The mosquito Anopheles gambiae s.s. is a widespread and efficient vector of malaria across sub-Saharan Africa. This species also is characterized by abundant chromosomal inversion polymorphisms that are considered important sources of ecological flexibility . Inversion polymorphisms are distributed non-randomly in the A. gambiae genome, occurring exclusively on chromosome 2 and predominantly on its right arm (6 of 7 most widely distributed polymorphic inversions, i.e. 2Rj, 2Rb, 2Rc, 2Rd, 2Ru, 2Rbk, 2La; ). This suggests that the inversions (or genes captured within them) are targets of selection, contributing to more efficient exploitation of the environment and ultimately increased malaria transmission .
Not only are inversions distributed non-randomly at the genome level, but also at the population level. In Mali, West Africa, the four most frequent polymorphic inversions (2Rj, b, c, and u) are not found in all possible combinations (karyotypes) even when sampled from the same village, counter to expectation based on random mating. Certain karyotypes are completely absent or significantly reduced in frequency. Based on these data, A. gambiae could be subdivided into three assortatively mating chromosomal forms each recognized by characteristic arrangements of 2R: j, b, cu and bcu for SAVANNA; bc and u for MOPTI; jcu and jbcu for BAMAKO [3, 4]. Moreover, ecological and physiological differences distinguish chromosomal forms. Whereas SAVANNA is associated with rain-dependent temporary pools far from rivers and irrigated zones and reproductively active only in the rainy season, MOPTI is associated with irrigated agricultural sites and remains reproductively active into the dry season where permanent or semi-permanent larval breeding sites are available. The MOPTI form is distributed mainly in the central part of West Africa, while SAVANNA is broadly distributed in the African savannas, extending east of the Rift Valley. BAMAKO is much more restricted in its range. It is associated with the Niger River and its tributaries in southern Mali and northern Guinea Conakry, and like SAVANNA is reproductively quiescent in the dry season. The individual inversions comprising karyotypic differences between chromosomal forms cannot contain reproductive barriers per se, as they are shared between forms by common ancestry as well as ongoing gene flow . However, taken together, the set of 2R arrangements typical of each chromosomal form may reflect the boundaries of different ecological preferences and/or tolerances which can lead to divergence and ultimately speciation.
An important goal driving population genomics studies of A. gambiae is to uncover mechanisms for ecological divergence and assortative mating. Addressing those questions requires the development of efficient methods for recognizing inter- and intraspecific subdivisions that do not rely on traditional karyotype analysis – a method limited both by paucity of cytogenetic expertise and availability of properly preserved samples at the correct developmental stage (half-gravid females). In 1997, Favia et al  reported fixed differences in the X-linked rDNA that distinguish MOPTI from SAVANNA and BAMAKO chromosomal forms in Mali; the latter were indistinguishable at this marker. The fixed rDNA differences defined molecular forms M and S of A. gambiae, now considered incipient species [6, 7]. The molecular forms are not characterized by form-specific inversion polymorphisms, although these are found at different frequencies in M and S depending on their geographical origin. The only known exception to this general observation concerns the 2Rj inversion, which is exclusive to the S-form according to available records . Within the S molecular form, the 2Rj inversion is present in the homozygous state in association with the 2Rcu arrangement in the chromosomal form BAMAKO (i.e., jcu/jcu, jcu/jbcu, jbcu/jbcu), while it may be polymorphic and associated with different chromosomal arrangements in some SAVANNA populations of West Africa .
To date, attempts to find fixed molecular differences between SAVANNA and BAMAKO chromosomal forms have failed, though molecular evidence for restricted gene flow exists [10, 11]. Based on the fact that the BAMAKO form is characterized by 2Rj in the homozygous state and that sympatric SAVANNA populations harbor only low levels of 2Rj polymorphism, the recently characterized breakpoint junctions of this inversion  were exploited to develop a PCR diagnostic assay for molecular determination of the 2Rj karyotype of A. gambiae specimens and thus identification of BAMAKO specimens in Mali and other Afrotropical sites where the BAMAKO chromosomal form is prevalent over the 2Rj-carrier SAVANNA. Here, the assay is presented together with the results of extensive validation involving field collections from Mali and three other West and western Central African countries. Samples were karyotyped using traditional and molecular methods in parallel. Overall, molecular and cytogenetic results were almost perfectly congruent for 2Rj standard and inverted homokaryotypes. Thus, in areas of sympatry between BAMAKO and SAVANNA populations with a low frequency of 2Rj (mainly in Mali), molecular karyotyping of 2Rj will be a rapid and effective approach to identify and specifically study the BAMAKO chromosomal form. However, 2Rj inversion heterozygotes were frequently misclassified as 2Rj standard by PCR-based analysis, possibly owing to molecular divergence (null alleles) of 2Rj chromosomes between isolated SAVANNA populations.