increase only made up a small proportion of the total variation between lines. There was no evidence of interaction between the various GUS copies and they appeared to segregate independently. The variation between progeny lines indicated that the number of gene copies was not a good guide to the expression of the gene product and hence that the expression of the gene in progeny from a multiple-copy parent could not be predicted. The importance of these findings in relation to plant breeding and the risk assessment process is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

The inheritance of days to flowering in broccoli (Brassica oleracea var. italica)

Summary Crosses between an early flowering inbred broccoli (Brassica oleracea L. var. italica) HS140 and four later maturing inbred lines, S301, S310, s318, and S258, were studied to determine the inheritance of earliness as expressed by days to first open flower. Mean days to first flower for F₁ and F₂ were almost identical, and were close to the mean of the two parents in three crosses, indicating additive inheritance. In the cross HS140×S258, for which no F₁ plants were available, the F₂ mean was closer to the mean of the early parent, but this was likely due to a distortion of the data caused by the very late maturation of S258, the latest maturing parent in the study. Frequency distribution for parents and progenies supported the conclusions from parent and progeny mean values and indicated that days to maturity is a quantitative character, with mostly additive inheritance.Oregon Agricultural Experiment Station Technical Paper No. 7578.     

GUS基因和NPTⅡ基因表达的相关性及其在转基因棉花检测研究中的应用

利用农杆菌介导转化法,将含有35S启动子驱动NPTII基因和GUS基因以及棉纤维特异表达启动子E6驱动目的基因FB的植物表达载体转入到常规棉花R15中.重点分析了GUS基因和NPTII基因在愈伤诱导阶段、T0代及T1代转基因棉花中的表达情况.综合两个基因的表达来进行转基因棉花的阳性鉴定,可以为转基因棉花后代的纯合选育提供双重保障.7个转基因株系选育到T3代共获得株行51个,卡那霉素检测多数株行阳性率在90%以上,其中21个株行阳性率达100%.     

Transge ne inheritance, segregation and expression in bread wheat

Transgene integration, inheritance and expression were studied in six transgenic wheat (Triticum aestivum) lines produced by co-bombardment with two plasmids containing marker genes and genes encoding HMW subunits of wheat glutenin, respectively. Transgene insertion number ranged from 1 to approximately 15. Within a transgenic locus the majority of plasmid copies were found at dispersed genomic sites separated by intervening DNA. However, evidence was obtained for the arrangement of introduced plasmid copies as concatamers and for plasmid truncation and rearrangement. Transgenes were frequently located in genetically unlinked chromosome sites, resulting in independent segregation of loci among progeny. In two lines this gave rise to progeny containing only the gene of interest. Transgenes were inherited in the T₁ generation as a dominant trait although Mendelian segregation ratios were not always observed. No evidence of co-suppression of endogenous HMW subunits was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance and field performance of transgenic Korean Bt rice lines resistant to rice yellow stem borer

Transgenic Korean rice plants containing the cry1Ab gene were developed for resistance against yellow stem borer (Scirpophaga incertulas, YSB). More than 100 independent transgenic lines from three Korean varieties (P-I, P-II and P-III) were generated. The amount of Cry1Ab in transgenic T₀ plants was as high as 2.88% of total soluble proteins. These levels were sufficient to cause 100% mortality of YSB larvae. The majority of T₁ transgenic lines originated from the varieties P-I and P-II followed a Mendelian fashion of segregation. Deviation from the expected segregation ratio was observed in a small number of the transgenic lines of P-I and P-II origins. However, this deviation was primarily observed in the P-III originated lines. Segregation analysis of the T₁ generation indicated that 1–3 copies of the cry1Ab gene were integrated into the genome of the majority of the transgenic lines originating from varieties P-I and P-II. Stunted and semi-fertile mutants were observed in some transgenic lines. These aberrations were either independent or closely linked to the introduced cry1Ab gene loci in different transgenic lines. Reduction in GUS expression levels and loss of toxicity against YSB larvae were found in some transgenic lines. The transgenic T₃ and T₄ lines causing 100% mortality of third instar YSB larvae in the lab were completely protected in the field. Analysis of important yield components on nine selected transgenic lines indicated that stem length, panicle length, grain number per panicle, and seed setting rates were reduced in transgenic plants compared to those in non-transgenic parental rice lines. Number of panicles per cluster, however, was significantly higher in transgenic plants. The numerical value of the average yield was in general greater in the controls than in all the transgenic lines, indicating some ‘yield drag’. Since some selected lines were highly resistant to the YSB with good yielding potential, they offer effective potential for use in insect resistance management programs.     

Genetical analysis of resistance to Mycosphaerella pinodes in pea seedlings

Summary In studies of the inheritance of resistance, pea seedlings of seven lines in which stems and leaves were both resistant to Mycosphaerella pinodes were crossed with a line in which they were both susceptible. With seven of the crosses resistance was dominant to susceptibility. When F₂ progenies of five crosses were inoculated on either stems or leaves independently, phenotypes segregated in a ratio of 3 resistant: 1 susceptible indicating that a single dominant gene controlled resistance. F₂ progenies of one other cross gave ratios with a better fit to 9 resistant: 7 susceptible indicating that two co-dominant genes controlled resistance. The F₂ progeny of another cross segregated in complex ratios indicating multigene resistance.When resistant lines JI 97 and JI 1089 were crossed with a susceptible line and leaves and stems of each F₂ plant were inoculated, resistance phenotypes segregated independently demonstrating that leaf and stem resistance were controlled by different genes. In two experiments where the F₂ progeny of the cross JI 97×JI 1089 were tested for stem and leaf resistance separately, both characters segregated in a ratio of 15 resistant:1 susceptible indicating that these two resistant lines contain two non-allelic genes for stem resistance (designated Rmp1 and Rmp2) and two for leaf resistance (designated Rmp3 and Rmp4). Evidence that the gene for leaf resistance in JI 1089 is located in linkage group 4 of Pisum sativum is presented.     

Interspecific hybridisation between Hibiscus syriacus, Hibiscus sinosyriacus and Hibiscus paramutabilis

Interspecific hybrids from crosses between H. syriacus × H. paramutabilis and H. syriacus × H. sinosyriacus were obtained. In both cases unilateral incongruity was observed and reciprocal crosses yielded no fruits. In vitro embryo rescue, 11 weeks after pollination, increased the germination capacity of H. syriacus × H. sinosyriacus embryos, while this was not the case for H. syriacus × H. paramutabilis embryos. However, a lot of the generated H. syriacus × H. sinosyriacus seedlings were lost due to variegated and total albinism. In the progeny of H. syriacus ‘Oiseau Bleu’ × H. paramutabilis about 95% of the seedlings had an intermediate flower and leaf morphology compared to both parent plants. Leaves on the adult F1 hybrids showed a polymorphism. In total four different leaf types could be observed on the same plant. This leaf polymorphism also was seen in the progeny of H. syriacus ‘Melwhite’ × H. sinosyriacus ‘Lilac Queen’. In this progeny about 50% of the seedlings had an intermediate flower and leaf morphology compared with the parent plants. The hybrid nature of the seedlings of both progenies was also confirmed by AFLP analysis. Despite the low germination vigour of the pollen of the hybrids, a small F2 generation was obtained from H. syriacus ‘Oiseau Bleu’ × H. paramutabilis.     

Inheritance of white flower colour and congested growth habit in certain Buddleia progenies

Summary Buddleia fallowiana var. alba (FA) was crossed with B. davidii Nanhoensis Alba (NA) and both were crossed with the B. davidii cultivars Pink Delight (PD) and Royal Red; seedlings from the FA x NA progeny were backcrossed to the two parents. The pattern of inheritance of white or coloured flowers indicates two loci controlling white flower colour. FA is homozygous for a recessive gene, labelled alb-1, and NA is heterozygous for a dominant gene, Alb-2.In most segregating progenies some of the white flowered seedlings had a congested habit of growth and this is tentatively ascribed to two dominant complementary genes. NA is heterozygous for Con-1, which is tightly linked with Alb-2. FA and PD are heterozygous for Con-2.     

转Xa23基因水稻的白叶枯病抗性及其遗传分析

在分离克隆抗白叶枯病基因Xa23研究中获得大量转基因水稻材料。为了系统研究转Xa23基因水稻的抗病稳定性和遗传模式, 本文通过逐株进行抗白叶枯病接种鉴定、PCR和Southern blot分子检测, 对一批转Xa23基因水稻植株进行了T₀代到T₂代的跟踪分析。结果表明, Xa23基因的整合和表达, 使感病受体品种牡丹江8号获得抗病性。由于Xa23基因插入受体基因组的位点不同, 同是单拷贝插入的转基因T₀代抗病植株, 其抗病程度有明显差异。T₀代植株的抗病程度, 可以准确、稳定地遗传到T₁代和T₂代。单拷贝转基因植株分离群体的抗感植株分离比接近3∶1, 表明转Xa23基因遵循孟德尔单基因遗传模式。已获得2个纯合的单拷贝转基因抗病株系, 它们的抗病程度稍有差别, 将用于外源基因插入位置效应分析和杂交稻抗病育种。     

Inheritance of slow rusting to stem rust in wheat

Summary The inheritance of the slow rusting character was studied on F₅ progenies from seven spring wheat cultivars (Triticum aestivum) crossed in all possible combinations without reciprocals. The cultivars and their progenies were evaluated for slow rusting in 1974 and 1975 in epidemics of Puccinia graminis f. sp. tritici, races 15 and 151, and traces of other races. Slow rusting varied significantly among the parents and among the F₅ progeny of each cross. Transgressive segregation occurred in each cross, i.e. some progeny rusted more slowly than the parents and some faster. In crosses with both Idaed 59 and Kenya 58 the progeny distributions were skewed towards slow rust development but the distributions in the other crosses were normal. The genetic control of slow rusting was predominantly additive, and narrow sense heritability was approximately 80 percent. The number of segregating genes having an effect on slow rusting was estimated to be 2 to 12 pairs depending on the cross. Correlation between slow rusting and maturity was usually negative but in most crosses the relationship was small.Contribution No. 9624 from the Agricultural Experiment Station, University of Minnesota, St. Paul, Minnesota 55108.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.     

Inheritance of neurotoxin (ODAP) content, flower and seed coat colour in grass pea (Lathyrus sativus L.)

Summary A strong epidemiological association is known to exist between the consumption of grass pea and lathyrism. A neurotoxin, -N-Oxalyl-L-, -diaminopropanoic acid (ODAP) has been identified as the causative principle. This study was undertaken to investigate the mode of inheritance of the neurotoxin ODAP, flower and seed coat colour in grass pea. Five grass pea lines with low to high ODAP concentration were inter-crossed in all possible combinations to study the inheritance of the neurotoxin. Parents, F₁ and F₂ progenies were evaluated under field condition and ODAP analyzed by an ortho-phthalaldehyde spectrophotometric method. Many of the progenies of low x low ODAP crosses were found to be low in ODAP concentration indicating the low ODAP lines shared some genes in common for seed ODAP content. The F₁ progenies of the low ODAP x high ODAP crosses were intermediate in ODAP concentration and the F₂ progenies segregated covering the entire parental range. This continuous variation, together with very close to normal distribution of the F₂ population both of low x low and low x high ODAP crosses indicated that ODAP content was quantitatively inherited. Reciprocal crosses, in some cases, produced different results indicating a maternal effect on ODAP concentration. Blue and white flower coloured lines of grass pea were inter-crossed to study the inheritance of flower colour. Blue flower colour was dominant over the white. The F₂ progenies segregated in a 13:3 ratio indicating involvement of two genes with inhibiting gene interactions. The gene symbol LB for blue flower colour and LW for white flower colour is proposed.     

Salt-tolerance in Brassica juncea L. I. In vitro selection,agronomic evaluation and genetic stability

Summary In vitro selection of salt tolerant plants of Brassica juncea L. (Indian mustard) cv. Prakash has been accomplished by screening highly morphogenic cotyledon explant cultures on high NaCl media. Out of a total of 2,620 cotyledons cultured on high salt medium, 3 survived, showed sustained growth and regenerated shoots. They were multiplied by axillary bud culture on NaCl free medium. The salt-selected shoots retained salt tolerance following 3 month of growth and multiplication on control medium. While two of these somaclones flowered and set seeds, third one grew slowly, had abnormal leaf morphology and was sterile. The seed of the two fertile plants were sown in the field to raise R₁ segregating generation. Data were recorded for field, other agronomic components and oil content. The somaclonal lines, both selected salt-tolerant and non-selected, showed tremendous amount of variation for all the characters studied. One of the two tolerant somaclones invariably showed reduced height, longer reproductive phase and higher 1000 seed weight. Based on the agronomic performance of R₁ plants of these somaclones, some plants were selected and their progeny were evaluated for agronomic performance under standard field conditions and salt-tolerance in the greenhouse using sand pot culture method. Most of the lines bred true for their specific characteristics. In the greenhouse, selected salt-tolerant somaclones (SR-2 and SR-3) performed better for plant growth, yield and other agronomic traits at higher salt treatments, indicating thereby that salt-tolerance trait selected in vitro was expressed in the whole plants and is genetically stable and transmitted onto the progeny. The two tolerant lines, however, differed in their salt-tolerance during vegetative and reproductive phases as indicated by their salt-tolerance and stress susceptibility indices. The mechanism of salt-tolerance is not clear and needs to be further investigated.     

Means and variances for Fusarium head blight resistance of F2-derived bulks from winter triticale and winter wheat crosses

Fusarium head blight (FHB), caused by Fusarium graminearum and Fusarium culmorum, is a devastating disease in cereals. This study was undertaken to estimate progeny means and variances in each of five winter triticale and winter wheat crosses using unselected F₂−derived lines in F₄ or F₅ generation bulked at harvest of the previous generation. Fifty (triticale) and 95 (wheat) progeny per cross were inoculated in two (triticale) or three (wheat) field environments. FHB rating was assessed on a whole-plot basis. Mean disease severities of the parents ranged from 2.3 to 6.4 in triticale and from 3.1 to 6.5 in wheat on a 1-to-9 scale (1 = symptomless, 9 = 100% infected). The midparent values generally resembled the means of their derived progeny. Significant (P < 0.01) genotypic variance was detected within each cross, but genotype × environment interaction and error variances were also high for both crops. Medium to high entry-mean heritabilities (0.6–0.8) underline the feasibility of selecting F₂-derived bulks on a plot basis in several environments. Phenotypic correlation of FHB resistance between generation F_2:4 and F_2:5 was r = 0.87 (P < 0.01) tested across 150 wheat bulks at two locations. Our estimates of selection gain are encouraging for breeders to improve FHB resistance in triticale and wheat by recurrent selection within adapted materials.     

Embryonal recombination and germline inheritance of recombined FRT loci mediated by constitutively expressed FLP in tobacco

FLP site-specific recombination has been shown in transgenic plants to excise DNA sequences between target FRT sites, and thereby activate transgenes in plants. In previous reports, crossing of tobacco plants expressing FLP recombinase from a CaMV-35s promoter with plants containing the target FRT sites, hybrid plants with deletion sectors were generated, which were infrequently transmitted to progeny. In this report we evaluate the occurrence of recombination in F₁ hybrid seed derived from crosses of different FLP and FRT-reporter target lines and the germinal transmission of recombined loci from these hybrids to F₂ progeny. Twenty hybrids were generated from crosses of independent five FLP-active lines and four FRT-reporter target lines. In one hybrid, FLP deletions occurred at an early stage, prior to seed maturation, and the deletions from this hybrid were more efficiently transmitted to F₂ progeny. The demonstration of FLP-mediated recombination activity and germinal inheritance of the recombined FRT loci are supported by both molecular and enzymatic evidence. This revised version was published online in August 2006 with corrections to the Cover Date.